p53-defective tumors with a functional apoptosome-mediated pathway: a new therapeutic target.

BACKGROUND Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear. METHODS We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests. RESULTS Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P<.001). Expression of ACSL5 suppressed Triacsin c-induced cytochrome c release and subsequent cell death (cell survival of Triacsin c-treated mock- versus ACSL5-transduced SF268 cells; means = 40% and 83%, respectively; difference = 43%, 95% CI = 39% to 47%; P<.001). ACS was also essential to the maintenance of cardiolipin levels. Finally, Triacsin c suppressed growth of xenograft tumors (relative tumor volume on day 21 of Triacsin c-treated versus untreated mice; means = 4.6 and 9.6, respectively; difference = 5.0, 95% CI = 2.1 to 7.9; P = .006). CONCLUSIONS Many p53-defective tumors retain activity of the apoptosome, which is therefore a potential target for cancer chemotherapy. Inhibition of ACS may be a novel strategy to induce the death of p53-defective tumor cells.

[1]  Scott W. Lowe,et al.  Apoptosis A Link between Cancer Genetics and Chemotherapy , 2002, Cell.

[2]  T. M. Lewin,et al.  Do long-chain acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways? , 2002, The Journal of nutrition.

[3]  N. Sakuragi,et al.  Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers , 2000, International journal of cancer.

[4]  D. Green,et al.  Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation , 2004, The Journal of cell biology.

[5]  Takashi Tsuruo,et al.  Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal , 2003, Cancer science.

[6]  Luca Scorrano,et al.  A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. , 2002, Cancer cell.

[7]  James A. Wells,et al.  Direct activation of the apoptosis machinery as a mechanism to target cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Dean P. Jones,et al.  The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore , 2004, Nature.

[9]  Tomoda Hiroshi,et al.  Inhibition of acyl-CoA synthetase by triacsins. , 1987 .

[10]  S. Lowe,et al.  Control of apoptosis by p53 , 2003, Oncogene.

[11]  Roger Brent,et al.  Cyclin D3 activates Caspase 2, connecting cell proliferation with cell death , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Mason R. Mackey,et al.  Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the Outer Mitochondrial Membrane , 2002, Cell.

[13]  Yongge Zhao,et al.  Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release. , 2004, Molecular biology of the cell.

[14]  T. Tsuruo,et al.  Modulation of Heat-shock Protein 27 (Hsp27) Anti-apoptotic Activity by Methylglyoxal Modification* , 2002, The Journal of Biological Chemistry.

[15]  G. Woldegiorgis,et al.  Studies on the interaction of palmitoyl coenzyme A with the adenine nucleotide translocase. , 1982, The Journal of biological chemistry.

[16]  Y. Lazebnik,et al.  Oncogene-dependent apoptosis is mediated by caspase-9. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[17]  V. Gogvadze,et al.  Cardiolipin Is Not Required for Bax-mediated Cytochrome c Release from Yeast Mitochondria* , 2004, Journal of Biological Chemistry.

[18]  Ramana V. Davuluri,et al.  Direct coupling of the cell cycle and cell death machinery by E2F , 2002, Nature Cell Biology.

[19]  S. Orrenius,et al.  The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis. , 2004, Archives of biochemistry and biophysics.

[20]  Marc Prentki,et al.  Saturated Fatty Acid-induced Apoptosis in MDA-MB-231 Breast Cancer Cells , 2003, Journal of Biological Chemistry.

[21]  L. Scorrano,et al.  Arachidonic Acid Causes Cell Death through the Mitochondrial Permeability Transition , 2000, The Journal of Biological Chemistry.

[22]  M. Lutter,et al.  Biochemical pathways of caspase activation during apoptosis. , 1999, Annual review of cell and developmental biology.

[23]  Y. Okuda,et al.  Intracellular fatty acid downregulates ob gene expression in 3T3-L1 adipocytes. , 2002, Biochemical and biophysical research communications.

[24]  T. Tsuruo,et al.  Potent antitumor activity of MS-247, a novel DNA minor groove binder, evaluated by an in vitro and in vivo human cancer cell line panel. , 1999, Cancer research.

[25]  S. B. Tove,et al.  Solubilization of a long chain fatty acyl-CoA synthetase from chicken adipose tissue microsomes. , 1974, Biochimica et biophysica acta.

[26]  A. Ruoho,et al.  Photoaffinity labeling of mitochondrial proteins with 2‐azido [32P]palmitoyl CoA , 1995, FEBS letters.

[27]  T. Yamori,et al.  Panel of human cancer cell lines provides valuable database for drug discovery and bioinformatics , 2003, Cancer Chemotherapy and Pharmacology.

[28]  T. Tsuruo,et al.  Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis , 1999, Oncogene.

[29]  R. Kanamaru,et al.  Screening the p53 status of human cell lines using a yeast functional assay , 1997, Molecular carcinogenesis.

[30]  T. Tsuruo,et al.  Dominant‐negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S‐S dependent homodimerization , 2002, International journal of cancer.

[31]  X. Liu,et al.  Increased expression of Apaf-1 and procaspase-3 and the functionality of intrinsic apoptosis apparatus in non-small cell lung carcinoma , 2004, Biological chemistry.

[32]  J C Reed,et al.  Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. , 1998, Science.

[33]  Takashi Tsuruo,et al.  Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide. , 2003, Cancer research.

[34]  K. Helin,et al.  Apaf-1 is a transcriptional target for E2F and p53 , 2001, Nature Cell Biology.

[35]  J N Weinstein,et al.  Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. , 1997, Cancer research.

[36]  Y. Soini,et al.  Expression of caspases 3, 6 and 8 is increased in parallel with apoptosis and histological aggressiveness of the breast lesion , 1999, British Journal of Cancer.

[37]  Y. Tsujimoto,et al.  Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Lowe,et al.  Dissecting p53 tumor suppressor functions in vivo. , 2002, Cancer cell.

[39]  Yong-Yeon Cho,et al.  Fatty acid induced glioma cell growth is mediated by the acyl-CoA synthetase 5 gene located on chromosome 10q25.1-q25.2, a region frequently deleted in malignant gliomas , 2000, Oncogene.

[40]  S. Prescott,et al.  Fatty acid CoA ligase 4 is up-regulated in colon adenocarcinoma. , 2001, Cancer research.

[41]  M. Wieckowski,et al.  Long‐chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore , 2000, FEBS letters.