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Li R, Zhang X, Tian X, Shen C, Zhang Q, Zhang Y, Wang Z, Wang F, Tao Y. Triptolide inhibits tumor growth by induction of cellular senescence. Oncol Rep 2016; 37:442-448. [PMID: 27878302 DOI: 10.3892/or.2016.5258] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Accepted: 10/26/2016] [Indexed: 11/06/2022] Open
Abstract
Cellular senescence, an irreversible growth arrest of cells, is involved in protection against cancer. Triptolide (TPL) plays an important role in immunosuppressive, anti-fertility, anti-cystogenesis and anticancer activities. However, effect and mechanism of TPL on cellular senescence-associated antitumor is rarely reported. Herein HepG2 cells were used to explore the effect of TPL on tumor growth and cellular senescence. We showed that TPL inhibited tumor cell proliferation and growth in vitro and in vivo, accelerated cellular senescence and arrested cells at G0/G1 phase. We further demonstrated that TPL accelerated HepG2 cell senescence by regulating the AKT pathway. In addition, TPL could also enhance cellular senescence and inhibit tumor growth by negatively regulating human telomerase reverse transcriptase (hTERT) signaling pathway. These findings reveal a regulatory mechanism of TPL on cellular senescence, indicating that TPL promotes HepG2 cell senescence through AKT pathway and hTERT pathway simultaneously. Altogether, TPL-induced senescence can be regarded as a promising strategy for anticancer therapy and drug development.
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Affiliation(s)
- Ruidong Li
- Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
| | - Xiaofei Zhang
- Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
| | - Xiaoying Tian
- Bioscience Research Center, Shanghai 200120, P.R. China
| | - Conghuan Shen
- Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
| | - Quanbao Zhang
- Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
| | - Yihong Zhang
- Bioscience Research Center, Shanghai 200120, P.R. China
| | - Zhengxin Wang
- Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
| | - Feifei Wang
- Bioscience Research Center, Shanghai 200120, P.R. China
| | - Yifeng Tao
- Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
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Rana C, Piplani H, Vaish V, Nehru B, Sanyal SN. Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer. Tumour Biol 2015; 36:5999-6010. [PMID: 25744732 DOI: 10.1007/s13277-015-3276-7] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2014] [Accepted: 02/18/2015] [Indexed: 12/31/2022] Open
Abstract
Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.
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Affiliation(s)
- Chandan Rana
- Department of Biophysics, Panjab University, Chandigarh, 160014, India
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3
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Wu XQ, Huang C, He X, Tian YY, Zhou DX, He Y, Liu XH, Li J. Feedback regulation of telomerase reverse transcriptase: new insight into the evolving field of telomerase in cancer. Cell Signal 2013; 25:2462-8. [DOI: 10.1016/j.cellsig.2013.08.009] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2013] [Accepted: 08/23/2013] [Indexed: 01/07/2023]
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4
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Fujie H, Tanaka T, Tagawa M, Kaijun N, Watanabe M, Suzuki T, Nakayama K, Numasaki M. Antitumor activity of type III interferon alone or in combination with type I interferon against human non-small cell lung cancer. Cancer Sci 2011; 102:1977-90. [PMID: 21883692 PMCID: PMC11158295 DOI: 10.1111/j.1349-7006.2011.02079.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
The antitumor activities of type III interferon (IFN) (interleukin [IL]-28 and IL-29) and the combination of type III IFN and type I IFN (IFN-α) were evaluated using human non-small cell lung cancer (NSCLC). The expression of type III and type I receptor complexes was detected in NSCLC lines. IL-29 significantly inhibited the in vitro growth of a wide range of NSCLC lines in a dose-dependent fashion. To a lesser degree, IL-28A also displayed growth inhibitory activity. Antitumor activity of type III IFN is associated with cell cycle arrest at the G1 phase and apoptosis. IL-29 upregulated cyclin-dependent kinase inhibitor p21Waf1/Cip1 in cells sensitive, but not insensitive, to antiproliferative activity, and knockdown of p21 with small interfering RNA largely attenuated the antiproliferative effect. Intratumoral and systemic administration of IL-29 inhibited OBA-LK1 and LK-1, but not A549, tumor growth in severe combined immunodeficiency mice. Immunohistochemical analyses demonstrated marked upregulated p21 and downregulated Ki-67 expression in tumors treated with IL-29. The interferon combination of IL-29 and IFN-α displayed a more effective antiproliferative effect and a more intense p21 expression than each reagent alone in vitro. Furthermore, interferon combination therapy suppressed in vivo NSCLC growth more effectively than interferon monotherapy. These findings demonstrate that type III IFN can mediate direct antitumor activities via increased p21 expression and induction of apoptosis and cooperate with type I IFN to elicit more efficient direct antitumor activities, and suggest the possibility that type III IFN might improve the efficacy and reduce the side-effects of type I IFN cancer therapy.
