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Gao L, Meng F, Yang Z, Lafuente-Merchan M, Fernández LM, Cao Y, Kusamori K, Nishikawa M, Itakura S, Chen J, Huang X, Ouyang D, Riester O, Deigner HP, Lai H, Pedraz JL, Ramalingam M, Cai Y. Nano-drug delivery system for the treatment of multidrug-resistant breast cancer: Current status and future perspectives. Biomed Pharmacother 2024; 179:117327. [PMID: 39216449 DOI: 10.1016/j.biopha.2024.117327] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Revised: 08/11/2024] [Accepted: 08/21/2024] [Indexed: 09/04/2024] Open
Abstract
Breast cancer (BC) is one of the most frequently diagnosed cancers in women. Chemotherapy continues to be the treatment of choice for clinically combating it. Nevertheless, the chemotherapy process is frequently hindered by multidrug resistance, thereby impacting the effectiveness of the treatment. Multidrug resistance (MDR) refers to the phenomenon in which malignant tumour cells develop resistance to anticancer drugs after one single exposure. It can occur with a broad range of chemotherapeutic drugs with distinct chemical structures and mechanisms of action, and it is one of the major causes of treatment failure and disease relapse. Research has long been focused on overcoming MDR by using multiple drug combinations, but this approach is often associated with serious side effects. Therefore, there is a pressing need for in-depth research into the mechanisms of MDR, as well as the development of new drugs to reverse MDR and improve the efficacy of breast cancer chemotherapy. This article reviews the mechanisms of multidrug resistance and explores the application of nano-drug delivery system (NDDS) to overcome MDR in breast cancer. The aim is to offer a valuable reference for further research endeavours.
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Affiliation(s)
- Lanwen Gao
- State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University / International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Ministry of Education (MOE) of China / Guangdong Key Lab of Traditional Chinese Medicine Information Technology / International Science and Technology Cooperation Base of Guangdong Province / School of Pharmacy, Jinan University, Guangdong, Guangzhou 510632, China.
| | - Fansu Meng
- Zhongshan Hospital of Traditional Chinese Medicine Affiliated to Guangzhou University of Traditional Chinese Medicine, Zhongshan 528400, China.
| | - Zhenjiang Yang
- Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, China.
| | - Markel Lafuente-Merchan
- NanoBioCel Group, Department of Pharmacy and Food Sciences, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz 01006, Spain; Bioaraba Health Research Institute, Jose Atxotegi, s/n, Vitoria-Gasteiz 01009, Spain; Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute of Health Carlos III, Madrid 28029, Spain.
| | - Laura Merino Fernández
- NanoBioCel Group, Department of Pharmacy and Food Sciences, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz 01006, Spain; Bioaraba Health Research Institute, Jose Atxotegi, s/n, Vitoria-Gasteiz 01009, Spain; Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute of Health Carlos III, Madrid 28029, Spain.
| | - Ye Cao
- State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University / International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Ministry of Education (MOE) of China / Guangdong Key Lab of Traditional Chinese Medicine Information Technology / International Science and Technology Cooperation Base of Guangdong Province / School of Pharmacy, Jinan University, Guangdong, Guangzhou 510632, China.
| | - Kosuke Kusamori
- Laboratory of Cellular Drug Discovery and Development, Faculty of Pharmaceutical Sciences Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
| | - Makiya Nishikawa
- Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
| | - Shoko Itakura
- Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
| | - Junqian Chen
- The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
| | - Xiaoxun Huang
- Zhongshan Hospital of Traditional Chinese Medicine Affiliated to Guangzhou University of Traditional Chinese Medicine, Zhongshan 528400, China.
| | - Dongfang Ouyang
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Charlestown, Boston, MA 02129, USA.
| | - Oliver Riester
- Institute of Precision Medicine, Medical and Life Sciences Faculty, Furtwangen University, Villingen-Schwenningen 78054, Germany.
| | - Hans-Peter Deigner
- Institute of Precision Medicine, Medical and Life Sciences Faculty, Furtwangen University, Villingen-Schwenningen 78054, Germany.
| | - Haibiao Lai
- Zhongshan Hospital of Traditional Chinese Medicine Affiliated to Guangzhou University of Traditional Chinese Medicine, Zhongshan 528400, China.
| | - Jose Luis Pedraz
- NanoBioCel Group, Department of Pharmacy and Food Sciences, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz 01006, Spain; Bioaraba Health Research Institute, Jose Atxotegi, s/n, Vitoria-Gasteiz 01009, Spain; Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute of Health Carlos III, Madrid 28029, Spain; Joint Research Laboratory (JRL) on Bioprinting and Advanced Pharma Development, A Joint Venture of TECNALIA (Basque Research and Technology Alliance), Centro de Investigación Lascaray Ikergunea, Avenida Miguel de Unamuno, Vitoria-Gasteiz 01006, Spain.
| | - Murugan Ramalingam
- NanoBioCel Group, Department of Pharmacy and Food Sciences, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz 01006, Spain; Bioaraba Health Research Institute, Jose Atxotegi, s/n, Vitoria-Gasteiz 01009, Spain; Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute of Health Carlos III, Madrid 28029, Spain; Joint Research Laboratory (JRL) on Bioprinting and Advanced Pharma Development, A Joint Venture of TECNALIA (Basque Research and Technology Alliance), Centro de Investigación Lascaray Ikergunea, Avenida Miguel de Unamuno, Vitoria-Gasteiz 01006, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao 48013, Spain; School of Basic Medical Sciences, Binzhou Medical University, Yantai 264003, China.
| | - Yu Cai
- State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University / International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Ministry of Education (MOE) of China / Guangdong Key Lab of Traditional Chinese Medicine Information Technology / International Science and Technology Cooperation Base of Guangdong Province / School of Pharmacy, Jinan University, Guangdong, Guangzhou 510632, China.
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Hanitrarimalala V, Bednarska I, Murakami T, Papadakos KS, Blom AM. Intracellular cartilage oligomeric matrix protein augments breast cancer resistance to chemotherapy. Cell Death Dis 2024; 15:480. [PMID: 38965233 PMCID: PMC11224260 DOI: 10.1038/s41419-024-06872-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2023] [Revised: 06/26/2024] [Accepted: 06/27/2024] [Indexed: 07/06/2024]
Abstract
Chemotherapy persists as the primary intervention for breast cancer, with chemoresistance posing the principal obstacle to successful treatment. Herein, we show that cartilage oligomeric matrix protein (COMP) expression leads to increased cancer cell survival and attenuated apoptosis under treatment with several chemotherapeutic drugs, anti-HER2 targeted treatment, and endocrine therapy in several breast cancer cell lines tested. The COMP-induced chemoresistance was independent of the breast cancer subtype. Extracellularly delivered recombinant COMP failed to rescue cells from apoptosis while endoplasmic reticulum (ER)-restricted COMP-KDEL conferred resistance to apoptosis, consistent with the localization of COMP in the ER, where it interacted with calpain. Calpain activation was reduced in COMP-expressing cells and maintained at a lower level of activation during treatment with epirubicin. Moreover, the downstream caspases of calpain, caspases -9, -7, and -3, exhibited significantly reduced activation in COMP-expressing cells under chemotherapy treatment. Chemotherapy, when combined with calpain activators, rendered the cells expressing COMP more chemosensitive. Also, the anti-apoptotic proteins phospho-Bcl2 and survivin were increased in COMP-expressing cells upon chemotherapy. Cells expressing a mutant COMP lacking thrombospondin repeats exhibited reduced chemoresistance compared to cells expressing full-length COMP. Evaluation of calcium levels in the ER, cytosol, and mitochondria revealed that COMP expression modulates intracellular calcium homeostasis. Furthermore, patients undergoing chemotherapy or endocrine therapy demonstrated significantly reduced overall survival time when tumors expressed high levels of COMP. This study identifies a novel role of COMP in chemoresistance and calpain inactivation in breast cancer, a discovery with potential implications for anti-cancer therapy.
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Affiliation(s)
| | - Izabela Bednarska
- Department of Translational Medicine, Lund University, Malmö, S-214 28, Sweden
| | - Takashi Murakami
- Department of Microbiology, Saitama Medical University, Saitama, 350-0495, Japan
| | | | - Anna M Blom
- Department of Translational Medicine, Lund University, Malmö, S-214 28, Sweden
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Sigdel I, Ofori-Kwafo A, Heizelman RJ, Nestor-Kalinoski A, Prabhakarpandian B, Tiwari AK, Tang Y. Biomimetic on-chip assay reveals the anti-metastatic potential of a novel thienopyrimidine compound in triple-negative breast cancer cell lines. Front Bioeng Biotechnol 2023; 11:1227119. [PMID: 37840664 PMCID: PMC10569307 DOI: 10.3389/fbioe.2023.1227119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Accepted: 09/19/2023] [Indexed: 10/17/2023] Open
Abstract
Introduction: This study presents a microfluidic tumor microenvironment (TME) model for evaluating the anti-metastatic efficacy of a novel thienopyrimidines analog with anti-cancer properties utilizing an existing commercial platform. The microfluidic device consists of a tissue compartment flanked by vascular channels, allowing for the co-culture of multiple cell types and providing a wide range of culturing conditions in one device. Methods: Human metastatic, drug-resistant triple-negative breast cancer (TNBC) cells (SUM159PTX) and primary human umbilical vein endothelial cells (HUVEC) were used to model the TME. A dynamic perfusion scheme was employed to facilitate EC physiological function and lumen formation. Results: The measured permeability of the EC barrier was comparable to observed microvessels permeability in vivo. The TNBC cells formed a 3D tumor, and co-culture with HUVEC negatively impacted EC barrier integrity. The microfluidic TME was then used to model the intravenous route of drug delivery. Paclitaxel (PTX) and a novel non-apoptotic agent TPH104c were introduced via the vascular channels and successfully reached the TNBC tumor, resulting in both time and concentration-dependent tumor growth inhibition. PTX treatment significantly reduced EC barrier integrity, highlighting the adverse effects of PTX on vascular ECs. TPH104c preserved EC barrier integrity and prevented TNBC intravasation. Discussion: In conclusion, this study demonstrates the potential of microfluidics for studying complex biological processes in a controlled environment and evaluating the efficacy and toxicity of chemotherapeutic agents in more physiologically relevant conditions. This model can be a valuable tool for screening potential anticancer drugs and developing personalized cancer treatment strategies.
