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Ahmad F, Ma L, Wei W, Liu Y, Hakim I, Daugherty A, Mujahid S, Radin AA, Chua MS, So S. Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma. Liver Int 2023; 43:2794-2807. [PMID: 37833852 DOI: 10.1111/liv.15756] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/20/2023] [Revised: 09/19/2023] [Accepted: 09/21/2023] [Indexed: 10/15/2023]
Abstract
BACKGROUND AND AIMS Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. METHODS We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its in vitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its in vivo efficacy. RESULTS CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, β-tubulin, glypican-3 (GPC3), β-catenin, and c-Myc. In vivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CONCLUSION CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, β-catenin, and c-Myc highlights a novel mechanism of action.
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Affiliation(s)
- Faiz Ahmad
- Asian Liver Center, Department of Surgery, School of Medicine, Stanford, California, USA
| | - Li Ma
- Asian Liver Center, Department of Surgery, School of Medicine, Stanford, California, USA
| | - Wei Wei
- Asian Liver Center, Department of Surgery, School of Medicine, Stanford, California, USA
| | - Yi Liu
- Asian Liver Center, Department of Surgery, School of Medicine, Stanford, California, USA
| | - Isaac Hakim
- Aria Pharmaceuticals, Palo Alto, California, USA
| | | | - Sana Mujahid
- Aria Pharmaceuticals, Palo Alto, California, USA
| | | | - Mei-Sze Chua
- Asian Liver Center, Department of Surgery, School of Medicine, Stanford, California, USA
| | - Samuel So
- Asian Liver Center, Department of Surgery, School of Medicine, Stanford, California, USA
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Qu WZ, Wang L, Chen JJ, Wang Y. Raf kinase inhibitor protein combined with phosphorylated extracellular signal-regulated kinase offers valuable prognosis in gastrointestinal stromal tumor. World J Gastroenterol 2023; 29:4200-4213. [PMID: 37475847 PMCID: PMC10354573 DOI: 10.3748/wjg.v29.i26.4200] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 05/20/2023] [Accepted: 06/02/2023] [Indexed: 07/10/2023] Open
Abstract
BACKGROUND Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Tyrosine kinase inhibitors, such as imatinib, have been used as first-line therapy for the treatment of GISTs. Although these drugs have achieved considerable efficacy in some patients, reports of resistance and recurrence have emerged. Extracellular signal-regulated kinase 1/2 (ERK1/2) protein, as a member of the mitogen-activated protein kinase (MAPK) family, is a core molecule of this signaling pathway. Nowadays, research reports on the important clinical and prognostic value of phosphorylated-ERK (P-ERK) and phosphorylated-MAPK/ERK kinase (P-MEK) proteins closely related to raf kinase inhibitor protein (RKIP) have gradually emerged in digestive tract tumors such as gastric cancer, colon cancer, and pancreatic cancer. However, literature on the expression of these downstream proteins combined with RKIP in GIST is scarce. This study will focus on this aspect and search for answers to the problem. AIM To detect the expression of RKIP, P-ERK, and P-MEK protein in GIST and to analyze their relationship with clinicopathological characteristics and prognosis of this disease. Try to establish a new prognosis evaluation model using RKIP and P-ERK in combination with analysis and its prognosis evaluation efficacy. METHODS The research object of our experiment was 66 pathologically diagnosed GIST patients with complete clinical and follow-up information. These patients received surgical treatment at China Medical University Affiliated Hospital from January 2015 to January 2020. Immunohistochemical method was used to detect the expression of RKIP, P-ERK, and P-MEK proteins in GIST tissue samples from these patients. Kaplan-Meier method was used to calculate the survival rate of 63 patients with complete follow-up data. A Nomogram was used to represent the new prognostic evaluation model. The Cox multivariate regression analysis was conducted separately for each set of risk evaluation factors, based on two risk classification systems [the new risk grade model vs the modified National Institutes of Health (NIH) 2008 risk classification system]. Receiver operating characteristic (ROC) curves were used for evaluating the accuracy and efficiency of the two prognostic evaluation systems. RESULTS In GIST tissues, RKIP protein showed positive expression in the cytoplasm and cell membrane, appearing as brownish-yellow or brown granules. The expression of RKIP was related to GIST tumor size, NIH grade, and mucosal invasion. P-ERK protein exhibited heterogeneous distribution in GIST cells, mainly in the cytoplasm, with occasional presence in the nucleus, and appeared as brownish-yellow granules, and the expression of P-ERK protein was associated with GIST tumor size, mitotic count, mucosal invasion, and NIH grade. Meanwhile, RKIP protein expression was negatively correlated with P-ERK expression. The results in COX multivariate regression analysis showed that RKIP protein expression was not an independent risk factor for tumor prognosis. However, RKIP combined with P-ERK protein expression were identified as independent risk factors for prognosis with statistical significance. Furthermore, we establish a new prognosis evaluation model using RKIP and P-ERK in combination and obtained the nomogram of the new prognosis evaluation model. ROC curve analysis also showed that the new evaluation model had better prognostic performance than the modified NIH 2008 risk classification system. CONCLUSION Our experimental results showed that the expression of RKIP and P-ERK proteins in GIST was associated with tumor size, NIH 2008 staging, and tumor invasion, and P-ERK expression was also related to mitotic count. The expression of the two proteins had a certain negative correlation. The combined expression of RKIP and P-ERK proteins can serve as an independent risk factor for predicting the prognosis of GIST patients. The new risk assessment model incorporating RKIP and P-ERK has superior evaluation efficacy and is worth further practical application to validate.
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Affiliation(s)
- Wen-Zhi Qu
- Department of General Surgery, The 4th Affiliated Hospital of China Medical University, Shenyang 110032, Liaoning Province, China
| | - Luan Wang
- Department of Emergency, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China
| | - Juan-Juan Chen
- Department of Medical Service, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China
| | - Yang Wang
- Department of General Surgery, The 4th Affiliated Hospital of China Medical University, Shenyang 110032, Liaoning Province, China
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Guo R, Liu T, Shasaltaneh MD, Wang X, Imani S, Wen Q. Targeting Adenylate Cyclase Family: New Concept of Targeted Cancer Therapy. Front Oncol 2022; 12:829212. [PMID: 35832555 PMCID: PMC9271773 DOI: 10.3389/fonc.2022.829212] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2021] [Accepted: 05/26/2022] [Indexed: 12/18/2022] Open
Abstract
The adenylate cyclase (ADCY) superfamily is a group of glycoproteins regulating intracellular signaling. ADCYs act as key regulators in the cyclic adenosine monophosphate (cAMP) signaling pathway and are related to cell sensitivity to chemotherapy and ionizing radiation. Many members of the superfamily are detectable in most chemoresistance cases despite the complexity and unknownness of the specific mechanism underlying the role of ADCYs in the proliferation and invasion of cancer cells. The overactivation of ADCY, as well as its upstream and downstream regulators, is implicated as a major potential target of novel anticancer therapies and markers of exceptional responders to chemotherapy. The present review focuses on the oncogenic functions of the ADCY family and emphasizes the possibility of the mediating roles of deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) in ADCY as a prognostic therapeutic target in modulating resistance to chemotherapy and immunotherapy. It assesses the mediating roles of ADCY and its counterparts as stress regulators in reprogramming cancer cell metabolism and the tumor microenvironment. Additionally, the well-evaluated inhibitors of ADCY-related signaling, which are under clinical investigation, are highlighted. A better understanding of ADCY-induced signaling and deleterious nsSNPs (p.E1003K and p.R1116C) in ADCY6 provides new opportunities for developing novel therapeutic strategies in personalized oncology and new approaches to enhance chemoimmunotherapy efficacy in treating various cancers.
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Affiliation(s)
- Rui Guo
- Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, China
| | - Tian Liu
- Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, China
| | | | - Xuan Wang
- China Regional Research Center, International Centre for Genetic Engineering and Biotechnology Taizhou, Jiangsu, China
| | - Saber Imani
- Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, China
- China Regional Research Center, International Centre for Genetic Engineering and Biotechnology Taizhou, Jiangsu, China
- *Correspondence: Saber Imani, ; QingLian Wen,
| | - QingLian Wen
- Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, China
- *Correspondence: Saber Imani, ; QingLian Wen,
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Deng J, Gutiérrez LG, Stoll G, Motiño O, Martins I, Núñez L, Bravo-San Pedro JM, Humeau J, Bordenave C, Pan J, Fohrer-Ting H, Souquere S, Pierron G, Hetz C, Villalobos C, Kroemer G, Senovilla L. Paradoxical implication of BAX/BAK in the persistence of tetraploid cells. Cell Death Dis 2021; 12:1039. [PMID: 34725331 PMCID: PMC8560871 DOI: 10.1038/s41419-021-04321-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 09/28/2021] [Accepted: 10/06/2021] [Indexed: 12/22/2022]
Abstract
Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of β-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.
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Affiliation(s)
- Jiayin Deng
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
| | - Lucía G Gutiérrez
- Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid - CSIC, Valladolid, Spain
| | - Gautier Stoll
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
| | - Omar Motiño
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
| | - Isabelle Martins
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
| | - Lucía Núñez
- Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid - CSIC, Valladolid, Spain
| | - José Manuel Bravo-San Pedro
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
- Facultad de Medicina, Departamento de Fisiología, Universidad Complutense de Madrid, Madrid, Spain
| | - Juliette Humeau
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
- Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, QC, Canada, H3C 3J7
- Department of Medicine, Université de Montréal, Montreal, QC, Canada, H3C 3J7
| | - Chloé Bordenave
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
| | - Juncheng Pan
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France
| | - Hélène Fohrer-Ting
- Centre de Recherche des Cordeliers, Center for Histology, Cell Imaging and Cytometry (CHIC), Sorbonne Université, Inserm, Université de Paris, F-75006, Paris, France
| | | | - Gerard Pierron
- CNRS, UMR9196, Gustave Roussy Cancer Campus, Villejuif, France
| | - Claudio Hetz
- Faculty of Medicine, Biomedical Neuroscience Institute (BNI), University of Chile, Santiago, 8380453, Chile
- Center for Geroscience, Brain Health and Metabolism (GERO), Santiago, 7800003, Chile
- The Buck Institute for Research in Aging, Novato, CA, 94945, USA
| | - Carlos Villalobos
- Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid - CSIC, Valladolid, Spain.
| | - Guido Kroemer
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France.
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France.
- Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, France.
| | - Laura Senovilla
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, F-75006, Paris, France.
- Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France.
- Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid - CSIC, Valladolid, Spain.
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Huang W, Hua H, Xiao G, Yang X, Yang Q, Jin L. ZC3HAV1 promotes the proliferation and metastasis via regulating KRAS in pancreatic cancer. Aging (Albany NY) 2021; 13:18482-18497. [PMID: 34319912 PMCID: PMC8351712 DOI: 10.18632/aging.203296] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Accepted: 06/19/2021] [Indexed: 12/14/2022]
Abstract
Proliferation and metastasis are important malignant features of pancreatic cancer (PC), but the underlying molecular mechanism is unclear. ZC3HAV1, a PARP family member of proteins-enzymes, has been considered to play a significant part in a variety of biological processes. Nonetheless, the functions of ZC3HAV1 in developing PC are still unknown. This research aims to explore the biological function and the expression of ZC3HAV1 shown in PC. In our study, PCR analysis suggested that ZC3HAV1 was expressed at a high level in PC tissues and cell lines, and high ZC3HAV1 expression was remarkably related to poor prognosis. The functional assays indicated that upregulated ZC3HAV1 accelerated PC cell proliferation along with colony formation capacities in vitro. Subsequently, ZC3HAV1 could upregulate cyclin D1 and CDK2 and also promote G1/S transition in cells of PC. What's more, we also discovered that ZC3HAV1 promotes the migration and the invasion of PC cells. It upregulates the expression of EMT (epithelial-mesenchymal transition) relevant markers. Conversely, the functional assays showed that ZC3HAV1 knockdown significantly reduced tumorigenesis. Using bioinformatics analysis and immunoprecipitation assays we found that ZC3HAV1 could directly bind to KRAS and positively regulate its expression. Furthermore, ZC3HAV1 overexpression activated MAPK signaling by increasing p-ERK levels. Conversely, knockdown of KRAS attenuated ZC3HAV1-mediated promotion of proliferation and invasion in cells of PC. The result indicated that ZC3HAV1 was in relation to poor prognosis and accelerated the proliferation and metastasis of PC cells by regulation of KRAS. Our research may offer brand-new evidence to diagnose and treat PC in clinic.
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Affiliation(s)
- Wei Huang
- Department of Gastrointestinal Surgery, The First People’s Hospital of Neijiang, Neijiang, Sichuan, China
| | - Hao Hua
- Department of Hepatic-Biliary-Pancreatic Surgery, The First People’s Hospital of Neijiang, Neijiang, Sichuan, China
| | - Guoliang Xiao
- Department of Gastrointestinal Surgery, The First People’s Hospital of Neijiang, Neijiang, Sichuan, China
| | - Xianjin Yang
- Department of Gastrointestinal Surgery, The First People’s Hospital of Neijiang, Neijiang, Sichuan, China
| | - Qin Yang
- Department of Gastroenterology, The First People’s Hospital of Neijiang, Neijiang, Sichuan, China
| | - Lu Jin
- Department of Pediatrics, The First People’s Hospital of Neijiang, Neijiang, Sichuan, China
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Hawash M, Kahraman DC, Cetin-Atalay R, Baytas SN. Induction of Apoptosis in Hepatocellular Carcinoma Cell Lines by Novel Indolylacrylamide Derivatives: Synthesis and Biological Evaluation. Chem Biodivers 2021; 18:e2001037. [PMID: 33713038 DOI: 10.1002/cbdv.202001037] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Accepted: 03/12/2021] [Indexed: 12/22/2022]
Abstract
Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and one of the leading causes of cancer associated death worldwide. This is due to the highly resistant nature of this malignancy and the lack of effective treatment options for advanced stage HCC patients. The hyperactivity of PI3K/Akt and Ras/Raf/MEK/ERK signaling pathways contribute to the cancer progression, survival, motility, and resistance mechanisms, and the interaction of these two pathways are responsible for the regulation of cancer cell growth and development. Therefore, it is vital to design and develop novel therapeutic options for HCC treatment targeting these hyperactive pathways. For this purpose, novel series of trans-indole-3-ylacrylamide derivatives originated from the lead compound, 3-(1H-indole-3-yl)-N-(3,4,5-trimethoxyphenyl)acrylamide, have been synthesized and analyzed for their bioactivity on cancer cells along with the lead compound. Based on the initial screening, the most potent compounds were selected to elucidate their effects on cellular signaling activity of HCC cell lines. Cell cycle analysis, immunofluorescence, and Western blot analysis revealed that lead compound and (E)-N-(4-tert-butylphenyl)-3-(1H-indole-3-yl)acrylamide induced cell cycle arrest at the G2/M phase, enhanced chromatin condensation and PARP-cleavage, addressing induction of apoptotic cell death. Additionally, these compounds decreased the activity of ERK signaling pathway, where phosphorylated ERK1/2 and c-Jun protein levels diminished significantly. Relevant to these findings, the lead compound was able to inhibit tubulin polymerization as well. To conclude, the novel trans-indole-3-ylacrylamide derivatives inhibit one of the critical pathways associated with HCC which results in cell cycle arrest and apoptosis in HCC cell lines.
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Affiliation(s)
- Mohammed Hawash
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey.,Present address, Department of Pharmacy, Faculty of Medicine and Health Sciences, An-Najah National University, Nablus, Palestine
| | - Deniz Cansen Kahraman
- Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, 06800, Ankara, Turkey
| | - Rengul Cetin-Atalay
- Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, 06800, Ankara, Turkey
| | - Sultan Nacak Baytas
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey
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Yao W, Lin Z, Wang G, Li S, Chen B, Sui Y, Huang J, Liu Q, Shi P, Lin X, Liu Q, Yao H. Delicaflavone induces apoptosis via mitochondrial pathway accompanying G2/M cycle arrest and inhibition of MAPK signaling cascades in cervical cancer HeLa cells. PHYTOMEDICINE : INTERNATIONAL JOURNAL OF PHYTOTHERAPY AND PHYTOPHARMACOLOGY 2019; 62:152973. [PMID: 31177019 DOI: 10.1016/j.phymed.2019.152973] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/07/2019] [Revised: 05/22/2019] [Accepted: 05/25/2019] [Indexed: 05/22/2023]
Abstract
BACKGROUND Cervical cancer (CCa) represents the fourth most common cause of cancer-related death in women worldwide. CCa therapy is still a major clinical challenge worldwide. Finding and developing new anti-CCa chemotherapeutic drugs is a very significant issue. Delicaflavone is a rare biflavonoid from Selaginella doederleinii Hieron, which has shown strong anti-cancer activities in our preliminary screening. PURPOSE The present study aimed to investigate the apoptotic effect and mechanism of delicaflavone against CCa. METHODS In this study, the effect and potential mechanism of delicaflavone against CCa were investigated in vitro and in vivo by MTT assay, TEM, flow cytometry, western blot assay, qPCR assay, immunofluorescence assay and the mouse xenograft tumor model. RESULTS It was confirmed that delicaflavone inhibited the proliferation of human CCa HeLa cells, and induced morphological changes, G2/M phase arrest and apoptosis in a dose- and time-dependent manner. HeLa cells treated with delicaflavone showed the loss of mitochondrial membrane potential, release of Cytochrome c, activation of caspases, alteration of Bax/Bcl-2 balance, and the inhibition of MAPK signaling cascades. Furthermore, delicaflavone significantly decreased tumor growth in a dose-dependent manner without apparent side effects in a xenograft tumor model of HeLa cells. Immunohistochemistry analysis confirmed the up-regulation of Caspase-9, Caspase-3, Bax protein and down-regulation of Bcl-2 protein in the xenografts tumors, which was consistent with the results in vitro. CONCLUSION The results of the current study show that apoptosis is induced by the mitochondrial pathway accompanying with G2/M cycle arrest and inhibition of MAPK signaling cascades in human CCa HeLa cells, which can be used as a promising therapeutic drug for CCa.
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Affiliation(s)
- Wensong Yao
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China; Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350122, China
| | - Zhen Lin
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China
| | - Gang Wang
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China; Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350122, China
| | - Shaoguang Li
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China
| | - Bing Chen
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China; Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350122, China
| | - Yuxia Sui
- Department of Pharmacy, Provincial Clinical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou 350001, China
| | - Jianyong Huang
- Department of Pharmaceutical, Fujian Medical University Union Hospital, Fuzhou 350001, China
| | - Qicai Liu
- Department of Reproductive Medicine Centre, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China
| | - Peiying Shi
- Department of Traditional Chinese Medicine Resource and Bee Products, Bee Science College, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Xinhua Lin
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China; Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350122, China.
| | - Qicai Liu
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China; Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350122, China
| | - Hong Yao
- Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China.
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Xie X, Zu X, Liu F, Wang T, Wang X, Chen H, Liu K, Wang P, Liu F, Zheng Y, Bode AM, Dong Z, Kim DJ. Purpurogallin is a novel mitogen-activated protein kinase kinase 1/2 inhibitor that suppresses esophageal squamous cell carcinoma growth in vitro and in vivo. Mol Carcinog 2019; 58:1248-1259. [PMID: 31100197 DOI: 10.1002/mc.23007] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2018] [Revised: 03/05/2019] [Accepted: 03/08/2019] [Indexed: 12/28/2022]
Abstract
Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.