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MESH Headings
- Animals
- Apoptosis/drug effects
- Carcinoma, Non-Small-Cell Lung/drug therapy
- Carcinoma, Non-Small-Cell Lung/metabolism
- Carcinoma, Non-Small-Cell Lung/pathology
- Cell Line, Tumor/drug effects
- Cyclin-Dependent Kinase Inhibitor p21/biosynthesis
- Cyclin-Dependent Kinase Inhibitor p21/genetics
- Dose-Response Relationship, Drug
- Drug Screening Assays, Antitumor
- Drug Synergism
- G1 Phase/drug effects
- Gene Expression Regulation, Neoplastic/drug effects
- Humans
- Interferon-alpha/pharmacology
- Interferon-alpha/therapeutic use
- Interferons
- Interleukins/pharmacology
- Interleukins/therapeutic use
- Lung Neoplasms/drug therapy
- Lung Neoplasms/metabolism
- Lung Neoplasms/pathology
- Mesothelioma/metabolism
- Mesothelioma/pathology
- Mice
- Mice, SCID
- Neoplasm Proteins/analysis
- Neoplasm Proteins/biosynthesis
- Neoplasm Proteins/drug effects
- Neoplasm Proteins/genetics
- Receptor, Interferon alpha-beta/analysis
- Receptor, Interferon alpha-beta/drug effects
- Receptors, Cytokine/analysis
- Receptors, Cytokine/biosynthesis
- Receptors, Cytokine/drug effects
- Receptors, Cytokine/genetics
- Tumor Stem Cell Assay
- Up-Regulation/drug effects
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Hitomi Fujie
- Department of Nutrition Physiology, Faculty of Pharmaceutical Sciences, Josai University, Sakado, Japan
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5
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Dynamic telomerase gene suppression via network effects of GSK3 inhibition. PLoS One 2009; 4:e6459. [PMID: 19649288 PMCID: PMC2714081 DOI: 10.1371/journal.pone.0006459] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2009] [Accepted: 06/30/2009] [Indexed: 01/15/2023] Open
Abstract
Background Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. Methodology/Principal Findings In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3′-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFκB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. Conclusions/Significance Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.