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Affiliation(s)
- Indira Sigdel
- Biofluidics Laboratory, Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH, United States
| | - Awurama Ofori-Kwafo
- Biofluidics Laboratory, Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH, United States
| | - Robert J. Heizelman
- Department of Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor, MI, United States
| | - Andrea Nestor-Kalinoski
- Department of Surgery, College of Medicine and Life Sciences, University of Toledo, Toledo, OH, United States
| | | | - Amit K. Tiwari
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Yuan Tang
- Biofluidics Laboratory, Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH, United States
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Zhu YX, Jia HR, Duan QY, Wu FG. Nanomedicines for combating multidrug resistance of cancer. WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY 2021; 13:e1715. [PMID: 33860622 DOI: 10.1002/wnan.1715] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 02/27/2021] [Accepted: 03/01/2021] [Indexed: 12/12/2022]
Abstract
Chemotherapy typically involves the use of specific chemodrugs to inhibit the proliferation of cancer cells, but the frequent emergence of a variety of multidrug-resistant cancer cells poses a tremendous threat to our combat against cancer. The fundamental causes of multidrug resistance (MDR) have been studied for decades, and can be generally classified into two types: one is associated with the activation of diverse drug efflux pumps, which are responsible for translocating intracellular drug molecules out of the cells; the other is linked with some non-efflux pump-related mechanisms, such as antiapoptotic defense, enhanced DNA repair ability, and powerful antioxidant systems. To overcome MDR, intense efforts have been made to develop synergistic therapeutic strategies by introducing MDR inhibitors or combining chemotherapy with other therapeutic modalities, such as phototherapy, gene therapy, and gas therapy, in the hope that the drug-resistant cells can be sensitized toward chemotherapeutics. In particular, nanotechnology-based drug delivery platforms have shown the potential to integrate multiple therapeutic agents into one system. In this review, the focus was on the recent development of nanostrategies aiming to enhance the efficiency of chemotherapy and overcome the MDR of cancer in a synergistic manner. Different combinatorial strategies are introduced in detail and the advantages as well as underlying mechanisms of why these strategies can counteract MDR are discussed. This review is expected to shed new light on the design of advanced nanomedicines from the angle of materials and to deepen our understanding of MDR for the development of more effective anticancer strategies. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.
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Affiliation(s)
- Ya-Xuan Zhu
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Hao-Ran Jia
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Qiu-Yi Duan
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
| | - Fu-Gen Wu
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
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Han Y, Qu YQ, Mok SWF, Chen J, Xia CL, He HQ, Li Z, Zhang W, Qiu CL, Liu L, Law BYK, Wong VKW. A Novel Drug Resistance Mechanism: Genetic Loss of Xeroderma Pigmentosum Complementation Group C ( XPC) Enhances Glycolysis-Mediated Drug Resistance in DLD-1 Colon Cancer Cells. Front Pharmacol 2019; 10:912. [PMID: 31551763 PMCID: PMC6746939 DOI: 10.3389/fphar.2019.00912] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2019] [Accepted: 07/18/2019] [Indexed: 02/04/2023] Open
Abstract
The pro-apoptotic proteins BAX and BAK are critical regulatory factors constituting the apoptosis machinery. Downregulated expression of BAX and BAK in human colorectal cancer lead to chemotherapeutic failure and poor survival rate in patients. In this study, isogenic DLD-1 colon cancer cells and the BAX and BAK double knockout counterpart were used as the cellular model to investigate the role of BAX/BAK-associated signaling network and the corresponding downstream effects in the development of drug resistance. Our data suggested that DLD-1 colon cancer cells with BAX/BAK double-knockout were selectively resistant to a panel of FDA-approved drugs (27 out of 66), including etoposide. PCR array analysis for the transcriptional profiling of genes related to human cancer drug resistance validated the altered level of 12 genes (3 upregulated and 9 downregulated) in DLD-1 colon cancer cells lack of BAX and BAK expression. Amongst these genes, XPC responsible for DNA repairment and cellular respiration demonstrated the highest tolerance towards etoposide treatment accompanying upregulated glycolysis as revealed by metabolic stress assay in DLD-1 colon cancer cells deficient with XPC. Collectively, our findings provide insight into the search of novel therapeutic strategies and pharmacological targets to against cancer drug resistance genetically associated with BAX, BAK, and XPC, for improving the therapy of colorectal cancer via the glycolytic pathway.
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Affiliation(s)
- Yu Han
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Yuan Qing Qu
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Simon Wing Fai Mok
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Juan Chen
- The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China
| | - Cheng Lai Xia
- Foshan Maternal and Child Health Research Institute, Foshan Women and Children's Hospital Affiliated to Southern Medical University, Foshan, China
| | - Hu Qiang He
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Zheng Li
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Wei Zhang
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Cong Ling Qiu
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Liang Liu
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Betty Yuen Kwan Law
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
| | - Vincent Kam Wai Wong
- State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
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Star-PAP, a poly(A) polymerase, functions as a tumor suppressor in an orthotopic human breast cancer model. Cell Death Dis 2017; 8:e2582. [PMID: 28151486 PMCID: PMC5386448 DOI: 10.1038/cddis.2016.199] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Revised: 06/03/2016] [Accepted: 06/08/2016] [Indexed: 12/21/2022]
Abstract
Star-PAP is a noncanonical poly(A) polymerase and required for the expression of a select set of mRNAs. However, the pathological role of Star-PAP in cancer largely remains unknown. In this study, we observed decreased expression of Star-PAP in breast cancer cell lines and tissues. Ectopic Star-PAP expression inhibited proliferation as well as colony-forming ability of breast cancer cells. In breast cancer patients, high levels of Star-PAP correlated with an improved prognosis. Moreover, by regulating the expression of BIK (BCL2-interacting killer), Star-PAP induced apoptosis of breast cancer cells through the mitochondrial pathway. The growth of breast cancer xenografts in NOD/SCID mice was also inhibited by the doxycycline-induced Star-PAP overexpression. Furthermore, Star-PAP sensitized breast cancer cells to chemotherapy drugs both in vitro and in vivo. In mammary epithelial cells, Star-PAP knockdown partially transformed these cells and induced them to undergo epithelial-mesenchymal transition (EMT). These findings suggested that Star-PAP possesses tumor-suppressing activity and can be a valuable target for developing new cancer therapeutic strategies.
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Overexpression of HOXB7 protein reduces sensitivity of oral cancer cells to chemo-radiotherapy. Cancer Gene Ther 2016; 23:419-424. [PMID: 27834359 DOI: 10.1038/cgt.2016.55] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2016] [Revised: 09/12/2016] [Accepted: 09/19/2016] [Indexed: 12/12/2022]
Abstract
The upregulation of homeobox-B7 (HOXB7) in cancers has been reported. However, its role in oral cancer progression remains to be investigated. In current study, our data revealed that reconstitution of HOXB7 expression by transient transfection resulted in increased cell growth, migration and invasion in vitro. In addition, apoptosis and clonogenic assay data showed that overexpression of HOXB7 decreased the sensitivity of oral cancer cells to vincristine-induced apoptosis of HSC-4 and KB/VCR cells. Furthermore, overexpression of HOXB7 promoted oral cancer cells' migration and invasion through activation of TGFβ2/SMAD3 signaling pathway. Moreover, forced expression of HOXB7 increased Bcl-2 to Bax ratio, which would promote cell survival and induce drug and radiotherapy resistance. Altogether, our findings support the role of HOXB7 in the progression of oral cancer. Therefore, HOXB7 potentially can be a therapeutic target for oral cancer.
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Dai HY, Chen HY, Lai WC, Hung MC, Li LY. Targeted expression of BikDD combined with metronomic doxorubicin induces synergistic antitumor effect through Bax activation in hepatocellular carcinoma. Oncotarget 2016; 6:23807-19. [PMID: 26247632 PMCID: PMC4695153 DOI: 10.18632/oncotarget.4278] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Accepted: 06/05/2015] [Indexed: 12/22/2022] Open
Abstract
Conventional chemotherapy is commonly used to treat advanced non-resectable hepatocellular carcinoma (HCC) but this treatment modality has not demonstrated convincing survival benefit in HCC patients. Our previous studies indicated that targeted expression of therapeutic BikDD driven by a liver cancer-specific α-fetoprotein promoter/enhancer (eAFP) in the VISA backbone (eAFP-VISA-BikDD) significantly and specifically kills HCC cells in multiple orthotopic animal models. To enhance its therapeutic efficacy, we combined eAFP-VISA-BikDD with chemotherapeutic agents and found that eAFP-VISA-BikDD plus doxorubicin (Dox) or 5-fluorouracil (5-FU) demonstrated synergistic cytotoxicity in HCC cells. Specifically, the combination of eAFP-VISA-BikDD plus Dox markedly induced apoptosis via increased Bax mitochondrial translocation and cytoplasmic cytochrome c release. Compared with either agent alone, a low dose of Dox combined with eAFP-VISA-BikDD induced better antitumor effect and prolonged longer survival of mice in two orthotopic liver cancer xenograft models. Our findings provide strong preclinical support for evaluating the combined therapy of eAFP-VISA-BikDD and Dox in a clinical setting as a treatment option for HCC.