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Affiliation(s)
- Xiaomeng Xie
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China
| | - Xueyin Zu
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China
| | - Feifei Liu
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China
| | - Ting Wang
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China
| | - Xiangyu Wang
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China
| | - Hanyong Chen
- The Hormel Institute, University of Minnesota, Austin, Minnesota
| | - Kangdong Liu
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China.,The Affiliated Cancer Hospital, Zhengzhou University, Zhengzhou, Henan, China.,The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou, Henan, China
| | - Penglei Wang
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China
| | - Fangfang Liu
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China
| | - Yan Zheng
- The Affiliated Cancer Hospital, Zhengzhou University, Zhengzhou, Henan, China
| | - Ann M Bode
- The Hormel Institute, University of Minnesota, Austin, Minnesota
| | - Zigang Dong
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China.,The Hormel Institute, University of Minnesota, Austin, Minnesota.,The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou, Henan, China.,International Joint Research Center of Cancer Chemoprevention, Zhengzhou, China
| | - Dong Joon Kim
- Department of Molecular and Cellular Biology, China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan, China.,Department of Pathophysiology, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China
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9
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Xu P, Chen AY, Ganaie SS, Cheng F, Shen W, Wang X, Kleiboeker S, Li Y, Qiu J. The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs. J Virol 2019; 93:e01464-18. [PMID: 30282717 PMCID: PMC6288338 DOI: 10.1128/jvi.01464-18] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2018] [Accepted: 10/01/2018] [Indexed: 12/27/2022] Open
Abstract
Lytic infection of human parvovirus B19 (B19V) takes place exclusively in human erythroid progenitor cells of bone marrow and fetal liver, which disrupts erythropoiesis. During infection, B19V expresses three nonstructural proteins (NS1, 11-kDa, and 7.5-kDa) and two structural proteins (VP1 and VP2). While NS1 is essential for B19V DNA replication, 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of 11-kDa in viral DNA replication and elucidated the underlying mechanism. We found that 11-kDa specially interacts with cellular growth factor receptor-bound protein 2 (Grb2) during virus infection and in vitro We determined a high affinity interaction between 11-kDa and Grb2 that has an equilibrium dissociation constant (KD ) value of 18.13 nM. In vitro, one proline-rich motif was sufficient for 11-kDa to sustain a strong interaction with Grb2. In consistence, in vivo during infection, one proline-rich motif was enough for 11-kDa to significantly reduce phosphorylation of extracellular signal-regulated kinase (ERK). Mutations of all three proline-rich motifs of 11-kDa abolished its capability to reduce ERK activity and, accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding a short hairpin RNA (shRNA) targeting Grb2 decreased the expression of Grb2 as well as the level of ERK phosphorylation, which resulted in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa protein interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of nonimmunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins, VP1 and VP2, and three nonstructural proteins, NS1, 11-kDa, and 7.5-kDa. While NS1 is essential, 11-kDa plays an enhancing role in viral DNA replication. Here, we elucidated a mechanism underlying 11-kDa protein-regulated B19V DNA replication. 11-kDa is tightly associated with cellular growth factor receptor-bound protein 2 (Grb2) during infection. In vitro, 11-kDa interacts with Grb2 with high affinity through three proline-rich motifs, of which at least one is indispensable for the regulation of viral DNA replication. 11-kDa and Grb2 interaction disrupts extracellular signal-regulated kinase (ERK) signaling, which mediates upregulation of B19V replication. Thus, our study reveals a novel mechanism of how a parvoviral small nonstructural protein regulates viral DNA replication by interacting with a host protein that is predominately expressed in the cytoplasm.
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Affiliation(s)
- Peng Xu
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Aaron Yun Chen
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Safder S Ganaie
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Fang Cheng
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Weiran Shen
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Xiaomei Wang
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
- Department of Biological Science and Technology, Wuhan University of Bioengineering, Wuhan, China
| | - Steve Kleiboeker
- Department of Research and Development, Viracor Eurofins Laboratories, Lee's Summit, Missouri, USA
| | - Yi Li
- Department of Biological Science and Technology, Wuhan University of Bioengineering, Wuhan, China
| | - Jianming Qiu
- Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA
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10
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Arellano VJ, Martinell García P, Rodríguez Plaza JG, Lara Ortiz MT, Schreiber G, Volkmer R, Klipp E, Rio GD. An Antimicrobial Peptide Induces FIG1-Dependent Cell Death During Cell Cycle Arrest in Yeast. Front Microbiol 2018; 9:1240. [PMID: 29963019 PMCID: PMC6010521 DOI: 10.3389/fmicb.2018.01240] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Accepted: 05/23/2018] [Indexed: 12/30/2022] Open
Abstract
Although most antibiotics act on cells that are actively dividing and non-dividing cells such as in microbe sporulation or cancer stem cells represent a new paradigm for the control of disease. In addition to their relevance to health, such antibiotics may promote our understanding of the relationship between the cell cycle and cell death. No antibiotic specifically acting on microbial cells arrested in their cell cycle has been identified until the present time. In this study we used an antimicrobial peptide derived from α-pheromone, IP-1, targeted against MATa Saccharomyces cerevisiae cells in order to assess its dependence on cell cycle arrest to kill cells. Analysis by flow cytometry and fluorescence microscopy of various null mutations of genes involved in biological processes activated by the pheromone pathway (the mitogen-activated protein kinase pathway, cell cycle arrest, cell proliferation, autophagy, calcium influx) showed that IP-1 requires arrest in G0/G1 in order to kill yeast cells. Isolating cells in different cell cycle phases by elutriation provided further evidence that entry into cell cycle arrest, and not into G1 phase, is necessary if our peptide is to kill yeast cells. We also describe a variant of IP-1 that does not activate the pheromone pathway and consequently does not kill yeast cells that express the pheromone’s receptor; the use of this variant peptide in combination with different cell cycle inhibitors that induce cell cycle arrest independently of the pheromone pathway confirmed that it is cell cycle arrest that is required for the cell death induced by this peptide in yeast. We show that the cell death induced by IP-1 differs from that induced by α-pheromone and depends on FIG1 in a way independent of the cell cycle arrest induced by the pheromone. Thus, IP-1 is the first molecule described that specifically kills microbial cells during cell cycle arrest, a subject of interest beyond the process of mating in yeast cells. The experimental system described in this study should be useful in the study of the mechanisms at play in the communication between cell cycle arrest and cell death on other organisms, hence promoting the development of new antibiotics.
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Affiliation(s)
- Vladimir J Arellano
- Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
| | - Paula Martinell García
- Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.,Theoretische Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany
| | | | - Maria T Lara Ortiz
- Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
| | | | - Rudolf Volkmer
- Institut für Medizinische Immunologie, Charité - Universitätsmedizin Berlin, Berlin, Germany.,Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany
| | - Edda Klipp
- Theoretische Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany
| | - Gabriel Del Rio
- Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
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11
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Ciocci M, Iorio E, Carotenuto F, Khashoggi HA, Nanni F, Melino S. H2S-releasing nanoemulsions: a new formulation to inhibit tumor cells proliferation and improve tissue repair. Oncotarget 2018; 7:84338-84358. [PMID: 27741519 PMCID: PMC5356665 DOI: 10.18632/oncotarget.12609] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Accepted: 10/07/2016] [Indexed: 12/16/2022] Open
Abstract
The improvement of solubility and/or dissolution rate of poorly soluble natural compounds is an ideal strategy to make them optimal candidates as new potential drugs. Accordingly, the allyl sulfur compounds and omega-3 fatty acids are natural hydrophobic compounds that exhibit two important combined properties: cardiovascular protection and antitumor activity. Here, we have synthesized and characterized a novel formulation of diallyl disulfide (DADS) and α-linolenic acid (ALA) as protein-nanoemulsions (BAD-NEs), using ultrasounds. BAD-NEs are stable over time at room temperature and show antioxidant and radical scavenging property. These NEs are also optimal H2S slow-release donors and show a significant anti-proliferative effect on different human cancer cell lines: MCF-7 breast cancer and HuT 78 T-cell lymphoma cells. BAD-NEs are able to regulate the ERK1/2 pathway, inducing apoptosis and cell cycle arrest at the G0/G1 phase. We have also investigated their effect on cell proliferation of human adult stem/progenitor cells. Interestingly, BAD-NEs are able to improve the Lin- Sca1+ human cardiac progenitor cells (hCPC) proliferation. This stem cell growth stimulation is combined with the expression and activation of proteins involved in tissue-repair, such as P-AKT, α-sma and connexin 43. Altogether, our results suggest that these antioxidant nanoemulsions might have potential application in selective cancer therapy and for promoting the muscle tissue repair.
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Affiliation(s)
- Matteo Ciocci
- Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome, Italy
| | - Egidio Iorio
- Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy
| | - Felicia Carotenuto
- Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy
| | - Haneen A Khashoggi
- Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome, Italy
| | - Francesca Nanni
- Department of Industrial Engineering, University of Rome Tor Vergata, Rome, Italy
| | - Sonia Melino
- Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome, Italy
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12
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Jayasooriya RGPT, Molagoda IMN, Park C, Jeong JW, Choi YH, Moon DO, Kim MO, Kim GY. Molecular chemotherapeutic potential of butein: A concise review. Food Chem Toxicol 2017; 112:1-10. [PMID: 29258953 DOI: 10.1016/j.fct.2017.12.028] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2017] [Revised: 12/14/2017] [Accepted: 12/15/2017] [Indexed: 12/13/2022]
Abstract
Butein is a biologically active flavonoid isolated from the bark of Rhus verniciflua Stokes, which is known to have therapeutic potential against various cancers. Notably, butein inhibits cancer cell growth by inducing G2/M phase arrest and apoptosis. Butein-induced G2/M phase arrest is associated with increased phosphorylation of ataxia telangiectasia mutated (ATM) and Chk1/2, and consequently, with reduced cdc25C levels. In addition, butein-induced apoptosis is mediated through the activation of caspase-3, which is associated with changes in the expression of Bcl-2 and Bax proteins. Intriguingly, butein sensitizes cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via ERK-mediated Sp1 activation, which promotes the transcription of specific death receptor 5. Butein also inhibits the migration and invasion of human cancer cells by suppressing nuclear factor-κB- and extracellular signal-regulated kinases 1/2-mediated expression of matrix metalloproteinase-9 and vascular endothelial growth factor. Additionally, butein downregulates the expression of human telomerase reverse transcriptase and causes a concomitant decrease in telomerase activity. These findings provide the basis for the pharmaceutical development of butein. The aim of this review is to provide an update on the mechanisms underlying the anticancer activity of butein, with a special focus on its effects on different cellular signaling cascades.
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Affiliation(s)
- Rajapaksha Gedara Prasad Tharanga Jayasooriya
- Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea; Department of Biological Sciences, Faculty of Applied Science, University of Rajarata, Mihintale 50300, Sri Lanka
| | | | - Cheol Park
- Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan 67340, Republic of Korea
| | - Jin-Woo Jeong
- Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47227, Republic of Korea
| | - Yung Hyun Choi
- Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47227, Republic of Korea
| | - Dong-Oh Moon
- Department of Biology Education, Daegu University, Jillyang, Gyeongsan, Gyeonsangbuk-do 38453, Republic of Korea
| | - Mun-Ock Kim
- Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang, Chungcheongbuk-do 28116, Republic of Korea
| | - Gi-Young Kim
- Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea.