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6
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The biological activity of G-quadruplex DNA binding papaverine-derived ligand in breast cancer cells. Invest New Drugs 2008; 27:289-96. [DOI: 10.1007/s10637-008-9173-9] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2008] [Accepted: 08/18/2008] [Indexed: 11/30/2022]
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7
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Ding L, Zhu S, Xie S, Wu X. Effect of exogenous bFGF on the proliferation of human adenoid cystic carcinoma ACC-2 cells. ACTA ACUST UNITED AC 2008; 28:227-9. [DOI: 10.1007/s11596-008-0229-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2007] [Indexed: 10/19/2022]
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8
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Ueda Y, Watanabe S, Tei S, Saitoh N, Kuratsu JI, Nakao M. High mobility group protein HMGA1 inhibits retinoblastoma protein-mediated cellular G0 arrest. Cancer Sci 2007; 98:1893-901. [PMID: 17877762 PMCID: PMC11160013 DOI: 10.1111/j.1349-7006.2007.00608.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2007] [Revised: 07/24/2007] [Accepted: 08/04/2007] [Indexed: 11/30/2022] Open
Abstract
Retinoblastoma protein (RB) acts as a tumor suppressor in many tissue types, by promoting cell arrest via E2F-mediated transcriptional repression. In addition to the aberrant forms of the RB gene found in different types of cancers, many viral oncoproteins including the simian virus 40 large T antigen target RB. However, cellular factors that inhibit RB function remain to be elucidated. Here, we report that RB interacts with the high mobility group protein A1 (HMGA1), a-non-histone architectural chromatin factor that is frequently overexpressed in cancer cells. HMGA1 binds the small pocket domain of RB, and competes with HDAC1. Subsequently, overexpression of HMGA1 abolishes the inhibitory effect of RB on E2F-activated transcription from the cyclin E promoter. Under serum starvation, T98G cells had been previously shown to be arrested in the G0 phase in an RB-mediated manner. The G0 phase was characterized by growth arrest and low levels of transcription, together with the hypophosphorylation of RB and the downregulation of HMGA1. In contrast, such serum-depleted G0 arrest was abrogated in T98G cells overexpressing HMGA1. The overexpressed HMGA1 was found to form complexes with cellular RB, suggesting that downregulation of HMGA1 is required for G0 arrest. There were no phenotypic changes in HMGA1-expressing T98G cells in the presence of serum, but the persistent expression of HMGA1 under serum starvation caused various nuclear abnormalities, which were similarly induced in T antigen-expressing T98G cells. Our present findings indicate that overexpression of HMGA1 disturbs RB-mediated cell arrest, suggesting a negative control of RB by HMGA1.
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Affiliation(s)
- Yasuaki Ueda
- Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University 2-2-1 Honjo, Kumamoto 860-0811, Japan
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9
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Ide T. [Mechanism of cell proliferation--cell cycle, oncogenes, and senescence]. YAKUGAKU ZASSHI 2007; 126:1087-115. [PMID: 17077613 DOI: 10.1248/yakushi.126.1087] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.
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Affiliation(s)
- Toshinori Ide
- Department of Cellular and Molecular Biology, Division of Integrated Medical Science, Graduated School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City 734-8551, Japan.
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10
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Zupanska A, Adach A, Dziembowska M, Kaminska B. Alternative pathway of transcriptional induction of p21WAF1/Cip1 by cyclosporine A in p53-deficient human glioblastoma cells. Cell Signal 2007; 19:1268-78. [PMID: 17321721 DOI: 10.1016/j.cellsig.2007.01.011] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2006] [Revised: 01/08/2007] [Accepted: 01/08/2007] [Indexed: 11/28/2022]
Abstract
The cyclin-dependent kinase inhibitor p21WAF1/CIP1, a critical regulator of the cell cycle, is mainly regulated by p53 tumour suppressor at the transcriptional level. Restoration of p21WAF1/Cip1 expression in p53-deficient malignant cells suppress tumour growth. Cyclosporine A (CsA) affects proliferation and survival of cultured malignant glioma cells and impairs growth of experimental gliomas. CsA induced p21WAF1/Cip1 expression de novo in human glioblastoma cells with p53 deficiency. We demonstrate that transcriptional activation of p21WAF1/Cip1 expression correlated with induction of ERK1/2 and c-Jun phosphorylation in CsA-treated glioblastoma cells. Pre-treatment with ERK pathway inhibitors or overexpression of dominant-negative mutants MKK1, ERK2 and c-Jun reduced activation of the p21WAF1/Cip1 promoter. Overexpression of tethered AP-1 dimers containing c-Jun was sufficient to activate the truncated -200 bp p21WAF1/Cip1 promoter, which does not contain p53 binding sites. Chromatin immunoprecipitation revealed that P-c-Jun is bound to the proximal part of p21WAF1/Cip1 promoter in CsA-treated glioblastoma cells. It suggests that CsA activates p53-independent, transcriptional activation p21WAF1/Cip1 expression, mediated by ERK/c-Jun/AP-1 signaling pathway.