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Affiliation(s)
- Huei-Yue Dai
- Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan
| | - Hui-Yu Chen
- Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan
| | - Wei-Chen Lai
- Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan
| | - Mien-Chie Hung
- Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan.,Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan.,Department of Biotechnology, Asia University, Taichung, Taiwan.,Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Long-Yuan Li
- Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan.,Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan.,Department of Biotechnology, Asia University, Taichung, Taiwan
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Wang S, Duan C, Zhang F, Wang X, Guo X. The Roles of the Interaction of BCL2-Antagonist/Killer 1, Apoptotic Peptidase Activating Factor 1 and Selenium in the Pathogenesis of Kashin-Beck Disease. Biol Trace Elem Res 2016; 170:17-24. [PMID: 26179084 DOI: 10.1007/s12011-015-0424-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Accepted: 06/26/2015] [Indexed: 01/25/2023]
Abstract
BCL2-antagonist/killer 1 (BAK1) and apoptotic peptidase activating factor 1 (APAF1) are significant genes in apoptosis signalling pathway of Kashin-Beck disease (KBD). We aimed to verify the protein expression levels of BAK1 and APAF1 in the cartilage and chondrocytes of patients with KBD. Additionally, we explored the relationship between the levels of these proteins and selenium concentration. Chondrocytes was cultured and treated with sodium selenite in vitro. Immunohistochemistry and Western blotting were used to verify the expression levels of BAK1 and APAF1. Compared with the control samples, APAF1 was upregulated and BAK1 was downregulated in the cartilage and chondrocytes of KBD patients. APAF1 expression was higher in the middle and deep zone in the KBD cartilage. APAF1 levels decreased gradually with the increasing selenium concentration (0.05, 0.10 and 0.25 mg/L). BAK1 expression in the 0.25 mg/L selenium group was lower than that of the control group. Different selenium concentrations had varying effects on BAK1 and APAF1 levels. APAF1 may play an important role in the pathogenesis of KBD. APAF1-related apoptosis was more pronounced in the middle and deep zones of the KBD cartilage. APAF may represent a potentially novel molecular target, which may be a biomarker of the role of selenium on the prevention and treatment of KBD. The role of BAK1 in the pathogenesis of KBD requires further study.
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Affiliation(s)
- Sen Wang
- School of Public Health, Health Science Center of Xi'an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, No. 76 Yanta West Road, Xi'an, Shaanxi, 710061, People's Republic of China
| | - Chen Duan
- School of Public Health, Health Science Center of Xi'an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, No. 76 Yanta West Road, Xi'an, Shaanxi, 710061, People's Republic of China
| | - Feng Zhang
- School of Public Health, Health Science Center of Xi'an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, No. 76 Yanta West Road, Xi'an, Shaanxi, 710061, People's Republic of China
| | - Xi Wang
- School of Public Health, Health Science Center of Xi'an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, No. 76 Yanta West Road, Xi'an, Shaanxi, 710061, People's Republic of China
| | - Xiong Guo
- School of Public Health, Health Science Center of Xi'an Jiaotong University, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission, No. 76 Yanta West Road, Xi'an, Shaanxi, 710061, People's Republic of China.
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Yuan Z, Wang H, Hu Z, Huang Y, Yao F, Sun S, Wu B. Quercetin Inhibits Proliferation and Drug Resistance in KB/VCR Oral Cancer Cells and Enhances Its Sensitivity to Vincristine. Nutr Cancer 2014; 67:126-36. [DOI: 10.1080/01635581.2015.965334] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
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11
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Zhao D, Yuan H, Yi F, Meng C, Zhu Q. Autophagy prevents doxorubicin‑induced apoptosis in osteosarcoma. Mol Med Rep 2014; 9:1975-81. [PMID: 24639013 DOI: 10.3892/mmr.2014.2055] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2013] [Accepted: 02/10/2014] [Indexed: 11/05/2022] Open
Abstract
Autophagy is a process of selective degradation of cellular components. Autophagy is an adaptive process in the majority of tumor cells; it provides sufficient nutrients by degrading cellular components to enhance the survival of tumors. Osteosarcoma is the most common type of primary malignant bone tumor in children and adolescents. Identification of an improved therapeutic strategy for the treatment of osteosarcoma is urgently required. Osteosarcoma has been primarily treated by chemotherapy and the phenomena of resistance to the therapy has become increasingly common. Doxorubicin (Dox) is a classic chemotherapeutic drug for the treatment of osteosarcoma, and certain studies have suggested that Dox induces autophagy. On the basis of the protective effect of autophagy for tumors, the present study investigated whether U2OS and Saos-2 osteosarcoma cells activate autophagy to reduce Dox-induced apoptosis. Dox was observed to inhibit the growth of U2OS and Saos-2 osteosarcoma cells in a concentration-dependent manner. The results of the western blot analysis demonstrated that Dox induced increased expression levels of the apoptosis-related proteins cleaved caspase-3 and cytochrome c and loss of mitochondrial membrane potential (MMP) in the U2OS and Saos-2 osteosarcoma cells. Furthermore, the results of the western blot analysis also revealed that Dox increased the expression levels of the autophagy-related protein microtubule-associated protein 1 light chain 3 and reduced those of p62 in the U2OS and Saos-2 osteosarcoma cells. In order to determine the effect of autophagy on the apoptosis induced by Dox in the U2OS and Saos-2 osteosarcoma cells, autophagy-related protein (Atg)7 small interfering (si) RNA or the autophagy inhibitor 3-methyladenine (3-MA) alone or combined with Dox was used in U2OS and Saos-2 osteosarcoma cells. The results identified that Atg7 siRNA and the autophagy inhibitor 3-MA significantly elevated the levels of growth inhibition by Dox and markedly increased the expression levels of the apoptosis‑related proteins cleaved caspase-3 and cytochrome c, and reduced the levels of MMP in the U2OS and Saos-2 osteosarcoma cells, which were treated with Dox. These results indicated that autophagy was the protective mechanism used by U2OS and Saos-2 osteosarcoma against Dox-induced apoptosis. The inhibition of autophagy notably increases the levels of apoptosis induced by Dox. This suggested that Dox used in combination with autophagy inhibitors may effectively treat osteosarcoma.
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Affiliation(s)
- Dongxu Zhao
- Department of Spine Surgery, The First Bethune Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Hongping Yuan
- Department of Nephrology, The Fourth Bethune Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Fei Yi
- Department of Spine Surgery, The First Bethune Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Chunyang Meng
- Department of Spine Surgery, The First Bethune Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
| | - Qingsan Zhu
- Department of Spine Surgery, The First Bethune Hospital, Jilin University, Changchun, Jilin 130021, P.R. China
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Small activating RNA restores the activity of the tumor suppressor HIC-1 on breast cancer. PLoS One 2014; 9:e86486. [PMID: 24489730 PMCID: PMC3904905 DOI: 10.1371/journal.pone.0086486] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2013] [Accepted: 12/09/2013] [Indexed: 12/20/2022] Open
Abstract
HIC-1 is a gene that is hypermethylated in cancer, and commonly downregulated in human breast cancer. However, the precise mechanisms and molecular pathways regulated by HIC-1 remain unclear. We assessed HIC-1 expression on a tissue microarray containing 80 cases of breast cancer. We also analyzed its biological function by restoring HIC-1 expression using 5-aza-2′ deoxycytidine (5-CdR) and small-activating RNAs for the reversal of HIC-1 tumor suppressive effects on MCF-7 and MDA-MB-231 cell lines. An Agilent Q44h global expressing microarray was probed after restoring the expression of HIC-1. Data demonstrated that HIC-1 expression was reduced significantly in breast cancer tissues. HIC-1 immunohistochemistry resulted in mean staining scores in cancer tissue and normal ductal epithelia of 3.54 and 8.2, respectively (p<0.01). 5-CdR partially reversed HIC-1 expression, and modulated cell growth and apoptosis. dsHIC1-2998, an saRNA, showed activating efficacy in breast cancer cells. A group of differentially expressed genes were characterized by cDNA microarray. Upon saRNA treatment, genes upregulated included those involved in immune activation, cell cycle interference, the induction of apoptosis, anti-metastasis, and cell differentiation. Downregulated genes included oncogenes and those that play roles in cell invasion, cell growth, and cell division. Our findings may provide valuable resources not only for gene functional studies, but also for potential clinical applications to develop novel drug targets.
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Wang KL, Hsia SM, Yeh JY, Cheng SC, Wang PS, Wang SW. Anti-Proliferative Effects of Evodiamine on Human Breast Cancer Cells. PLoS One 2013; 8:e67297. [PMID: 23840656 PMCID: PMC3694052 DOI: 10.1371/journal.pone.0067297] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2013] [Accepted: 05/17/2013] [Indexed: 11/24/2022] Open
Abstract
Endocrine sensitivity, assessed by the expression of estrogen receptor (ER), has long been the predict factor to guide therapeutic decisions. Tamoxifen has been the most successful hormonal treatment in endocrine-sensitive breast cancer. However, in estrogen-insensitive cancer tamoxifen showed less effectiveness than in estrogen-sensitive cancer. It is interesting to develop new drugs against both hormone-sensitive and insensitive tumor. In this present study we examined anticancer effects of evodiamine extracted from the Chinese herb, Evodiae fructus, in estrogen-dependent and –independent human breast cancer cells, MCF-7 and MDA-MB-231 cells, respectively. Evodiamine inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner with concentration of 1×10−6 and 1×10−5 M. Evodiamine also induced apoptosis via up-regulation of caspase 7 activation, PARP cleavage (Bik and Bax expression). The expression of ER α and β in protein and mRNA levels was down-regulated by evodiamine according to data from immunoblotting and RT-PCR analysis. Overall, our results indicate that evodiamine mediates degradation of ER and induces caspase-dependent pathway leading to inhibit proliferation of breast cancer cell lines. It suggests that evodiamine may in part mediate through ER-inhibitory pathway to inhibit breast cancer cell proliferation.
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Affiliation(s)
- Kai-Lee Wang
- Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
- The Center of General Education, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan, Republic of China
| | - Shih-Min Hsia
- School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan, Republic of China
| | - Jiun-Yih Yeh
- Division of Quality Assurance, Mithra Biotechnology Inc., Taipei, Taiwan, Republic of China
| | - Shao-Chi Cheng
- Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
| | - Paulus S. Wang
- Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
- Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China
- Ph. D. Program of Aging, College of Medicine, China Medical University, Taichung, Taiwan, Republic of China
- Department of Biotechnology, Asia University, Taichung, Taiwan, Republic of China
- * E-mail: (PSW); (SWW)
| | - Shyi-Wu Wang
- Department of Physiology and Pharmacology, College of Medicine, Chang-Gung University, Taoyuan, Taiwan, Republic of China
- * E-mail: (PSW); (SWW)
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He LY, Wei X, Du L, Liu L, Xu F, Min J, Li C, Tao DD, Chen Q, Hu JB, Gong JP. Remarkably reduced expression of FoxO3a in metaplastic colorectum, primary colorectal cancer and liver metastasis. ACTA ACUST UNITED AC 2013; 33:205-211. [PMID: 23592131 DOI: 10.1007/s11596-013-1098-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2012] [Indexed: 12/14/2022]
Abstract
The forkhead family members of transcription factors (FoxOs) are expected to be potential cancer-related drug targets and thus are being extremely studied recently. In the present study, FoxO3a, one major member of this family, was identified to be down-regulated in colorectal cancer through micro-array analysis, which was confirmed by RT-PCR and Western blot in 28 patients. Moreover, immunohistochemistry (IHC) showed that the expression levels of FoxO3a were remarkably reduced in 99 cases of primary colorectal cancer, liver metastasis, and even in metaplastic colorectal tissue. IHC also revealed an exclusion of FoxO3a from the nucleus of most cells of tumor-associated tissues. Silencing FoxO3a by siRNA led to elevation of G2-M phase cells. We conclude that the downregulation of FoxO3a may greatly contribute to tumor development, and thus FoxO3a may represent a novel therapeutic target in colorectal cancer.
