Hsp60 Regulation of Tumor Cell Apoptosis 论文

摘要

Molecular chaperones may promote cell survival, but how this process is regulated, especially in cancer, is not well understood. Using high throughput proteomics screening, we identified the cell cycle regulator and apoptosis inhibitor survivin as a novel protein associated with the molecular chaperone Hsp60. Acute ablation of Hsp60 by small interfering RNA destabilizes the mitochondrial pool of survivin, induces mitochondrial dysfunction, and activates caspase-dependent apoptosis. This response involves disruption of an Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and Bax-dependent apoptosis. In vivo, Hsp60 is abundantly expressed in primary human tumors, as compared with matched normal tissues, and small interfering RNA ablation of Hsp60 in normal cells is well tolerated and does not cause apoptosis. Therefore, Hsp60 orchestrates a broad cell survival program centered on stabilization of mitochondrial survivin and restraining of p53 function, and this process is selectively exploited in cancer. Hsp60 inhibitors may function as attractive anticancer agents by differentially inducing apoptosis in tumor cells. Molecular chaperones may promote cell survival, but how this process is regulated, especially in cancer, is not well understood. Using high throughput proteomics screening, we identified the cell cycle regulator and apoptosis inhibitor survivin as a novel protein associated with the molecular chaperone Hsp60. Acute ablation of Hsp60 by small interfering RNA destabilizes the mitochondrial pool of survivin, induces mitochondrial dysfunction, and activates caspase-dependent apoptosis. This response involves disruption of an Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and Bax-dependent apoptosis. In vivo, Hsp60 is abundantly expressed in primary human tumors, as compared with matched normal tissues, and small interfering RNA ablation of Hsp60 in normal cells is well tolerated and does not cause apoptosis. Therefore, Hsp60 orchestrates a broad cell survival program centered on stabilization of mitochondrial survivin and restraining of p53 function, and this process is selectively exploited in cancer. Hsp60 inhibitors may function as attractive anticancer agents by differentially inducing apoptosis in tumor cells. Molecular chaperones, especially members of the Heat Shock Protein (Hsp) 2The abbreviations used are: Hspheat shock proteinsiRNAsmall interfering RNAWTwild typeCCCPcarbonyl cyanide p-chlorophenylhydrazoneGSTglutathione S-transferaseCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. gene family (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 631-677Crossref PubMed Scopus (4438) Google Scholar), assist in protein folding quality control, protein degradation, and protein trafficking among subcellular compartments (2Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2799) Google Scholar). This involves periodic cycles of ATPase activity, recruitment of additional chaperones, and compartmentalization in subcellular microdomains, including mitochondria (3Young J.C. Hoogenraad N.J. Hartl F.U. Cell. 2003; 112: 41-50Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). Molecular chaperones have often been associated with enhanced cell survival (4Beere H.M. J. Cell Sci. 2004; 117: 2641-2651Crossref PubMed Scopus (519) Google Scholar) via suppression of apoptosome-initiated mitochondrial cell death (5Paul C. Manero F. Gonin S. Kretz-Remy C. Virot S. Arrigo A.P. Mol. Cell. Biol. 2002; 22: 816-834Crossref PubMed Scopus (376) Google Scholar), increased stability of survival effectors (6Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (846) Google Scholar), and inactivation of p53 (7Wadhwa R. Takano S. Robert M. Yoshida A. Nomura H. Reddel R.R. Mitsui Y. Kaul S.C. J. Biol. Chem. 1998; 273: 29586-29591Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). As typically represented by Hsp90, the chaperone anti-apoptotic function may become selectively exploited in cancer (8Whitesell L. Lindquist S.L. Nat. Rev. Cancer. 