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Affiliation(s)
- Agata Zupanska
- Laboratory of Transcription Regulation, Department Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str., 02-093, Warsaw, Poland
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11
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Lai SR, Cunningham AP, Huynh VQ, Andrews LG, Tollefsbol TO. Evidence of extra-telomeric effects of hTERT and its regulation involving a feedback loop. Exp Cell Res 2007; 313:322-30. [PMID: 17134697 PMCID: PMC1829489 DOI: 10.1016/j.yexcr.2006.10.014] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2006] [Revised: 09/29/2006] [Accepted: 10/16/2006] [Indexed: 10/24/2022]
Abstract
The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the enzyme telomerase which is responsible for telomeric maintenance and extension. Using RNA interference to knock down hTERT mRNA expression, we provide evidence that hTERT exerts extra-telomeric effects on the cell cycle and on its own regulatory proteins, specifically: p53 and p21. We tested our hypothesis that hTERT regulates its own expression through effects on upstream regulatory genes using transformed human embryonic kidney (HEK 293) cells, p53 and p16(INK4a) null human ovarian cancer SKOV-3 cells, and p53-null MDA-MB-157 human mammary cancer cells. In HEK 293 cells, hTERT knockdown resulted in elevated p53 and p21 transcription and a decrease in cellular proliferation. Similar results were observed in the MDA-MB-157 cell line where p21 was upregulated, correlating with cell growth inhibition. In contrast, we observed a decrease in expression of p21 in SKOV-3 cells with hTERT knockdown and cell growth appeared to be unaffected. These findings suggest that hTERT may be involved in a feedback loop system, thereby playing a role in its own regulation.
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Affiliation(s)
- Serene R. Lai
- Department of Biology, University of Alabama at Birmingham, AL 35294, USA
| | | | - Vu Q. Huynh
- Department of Biology, University of Alabama at Birmingham, AL 35294, USA
- Center for Aging, University of Alabama at Birmingham, AL 35294, USA
- Comprehensive Cancer Center, University of Alabama at Birmingham, AL 25294, USA
| | - Lucy G. Andrews
- Department of Biology, University of Alabama at Birmingham, AL 35294, USA
| | - Trygve O. Tollefsbol
- Department of Biology, University of Alabama at Birmingham, AL 35294, USA
- Center for Aging, University of Alabama at Birmingham, AL 35294, USA
- Comprehensive Cancer Center, University of Alabama at Birmingham, AL 25294, USA
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12
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Zhu B, Zhang LH, Zhao YM, Cui JR, Strada SJ. 8-chloroadenosine induced HL-60 cell growth inhibition, differentiation, and G(0)/G(1) arrest involves attenuated cyclin D1 and telomerase and up-regulated p21(WAF1/CIP1). J Cell Biochem 2006; 97:166-77. [PMID: 16173047 DOI: 10.1002/jcb.20630] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
8-Chloroadenosine, an active dephosphorylated metabolite of the antineoplastic agent 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), induces growth inhibition in multiple carcinomas. Here we report that 8-chloroadenosine inhibits growth in human promyelocytic leukemia HL-60 cells by a G(0)/G(1) phase arrest and terminates cell differentiation along the granulocytic lineage. The mechanism of 8-chloroadenosine-induced G(0)/G(1) arrest is independent of apoptosis. The expressions of cyclin D1 and c-myc in HL-60 are suppressed by 8-chloroadenosine, whereas the cyclin-dependent kinases inhibitor p21(WAF1/CIP1) is up-regulated. 8-Chloroadenosine has less effect on the expressions of cyclin-dependent kinase (cdk)2 and cdk4, G(1) phase cyclin-dependent kinases, and only moderately induces the expression of transforming growth factor beta1 (TGFbeta1) and the mitotic inhibitor p27(KIP1). Telomerase activity is reduced in extracts of 8-chloroadenosine treated HL-60 cells, but 8-chloroadenosine does not directly inhibit the catalytic activity of telomerase in vitro. Therefore, anti-proliferation of HL-60 cells by 8-chloroadenosine involves coordination of cyclin D1 suppression, reduction of telomerase activity, and up-regulation of p21(WAF1/CIP1) that arrest cell-cycle progression at G(0)/G(1) phase and terminate cell differentiation.