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13
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Bai C, Gao Y, Zhang X, Yang W, Guan W. Melatonin promotes self-renewal of nestin-positive pancreatic stem cells through activation of the MT2/ERK/SMAD/nestin axis. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2017; 46:62-74. [PMID: 29037070 DOI: 10.1080/21691401.2017.1389747] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promotion of self-renewal in pancreatic stem cells remain unknown. In this study, we incubated murine pancreatic stem cells (PSCs) with various concentrations of melatonin (0.01, 0.1, 1, 10 or 100 μM) to screen for the optimum culture medium for increasing cell proliferation. We found that 10 μM melatonin can significantly increase proliferation and enhance expression of a stem cell marker, nestin, in PSCs via melatonin receptor 2 (MT2). Thus, we used 10 μM melatonin to study the melatonin-mediated molecular mechanisms of cell proliferation in PSCs. We applied extracellular signal-regulated kinase (ERK) pathway inhibitor SCH772984 and transforming growth factor beta (TGF-β) pathway inhibitor SB431542, along with interfering RNAs siERK1, siERK2, siSmad2, siSmad3, siSmad4 and siNestin, to melatonin-treated PSCs to research the roles of these genes in self-renewal. The results revealed a novel molecular mechanism by which melatonin promotes self-renewal of PSCs: a chain reaction in the MT2/ERK/SMAD/nestin axis promoted the aforementioned self-renewal as well as inhibited differentiation. In addition, upregulation of nestin created a positive feedback loop in the regulation of the transforming growth factor beta 1 (TGF-β1)/SMADs pathway by promoting expression of Smad4. Conversely, knockdown of nestin significantly suppressed the proliferative effect in melatonin-treated PSCs. These are all novel mechanisms through which the ERK pathway cooperatively crosstalks with the SMAD pathway to regulate nestin expression, thereby enhancing self-renewal in PSCs.
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Affiliation(s)
- Chunyu Bai
- a Key Laboratory of Precision Oncology of Shandong Higher Education , Institute of precision medicine , Jining , Shandong Province , P. R. China.,b Institute of Animal Sciences , Chinese Academy of Agricultural Sciences , Beijing , P. R. China
| | - Yuhua Gao
- b Institute of Animal Sciences , Chinese Academy of Agricultural Sciences , Beijing , P. R. China.,c College of Basic Medicine , Jining Medical University , Jining , Shandong Province , P. R. China
| | - Xiangyang Zhang
- c College of Basic Medicine , Jining Medical University , Jining , Shandong Province , P. R. China
| | - Wancai Yang
- a Key Laboratory of Precision Oncology of Shandong Higher Education , Institute of precision medicine , Jining , Shandong Province , P. R. China.,d Department of Pathology , University of Illinois at Chicago , Chicago , IL , USA
| | - Weijun Guan
- b Institute of Animal Sciences , Chinese Academy of Agricultural Sciences , Beijing , P. R. China
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14
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Mechanical stretch triggers rapid epithelial cell division through Piezo1. Nature 2017; 543:118-121. [PMID: 28199303 PMCID: PMC5334365 DOI: 10.1038/nature21407] [Citation(s) in RCA: 530] [Impact Index Per Article: 66.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Accepted: 01/23/2017] [Indexed: 12/13/2022]
Abstract
Despite acting as a barrier for the organs they encase, epithelial cells turnover at some of the fastest rates in the body. Yet, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How do the number of dying cells match those dividing to maintain constant numbers? We previously found that when epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die1. Conversely, what controls epithelial cell division to balance cell death at steady state? Here, we find that cell division occurs in regions of low cell density, where epithelial cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the same Piezo1 channel. To do so, stretch triggers cells paused in early G2 to activate calcium-dependent ERK1/2 phosphorylation that activates cyclin B transcription necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at steady state, the type of mechanical force controls the outcome: stretch induces cell division whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated since it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions where cells divide, Piezo1 localizes to the plasma membrane and cytoplasm whereas in dense regions where cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion/apoptosis in crowded regions and cell division in sparse regions.
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15
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Huang HL, Chao MW, Chen CC, Cheng CC, Chen MC, Lin CF, Liou JP, Teng CM, Pan SL. LTP-1, a novel antimitotic agent and Stat3 inhibitor, inhibits human pancreatic carcinomas in vitro and in vivo. Sci Rep 2016; 6:27794. [PMID: 27278358 PMCID: PMC4899784 DOI: 10.1038/srep27794] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2016] [Accepted: 05/23/2016] [Indexed: 01/05/2023] Open
Abstract
Pancreatic cancer is the leading cause of cancer death worldwide with a poor survival rate. The objective of this study was to determine the mechanism of action of a novel antimitotic and Stat3 inhibitor, LTP-1, on human pancreatic cancer in vitro and in vivo. We found that LTP-1 inhibited pancreatic cancer cell growth and viability with significant G2/M arrest and disruption of microtubule dynamics. LTP-1 also caused G2/M arrest-independent Stat3 dephosphorylation along with ERK activation, which indicated the possible dual function of LTP-1. Long-term treatment of LTP-1 also induced polyploidy, activated caspases, induced subG1 cell population, and therefore, triggered pancreatic cancer cell apoptosis. Finally, we used an in vivo xenograft model to demonstrate that LTP-1 suppressed the growth of pancreatic adenocarcinoma. In summary, our data suggest that LTP-1 may alter microtubule dynamics, which ultimately causes polyploidy and apoptosis, thereby inhibiting pancreatic cancer growth in vitro and in vivo. This study provides evidence that LTP-1 could be a potential therapeutic agent for further development of pancreatic cancer treatment.
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Affiliation(s)
- Han-Li Huang
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Min-Wu Chao
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Chung-Chun Chen
- Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chun-Chun Cheng
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
| | - Mei-Chuan Chen
- Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
- Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Chao-Feng Lin
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Department of Internal Medicine, Division of Cardiology, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan
| | - Jing-Ping Liou
- School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Che-Ming Teng
- Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Shiow-Lin Pan
- The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
- Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan
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16
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Sauvanet C, Garbett D, Bretscher A. The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation. Mol Biol Cell 2015; 26:3615-27. [PMID: 26310448 PMCID: PMC4603932 DOI: 10.1091/mbc.e15-07-0498] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Accepted: 08/18/2015] [Indexed: 11/11/2022] Open
Abstract
We examine the dynamics and function of the apical scaffolding protein E3KARP/NHERF2, which consists of two PDZ domains and a tail containing an ezrin-binding domain. The exchange rate of E3KARP is greatly enhanced during mitosis due to phosphorylation at Ser-303 in its tail region. Whereas E3KARP can substitute for the function of the closely related scaffolding protein EBP50/NHERF1 in the formation of interphase microvilli, E3KARP S303D cannot. Moreover, the S303D mutation enhances the in vivo dynamics of the E3KARP tail alone, whereas in vitro the interaction of E3KARP with active ezrin is unaffected by S303D, implicating another factor regulating dynamics in vivo. A-Raf is found to be required for S303 phosphorylation in mitotic cells. Regulation of the dynamics of EBP50 is known to be dependent on its tail region but modulated by PDZ domain occupancy, which is not the case for E3KARP. Of interest, in both cases, the mechanisms regulating dynamics involve the tails, which are the most diverged region of the paralogues and probably evolved independently after a gene duplication event that occurred early in vertebrate evolution.
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Affiliation(s)
- Cécile Sauvanet
- Department of Molecular Biology and Genetics, Weill Institute for Molecular and Cell Biology, Cornell University, Ithaca, NY 14853
| | - Damien Garbett
- Department of Molecular Biology and Genetics, Weill Institute for Molecular and Cell Biology, Cornell University, Ithaca, NY 14853
| | - Anthony Bretscher
- Department of Molecular Biology and Genetics, Weill Institute for Molecular and Cell Biology, Cornell University, Ithaca, NY 14853
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17
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Zhang Y, Guo Z, Xu L. Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2014; 37:812-818. [PMID: 24632106 DOI: 10.1016/j.etap.2014.02.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/18/2013] [Revised: 01/28/2014] [Accepted: 02/10/2014] [Indexed: 06/03/2023]
Abstract
The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades.
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Affiliation(s)
- Yali Zhang
- Department of Biochemistry, School of Medicine, Nantong University, 19 Qi Xiu Rode, 226001 Nantong, China; Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, 388 Yu Hang Tang Road, 310058 Hangzhou, China.
| | - Zonglou Guo
- Department of Biosystem Engineering, College of Biosystem Engineering and Food Science, Zhejiang University, 388 Yu Hang Tang Road, 310058 Hangzhou, China.
| | - Lihong Xu
- Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, 388 Yu Hang Tang Road, 310058 Hangzhou, China.
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18
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Cullis J, Meiri D, Sandi MJ, Radulovich N, Kent OA, Medrano M, Mokady D, Normand J, Larose J, Marcotte R, Marshall CB, Ikura M, Ketela T, Moffat J, Neel BG, Gingras AC, Tsao MS, Rottapel R. The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1. Cancer Cell 2014; 25:181-95. [PMID: 24525234 DOI: 10.1016/j.ccr.2014.01.025] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2013] [Revised: 11/26/2013] [Accepted: 01/23/2014] [Indexed: 10/25/2022]
Abstract
Cellular transformation by oncogenic RAS engages the MAPK pathway under strict regulation by the scaffold protein KSR-1. Here, we report that the guanine nucleotide exchange factor GEF-H1 plays a critical role in a positive feedback loop for the RAS/MAPK pathway independent of its RhoGEF activity. GEF-H1 acts as an adaptor protein linking the PP2A B' subunits to KSR-1, thereby mediating the dephosphorylation of KSR-1 S392 and activation of MAPK signaling. GEF-H1 is important for the growth and survival of HRAS(V12)-transformed cells and pancreatic tumor xenografts. GEF-H1 expression is induced by oncogenic RAS and is correlated with pancreatic neoplastic progression. Our results, therefore, identify GEF-H1 as an amplifier of MAPK signaling and provide mechanistic insight into the progression of RAS mutant tumors.
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Affiliation(s)
- Jane Cullis
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - David Meiri
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Maria Jose Sandi
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Nikolina Radulovich
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Oliver A Kent
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Mauricio Medrano
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Daphna Mokady
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Josee Normand
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Jose Larose
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Richard Marcotte
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Christopher B Marshall
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada
| | - Mitsuhiko Ikura
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Troy Ketela
- Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Donnelly Centre and Banting and Best Department of Medical Research, 160 College Street, Room 8-804, University of Toronto, Toronto, ON M5S 3E1, Canada
| | - Jason Moffat
- Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Donnelly Centre and Banting and Best Department of Medical Research, 160 College Street, Room 8-804, University of Toronto, Toronto, ON M5S 3E1, Canada
| | - Benjamin G Neel
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Anne-Claude Gingras
- Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Room 992A, Toronto, ON M5G 1X5, Canada
| | - Ming-Sound Tsao
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada
| | - Robert Rottapel
- Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medicine, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Immunology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Division of Rheumatology, St. Michael's Hospital, 30 Bond Street, Toronto, ON M5B 1W8, Canada.