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Affiliation(s)
- Le-Ya He
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Xin Wei
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Lei Du
- The Laboratory of Apoptosis and Cancer Biology, the State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Lu Liu
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Feng Xu
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Jiang Min
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Chuan Li
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - De-Ding Tao
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Quan Chen
- The Laboratory of Apoptosis and Cancer Biology, the State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Jun-Bo Hu
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Jian-Ping Gong
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
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Yue B, Zhao CR, Xu HM, Li YY, Cheng YN, Ke HN, Yuan Y, Wang RQ, Shi YQ, Lou HX, Qu XJ. Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies. Biochim Biophys Acta Gen Subj 2013; 1830:2194-203. [DOI: 10.1016/j.bbagen.2012.10.011] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2012] [Revised: 10/04/2012] [Accepted: 10/11/2012] [Indexed: 01/08/2023]
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Wang S, Guo X, Wang W, Wang S. Genome-wide study identifies the regulatory gene networks and signaling pathways from chondrocyte and peripheral blood monocyte of Kashin-Beck disease. Genes Cells 2012; 17:619-32. [PMID: 22776318 DOI: 10.1111/j.1365-2443.2012.01620.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2012] [Accepted: 04/11/2012] [Indexed: 12/01/2022]
Abstract
This investigation was designed to unravel gene networks in Kashin-Beck disease (KBD) and better identify target genes of KBD for gene therapy development. RNA was isolated separately from cartilage and peripheral blood samples of patients with KBD and healthy controls. Agilent 44K human whole-genome oligonucleotide microarrays were used to detect differentially expressed genes. Three significant canonical pathways and nine chondrocyte networks from chondrocytic gene expression profiles were screened using ingenuity pathway analysis (IPA), but only one network and no canonical pathways from peripheral blood monocytic gene profile were identified. Bak1, APAF-1, CASP6, IGFBP2, Col5a2 and TGFBI extracted from significant genes that involved in chondrocytic canonical pathways and networks may have closer relationship with the etiopathogenesis of KBD. Those genes may be potential targets for gene diagnosis and treatment. Six physiological functions were predominant and unique to the chondrocytic genes, whereas two were unique to peripheral blood monocytic genes. The identified genes may represent a source of potentially novel molecular targets, which may provide a better understanding of the molecular details in KBD pathogenesis and also provide useful pathways and network maps for the future research in osteochondrosis.
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Affiliation(s)
- Sen Wang
- Medicine College of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Key Laboratory of Trace Elements and Endemic Diseases, Ministry of Health, Xi'an, Shaanxi 710061, China
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17
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Yang ZR, Liu M, Peng XL, Lei XF, Zhang JX, Dong WG. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro. Biochem Biophys Res Commun 2012; 421:627-33. [PMID: 22546556 DOI: 10.1016/j.bbrc.2012.04.079] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2012] [Accepted: 04/15/2012] [Indexed: 01/06/2023]
Abstract
Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.
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Affiliation(s)
- Zi-Rong Yang
- Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, PR China
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18
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Yang ZR, Dong WG, Lei XF, Liu M, Liu QS. Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer. World J Gastroenterol 2012; 18:1590-601. [PMID: 22529687 PMCID: PMC3325524 DOI: 10.3748/wjg.v18.i14.1590] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2011] [Revised: 09/23/2011] [Accepted: 09/30/2011] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the mechanisms of the biological roles of Dickkopf-3 (Dkk-3) in cell invasion, survival and apoptosis in colon cancer cells.
METHODS: Three human colon cancer cell lines, i.e., HT-29, LoVo and SW480, were used. Overexpression of Dkk-3 induced by pEGFP-N1-Dkk-3-GFP plasmid in LoVo cells was performed using Lipofectamine 2000 reagent. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of Dkk-3, respectively. Cell proliferation assay, cell cycle analysis, hoechst 33258 assay and Matrigel invasion assay were performed on Dkk-3 overexpressing transfectants.
RESULTS: The mRNA and protein expressions of Dkk-3 in HT-29 (mRNA: 0.06 ± 0.02, protein: 0.06 ± 0.01) and LoVo (mRNA: 0.07 ± 0.02, protein: 0.07 ± 0.02) cells were significantly lower than that in SW480 cells (mRNA: 0.92 ± 0.04, protein: 0.69 ± 0.13; all P < 0.05), and the greatest levels of invasiveness was in LoVo cells. Dkk-3 overexpression inhibited the proliferation and invasion of LoVo cells and induced cell cycle arrest at G0/G1 phase and subsequent apoptosis, as indicated by increased chromatin condensation and fragments, upregulated Bax and cytochrome c protein, downregulated survivin and Bcl-2 protein, and the activation of caspase-3 and caspase-9. Furthermore, Dkk-3 overexpression reduced the accumulation of cytosolic fraction of β-catenin.
CONCLUSION: Dkk-3 overexpression induced apoptosis in human colon cancer possibly through the mitochondrial pathway. Dkk-3 may be involved in the Wnt/β-catenin signaling pathways in colon cancer.
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Hsu JL, Chao CH, Xie X, Hung MC. Advances in Liposome-Based Targeted Gene Therapy of Cancer. RECENT ADVANCES IN CANCER RESEARCH AND THERAPY 2012:113-133. [DOI: 10.1016/b978-0-12-397833-2.00005-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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20
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Mebratu YA, Schwalm K, Smith KR, Schuyler M, Tesfaigzi Y. Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia. Am J Respir Crit Care Med 2011; 183:1531-8. [PMID: 21317312 PMCID: PMC3137142 DOI: 10.1164/rccm.201011-1930oc] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2010] [Accepted: 02/11/2010] [Indexed: 12/16/2022] Open
Abstract
RATIONALE Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. OBJECTIVES To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. METHODS We screened for dysregulated expression of the Bcl-2 family members. MEASUREMENTS AND MAIN RESULTS We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. CONCLUSIONS These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.
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Straza MW, Paliwal S, Kovi RC, Rajeshkumar B, Trenh P, Parker D, Whalen GF, Lyle S, Schiffer CA, Grossman SR. Therapeutic targeting of C-terminal binding protein in human cancer. Cell Cycle 2010; 9:3740-50. [PMID: 20930544 DOI: 10.4161/cc.9.18.12936] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
The CtBP transcriptional corepressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is antagonized by p14/p19(ARF) tumor suppressors. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells. MTOB induced apoptosis was p53-independent, correlated with the derepression of the proapoptotic CtBP repression target Bik, and was rescued by CtBP overexpression or Bik silencing. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy. In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer, the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP may represent a useful therapeutic strategy in human malignancies.
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Affiliation(s)
- Michael W Straza
- Department of Cancer Biology, University of Massachusetts Medical School and UMass Memorial Cancer Center, Worcester, MA, USA
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BIK, the founding member of the BH3-only family proteins: mechanisms of cell death and role in cancer and pathogenic processes. Oncogene 2009; 27 Suppl 1:S20-9. [PMID: 19641504 DOI: 10.1038/onc.2009.40] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
BIK is the founding member of the BH3-only family pro-apoptotic proteins. BIK is predominantly localized in the ER and induces apoptosis through the mitochondrial pathway by mobilizing calcium from the ER to the mitochondria and remodeling the mitochondrial cristae. BIK-mediated apoptosis is mediated by selective activation of BAX. BIK also induces non-apoptotic cell death in certain cell types by unknown mechanisms. BIK is non-essential for animal development, but appears to be functionally redundant for certain developmental functions with BIM. BIK is implicated in the selection of mature B cells in humans. BIK is a pro-apoptotic tumor suppressor in several human tissues and its expression in cancers is prevented by chromosomal deletions encompassing the Bik locus or by epigenetic silencing. BIK appears to be a critical effector in apoptosis induced by toxins, cytokines and virus infection. Several anti-cancer drugs transcriptionally activate Bik gene expression through transcriptional pathways dependent on factors such as E2F and p53 or by removal of epigenetic marks on the chromatin. BIK appears to be a prominent target for anti-cancer drugs that inhibit proteasomal functions. BIK has also been used as a therapeutic molecule in gene therapy-based approaches to treat difficult cancers.
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Wesarg E, Hoffarth S, Wiewrodt R, Kröll M, Biesterfeld S, Huber C, Schuler M. Targeting BCL-2 family proteins to overcome drug resistance in non-small cell lung cancer. Int J Cancer 2007; 121:2387-94. [PMID: 17688235 DOI: 10.1002/ijc.22977] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Cytotoxic chemotherapies are standard of care for patients suffering from advanced non-small cell lung cancer (NSCLC). However, objective responses are only achieved in 20% of cases and long-term survival is rarely observed. Clinically applied anticancer drugs exert at least some of their activities by inducing apoptosis. A critical step in apoptotic signal transduction is the permeabilization of the mitochondrial outer membrane (MOM), which is regulated by the BCL-2 family of proteins. Hence, therapeutic targeting of BCL-2 proteins is a promising approach to increase the drug-sensitivity of cancers. To this end we have assessed the impact of conditional expression of the proapoptotic multidomain (BH1-2-3) protein BAK, which directly permeabilizes the MOM, and the BH3-mimetic ABT-737, which acts indirectly by derepressing BH1-2-3 proteins, on apoptosis and drug sensitivity of NSCLC cells. Conditionally expressed BAK sensitized resistant NSCLC cells to drug-induced apoptosis. In contrast, ABT-737 was ineffective in those NSCLC cells expressing high levels of the anti-apoptotic MCL-1 protein. Tissue microarray analysis of tumor samples from 84 chemotherapy-naïve NSCLC patients revealed MCL-1 expression in 56% of cases, thus supporting the relevance of this resistance factor in a clinical setting. Enforced expression of the BH3-only protein NOXA, which targets MCL-1, overcame resistance to ABT-737. Moreover, combining conditionally expressed BAK with ABT-737 enhanced apoptosis in NSCLC cells independently of their MCL-1 status. In conclusion, the heterogeneity of apoptosis defects observed in drug-resistant NSCLC demands individually tailored molecular therapies. Targeting the MOM permeabilizer BAK appears to have a broader apoptogenic activity than the BH3-only mimetic ABT-737.