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Affiliation(s)
- Bing Zhu
- Department of Pharmacology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
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Chen GG, Sin FLF, Leung BCS, Ng HK, Poon WS. Glioblastoma cells deficient in DNA-dependent protein kinase are resistant to cell death. J Cell Physiol 2005; 203:127-32. [PMID: 15493013 DOI: 10.1002/jcp.20230] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli.
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Affiliation(s)
- George G Chen
- Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong.
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14
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Kunisada M, Budiyanto A, Bito T, Nishigori C, Ueda M. Retinoic acid suppresses telomerase activity in HSC-1 human cutaneous squamous cell carcinoma. Br J Dermatol 2005; 152:435-43. [PMID: 15787811 DOI: 10.1111/j.1365-2133.2005.06471x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND Activation of telomerase is crucial for the continued growth and progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumours. OBJECTIVE Because retinoic acid (RA) plays an important role in the growth and differentiation of keratinocytes and as RA has some preventive and therapeutic effects on human skin cancers, we examined the effect of RA on the telomerase activity of HSC-1 human cutaneous squamous cell carcinoma cells. RESULTS Treatment of HSC-1 cells with all-trans RA (ATRA) significantly suppressed their telomerase activity. The suppression of telomerase activity was obvious at day 4 and was maximal at day 5 after the start of treatment with RA. This suppression was reversible as removal of ATRA allowed the recovery of telomerase activity. The suppression of telomerase activity correlated with the decreased expression of mRNA of human telomerase catalytic subunit (hTERT), the rate-limiting determinant of enzyme activity. The production of c-myc and of Sp1 proteins, transcription factors regulating hTERT expression, was not suppressed in HSC-1 cells by ATRA, but phosphorylation of extracellular signal-regulated kinases (ERK)1/2 and of the serine/threonine kinase Akt was significantly suppressed. Phosphorylation of the epidermal growth factor receptor, which regulates hTERT expression in HSC-1 cells, was not altered by ATRA. CONCLUSIONS These data indicate that RA is effective in inhibiting telomerase activity in HSC-1 cells. Suppression of ERK1/2 and Akt activation is presumed to be involved in the RA-induced suppression of hTERT.
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Affiliation(s)
- M Kunisada
- Division of Dermatology, Clinical Molecular Medicine, Faculty of Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
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15
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Zhang WG, Yu JP, Wu QM, Tong Q, Li SB, Wang XH, Xie GJ. Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells. World J Gastroenterol 2004; 10:2779-84. [PMID: 15334669 PMCID: PMC4572101 DOI: 10.3748/wjg.v10.i19.2779] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells.
METHODS: Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by 3H-thymidine (3H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27kip1 was detected by immunocytochemical technique.
RESULTS: After exposed to MG-132, the growth and value of 3H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 μmol/L of MG-132 for 24, 48, 72 and 96 h (P < 0.01). The percentage of cells at G0/G1 phase was increased and that at S and G2/M was decreased (P < 0.01). The rate of apoptotic cells treated with 5 μmol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27kip1 were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group.
CONCLUSION: MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27kip1 expression, G1 arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.
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Affiliation(s)
- Wei-Guo Zhang
- Digestive Department, Taihe Hospital, Yunyang Medical College, Shiyan 442000, Hubei Province, China.