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Raghunandan R, Frissora FW, Muthusamy N. Modulation of Ets-1 expression in B lymphocytes is dependent on the antigen receptor-mediated activation signals and cell cycle status. Scand J Immunol 2013; 77:75-83. [PMID: 23216019 DOI: 10.1111/sji.12012] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2012] [Accepted: 10/29/2012] [Indexed: 01/30/2023]
Abstract
In this report, we tested the hypothesis that Ets-1 transcription factor is modulated at the mRNA level during B cell antigen receptor (BCR)-induced cell-signalling events. Quiescent B cells express high levels of Ets-1 mRNA. Stimulation through the BCR results in time-dependent inhibition of Ets-1 mRNA expression in primary splenic B cells with maximal inhibition observed by 16-h post-stimulation. Inhibition of Ets-1 expression is specific to antigen receptor but not CD40-mediated activation. Antigen receptor-induced inhibition of Ets-1 mRNA can be mimicked by phorbol myristate acetate (PMA) and/or ionomycin. PMA but not ionomycin-induced inhibition of Ets-1 expression is rescued by the inhibitors of protein kinase C and MEK. Extended time-course analysis revealed a time-dependent cyclical pattern in the re-expression of Ets-1 mRNA. While resting cells revealed maximal Ets-1 mRNA expression, activation events that induced exit from G(0) /G(1) or cells blocked in early S phase exhibited decreased Ets-1 mRNA levels. Interestingly, cells arrested at late G2 or M phase of the cell cycle failed to down modulate Ets-1 mRNA expression. Overexpression of Ets-1 in 70Z/3 B cell line caused abnormal accumulation of cells in S phase associated with increased cyclin A expression. Consistent with a requirement for Ets-1 in BCR-induced cell cycle entry, splenic B cells from mice deficient in Ets-1 showed defective antigen receptor-induced DNA synthesis and S phase entry. These results suggest a critical role for Ets-1 regulation during B cell activation and cell cycle entry.
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Affiliation(s)
- R Raghunandan
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
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20
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Flacke JP, Flacke H, Appukuttan A, Palisaar RJ, Noldus J, Robinson BD, Reusch HP, Zippin JH, Ladilov Y. Type 10 soluble adenylyl cyclase is overexpressed in prostate carcinoma and controls proliferation of prostate cancer cells. J Biol Chem 2012; 288:3126-35. [PMID: 23255611 DOI: 10.1074/jbc.m112.403279] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
cAMP signaling plays an essential role in modulating the proliferation of different cell types, including cancer cells. Until now, the regulation of this pathway was restricted to the transmembrane class of adenylyl cyclases. In this study, significant overexpression of soluble adenylyl cyclase (sAC), an alternative source of cAMP, was found in human prostate carcinoma, and therefore, the contribution of this cyclase was investigated in the prostate carcinoma cell lines LNCaP and PC3. Suppression of sAC activity by treatment with the sAC-specific inhibitor KH7 or by sAC-specific knockdown mediated by siRNA or shRNA transfection prevented the proliferation of prostate carcinoma cells, led to lactate dehydrogenase release, and induced apoptosis. Cell cycle analysis revealed a significant rise in the G(2) phase population 12 h after sAC inhibition, which was accompanied by the down-regulation of cyclin B(1) and CDK1. sAC-dependent regulation of proliferation involves the EPAC/Rap1/B-Raf signaling pathway. In contrast, protein kinase A does not play a role. In conclusion, this study suggests a novel sAC-dependent signaling pathway that controls the proliferation of prostate carcinoma cells.
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Affiliation(s)
- Jan-Paul Flacke
- Department of Clinical Pharmacology, Ruhr University Bochum, 44801 Bochum, Germany
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21
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Role of JNK and p38 MAPK in Taiwanin A-induced cell death. Life Sci 2012; 91:1358-65. [PMID: 23123629 DOI: 10.1016/j.lfs.2012.10.007] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2012] [Revised: 10/15/2012] [Accepted: 10/18/2012] [Indexed: 11/23/2022]
Abstract
AIM The lignan compound Taiwanin A is cytotoxic for human cancer cells. Taiwanin A has been previously shown to damage microtubules, induce mitotic arrest and cause apoptosis in cancer cells. The goal of the current study is to identify intracellular signaling pathways that are involved in Taiwanin A-mediated apoptosis. MAIN METHODS We examined the activation of three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), in HepG2 cells after Taiwanin A treatment. The role of MAPK activation in Taiwanin A-induced apoptosis was examined using Western blotting, caspase activity assays combined with specific MAPK inhibitors and shRNA treatment to knockdown JNK. KEY FINDINGS Taiwanin A activated all three MAPKs (ERK, p38 and JNK). Cytotoxicity was blocked by the p38 MAPK inhibitor SB203580 and the JNK inhibitor SP600125 but not by the ERK inhibitor PD98059. A combined treatment of SB203580 and SP600125 showed increased effects on the inhibition of Taiwanin A cytotoxicity, suggesting that both JNK and p38 play a role in Taiwanin A-induced apoptosis. Inhibition of p38 activity reduced Taiwanin A-induced p53 phosphorylation on Ser15. Direct interaction of Taiwanin A-activated p38 and p53 was demonstrated by immunoprecipitation. In addition, inhibition of JNK by SP600125 or silencing of the JNK scaffold protein JIP2 reduced phosphorylation of Bcl-2, which may help to promote anti-apoptotic pathways. SIGNIFICANCE We demonstrated for the first time that two distinct apoptotic pathways, the p38-p53 and JNK-Bcl-2 pathways, were triggered by the anti-microtubule compound Taiwanin A.
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22
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Kamal A, Mallareddy A, Janaki Ramaiah M, Pushpavalli S, Suresh P, Kishor C, Murty J, Rao NS, Ghosh S, Addlagatta A, Pal-Bhadra M. Synthesis and biological evaluation of combretastatin-amidobenzothiazole conjugates as potential anticancer agents. Eur J Med Chem 2012; 56:166-78. [DOI: 10.1016/j.ejmech.2012.08.021] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2012] [Revised: 07/06/2012] [Accepted: 08/15/2012] [Indexed: 12/13/2022]
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Tang D, Yuan H, Vielemeyer O, Perez F, Wang Y. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly. Biol Open 2012; 1:1204-14. [PMID: 23259055 PMCID: PMC3522882 DOI: 10.1242/bio.20122659] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2012] [Accepted: 09/04/2012] [Indexed: 01/30/2023] Open
Abstract
GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.
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Affiliation(s)
- Danming Tang
- Department of Molecular, Cellular and Developmental Biology, University of Michigan , 830 North University Avenue, Ann Arbor, MI 48109-1048 , USA
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24
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Illert AL, Zech M, Moll C, Albers C, Kreutmair S, Peschel C, Bassermann F, Duyster J. Extracellular signal-regulated kinase 2 (ERK2) mediates phosphorylation and inactivation of nuclear interaction partner of anaplastic lymphoma kinase (NIPA) at G2/M. J Biol Chem 2012; 287:37997-8005. [PMID: 22955283 DOI: 10.1074/jbc.m112.373464] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
NIPA is an F-box-like protein that contributes to the timing of mitotic entry. It targets nuclear cyclin B1 for ubiquitination in interphase, whereas in G(2)/M phase, NIPA is inactivated by phosphorylation to allow for cyclin B1 accumulation, a critical event for proper G(2)/M transition. We recently specified three serine residues of NIPA and demonstrated a sequential phosphorylation at G(2)/M, where initial Ser-354 and Ser-359 phosphorylation is most crucial for SCF(NIPA) inactivation. In this study, we identified ERK2 as the kinase responsible for this critical initial phosphorylation step. Using in vitro kinase assays, we found that both ERK1 and ERK2 phosphorylated NIPA with high efficiency. Mutation of either Ser-354 or Ser-359 abolished ERK-dependent NIPA phosphorylation. Pharmacologic inhibition of ERK1/2 in cell lines resulted in decreased NIPA phosphorylation at G(2)/M. By combining cell cycle synchronization with stable expression of shRNA targeting either ERK1 or ERK2, we showed that ERK2 but not ERK1 mediated NIPA inactivation at G(2)/M. ERK2 knockdown led to a delay at the G(2)/M transition, a phenotype also observed in cells expressing a phospho-deficient mutant of NIPA. Thus, our data add to the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation, which may be due in part to the differential ability of these kinases to phosphorylate and inactivate NIPA at G(2)/M.
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Affiliation(s)
- Anna Lena Illert
- Department of Internal Medicine III, Technical University of Munich, 81675 Munich, Germany
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25
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Liu L, Zhu Y, Xu Y, Reiter RJ. Prevention of ERK activation involves melatonin-induced G(1) and G(2) /M phase arrest in the human osteoblastic cell line hFOB 1.19. J Pineal Res 2012; 53:60-6. [PMID: 21988060 DOI: 10.1111/j.1600-079x.2011.00971.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Melatonin regulates mitogen-activated protein kinase (MAPK) and Akt signaling pathways. The MAPK family mainly includes extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Our previous study documented that melatonin delays osteoblast proliferation; however, the mechanism of action of melatonin remains unclear. Here, we demonstrate that melatonin significantly inhibited phosphorylation of ERK but not p38, JNK, or Akt in a human osteoblastic cell line 1.19 (hFOB), as measured by western blot. The expression of ERK, p38, JNK, and Akt was not altered. PD98059 (a selective inhibitor of MEK that disrupts downstream activation of ERK) and melatonin alone, and especially in combination, significantly induced an antiproliferative effect, G(1) and G(2) /M phase arrest of the cell cycle, and downregulation of the expression at both the protein and mRNA levels of cyclin D1 and CDK4, related to the G(1) phase, and of cyclin B1 and CDK1, related to the G(2) /M phase, as measured by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method, flow cytometry after propidium iodide staining, and both western blot and real-time PCR, respectively. Moreover, the combination of PD98059 and melatonin synergistically and markedly augmented the action of either agent alone. Coimmunoprecipitation further confirmed that there was an interaction between phosphorylation of ERK and cyclin D1, CDK4, cyclin B1, or CDK1, which was weaken in the presence of melatonin or PD98059. These results suggest that the prevention of ERK activation is involved in melatonin-induced G(1) and G(2) /M phase arrest, and this inhibitory effect is potentially via the ERK, but not p38, JNK, or Akt, pathway.