2005; 5: 761-772Crossref PubMed Scopus (1985) Google Scholar) and may play a central role in tumor cell maintenance (9Isaacs J.S. Xu W. Neckers L. Cancer Cell. 2003; 3: 213-217Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar), but how general this paradigm is for other chaperones is not well understood. heat shock protein small interfering RNA wild type carbonyl cyanide p-chlorophenylhydrazone glutathione S-transferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. In particular, Hsp60, together with its associated chaperonin, Hsp10, has been recognized as an evolutionarily conserved stress response chaperone (10Zhao Q. Wang J. Levichkin I.V. Stasinopoulos S. Ryan M.T. Hoogenraad N.J. EMBO J. 2002; 21: 4411-4419Crossref PubMed Scopus (724) Google Scholar), largely but not exclusively compartmentalized in mitochondria (11Soltys B.J. Gupta R.S. Int. Rev. Cytol. 2000; 194: 133-196Crossref PubMed Scopus (95) Google Scholar) and with critical roles in organelle biogenesis and folding/refolding of imported preproteins (12Deocaris C.C. Kaul S.C. Wadhwa R. Cell Stress Chaperones. 2006; 11: 116-128Crossref PubMed Scopus (157) Google Scholar). However, whether Hsp60 also contributes to cell survival is controversial, with data suggesting a pro-apoptotic function via enhanced caspase activation (13Samali A. Cai J. Zhivotovsky B. Jones D.P. Orrenius S. EMBO J. 1999; 18: 2040-2048Crossref PubMed Scopus (463) Google Scholar, 14Xanthoudakis S. Roy S. Rasper D. Hennessey T. Aubin Y. Cassady R. Tawa P. Ruel R. Rosen A. Nicholson D.W. EMBO J. 1999; 18: 2049-2056Crossref PubMed Scopus (269) Google Scholar) or, conversely, an anti-apoptotic mechanism involving sequestration of Bax-containing complexes (15Shan Y.X. Liu T.J. Su H.F. Samsamshariat A. Mestril R. Wang P.H. J. Mol. Cell. Cardiol. 2003; 35: 1135-1143Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). A role of Hsp60 in cancer is equally uncertain, as up-regulation (16Thomas X. Campos L. Mounier C. Cornillon J. Flandrin P. Le Q.H. Piselli S. Guyotat D. Leuk. 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Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar), one of the most “cancer-specific” genes (21Velculescu V.E. Madden S.L. Zhang L. Lash A.E. Yu J. Rago C. Lal A. Wang C.J. Beaudry G.A. Ciriello K.M. Cook B.P. Dufault M.R. Ferguson A.T. Gao Y. He T.C. Hermeking H. Hiraldo S.K. Hwang P.M. Lopez M.A. Luderer H.F. Mathews B. Petroziello J.M. Polyak K. Zawel L. Zhang W. Zhang X. Zhou W. Haluska F.G. Jen J. Sukumar S. Landes G.M. Riggins G.J. Vogelstein B. Kinzler K.W. Nat. Genet. 1999; 23: 387-388Crossref PubMed Scopus (619) Google Scholar) involved in protection from apoptosis and control of mitosis in transformed cells. Accordingly, binding of survivin to Hsp90 (22Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar) or the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) (23Kang B.H. Altieri D.C. J. Biol. Chem. 2006; 281: 24721-24727Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) maintains survivin stability against proteasome-dependent destruction (22Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar, 23Kang B.H. Altieri D.C. J. Biol. Chem. 2006; 281: 24721-24727Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and preserves an anti-apoptotic threshold in tumor cells (24Plescia J. Salz W. Xia F. Pennati M. Zaffaroni N. Daidone M.G. Meli M. Dohi T. Fortugno P. Nefedova Y. Gabrilovich D.I. Colombo G. Altieri D.C. Cancer Cell. 2005; 7: 457-468Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). In this study, we used high throughput proteomics screening to identify novel binding partners of survivin that potentially contribute to its tumorigenic role. We found that Hsp60 (12Deocaris C.C. Kaul S.C. Wadhwa R. Cell Stress Chaperones. 