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Budiyanto A, Bito T, Kunisada M, Ashida M, Ichihashi M, Ueda M. Inhibition of the epidermal growth factor receptor suppresses telomerase activity in HSC-1 human cutaneous squamous cell carcinoma cells. J Invest Dermatol 2004; 121:1088-94. [PMID: 14708611 DOI: 10.1046/j.1523-1747.2003.12529.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Activation of telomerase, which stabilizes the telomere length of chromosomes, is crucial for the continued growth or progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumors. Because epidermal growth factor plays an important role during the tumorigenesis of epithelial tissue, we have now examined the role of epidermal growth factor signaling in regulating telomerase activity using HSC-1 human cutaneous squamous cell carcinoma cells. Treatment of HSC-1 cells with AG 1478, an inhibitor of the epidermal growth factor receptor, or with a neutralizing antibody to the epidermal growth factor receptor, significantly suppressed their telomerase activity, in association with inhibiting their growth. The suppression of telomerase activity was obvious at day 3 and was maximal at day 5 after treatment with AG 1478. The suppression of telomerase activity correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. The expression of c-Myc and of Sp1 proteins, transcription factors for hTERT, were also suppressed by AG 1478 in HSC-1 cells, but the expression of Ets-2 protein, another transcription factor, was not affected. The expression of Mad-1, a competitor of c-Myc, was increased. Inhibition of ERK, Src, or Akt suppressed telomerase activity in HSC-1 cells, but to a lesser extent than did treatment with AG 1478. Serum starvation suppressed telomerase activity, but addition of epidermal growth factor or transforming growth factor alpha did not increase it, indicating the involvement of other epidermal growth factor receptor ligands in the activation of telomerase in HSC-1 cells. These data indicate that blockade of the epidermal growth factor receptor might be effective in inhibiting telomerase activity of squamous cell carcinomas, which would lead to the suppression of tumor growth.
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Affiliation(s)
- Arief Budiyanto
- Division of Dermatology, Clinical Molecular Medicine, Faculty of Medicine, Kobe University Graduate School of Medicine, Japan
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Kim A, Lee EH, Choi SH, Kim CK. In vitro and in vivo transfection efficiency of a novel ultradeformable cationic liposome. Biomaterials 2004; 25:305-13. [PMID: 14585718 DOI: 10.1016/s0142-9612(03)00534-9] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Cationic lipids have been often used as one of the major components in making most promising non-viral gene delivery systems, whereas sodium cholate, a surfactant so-called edge activator has been used in preparing ultradeformable and ultraflexible liposomes called Transfersomes. Using both a cationic lipid, DOTAP and sodium cholate, a novel formulation of ultradeformable cationic liposome (UCL) has been prepared. The average particle size of this formulation was approximately 80 nm. The physical and chemical stabilities at two different temperatures (4 degrees C and 20 degrees C) were also evaluated for 60 days. The ultradeformability of new formulation was also assessed, and it has been proved that the formulation is deformable. In vitro transfection efficiency of plasmid DNA/UCL was assessed by the expression of green fluorescent protein (GFP) in four cell lines, OVCAR-3 (human ovarian carcinoma cells), HepG2 (human hepatoma cells), H-1299 (human lung carcinoma cells) and T98G (human brain carcinoma cells). The optimal ratio of DNA to liposome for maximal transfection efficiency was 1:14 (w/w) in all the cell lines except for the human brain carcinoma cells. The same formulation was tested for in vivo transfection efficiency and its retention time within the organs by applying the DNA/UCL complexes on hair-removed dorsal skin of mice non-invasively. It was found that genes were transported into several organs for 6 days once applied on intact skin.