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Affiliation(s)
- Emmanuelle Wesarg
- Gene Therapy Laboratory, Johannes Gutenberg University, Mainz, Germany
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Shimazu T, Degenhardt K, Nur-E-Kamal A, Zhang J, Yoshida T, Zhang Y, Mathew R, White E, Inouye M. NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes Dev 2007; 21:929-41. [PMID: 17403773 PMCID: PMC1847711 DOI: 10.1101/gad.1522007] [Citation(s) in RCA: 114] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Ribonucleases, antibiotics, bacterial toxins, and viruses inhibit protein synthesis, which results in apoptosis in mammalian cells. How the BCL-2 family of proteins regulates apoptosis in response to the shutoff of protein synthesis is not known. Here we demonstrate that an Escherichia coli toxin, MazF, inhibited protein synthesis by cleavage of cellular mRNA and induced apoptosis in mammalian cells. MazF-induced apoptosis required proapoptotic BAK and its upstream regulator, the proapoptotic BH3-only protein NBK/BIK, but not BIM, PUMA, or NOXA. Interestingly, in response to MazF induction, NBK/BIK activated BAK by displacing it from anti-apoptotic proteins MCL-1 and BCL-X(L) that sequester BAK. Furthermore, NBK/BIK- or BAK-deficient cells were resistant to cell death induced by pharmacologic inhibition of translation and by virus-mediated shutoff of protein synthesis. Thus, the BH3-only protein NBK/BIK is the apical regulator of a BAK-dependent apoptotic pathway in response to shutoff of protein synthesis that functions to displace BAK from sequestration by MCL1 and BCL-X(L). Although NBK/BIK is dispensable for development, it is the BH3-only protein targeted for inactivation by viruses, suggesting that it plays a role in pathogen/toxin response through apoptosis activation.
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Affiliation(s)
- Tsutomu Shimazu
- Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
| | - Kurt Degenhardt
- Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854, USA
| | - Alam Nur-E-Kamal
- Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
| | - Junjie Zhang
- Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
| | - Takeshi Yoshida
- Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
| | - Yonglong Zhang
- Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
| | - Robin Mathew
- Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854, USA
| | - Eileen White
- Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854, USA
- Cancer Institute of New Jersey, New Brunswick, New Jersey 08903, USA
- E-MAIL ; FAX (732) 235-5759
| | - Masayori Inouye
- Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
- Corresponding authors.E-MAIL ; FAX (732) 235-4559
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Aneja R, Zhou J, Zhou B, Chandra R, Joshi HC. Treatment of hormone-refractory breast cancer: apoptosis and regression of human tumors implanted in mice. Mol Cancer Ther 2006; 5:2366-77. [PMID: 16985071 DOI: 10.1158/1535-7163.mct-06-0205] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Following surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. Another chemotherapeutic strategy uses microtubule inhibitors such as taxanes. Unfortunately, these agents elicit toxicities such as leukocytopenia, diarrhea, alopecia, and peripheral neuropathies and are also associated with the emergence of drug resistance. We have previously described a tubulin-binding, natural compound, noscapine, that was nontoxic and triggered apoptosis in many cancer types albeit at 10 mumol/L or higher concentrations depending on the cell type. We now show that a synthetic analogue of noscapine, 9-bromonoscapine, is approximately 10-fold to 15-fold more potent than noscapine in inhibiting cell proliferation and induces apoptosis following G2-M arrest in hormone-insensitive human breast cancers (MDA-MB-231). Furthermore, a clear loss of mitochondrial membrane potential, release of cytochrome c, activation of the terminal caspase-3, and the cleavage of its substrates such as poly(ADP-ribose) polymerase, suggest an intrinsic apoptotic mechanism. Taken together, these data point to a mitochondrially mediated apoptosis of hormone-insensitive breast cancer cells. Human tumor xenografts in nude mice showed significant tumor volume reduction and a surprising increase in longevity without signs of obvious toxicity. Thus, our data provide compelling evidence that 9-bromonoscapine can be useful for the therapy of hormone-refractory breast cancer.
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Affiliation(s)
- Ritu Aneja
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
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26
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Sturm I, Stephan C, Gillissen B, Siebert R, Janz M, Radetzki S, Jung K, Loening S, Dörken B, Daniel PT. Loss of the tissue-specific proapoptotic BH3-only protein Nbk/Bik is a unifying feature of renal cell carcinoma. Cell Death Differ 2006; 13:619-27. [PMID: 16322756 DOI: 10.1038/sj.cdd.4401782] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
We report for the first time inactivation of a tissue-specific Bcl-2 homology domain 3 (BH3)-only protein as a common aspect in human cancer. In detail, we show that loss of the BH3-only protein natural born killer (Nbk)/Bcl-2-interacting killer (Bik) is a common feature of clear-cell renal cell carcinoma (RCC). While strong Nbk expression is found in the renal tubuli and the epithelial lining of the glomerula, a consistent loss of Nbk expression was observed in primary RCC tissue and RCC cell lines. Mutation of Nbk is, however, rare, whereas deletion of the Nbk gene at 22q13.2 is frequent. In addition to loss of heterozygosity (LOH), DNA methylation mediates transcriptional silencing of the Nbk gene. The conditional restoration of Nbk/Bik expression led to apoptotic death of RCC but not of nonmalignant renal epithelia. A broader expression analysis of RCC cell lines for BH3-only proteins revealed that loss of Nbk coincides with failure to express Bim, whereas Puma, Bid and BNIP3 are readily detectable and, in case of Puma, inducible by p53. These data delineate a role for defects in BH3-only proteins as tumor suppressors in RCC and may explain at the same time the impressive clinical apoptosis resistance of RCC.
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Affiliation(s)
- I Sturm
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Berlin, Germany
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MDR1/P-glycoprotein overexpression in bladder transitional cell carcinoma and its correlation with expression of survivin and Fas. ACTA ACUST UNITED AC 2006. [DOI: 10.1007/s11805-006-0117-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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28
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Sturm I, Rau B, Schlag PM, Wust P, Hildebrandt B, Riess H, Hauptmann S, Dörken B, Daniel PT. Genetic dissection of apoptosis and cell cycle control in response of colorectal cancer treated with preoperative radiochemotherapy. BMC Cancer 2006; 6:124. [PMID: 16686938 PMCID: PMC1525199 DOI: 10.1186/1471-2407-6-124] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2005] [Accepted: 05/10/2006] [Indexed: 02/05/2023] Open
Abstract
Background In previous analyses we identified therapy-induced upregulation of the CDK inhibitor p21CIP/WAF-1 and consequently decreased tumor cell proliferation or loss of Bax as adverse factors for survival in rectal cancer treated with radiochemotherapy. Here, we address the individual role of p53 and its transcriptional targets, p21CIP/WAF-1 and Bax, on apoptosis induced by individual components of multimodal anticancer therapy, i.e. 5-fluorouracil (5-FU), ionising γ-radiation (IR) and heat shock/hyperthermia. Methods We analysed tumor samples 66 patients with rectal carcinoma treated by a neoadjuvant approach with radiochemotherapy ± heat shock/hyperthermia for the expression and mutation of p53 and the expression of p21CIP/WAF-1 and Bax. These data were correlated with the tumor response. The functional relevance of p53, p21CIP/WAF-1 and Bax was investigated in isogeneic HCT116 cell mutants treated with 5-FU, IR and heat shock. Results Rectal carcinoma patients who received an optimal heat shock treatment showed a response that correlated well with Bax expression (p = 0.018). Local tumor response in the whole cohort was linked to expression of p21CIP/WAF-1 (p < 0.05), but not p53 expression or mutation. This dichotomy of p53 pathway components regulating response to therapy was confirmed in vitro. In isogeneic HCT116 cell mutants, loss of Bax but not p53 or p21CIP/WAF-1 resulted in resistance against heat shock. In contrast, loss of p21CIP/WAF-1 or, to a lesser extent, p53 sensitized predominantly for 5-FU and IR. Conclusion These data establish a different impact of p53 pathway components on treatment responses. While chemotherapy and IR depend primarily on cell cycle control and p21, heat shock depends primarily on Bax. In contrast, p53 status poorly correlates with response. These analyses therefore provide a rational approach for dissecting the mode of action of single treatment modalities that may be employed to circumvent clinically relevant resistance mechanisms in rectal cancer.
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Affiliation(s)
- Isrid Sturm
- University Medical Center Charité, Campus Virchow Klinikum, Department of Hematology and Oncology, 13353 Berlin, Germany
| | - Beate Rau
- University Medical Center Charité, Campus Buch, Department of Surgery and Surgical Oncology, 13125 Berlin, Germany
| | - Peter M Schlag
- University Medical Center Charité, Campus Buch, Department of Surgery and Surgical Oncology, 13125 Berlin, Germany
| | - Peter Wust
- University Medical Center Charité, Campus Virchow Klinikum, Department of Radiology, 13353 Berlin, Germany
| | - Bert Hildebrandt
- University Medical Center Charité, Campus Virchow Klinikum, Department of Hematology and Oncology, 13353 Berlin, Germany
| | - Hanno Riess
- University Medical Center Charité, Campus Virchow Klinikum, Department of Hematology and Oncology, 13353 Berlin, Germany
| | - Steffen Hauptmann
- University of Halle-Wittenberg, Institute of Pathology, 06097 Halle, Germany
| | - Bernd Dörken
- University Medical Center Charité, Campus Virchow Klinikum, Department of Hematology and Oncology, 13353 Berlin, Germany
| | - Peter T Daniel
- University Medical Center Charité, Campus Virchow Klinikum, Department of Hematology and Oncology, 13353 Berlin, Germany
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Wendt J, Radetzki S, von Haefen C, Hemmati PG, Güner D, Schulze-Osthoff K, Dörken B, Daniel PT. Induction of p21CIP/WAF-1 and G2 arrest by ionizing irradiation impedes caspase-3-mediated apoptosis in human carcinoma cells. Oncogene 2006; 25:972-80. [PMID: 16331277 DOI: 10.1038/sj.onc.1209031] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon gamma-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21-/-) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to gamma-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.