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Affiliation(s)
- Lifeng Liu
- Department of Orthopaedics, First Hospital, China Medical University, Shenyang, Liaoning, China
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26
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Brobeil A, Graf M, Eiber M, Wimmer M. Interaction of PTPIP51 with Tubulin, CGI-99 and Nuf2 During Cell Cycle Progression. Biomolecules 2012; 2:122-42. [PMID: 24970130 PMCID: PMC4030868 DOI: 10.3390/biom2010122] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2011] [Revised: 02/04/2012] [Accepted: 02/14/2012] [Indexed: 02/07/2023] Open
Abstract
Protein tyrosine phosphatase interacting protein 51 (PTPIP51), also known as regulator of microtubule dynamics protein 3, was identified as an in vitro and in vivo interaction partner of CGI-99 and Nuf-2. PTPIP51 mRNA is expressed in all stages of the cell cycle; it is highly expressed six hours post-nocodazole treatment and minimally expressed one hour post-nocodazole treatment. Recent investigations located PTPIP51 protein at the equatorial plate. This study reports the localization of the PTPIP51/CGI-99 and the PTPIP51/Nuf-2 complex at the equatorial region during mitosis. Moreover, Duolink proximity ligation assays revealed an association of PTPIP51 with the microtubular cytoskeleton and the spindle apparatus. High amounts of phosphorylated PTPIP51 associated with the spindle poles was seen by confocal microscopy. In parallel a strong interaction of PTPIP51 with the epidermal growth factor receptor phosphorylating PTPIP51 at the tyrosine 176 residue was seen. In the M/G1 transition a high level of interaction between PTPIP51 and PTP1B was registered, thus restoring the interaction of PTPIP51 and Raf-1, depleted in mitotic cells. Summarizing these new facts, we conclude that PTPIP51 is necessary for normal mitotic processes, impacting on chromosomal division and control of the MAPK pathway activity.
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Affiliation(s)
- Alexander Brobeil
- Institute of Anatomy and Cell Biology, Justus-Liebig-University, 35392 Giessen, Germany.
| | - Michaela Graf
- Institute of Anatomy and Cell Biology, Justus-Liebig-University, 35392 Giessen, Germany.
| | - Moritz Eiber
- Institute of Anatomy and Cell Biology, Justus-Liebig-University, 35392 Giessen, Germany.
| | - Monika Wimmer
- Institute of Anatomy and Cell Biology, Justus-Liebig-University, 35392 Giessen, Germany.
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The MEK1/2 inhibitor, selumetinib (AZD6244; ARRY-142886), enhances anti-tumour efficacy when combined with conventional chemotherapeutic agents in human tumour xenograft models. Br J Cancer 2012; 106:858-66. [PMID: 22343622 PMCID: PMC3305954 DOI: 10.1038/bjc.2012.8] [Citation(s) in RCA: 89] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Background: The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy. Methods: The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations. Results: Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib. Conclusion: The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy.
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Xu Y, Liu L, Qiu X, Jiang L, Huang B, Li H, Li Z, Luo W, Wang E. CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells. PLoS One 2011; 6:e21119. [PMID: 21698152 PMCID: PMC3116867 DOI: 10.1371/journal.pone.0021119] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2011] [Accepted: 05/19/2011] [Indexed: 01/13/2023] Open
Abstract
C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G2/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G0/G1 and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G2/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G0/G1 and G1/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.
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Affiliation(s)
- Ying Xu
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
| | - Lifeng Liu
- Department of Orthopaedics, First Affiliated Hospital of China Medical University, Shenyang, Liaoning, China
| | - Xueshan Qiu
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
- * E-mail:
| | - Lili Jiang
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
| | - Bo Huang
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
| | - Haiying Li
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
| | - Zixuan Li
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
| | - Wenting Luo
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
| | - Enhua Wang
- Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China
- Institute of Pathology and Pathophysiology, China Medical University, Shenyang, Liaoning, China
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O'Donnell JJ, Zhuge Y, Holian O, Cheng F, Thomas LL, Forsyth CB, Lum H. Loss of p120 catenin upregulates transcription of pro-inflammatory adhesion molecules in human endothelial cells. Microvasc Res 2011; 82:105-12. [PMID: 21554891 DOI: 10.1016/j.mvr.2011.04.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2011] [Accepted: 04/23/2011] [Indexed: 12/23/2022]
Abstract
P120 catenin (p120ctn) is an adherens junction protein recognized to regulate barrier function, but emerging evidence indicates that p120ctn may also exert control on other cellular functions such as transcriptional suppression of genes. We investigated the hypothesis that loss of p120ctn in human endothelial cells activates transcription of pro-inflammatory adhesion molecules. For study, siRNA targeted to p120ctn was transfected into brain microvascular (HBMECs) or pulmonary artery endothelial cells (HPAECs) for 24-120h, which depleted 50-80% of endogenous p120ctn. This loss of p120ctn resulted in increased promoter reporter activity of transcription factors, NFκB, AP-1, and Kaiso, as well as of target genes, MMP-1 and ICAM-1. Real-time RT-PCR analysis indicated that the mRNA for ICAM-1, VCAM-1, and E- and P-selectins were all upregulated during the period of 24-120h of p120ctn depletion, although the time-course and extent of the expression profiles differed. The upregulated mRNA of adhesion molecules corresponded with increased PMN adhesion to the EC surface and elevated ICAM-1 protein expression. We further explored the role of ERK1/2 as a potential signaling mechanism responsible for regulation of transcriptional activities by p120ctn. Results indicated that loss of p120ctn increased phosphorylated ERK1/2, and a MEK1 inhibitor (PD98059) prevented NFκB nuclear translocation. This implicates ERK1/2 in signaling the NFκB activation induced by p120ctn loss. The findings provide strong evidence that deficiency in p120ctn expression in endothelial cells is a potent stimulus for transcriptional upregulation of multiple adhesion molecules. We conclude that p120ctn functions to suppress transcription, which is an important and novel regulation in vascular endothelium.
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Affiliation(s)
- James J O'Donnell
- Department of Pharmacology, Rush University Medical Center, Chicago, IL, USA
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30
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Wu YG, Zhou P, Lan GC, Gao D, Li Q, Wei DL, Wang HL, Tan JH. MPF governs the assembly and contraction of actomyosin rings by activating RhoA and MAPK during chemical-induced cytokinesis of goat oocytes. PLoS One 2010; 5:e12706. [PMID: 20856880 PMCID: PMC2938347 DOI: 10.1371/journal.pone.0012706] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2010] [Accepted: 08/19/2010] [Indexed: 01/09/2023] Open
Abstract
The interplay between maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK) and Rho GTPase during actin-myosin interactions has yet to be determined. The mechanism by which microtubule disrupters induce the formation of ooplasmic protrusion during chemical-assisted enucleation of mammalian oocytes is unknown. Moreover, a suitable model is urgently needed for the study of cytokinesis. We have established a model of chemical-induced cytokinesis and have studied the signaling events leading to cytokinesis using this model. The results suggested that microtubule inhibitors activated MPF, which induced actomyosin assembly (formation of ooplasmic protrusion) by activating RhoA and thus MAPK. While MAPK controlled actin recruitment on its own, MPF promoted myosin enrichment by activating RhoA and MAPK. A further chemical treatment of oocytes with protrusions induced constriction of the actomyosin ring by inactivating MPF while activating RhoA. In conclusion, the present data suggested that the assembly and contraction of the actomyosin ring were two separable steps: while an increase in MPF activity promoted the assembly through RhoA-mediated activation of MAPK, a decrease in MPF activity triggered contraction of the ring by activating RhoA.
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Affiliation(s)
- Yan-Guang Wu
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Ping Zhou
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Guo-Cheng Lan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Da Gao
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Qing Li
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - De-Li Wei
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Hui-Li Wang
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Jing-He Tan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
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Cao JN, Shafee N, Vickery L, Kaluz S, Ru N, Stanbridge EJ. Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells. Cancer Res 2010; 70:6004-14. [PMID: 20570892 PMCID: PMC2938962 DOI: 10.1158/0008-5472.can-09-4490] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability.
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Affiliation(s)
- Jia-ning Cao
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA
| | - Norazizah Shafee
- Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia
| | - Larry Vickery
- Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, CA 92697, USA
| | - Stefan Kaluz
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA
| | - Ning Ru
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA
| | - Eric J. Stanbridge
- Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA
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Combined molecular analysis of BRAF and IDH1 distinguishes pilocytic astrocytoma from diffuse astrocytoma. Acta Neuropathol 2009; 118:401-5. [PMID: 19543740 DOI: 10.1007/s00401-009-0550-z] [Citation(s) in RCA: 191] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2009] [Revised: 05/20/2009] [Accepted: 05/20/2009] [Indexed: 01/12/2023]
Abstract
Separation of pilocytic astrocytoma from diffuse astrocytomas frequently poses problems mostly related to small sample size. Precise classification and grading are essential due to different therapeutic strategies prompted by diagnoses of pilocytic astrocytoma WHO grade I, diffuse astrocytomas WHO grade II or anaplastic astrocytoma WHO grade III. Recently, genomic aberrations with a high specificity for distinct glioma entities have been described. Pilocytic astrocytomas carry a duplication at chromosome band 7q34 containing a BRAF-KIAA1549 gene fusion in the majority of cases. IDH1 mutations are observed very frequently in adult astrocytomas and IDH2 mutations have been reported in some astrocytomas. We examined a series of 120 astrocytomas including 70 pilocytic astrocytomas WHO grade I and 50 diffuse astrocytomas WHO grade II for both, BRAF-KIAA1549 fusion with a newly developed FISH assay and mutations in IDH1 and IDH2 by direct sequencing. Pilocytic astrocytomas contained the BRAF fusion in 49 cases (70%) but neither IDH1 nor IDH2 mutations. Astrocytomas WHO grade II exhibited IDH1 mutations in 38 cases (76%) but neither IDH2 mutations nor BRAF fusions. Thus, combined molecular analysis of BRAF and IDH1 is a sensitive and highly specific approach to separate pilocytic astrocytoma from diffuse astrocytoma.