2006; 11: 116-128Crossref PubMed Scopus (157) Google Scholar) associates with survivin, and this recognition contributes to a broad anti-apoptotic program differentially exploited in tumors in vivo. Cell Culture and Reagents–Breast adenocarcinoma cells MCF-7 and MDA-MB-231, colon adenocarcinoma cells HCT116, normal intestinal epithelial cells Fhs 74INT, and primary WS-1 or HFF normal fibroblasts were obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture as recommended by the supplier. Wild-type (WT), p53-/-, and Bax-/- HCT116 cells were kindly provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD). To generate a cell line stably expressing survivin, MCF-7 cells were transfected with survivin cDNA by Lipofectamine (Invitrogen) and selected in a medium containing 1 mg/ml G418 (Invitrogen), and the colonies were picked after 2-3 weeks. Transfected clones were expanded and confirmed for overexpression of survivin (3-4-fold over endogenous levels) by Western blotting. Antibodies against Hsp60 (BD Transduction Laboratories), survivin (NOVUS Biologicals), p53 (Oncogene Research Products and Santa Cruz Biotechnology), p21 (Oncogene), Bax (Oncogene), Mdm-2 (Santa Cruz), cleaved caspase-3 (Cell Signaling Technology), β-actin (BD Transduction Laboratories), and Hsp27 (Cell Signaling) were used. Proteomics Screening–MCF-7 cells were lysed in 25 mm HEPES, pH 7.5, 100 mm KCl, 1% Triton X-100, plus protease inhibitors (Roche Applied Science) for 30 min at 4 °C. The cell extract was precleared with glutathione-agarose beads (Sigma-Aldrich) for 4 h at 4 °C, mixed with GST or GST-survivin beads, and washed with a 20-bead volume of phosphate-buffered saline, and bound proteins were eluted in 20 mm Tris, pH 7.4, 2 mm EDTA, 0.1% CHAPS, and 1 m NaCl. After concentration with a ProteoExtract protein precipitation kit (Calbiochem), samples were separated by two-dimensional gel electrophoresis and visualized by silver staining (Genomine, Pohang, Kyungbuk, South Korea). Gel image analysis was performed using PDQuest software (Bio-Rad), and images obtained from GST or GST-survivin eluates were matched after normalization of spot intensities. Spots detected in the GST-survivin eluate were excised from the gel and digested with trypsin (Promega), and peptides were analyzed using an Ettan matrix-assisted laser desorption ionization time-of-flight were matched to the Protein and data using by small interfering RNA was by of or RNA using as E. Plescia J. J.C. Duckett C.S. Altieri D.C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were transfected with control or to Hsp60 by cells were with control or at of survivin in mitochondria was as (3Young J.C. Hoogenraad N.J. Hartl F.U. Cell. 2003; 112: 41-50Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). of in and survivin were with 1 volume of containing mm mm 20 mm pH 7.5, mm and mixed in a volume of with mitochondria for 1 h at 30 in the or of of the of the mitochondria were by at for and the protein in mitochondria was by of cells were after h and with a caspase inhibitor and for of and of by as J.C. Dohi T. Altieri D.C. Cancer Res. 2006; PubMed Scopus Google Scholar). were analyzed using To mitochondrial transfected cells were with the Molecular and analyzed for in by with for as a and were from tumor cells as T. Beltrami E. Plescia J. Altieri D.C. J. 2004; PubMed Scopus Google Scholar). and GST proteins were mixed with mitochondrial from MCF-7 or HCT116 and bound proteins were analyzed by Western as (22Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar). survivin or Hsp60 was mixed with or and bound proteins were detected by or mitochondrial from HCT116 or MCF-7 cells were with or an to survivin or and the complexes were by the of protein beads in mm Tris, pH 7.5, 0.1% 1% 0.1% mm 1 mm protease inhibitors (Roche Applied and 1 mm After or were separated by electrophoresis and analyzed by Western blotting. of Hsp60 in various with of adenocarcinoma of colon E. C. R. X. G. C. M. D. P. E. C. S. Biol. 2006; 7: PubMed Scopus Google Scholar), A. J. Li C. S. P. C. J. R. M. M. G. Wong W. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), D. K. J. C. P. M. Cancer Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), L. Su Q. A. Y. S. A. J. J. R. M. T. Cancer Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), or matched normal were analyzed using Yu J. K. N. R. D. T. 