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Affiliation(s)
- J Wendt
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University Berlin, Berlin, Germany
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Oppermann M, Geilen CC, Fecker LF, Gillissen B, Daniel PT, Eberle J. Caspase-independent induction of apoptosis in human melanoma cells by the proapoptotic Bcl-2-related protein Nbk / Bik. Oncogene 2005; 24:7369-80. [PMID: 16007125 DOI: 10.1038/sj.onc.1208890] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk/Bik) has been described to inhibit Bcl-2 and Bcl-xL, thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk/Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk/Bik only weakly at the mRNA and protein level. Conditional expression of Nbk/Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk/Bik in vivo, stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk/Bik expression. By investigating the mechanism of Nbk/Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk/Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk/Bik.
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Affiliation(s)
- Malte Oppermann
- Department of Dermatology and Allergy, Skin cancer center, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
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31
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Wendt J, von Haefen C, Hemmati P, Belka C, Dörken B, Daniel PT. TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway. Oncogene 2005; 24:4052-64. [PMID: 15806163 DOI: 10.1038/sj.onc.1208580] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.
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Affiliation(s)
- Jana Wendt
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Humboldt University, Berlin, Germany
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32
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Fecker LF, Geilen CC, Hossini AM, Schwarz C, Fechner H, Bartlett DL, Orfanos CE, Eberle J. Selective induction of apoptosis in melanoma cells by tyrosinase promoter-controlled CD95 ligand overexpression. J Invest Dermatol 2005; 124:221-8. [PMID: 15654977 DOI: 10.1111/j.0022-202x.2004.23572.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Induction of apoptosis has been demonstrated previously by overexpression of CD95 ligand (CD95L) in cultured human melanoma cells. For in vivo approaches based on CD95L, however, targeted expression is a prerequisite and tyrosinase promoters have been considered for selection. Luciferase reporter gene assays performed for a representative panel of melanoma cell lines characterized by strong (SK-Mel-19), moderate (SK-Mel-13, MeWo), weak (A-375), and missing expression (M-5) of endogenous tyrosinase revealed high tyrosinase promoter activities in SK-Mel-19, SK-Mel-13, and MeWo, but only weak activities in A-375 and M-5 as well as in non-melanoma cell lines. After transfection of a CMV promoter CD95L expression construct, melanoma cells were found highly sensitive, as compared with non-melanoma cells. By applying a tyrosinase promoter CD95L construct, apoptosis was selectively induced in SK-Mel-19, SK-Mel-13, MeWo as well as in A-375, which was characterized by high CD95 surface expression and high sensitivity to agonistic CD95 activation. M5 and non-melanoma cell lines remained uninfluenced. Also, resistance to agonistic CD95 activation seen in MeWo characterized by weak CD95 surface expression was overcome by overexpression of CD95L. Our investigations provide evidence that tyrosinase promoter CD95L constructs may be of value for selective induction of apoptosis in therapeutic strategies for melanoma.
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Affiliation(s)
- Lothar F Fecker
- Department of Dermatology, Charite-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
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33
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Normand G, Hemmati PG, Verdoodt B, von Haefen C, Wendt J, Güner D, May E, Dörken B, Daniel PT. Retracted: p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity. J Biol Chem 2005; 280:7118-30. [PMID: 15582998 DOI: 10.1074/jbc.m412330200] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.
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Affiliation(s)
- Guillaume Normand
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, D-13125 Berlin-Buch, Germany
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34
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Scholz C, Wieder T, Stärck L, Essmann F, Schulze-Osthoff K, Dörken B, Daniel PT. Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway. Oncogene 2005; 24:1904-13. [PMID: 15674346 DOI: 10.1038/sj.onc.1208233] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes pathways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As2O3), a clinically efficient agent in anticancer therapy. As2O3-induced cell death coincides with cytochrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x(L), indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As2O3-induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As2O3-mediated cell death. Interestingly, As2O3-induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As2O3-mediated necrosis-like cell death without increasing caspase-3 cleavage. As2O3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As2O3-triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As2O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy.
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Affiliation(s)
- Christian Scholz
- Department of Hematology, Oncology and Tumor Immunology, Charité, Campus Berlin-Buch, Robert-Rössle-Klinik, Humboldt Universität, Berlin, Germany
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35
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von Haefen C, Gillissen B, Hemmati PG, Wendt J, Güner D, Mrozek A, Belka C, Dörken B, Daniel PT. Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway. Oncogene 2004; 23:8320-32. [PMID: 15467752 DOI: 10.1038/sj.onc.1207971] [Citation(s) in RCA: 72] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.
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Affiliation(s)
- Clarissa von Haefen
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Humboldt University, Berlin
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36
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Klöpfer A, Hasenjäger A, Belka C, Schulze-Osthoff K, Dörken B, Daniel PT. Adenine deoxynucleotides fludarabine and cladribine induce apoptosis in a CD95/Fas receptor, FADD and caspase-8-independent manner by activation of the mitochondrial cell death pathway. Oncogene 2004; 23:9408-18. [PMID: 15516989 DOI: 10.1038/sj.onc.1207975] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The adenine deoxynucleosides cladribine (2CdA) and fludarabine (FAraA) are DNA-damaging agents that interfere with DNA repair and induce apoptosis in nonproliferating lymphoid cells. Although both drugs are clinically used for the treatment of indolent lymphoproliferative diseases, the pathways of apoptosis induction remain largely unknown. In the present work, we demonstrate that both drugs induce apoptosis independently of death receptor signaling but activate the mitochondrial cell death pathway. To dissect the signaling pathways, we employed Jurkat cells either deficient for FADD or caspase-8 or overexpressing Bcl-2. In Bcl-2 overexpressing cells, apoptosis and cytochrome c release were blocked whereas processing of caspase-9, -3 and -8 was partially inhibited. In contrast, neither the deficiency of FADD or caspase-8 nor the interference with death receptor signaling by neutralizing anti-CD95/Fas antibodies affected cell death. Inhibitor experiments revealed that caspase-8 is processed by caspase-3-like caspases. Moreover, cytochrome c release and processing of caspase-9 and -3 occurred to an equal extent in wild-type FADD -/- and caspase-8 -/- Jurkat cells. Likewise, apoptosis induction by cladribine or fludarabine was not hampered upon inhibition of caspase-8 in MOLT-3 and MOLT-4 cells or overexpression of a dominant-negative FADD mutant in BJAB cells. Thus, we conclude that apoptosis induced by nucleoside analogues is independent from death receptor signaling as well as from a proposed direct effect on APAF-1, but rather follows the mitochondrial signaling pathway of cytochrome c release and subsequent processing of caspase-9 and -3.
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Affiliation(s)
- Antje Klöpfer
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany
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Schmelz K, Wieder T, Tamm I, Müller A, Essmann F, Geilen CC, Schulze-Osthoff K, Dörken B, Daniel PT. Tumor necrosis factor alpha sensitizes malignant cells to chemotherapeutic drugs via the mitochondrial apoptosis pathway independently of caspase-8 and NF-kappaB. Oncogene 2004; 23:6743-59. [PMID: 15273737 DOI: 10.1038/sj.onc.1207848] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The Hodgkin cell line HD-MyZ is resistant to apoptosis induced by tumor necrosis factor alpha (TNFalpha). In the present work, we show that pretreatment with TNFalpha sensitized the cells to apoptosis induced by antineoplastic agents and ceramide. TNFalpha pretreatment resulted in enhanced cleavage and activity of caspase-3 upon addition of etoposide, epirubicin or ceramide. No caspase-8 activation was detectable, although caspase-8 could be activated in cell-free extracts. Inhibition of caspase-8 by z-IETD-fmk did not block the sensitizing effect of TNFalpha. Furthermore, exogenous ceramide, a mediator of TNFalpha signaling, could not substitute for TNFalpha in sensitization to drug-induced apoptosis. In contrast, we observed mitochondrial changes following cotreatment of cells with TNFalpha and drugs. Mitochondrial permeability transition, cytochrome c release and subsequent processing of caspase-9 preceded the onset of apoptosis, and were enhanced by TNFalpha pretreatment. Interestingly, although transcription factor NF-kappaB protected HD-MyZ cells from drug-induced apoptosis, TNFalpha-mediated sensitization was independent of NF-kappaB, since overexpressing a dominant-negative IkappaB mutant did not alter the TNFalpha effect. Sensitization for drug-induced apoptosis by TNFalpha was abrogated by Bcl-x(L). Thus, the sensitizing effect of TNFalpha is mediated by the mitochondrial pathway and involves processing of caspase-2, -3 and -9, but appears to be independent of caspase-8 processing, Bid cleavage and NF-kappaB signaling. Therefore, sensitization by TNFalpha is mediated at least in part through different pathways, as reported for TRAIL. There, sensitization occurs through a FADD/caspase-8-dependent mechanism. Regarding TNFalpha, the sensitizing effect was also observed in myeloid leukemia cells. Therefore, TNFalpha or alternate molecules activating its pathways might be useful as sensitizers for chemotherapy in hematological malignancies.
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Affiliation(s)
- Karin Schmelz
- Department of Hematology, Oncology, and Tumor Immunology, University Medical Center Charité-Campus CBB, Humboldt University, Berlin, Germany
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Hasenjäger A, Gillissen B, Müller A, Normand G, Hemmati PG, Schuler M, Dörken B, Daniel PT. Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-x(L) in a strictly caspase-3-dependent manner in human carcinoma cells. Oncogene 2004; 23:4523-35. [PMID: 15064710 DOI: 10.1038/sj.onc.1207594] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.