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Affiliation(s)
- Adele Kim
- National Research Lab for Drug and Gene Delivery, College of Pharmacy, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea
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Sasakawa Y, Naoe Y, Inoue T, Sasakawa T, Matsuo M, Manda T, Mutoh S. Effects of FK228, a novel histone deacetylase inhibitor, on tumor growth and expression of p21 and c-myc genes in vivo. Cancer Lett 2003; 195:161-8. [PMID: 12767524 DOI: 10.1016/s0304-3835(03)00184-8] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
In this study, we examined the effects of FK228 (FR901228, depsipeptide) on tumor growth and expression of p21 and c-myc genes in vivo. FK228 induced the expression of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive to FK228. However, FK228 did not sufficiently modulate the expression of p21 mRNA and increased the expression of c-myc in tumor xenograft less sensitive to FK228. The modulation of p21 and/or c-myc genes may be critical for the marked antitumor activity of FK228 in vivo.
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MESH Headings
- Acetylation/drug effects
- Adenocarcinoma/drug therapy
- Adenocarcinoma/metabolism
- Adenocarcinoma/pathology
- Adrenal Gland Neoplasms/metabolism
- Adrenal Gland Neoplasms/pathology
- Animals
- Carcinoma, Non-Small-Cell Lung/metabolism
- Carcinoma, Non-Small-Cell Lung/pathology
- Carcinoma, Renal Cell/metabolism
- Carcinoma, Renal Cell/pathology
- Carcinoma, Small Cell/metabolism
- Carcinoma, Small Cell/pathology
- Cyclin-Dependent Kinase Inhibitor p21
- Cyclins/biosynthesis
- Cyclins/genetics
- Depsipeptides
- Enzyme Inhibitors/pharmacology
- Enzyme Inhibitors/therapeutic use
- Gene Expression Regulation, Neoplastic/drug effects
- Genes, myc
- Histone Deacetylase Inhibitors
- Humans
- Kidney Neoplasms/metabolism
- Kidney Neoplasms/pathology
- Lung Neoplasms/metabolism
- Lung Neoplasms/pathology
- Male
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Neoplasm Proteins/antagonists & inhibitors
- Neoplasm Proteins/biosynthesis
- Neoplasm Proteins/genetics
- Peptides, Cyclic/pharmacology
- Peptides, Cyclic/therapeutic use
- Pheochromocytoma/metabolism
- Pheochromocytoma/pathology
- Prostatic Neoplasms/metabolism
- Prostatic Neoplasms/pathology
- Protein Processing, Post-Translational/drug effects
- Proto-Oncogene Proteins c-myc/biosynthesis
- RNA, Messenger/biosynthesis
- RNA, Neoplasm/biosynthesis
- Tumor Cells, Cultured/drug effects
- Tumor Cells, Cultured/metabolism
- Tumor Cells, Cultured/transplantation
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Yuka Sasakawa
- Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka, 532-8514, Japan.
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Xu W, Yan M, Sun L, Zheng Z, Liu X. Ref-1 protein enhances the IL-2-stimulated telomerase activity. J Cell Biochem 2003; 88:1120-8. [PMID: 12647295 DOI: 10.1002/jcb.10462] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Telomerase is an important ribonucleoprotein enzyme involved in cellular proliferation and senescence. Activation of telomerase has been detected in a vast majority of human cancer cells. In this article, we demonstrated that Interleukin-2 (IL-2) which is the pivotal cytokine in the immune system could stimulate the activity of telomerase in the cultured BA/F3beta cells. It was also found that the level of IL-2-induced telomerase activity was decreased by the treatment with chemical oxidant in vitro. Since IL-2 stimulation produces a oxidative shift of the intracellular environment, the activation and maintenance of telomerase in this oxidative circumstance requires particular protection. Here we proved the redox factor-1 (Ref-1) protein was involved in this process. The addition of GST-Ref-1 protein increased the level of IL-2-induced telomerase activity in the TRAP assay, while elimination of the endogenous Ref-1 protein by immunodepletion decreased it. Consistent with these in vitro results, IL-2-induced telomerase activity could be enhanced by transient overexpression of Ref-1 protein in BA/F3beta cells. Taken together, these findings proved that Ref-1 protein benefits the activation of telomerase activity in the oxidative microenvironment of the BA/F3beta cells stimulated by IL-2.
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Affiliation(s)
- Weijing Xu
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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