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Waxman EA, Covy JP, Bukh I, Li X, Dawson TM, Giasson BI. Leucine-rich repeat kinase 2 expression leads to aggresome formation that is not associated with alpha-synuclein inclusions. J Neuropathol Exp Neurol 2009; 68:785-96. [PMID: 19535993 DOI: 10.1097/nen.0b013e3181aaf4fd] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common known cause of Parkinson disease, but how this protein results in the pathobiology of Parkinson disease is unknown. Moreover, there is variability in pathology among cases, and alpha-synuclein (alpha-syn) neuronal inclusions are often present, but whether LRRK2 is present in these pathological inclusions is controversial. This study characterizes novel LRRK2 antibodies, some of which preferentially recognize an aggregated form of LRRK2, as observed in cell culture models. Large perinuclear aggregates containing LRRK2 were promoted by proteasome inhibition and prevented by microtubule polymerization inhibition. Furthermore, they were vimentin- and gamma-tubulin- but not lamp1-immunoreactive, suggesting that these structures fit the definition of aggresomes. Inhibition of heat shock protein 90 led to the degradation of only the soluble/cytosolic pool of LRRK2, suggesting that the aggresomes formed independent of the stability provided by the heat shock protein 90. Although these novel anti-LRRK2 antibodies identified aggregates in model cell systems, they did not immunostain pathological inclusions in human brains. Furthermore, coexpression of LRRK2 and alpha-syn did not recruit alpha-syn into aggresomes in cultured cells, even in the presence of proteasome inhibition. Thus, although LRRK2 is a model system for aggresome formation, LRRK2 is not present in alpha-syn pathological inclusions.
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Affiliation(s)
- Elisa A Waxman
- Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6084, USA
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34
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Mansell JP, Farrar D, Jones S, Nowghani M. Cytoskeletal reorganisation, 1alpha,25-dihydroxy vitamin D3 and human MG63 osteoblast maturation. Mol Cell Endocrinol 2009; 305:38-46. [PMID: 19433260 DOI: 10.1016/j.mce.2009.02.032] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/27/2008] [Revised: 02/27/2009] [Accepted: 02/27/2009] [Indexed: 01/11/2023]
Abstract
Bone tissue is especially receptive to physical stimulation and agents with the capacity to mimic the signalling incurred via mechanical loading on osteoblasts may find an application in a bone regenerative setting. Recently this laboratory revealed that the major serum lipid, lysophosphatidic acid (LPA), co-operated with 1alpha,25-dihydroxy vitamin D3 (D3) in stimulating human osteoblast maturation. Actin stress fiber accrual in LPA treated osteoblasts would have generated peripheral tension which in turn may have heightened the maturation response of these cells to D3. To test this hypothesis we examined if other agents known to trigger stress fiber accumulation co-operated with D3 in stimulating human osteoblast maturation. Colchicine, nocodazole and LPA all co-operated with D3 to promote MG63 maturation in a MEK dependent manner. In contrast, calpeptin, a direct activator of Rho kinase and stress fiber accumulation did not act with D3 to secure MG63 differentiation. Herein we describe how the signalling elicited via microtubule disruption cooperates with D3 in the development of mature osteoblasts.
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Affiliation(s)
- Jason Peter Mansell
- Department of Oral & Dental Science, University of Bristol Dental School, Lower Maudlin St., Bristol, BS1 2LY, UK.
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35
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The activity of extracellular signal-regulated kinase is required during G2/M phase before metaphase–anaphase transition in synchronized leukemia cell lines. Int J Hematol 2009; 89:159-166. [DOI: 10.1007/s12185-008-0248-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2008] [Revised: 12/04/2008] [Accepted: 12/15/2008] [Indexed: 01/04/2023]
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36
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Pfister S, Janzarik WG, Remke M, Ernst A, Werft W, Becker N, Toedt G, Wittmann A, Kratz C, Olbrich H, Ahmadi R, Thieme B, Joos S, Radlwimmer B, Kulozik A, Pietsch T, Herold-Mende C, Gnekow A, Reifenberger G, Korshunov A, Scheurlen W, Omran H, Lichter P. BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas. J Clin Invest 2008; 118:1739-49. [PMID: 18398503 DOI: 10.1172/jci33656] [Citation(s) in RCA: 372] [Impact Index Per Article: 21.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2007] [Accepted: 02/13/2008] [Indexed: 12/30/2022] Open
Abstract
The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.
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Affiliation(s)
- Stefan Pfister
- Division Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany
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37
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Hamed H, Hawkins W, Mitchell C, Gilfor D, Zhang G, Pei XY, Dai Y, Hagan MP, Roberts JD, Yacoub A, Grant S, Dent P. Transient exposure of carcinoma cells to RAS/MEK inhibitors and UCN-01 causes cell death in vitro and in vivo. Mol Cancer Ther 2008; 7:616-629. [PMID: 18347148 DOI: 10.1158/1535-7163.mct-07-2376] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The present studies were initiated to determine in greater molecular detail how MEK1/2 inhibitors [PD184352 and AZD6244 (ARRY-142886)] interact with UCN-01 (7-hydroxystaurosporine) to kill mammary carcinoma cells in vitro and radiosensitize mammary tumors in vitro and in vivo and whether farnesyl transferase inhibitors interact with UCN-01 to kill mammary carcinoma cells in vitro and in vivo. Expression of constitutively activated MEK1 EE or molecular suppression of JNK and p38 pathway signaling blocked MEK1/2 inhibitor and UCN-01 lethality, effects dependent on the expression of BAX, BAK, and, to a lesser extent, BIM and BID. In vitro colony formation studies showed that UCN-01 interacted synergistically with the MEK1/2 inhibitors PD184352 or AZD6244 and the farnesyl transferase inhibitors FTI277 and R115,777 to kill human mammary carcinoma cells. Athymic mice carrying approximately 100 mm(3) MDA-MB-231 cell tumors were subjected to a 2-day exposure of either vehicle, R115,777 (100 mg/kg), the MEK1/2 inhibitor PD184352 (25 mg/kg), UCN-01 (0.2 mg/kg), or either of the drugs in combination with UCN-01. Transient exposure of tumors to R115,777, PD184352, or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15 to 30 days after drug administration. In contrast, combined treatment with R115,777 and UCN-01 or with PD184352 and UCN-01 significantly reduced tumor growth. Tumor cells isolated after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Irradiation of mammary tumors after drug treatment, but not before or during treatment, significantly enhanced the lethal effects of UCN-01 and MEK1/2 inhibitor treatment. These findings argue that UCN-01 and multiple inhibitors of the RAS-MEK pathway have the potential to suppress mammary tumor growth, and to interact with radiation, in vitro and in vivo.
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Affiliation(s)
- Hossein Hamed
- Department of Biochemistry, Virginia Commonwealth University, Richmond VA 23298-0035, USA
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38
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O’Neill C. The potential roles for embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 2008; 14:275-88. [DOI: 10.1093/humupd/dmn002] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
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39
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Kar B, Reichman CT, Singh S, O'Connor JP, Birge RB. Proapoptotic function of the nuclear Crk II adaptor protein. Biochemistry 2007; 46:10828-40. [PMID: 17764157 DOI: 10.1021/bi700537e] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Crk II and Crk L have both cytosolic and nuclear functions. While Crk L is a bona fide nuclear signaling protein because of its ability to bind tyrosine-phosphorylated STAT5 and act as a transcriptional coactivator, the function of nuclear Crk II is less well understood. The present study was undertaken to investigate whether Crk II is in the nucleus, how Crk II translocates into the nucleus, whether it possesses a functional NES, and to determine if nuclear Crk II affects cell cycle checkpoints and promotes apoptosis. Toward this goal, we used several independent techniques to show that a significant percentage of the total endogenous Crk II partitions in the nucleus in mammalian cells, where it forms distinct complexes with DOCK180, Wee1, and Abl. We found no evidence that Crk II bound to Crm1 nor that the localization of GFP-Crk II was sensitive to LMB, an inhibitor of Crm1. To better define the significance of nuclear Crk II localization, we generated a GFP-Crk II protein (GFP-Crk-nuc) fused to three tandem nuclear localization signals derived from the SV40 large T-antigen. GFP-Crk-nuc exhibited exclusive nuclear localization, and in contrast to wild-type Crk, GFP-Crk-nuc expressing cells could not be propagated upon selection in G418-containing media, suggesting nuclear accumulation of Crk II caused either growth arrest or apoptosis. When transiently transfected cells were FACS sorted, GFP-expressing cells showed defective cell adhesion on tissue culture surfaces and showed an increased level of apoptosis assessed by pycnotic nuclei, annexin V staining, and PARP cleavage. Although we found that Crk II bound to the cell cycle protein Wee1, expression of GFP-Crk-nuc did not induce a G2/M cell cycle block or cause increased Cdc2 Tyr15 phosphorylation. Finally, upon UV stimulation, we found that endogenous Crk II translocated to the nucleus and potentiated the extent of UV-inducible apoptosis after 4 h. These data suggest that nuclear compartmentalization of Crk II antagonizes its cytoskeletal functions and assign a proapoptotic role to the nuclear pool of Crk II.
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Affiliation(s)
- Bishnupriya Kar
- Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA
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Meloche S, Pouysségur J. The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene 2007; 26:3227-39. [PMID: 17496918 DOI: 10.1038/sj.onc.1210414] [Citation(s) in RCA: 834] [Impact Index Per Article: 46.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The Ras-dependent extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway plays a central role in cell proliferation control. In normal cells, sustained activation of ERK1/ERK2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes. In cells expressing activated Ras or Raf mutants, hyperactivation of the ERK1/2 pathway elicits cell cycle arrest by inducing the accumulation of cyclin-dependent kinase inhibitors. In this review, we discuss the mechanisms by which activated ERK1/ERK2 regulate growth and cell cycle progression of mammalian somatic cells. We also highlight the findings obtained from gene disruption studies.
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Affiliation(s)
- S Meloche
- Departments of Pharmacology and Molecular Biology, Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec, Canada.
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Morgan-Lappe SE, Tucker LA, Huang X, Zhang Q, Sarthy AV, Zakula D, Vernetti L, Schurdak M, Wang J, Fesik SW. Identification of Ras-Related Nuclear Protein, Targeting Protein for Xenopus Kinesin-like Protein 2, and Stearoyl-CoA Desaturase 1 as Promising Cancer Targets from an RNAi-Based Screen. Cancer Res 2007; 67:4390-8. [PMID: 17483353 DOI: 10.1158/0008-5472.can-06-4132] [Citation(s) in RCA: 113] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.