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Affiliation(s)
- Anne Hasenjäger
- Department of Hematology Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany
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Hur J, Chesnes J, Coser KR, Lee RS, Geck P, Isselbacher KJ, Shioda T. The Bik BH3-only protein is induced in estrogen-starved and antiestrogen-exposed breast cancer cells and provokes apoptosis. Proc Natl Acad Sci U S A 2004; 101:2351-6. [PMID: 14983013 PMCID: PMC356954 DOI: 10.1073/pnas.0307337101] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Evidence has been accumulating that some estrogen-dependent human breast cancers require estrogen for not only proliferation but also survival. To obtain insights into the molecular mechanisms of apoptosis of breast cancer cells subjected to estrogen starvation or exposed to antiestrogens, we characterized changes in the gene expression profile of MCF-7/BUS human breast cancer cells and revealed a strong induction of Bik, a member of the BH3-only proapoptotic proteins. The Bik mRNA transcript and protein were strongly induced by estrogen starvation or exposure to fulvestrant, a pure antiestrogen that competes with the natural estrogens for binding to the estrogen receptors. This Bik induction preceded apoptotic cell death, which was blocked by zVAD-fmk, a pancaspase inhibitor. Amounts of the Bcl-2-related proteins, such as Bcl-2, Bcl-XL, or Bax, showed only marginal changes in the presence or absence of estrogens or antiestrogens. Suppression of Bik expression by using the small interfering RNA effectively blocked the fulvestrant-induced breast cancer cell apoptosis. These results indicate that Bik is induced in MCF-7/BUS cells in the absence of estrogen signaling and plays a critical role in the antiestrogen-provoked breast cancer cell apoptosis.
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Affiliation(s)
- Jingyung Hur
- Department of Tumor Biology, Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA
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40
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Eberle J, Fecker LF, Hossini AM, Wieder T, Daniel PT, Orfanos CE, Geilen CC. CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants. Oncogene 2003; 22:9131-41. [PMID: 14668794 DOI: 10.1038/sj.onc.1207228] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The significance of CD95/Fas ligand expression by melanoma cells has remained a controversial matter in recent years. On the other hand, CD95 activation may represent a powerful tool for eliminating tumor cells. Here, we demonstrate expression of CD95 in 15/17 human melanoma cell lines analysed, but complete lack of CD95 ligand (CD95L). Overexpression of CD95 in a tetracycline-inducible expression system enhanced melanoma cell sensitivity to CD95 ligation but was unable to trigger apoptosis by itself. In clear contrast, all melanoma cells tested responded with increased apoptosis to conditional expression of CD95L (2-10-fold), both after transient and after stable transfection. Activation of caspase-8, Bid cleavage, cytochrome c release and caspase-3 activation followed after CD95L induction indicating a functional CD95-signaling cascade. CD95L was also able to enhance the proapoptotic effect of chemotherapeutics applied in parallel. Nude mouse experiments revealed that tumorigenicity was lost when melanoma xenografts were triggered to express CD95L. In addition, further progression of pre-existing melanomas was inhibited and even regression was seen after induction of CD95L expression. Due to these data, transfection of CD95L proofs as a highly efficient tool against melanoma cells in vitro and in vivo, and targeted expression of CD95L may thus represent a suitable strategy for melanoma therapy.
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Affiliation(s)
- Jürgen Eberle
- Department of Dermatology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, 14195 Berlin, Germany.
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41
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Prokop A, Wrasidlo W, Lode H, Herold R, Lang F, Henze G, Dörken B, Wieder T, Daniel PT. Induction of apoptosis by enediyne antibiotic calicheamicin ϑII proceeds through a caspase-mediated mitochondrial amplification loop in an entirely Bax-dependent manner. Oncogene 2003; 22:9107-20. [PMID: 14647446 DOI: 10.1038/sj.onc.1207196] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Calicheamicin thetaII is a member of the enediyne class of antitumor antibiotics that bind to DNA and induce apoptosis. These compounds differ, however, from conventional anticancer drugs as they bind in a sequence-specific manner noncovalently to DNA and cause sequence-selective oxidation of deoxyriboses and bending of the DNA helix. Calicheamicin is clinically employed as immunoconjugate to antibodies directed against, for example, CD33 in the case of gemtuzumab ozogamicin. Here, we show by the use of the unconjugated drug that calicheamicin-induced apoptosis is independent from death-receptor/FADD-mediated signals. Moreover, calicheamicin triggers apoptosis in a p53-independent manner as shown by the use of p53 knockout cells. Cell death proceeds via activation of mitochondrial permeability transition, cytochrome c release and activation of caspase-9 and -3. The overexpression of Bcl-x(L) or Bcl-2 strongly inhibited calicheamicin-induced apoptosis. Knockout of Bax abrogated cell death after calicheamicin treatment. Thus, the activation of mitochondria and execution of cell death occur through a fully Bax-dependent mechanism. Interestingly, caspase inhibition by the pancaspase-inhibitor zVAD-fmk interfered with mitochondrial activation by calicheamicin. This places caspase activation upstream of the mitochondria and indicates that calicheamicin-triggered apoptosis is enhanced through death receptor-independent activation of the caspase cascade, that is, an amplification loop that is required for full activation of the mitochondrial pathway.
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Affiliation(s)
- Aram Prokop
- Department of Pediatric Oncology/Hematology, University Medical Center Charité, Humboldt University of Berlin, Berlin 13353, Germany
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42
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Rau B, Sturm I, Lage H, Berger S, Schneider U, Hauptmann S, Wust P, Riess H, Schlag PM, Dörken B, Daniel PT. Dynamic expression profile of p21WAF1/CIP1 and Ki-67 predicts survival in rectal carcinoma treated with preoperative radiochemotherapy. J Clin Oncol 2003; 21:3391-401. [PMID: 12885834 DOI: 10.1200/jco.2003.07.077] [Citation(s) in RCA: 105] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
PURPOSE We investigated p53 and its downstream effectors p21WAF1/CIP1, BAX, and hMSH2 as well as the proliferation marker Ki-67 (mki-67/MIB-1) in patients undergoing preoperative radiochemotherapy for rectal carcinoma to identify prognostic and predictive factors. The focus of this study was on the dynamics of these genetic markers in a longitudinal study-that is, before and after radiochemotherapy. PATIENTS AND METHODS Expression of p53, BAX, p21WAF1/CIP1, Ki-67, and hMSH2 was investigated by immunohistochemistry in pre- and posttherapeutic tumor samples in 66 patients. Tumor DNA was screened for p53 mutations by single-strand conformation polymorphism-polymerase chain reaction (SSCP-PCR). Paired tumor samples (pretherapy and posttherapy) were collected prospectively. RESULTS Patients with a decrease in p21 expression following radiochemotherapy had better disease-free survival (P =.03). Similarly, patients with an increase in proliferative activity as measured by increased Ki-67 expression posttherapy had better disease-free survival (P <.005). In addition, we observed a significantly better prognosis for patients with high hMSH2 expression. In contrast, pretherapeutic levels of p53, BAX, or p21 expression and p53 mutation had no prognostic value, indicating that the combination of radiotherapy and chemotherapy might override defects in these genes. CONCLUSION These findings are novel and support the clinical relevance of p21 in the suppression of both proliferation and apoptosis. Thus, the dynamic induction of p21WAF1/CIP1 was associated with a lower proliferative activity but an ultimately worse treatment outcome following neoadjuvant radiochemotherapy and tumor resection. Induction of p21, therefore, represents a novel resistance mechanism in rectal cancer undergoing preoperative radiochemotherapy.
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Affiliation(s)
- Beate Rau
- Charité Medical School, Campus Berlin-Buch, Humboldt University, Department of Surgery and Surgical Oncology, Robert-Roessle Klinik, Lindenberger Weg 80, 13125 Berlin, Germany.
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Naumann U, Schmidt F, Wick W, Frank B, Weit S, Gillissen B, Daniel P, Weller M. Adenoviral natural born killer gene therapy for malignant glioma. Hum Gene Ther 2003; 14:1235-46. [PMID: 12952595 DOI: 10.1089/104303403767740777] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Glioblastoma is a lethal neoplasm resistant to conventional radiotherapy and chemotherapy. Natural born killer (NBK), also known as Bcl-2-interacting killer (BIK), is a death-promoting Bcl-2 family protein sharing with Bcl-2 only the Bcl homology 3 (BH3) domain. We here report that an adenoviral vector encoding NBK (Ad-NBK) uniformly induces cell death in 12 human malignant glioma cell lines. Ad-NBK-induced cell death involves neither quantitative mitochondrial cytochrome c release nor caspase 8, 9, 7, or 3 processing and is unaffected by the viral caspase inhibitor, cytokine response modifier A (CRM-A), or selective caspase 8 or 9 inhibitors. In contrast, Ad-NBK-induced cell death is inhibited by the broad-range caspase inhibitor, zVAD-fmk, or by adenoviral gene transfer of the X-linked inhibitor of apoptosis protein (XIAP). Further, Ad-NBK-induced cell death is inhibited by Bcl-2 or Bcl-xL gene transfer. Interestingly, Bcl-2- and Bcl-xL-transfected glioma cells, which are partially protected from Ad-NBK-induced cell death, accumulate much higher levels of NBK than are ever observed in control-infected cells. This indicates that complex formation with Bcl-2 or Bcl-xL sequesters NBK in an inactive form and that free NBK, rather than an NBK-mediated depletion of free antiapoptotic Bcl-2 family proteins, is the proximate mediator of Ad-NBK-induced cell death. Conversely, proteasome inhibition-mediated accumulation of NBK strongly enhances Ad-NBK-induced cell death. Finally, Ad-NBK-infected LN-229 glioma cells are not tumorigenic in nude mice. Thus Ad-NBK triggers an XIAP- and zVAD-fmk-sensitive cell death pathway in glioma cells with potential therapeutic value, provided that NBK expression can be selectively targeted to cancer cells.
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Affiliation(s)
- Ulrike Naumann
- Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tübingen, School of Medicine, D-72076 Tübingen, Germany.
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Gillissen B, Essmann F, Graupner V, Stärck L, Radetzki S, Dörken B, Schulze-Osthoff K, Daniel PT. Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway. EMBO J 2003; 22:3580-90. [PMID: 12853473 PMCID: PMC165613 DOI: 10.1093/emboj/cdg343] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-specific BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a single- vector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-deficient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wild-type HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x(L) and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not sufficient to confer sensitivity to Nbk.