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Affiliation(s)
- Susan E Morgan-Lappe
- Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois 60064, USA.
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42
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Callera GE, Montezano AC, Yogi A, Tostes RC, Touyz RM. Vascular signaling through cholesterol-rich domains: implications in hypertension. Curr Opin Nephrol Hypertens 2007; 16:90-104. [PMID: 17293683 DOI: 10.1097/mnh.0b013e328040bfbd] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
PURPOSE OF REVIEW Lipid rafts are emerging as key players in the integration of cellular responses. Alterations in these highly regulated signaling cascades are important in structural, mechanical and functional abnormalities that underlie vascular pathological processes. The present review focuses on recent advances in signal transduction through caveolae/lipid rafts, implicated in hypertensive processes. RECENT FINDINGS Caveolae/lipid rafts function as sites of dynamic regulatory events in receptor-induced signal transduction. Mediators of vascular function, including G-protein coupled receptors, Src family tyrosine kinases, receptor tyrosine kinases, protein phosphatases and nitric oxide synthase, are concentrated within these microdomains. The assembly of functionally active nicotinamide adenine dinucleotide phosphate oxidase and subsequent reactive oxygen species production are also dependent on interactions within the caveolae/lipid rafts. Recent findings have also demonstrated the importance of actin-cytoskeleton and focal adhesion sites for protein interactions with caveolae/lipid raft. SUMMARY Many vascular signaling processes are altered in hypertension. Whether these events involve lipid rafts/caveolae remains unclear. A better understanding of how signaling molecules compartmentalize in lipid rafts/caveolae will provide further insights into molecular mechanisms underlying vascular damage in cardiovascular disease.
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Affiliation(s)
- Glaucia E Callera
- Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Canada.
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43
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Sarthy AV, Morgan-Lappe SE, Zakula D, Vernetti L, Schurdak M, Packer JCL, Anderson MG, Shirasawa S, Sasazuki T, Fesik SW. Survivin depletion preferentially reduces the survival of activated K-Ras-transformed cells. Mol Cancer Ther 2007; 6:269-76. [PMID: 17237286 DOI: 10.1158/1535-7163.mct-06-0560] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Abstract
To identify cancer-specific targets, we have conducted a synthetic lethal screen using a small interfering RNA (siRNA) library targeting ∼4,000 individual genes for enhanced killing in the DLD-1 colon carcinoma cell line that expresses an activated copy of the K-Ras oncogene. We found that siRNAs targeting baculoviral inhibitor of apoptosis repeat-containing 5 (survivin) significantly reduced the survival of activated K-Ras-transformed cells compared with its normal isogenic counterpart in which the mutant K-Ras gene had been disrupted (DKS-8). In addition, survivin siRNA induced a transient G2-M arrest and marked polyploidy that was associated with increased caspase-3 activation in the activated K-Ras cells. These results indicate that tumors expressing the activated K-Ras oncogene may be particularly sensitive to inhibitors of the survivin protein. [Mol Cancer Ther 2007;6(1):269–76]
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Affiliation(s)
- Aparna V Sarthy
- Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA.
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44
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Feinstein TN, Linstedt AD. Mitogen-activated protein kinase kinase 1-dependent Golgi unlinking occurs in G2 phase and promotes the G2/M cell cycle transition. Mol Biol Cell 2006; 18:594-604. [PMID: 17182854 PMCID: PMC1783781 DOI: 10.1091/mbc.e06-06-0530] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G(2)/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle "checkpoint." A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G(2) to control G(2)/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G(2) Golgi unlinking and the G(2)/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.
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Affiliation(s)
- Timothy N. Feinstein
- Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213
| | - Adam D. Linstedt
- Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213
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Shinohara M, Mikhailov AV, Aguirre-Ghiso JA, Rieder CL. Extracellular signal-regulated kinase 1/2 activity is not required in mammalian cells during late G2 for timely entry into or exit from mitosis. Mol Biol Cell 2006; 17:5227-40. [PMID: 17035635 PMCID: PMC1679686 DOI: 10.1091/mbc.e06-04-0284] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
Extracellular signal-regulated kinase (ERK)1/2 activity is reported to be required in mammalian cells for timely entry into and exit from mitosis (i.e., the G2-mitosis [G2/M] and metaphase-anaphase [M/A] transitions). However, it is unclear whether this involvement reflects a direct requirement for ERK1/2 activity during these transitions or for activating gene transcription programs at earlier stages of the cell cycle. To examine these possibilities, we followed live cells in which ERK1/2 activity was inhibited through late G2 and mitosis. We find that acute inhibition of ERK1/2 during late G2 and through mitosis does not affect the timing of the G2/M or M/A transitions in normal or transformed human cells, nor does it impede spindle assembly, inactivate the p38 stress-activated checkpoint during late G2 or the spindle assembly checkpoint during mitosis. Using CENP-F as a marker for progress through G2, we also show that sustained inhibition of ERK1/2 transiently delays the cell cycle in early/mid-G2 via a p53-dependent mechanism. Together, our data reveal that ERK1/2 activity is required in early G2 for a timely entry into mitosis but that it does not directly regulate cell cycle progression from late G2 through mitosis in normal or transformed mammalian cells.
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Affiliation(s)
- Mio Shinohara
- *Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201
- Department of Biomedical Sciences, School of Public Health, and
| | - Alexei V. Mikhailov
- *Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201
- Department of Biomedical Sciences, School of Public Health, and
| | - Julio A. Aguirre-Ghiso
- Department of Biomedical Sciences, School of Public Health, and
- Gen*NY*Sis Center for Excellence in Cancer Genomics, State University of New York, Albany, NY 12144; and
| | - Conly L. Rieder
- *Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201
- Department of Biomedical Sciences, School of Public Health, and
- Marine Biology Laboratory, Woods Hole, MA 02543
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Eves EM, Shapiro P, Naik K, Klein UR, Trakul N, Rosner MR. Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint. Mol Cell 2006; 23:561-74. [PMID: 16916643 PMCID: PMC1626587 DOI: 10.1016/j.molcel.2006.07.015] [Citation(s) in RCA: 116] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2005] [Revised: 04/19/2006] [Accepted: 07/17/2006] [Indexed: 02/07/2023]
Abstract
Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.
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Affiliation(s)
- Eva M Eves
- Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637, USA
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Dangi S, Chen FM, Shapiro P. Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1. Cell Prolif 2006; 39:261-79. [PMID: 16872362 PMCID: PMC2839891 DOI: 10.1111/j.1365-2184.2006.00388.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.
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Affiliation(s)
- S Dangi
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA
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Neuhof T, Seibold M, Thewes S, Laue M, Han CO, Hube B, von Döhren H. Comparison of susceptibility and transcription profile of the new antifungal hassallidin A with caspofungin. Biochem Biophys Res Commun 2006; 349:740-9. [PMID: 16949033 DOI: 10.1016/j.bbrc.2006.08.110] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2006] [Accepted: 08/16/2006] [Indexed: 11/23/2022]
Abstract
This is the first report on the antifungal effects of the new glycolipopeptide hassallidin A. Due to related molecular structure moieties between hassallidin A and the established antifungal drug caspofungin we assumed parallels in the effects on cell viability. Therefore we compared hassallidin A with caspofungin by antifungal susceptibility testing and by analysing the genome-wide transcriptional profile of Candida albicans. Furthermore, we examined modifications in ultracellular structure due to hassallidin A treatment by electron microscopy. Hassallidin A was found to be fungicidal against all tested Candida species and Cryptococcus neoformans isolates. MICs ranged from 4 to 8 microg/ml, independently from the species. Electron microscopy revealed noticeable ultrastructural changes in C. albicans cells exposed to hassallidin A. Comparing the transcriptional profile of C. albicans cells treated with hassallidin A to that of cells exposed to caspofungin, only 20 genes were found to be similarly up- or down-regulated in both assays, while 227 genes were up- or down-regulated induced by hassallidin A specifically. Genes up-regulated in cells exposed to hassallidin A included metabolic and mitotic genes, while genes involved in DNA repair, vesicle docking, and membrane fusion were down-regulated. In summary, our data suggest that, although hassallidin A and caspofungin have similar structures, however, the effects on susceptibility and transcriptional response to yeasts seem to be different.
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Affiliation(s)
- Torsten Neuhof
- Technische Universität Berlin, Institut für Chemie, FG Biochemie und Molekulare Biologie, 10587 Berlin, Germany.
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Borysov SI, Cheng AWM, Guadagno TM. B-Raf Is Critical For MAPK Activation during Mitosis and Is Regulated in an M Phase-dependent Manner in Xenopus Egg Extracts. J Biol Chem 2006; 281:22586-96. [PMID: 16762920 DOI: 10.1074/jbc.m601432200] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.
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Affiliation(s)
- Sergiy I Borysov
- Molecular Oncology Program, Department of Interdisciplinary Oncology, H. Lee Moffitt Comprehensive Cancer Center and Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, Florida 33612, USA
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Kawabe JI, Okumura S, Nathanson MA, Hasebe N, Ishikawa Y. Caveolin regulates microtubule polymerization in the vascular smooth muscle cells. Biochem Biophys Res Commun 2006; 342:164-9. [PMID: 16480946 DOI: 10.1016/j.bbrc.2006.01.125] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2006] [Accepted: 01/16/2006] [Indexed: 10/25/2022]
Abstract
Microtubule and caveolin have common properties in intracellular trafficking and the regulation of cellular growth. Overexpression of caveolin in vascular smooth muscle cells increased the polymer form of microtubule without changing in the total amount of tubulin, and downregulation of caveolin decreased the polymer form of microtubule. Fractionation of cellular proteins followed by immunodetection as well as immunostaining of caveolin and microtubule revealed that caveolin and a portion of microtubule were co-localized in caveolar fractions. A caveolin scaffolding domain peptide, which mimics caveolin function, did not alter the polymerization of microtubule in vitro, but dramatically inhibited the depolymerization of microtubule induced by stathmin, a microtubule destabilizing protein, which was also found in caveolar fractions. Accordingly, it is most likely that caveolin increased the polymer form of microtubule through the inhibition of a microtubule destabilizer, stathmin, suggesting a novel role of caveolin in regulating cellular network and trafficking.
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Affiliation(s)
- Jun-ichi Kawabe
- Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.
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