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Affiliation(s)
- Bernhard Gillissen
- Department of Hematology, Charité-Campus Berlin-Buch, Humboldt University, D-13125 Berlin-Buch, Germany
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von Haefen C, Wieder T, Essmann F, Schulze-Osthoff K, Dörken B, Daniel PT. Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent, caspases-3/-8-driven mitochondrial amplification loop. Oncogene 2003; 22:2236-47. [PMID: 12700660 DOI: 10.1038/sj.onc.1206280] [Citation(s) in RCA: 140] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Caspase-8 is a key effector of death-receptor-triggered apoptosis. In a previous study, we demonstrated, however, that caspase-8 can also be activated in a death receptor-independent manner via the mitochondrial apoptosis pathway, downstream of caspase-3. Here, we show that caspases-3 and -8 mediate a mitochondrial amplification loop that is required for the optimal release of cytochrome c, mitochondrial permeability shift transition, and cell death during apoptosis induced by treatment with the microtubule-damaging agent paclitaxel (Taxol). In contrast, Smac release from mitochondria followed a different pattern, and therefore seems to be regulated independently from cytochrome c release. Taxol-induced cell death was inhibited by the use of synthetic, cell-permeable caspase-3- (zDEVD-fmk) or caspase-8-specific (zIETD-fmk) inhibitors. Apoptosis signaling was not affected by a dominant-negative FADD mutant (FADD-DN), thereby excluding a role of death receptor signaling in the amplification loop and drug-induced apoptosis. The inhibitor experiments were corroborated by the use of BJAB cells overexpressing the natural serpin protease inhibitor, cytokine response modifier A. These data demonstrate that the complete activation of mitochondria, release of cytochrome c, and execution of drug-induced apoptosis require a mitochondrial amplification loop that depends on caspases-3 and -8 activation. In addition, this is the first report to demonstrate death receptor-independent caspase-8 autoprocessing in vivo.
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Affiliation(s)
- Clarissa von Haefen
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, University of Berlin, Germany
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Mrózek A, Petrowsky H, Sturm I, Kraus J, Hermann S, Hauptmann S, Lorenz M, Dörken B, Daniel PT. Combined p53/Bax mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability. Cell Death Differ 2003; 10:461-7. [PMID: 12719723 DOI: 10.1038/sj.cdd.4401193] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
Gastric cancer is highly refractory to DNA-damaging therapies. We therefore studied both gene mutation and protein expression of p53 and Bax in a cohort of 116 patients with gastric cancer who underwent R0-resection with a curative intent. Bax mutation was independent from severe microsatellite instability (MSI), that is, global mismatch repair deficiency as determined by analysis of BAT-25/BAT-26 microsatellite markers. Thus, Bax-frameshift mutation is a feature of tumors with low MSI. In contrast and as expected, no p53 mutations were observed in the microsatellite instable tumors. p53 Mutation or p53 overexpression did not have an impact on disease prognosis. p53-Inactivation was, however, associated with an extremely poor prognosis in the subgroup of patients with Bax-mutated tumors. Thus, we show for the first time that the combined mutation of p53 and Bax, two key regulators of the mitochondrial apoptosis pathway, results in an extremely aggressive tumor biology and poor clinical prognosis.
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Affiliation(s)
- A Mrózek
- 1Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Humboldt University, Berlin, Germany
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Sturm I, Bosanquet AG, Hermann S, Güner D, Dörken B, Daniel PT. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death Differ 2003; 10:477-84. [PMID: 12719725 DOI: 10.1038/sj.cdd.4401194] [Citation(s) in RCA: 109] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Inactivation of p53 has been shown to correlate with poor prognosis and drug resistance in malignant tumors. Nevertheless, few reports have directly shown such effects in primary tumor cells. Here, we investigated the p53 mutational status in 138 B-CLL samples and compared these findings with drug and gamma-irradiation sensitivity profiles. p53 mutations resulted not only in a shorter survival but, notably also in selective resistance to alkylating agents, fludarabine and gamma-irradiation. In contrast, no such effect was observed for vincristine, anthracyclines and glucocorticoids. Thus, these latter compounds induce cell death at least in part by p53-independent pathways. Interestingly, p53 mutations clustered in patients who had received prior chemotherapy. In fact, we show for the first time that treatment with DNA-damaging alkylating agents correlates with occurrence of p53 mutations in a clinical setting. This finding may explain at least to some extent the development of resistance to second-line anticancer chemotherapy.
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MESH Headings
- Antineoplastic Agents, Alkylating/adverse effects
- Apoptosis/drug effects
- Apoptosis/genetics
- Chlorambucil/adverse effects
- Cyclophosphamide/adverse effects
- DNA Damage/drug effects
- DNA Damage/genetics
- DNA Mutational Analysis
- Drug Resistance, Neoplasm/genetics
- Female
- Gamma Rays/therapeutic use
- Humans
- Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy
- Leukemia, Lymphocytic, Chronic, B-Cell/genetics
- Leukemia, Lymphocytic, Chronic, B-Cell/metabolism
- Male
- Middle Aged
- Mutation/drug effects
- Risk Factors
- Survival Rate
- Tumor Suppressor Protein p53/drug effects
- Tumor Suppressor Protein p53/genetics
- Vidarabine/adverse effects
- Vidarabine/analogs & derivatives
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Affiliation(s)
- I Sturm
- Department of Hematology, Oncology and Tumor Immunology, Charité-Campus Berlin-Buch, Humboldt University, 13125 Berlin-Buch, Germany
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Güner D, Sturm I, Hemmati P, Hermann S, Hauptmann S, Wurm R, Budach V, Dörken B, Lorenz M, Daniel PT. Multigene analysis of Rb pathway and apoptosis control in esophageal squamous cell carcinoma identifies patients with good prognosis. Int J Cancer 2003; 103:445-54. [PMID: 12478659 DOI: 10.1002/ijc.10850] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Deregulation of cell-cycle G(1)-restriction point control by disruption of Rb-pathway components is a frequent event in cancer. In concert with the inactivation of cell death pathways, such events not only contribute to tumor development but also determine the intrinsic and acquired resistance to cancer therapy and, ultimately, disease prognosis. We previously observed that the cyclin-dependent kinase inhibitor p16(INK4a) and the proapoptotic Bcl-2 homolog Bax are positive prognostic factors and identify patients with good prognosis in esophageal squamous cell carcinoma (SCC). In the present study, we therefore extend our analysis to additional genes controlling the G(1) restriction point and apoptosis, respectively. This retrospective analysis was performed in a cohort of 53 patients undergoing surgery for esophageal SCC with curative intent, i.e., R0 resection. Protein expression profiles of cyclin D1, p16(INK4a), Rb, p21(CIP/WAF-1), p53, Bax and Bcl-2 were analyzed by immunohistochemistry and compared to p53 mutational status, as determined by SSCP-PCR of exons 5-8. Loss of p16(INK4a), Rb, p21(CIP/WAF-1) or Bax and overexpression of cyclin D1 were associated individually with shorter overall survival, while Bcl-2 expression and p53 mutation were not of prognostic relevance. The longest survival was observed in a subgroup of patients whose tumors bore a combination of favorite genotypes, i.e., low cyclin D1 and high Rb, p21(CIP/WAF-1), p16(INK4a) and Bax protein expression. These results show that multigene analyses based on limited sets of functionally linked genes reliably identify patients with good vs. poor prognosis.
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Affiliation(s)
- Dilek Güner
- Department of Radiation Oncology, University Medical Center Charité, Humboldt University, Lindenberger Weg 80, 13125 Berlin-Buch, Germany
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Zhou CZ, Peng ZH, Zhang F, Qiu GQ, He L. Loss of heterozygosity on long arm of chromosome 22 in sporadic colorectal carcinoma. World J Gastroenterol 2002; 8:668-73. [PMID: 12174376 PMCID: PMC4656318 DOI: 10.3748/wjg.v8.i4.668] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: The loss of heterozygosity (LOH) on tumor suppressor genes is believed to play a key role in carcinogenesis of colorectal cancer. In this study, we analyzed the LOH at 5 loci on the long arm of chromosome 22 in sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis.
METHODS: Five polymorphic microsatellite markers were analyzed in 83 cases of colorectal and normal DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer; Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological data were performed by χ² test. P < 0.05 was considered as statistically significant.
RESULTS: The average LOH frequency on chromosome 22q was 28.38%. The region between markers D22S280 and D22S274 (22q12.2-q13.33) exhibited relatively high LOH frequency. The two highest LOH loci with frequencies of 35.09% and 34.04% was identified on D22S280 (22q12.2-12.3) and D22S274 (22q13.32-13.33).8 cases showed LOH at all informative loci, suggesting that one chromosome 22q had been completely lost. On D22S274 locus, LOH frequency of rectal cancer was 50% (9/18), which was higher than that of proximal colon cancer (12%, 2/17) (P = 0.018). The frequency of distal colon cancer was 42% (5/12), also higher than that of proximal colon cancer. But there was no statistical significance. Putting both the tumors in distal colon and rectum together into consideration, the frequency, 47% (14/30), was higher than that of proximal colon cancer (P = 0.015), suggesting the mechanism of carcinogenisis was different in both groups.
CONCLUSIONS: This study provided evidence for the involvement of putative tumor suppressor genes related to the sporadic colorectal carcinoma on chromosome 22q. The tumor-suppressor-gene (s) might locate on the 22q12.2-12.3 and/or 22q13.32-13.33.
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Affiliation(s)
- Chong-Zhi Zhou
- Department of General Surgery, Shanghai First People Hospital, 85 Wujin Road, Shanghai 200080, China
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50
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von Haefen C, Wieder T, Gillissen B, Stärck L, Graupner V, Dörken B, Daniel PT. Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells. Oncogene 2002; 21:4009-19. [PMID: 12037683 DOI: 10.1038/sj.onc.1205497] [Citation(s) in RCA: 154] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2001] [Revised: 03/06/2002] [Accepted: 03/18/2002] [Indexed: 12/26/2022]
Abstract
The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.
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Affiliation(s)
- Clarissa von Haefen
- Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Humboldt University of Berlin, 13125, Germany
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