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Hsp60 a identify novel proteins that survivin, we used high throughput proteomics screening with of MCF-7 cell over GST-survivin and of bound proteins by two-dimensional gel electrophoresis and A protein spot with an molecular of and a of was detected in eluates of but not GST and identified as Hsp60 by of the matched and protein In GST-survivin associated with in and Hsp60, GST was survivin with or a associated with but not GST In survivin from but not Hsp60 in vivo, complexes with from subcellular not with Hsp60 or survivin Hsp60 of or of MCF-7 cells with on Hsp60 or survivin of tumor cells with a or pool to Hsp60 in of Hsp60 expression This was associated with of survivin in a that was in MCF-7 cells transfected with to Hsp60 In control on the expression of an Hsp27 in its mitochondrial and (11Soltys B.J. Gupta R.S. Int. Rev. Cytol. 2000; 194: 133-196Crossref PubMed Scopus (95) Google Scholar) compartments of Hsp60 were with a of To the of survivin after Hsp60 we performed Hsp60 in and destruction of survivin as compared with control transfected with Hsp60 of MCF-7 or HCT116 cells with control not in or a in cell by as compared with In of Hsp60 in tumor cell of and increased caspase In Hsp60 in tumor cells in of mitochondrial of mitochondrial in the and of caspase-3 to of and In control on mitochondrial or in MCF-7 cells To whether of survivin after Hsp60 to this cell death we MCF-7 clones stably transfected to survivin MCF-7 Hsp60 in cells not caspase or of as compared with transfected MCF-7 cells Hsp60 of pool of survivin to mitochondria is to and this mechanism tumor in T. Xia F. Altieri D.C. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). To whether cell death by Hsp60 involved the mitochondrial pool of survivin, we mitochondrial in survivin was imported in mitochondria in a by survivin of subcellular that Hsp60 in a of mitochondrial survivin the pool of survivin was largely In ablation of Hsp60 by was also associated with increased expression of p53 in and mitochondrial as compared with have been to p53 R. T. Mitsui Y. Reddel R.R. Kaul S.C. Cell Res. 2002; PubMed Scopus Google Scholar, Cancer Res. PubMed Scopus Google Scholar), we of p53 expression by Hsp60. of Hsp60 in HCT116 cells in increased expression of p53 and in up-regulation of Bax, to a p21 In in Bax expression were in HCT116 cells transfected with control or Hsp60 with cells of HCT116 or p53 cells with not in caspase-dependent cell death or of as compared with or cells transfected with To the of p53 for cell death by Hsp60 we analyzed HCT116 cells in Bax, a p53 gene by Hsp60 In of Bax-/- HCT116 cells with on caspase or and not in of mitochondrial A control, not cell or mitochondrial of Bax-/- HCT116 cells and of Hsp60-p53 whether Hsp60 associated with potentially restraining its In but not GST associated with p53 in from HCT116 cells In p53 that was from or mitochondrial of HCT116 cells Hsp60 In control complexes that were with not Hsp60 We at in the expression of the p53 in transfected cells. of MCF-7 or HCT116 cells with not Mdm-2 as compared with control transfected with Hsp60 in whether Hsp60 was exploited in cancer, we its expression and function in normal tumor cell Hsp60 was abundantly in mitochondrial and (11Soltys B.J. Gupta R.S. Int. Rev. Cytol. 2000; 194: 133-196Crossref PubMed Scopus (95) Google Scholar), of MCF-7 and HCT116 cells In primary WS-1 and HFF human fibroblasts of Hsp60 in subcellular compartments Hsp60 was or expressed at in normal of and in In Hsp60 was abundantly expressed in the tumor cell of of the and In control not normal or tumor not with of data that Hsp60 expression was in samples of adenocarcinoma of and as compared with matched normal To whether Hsp60 was selectively in tumor we Hsp60 expression in normal cell and analyzed cell of normal human epithelial cells or WS-1 primary human fibroblasts with Hsp60 a was not with the results obtained with tumor cell ablation of Hsp60 in normal cells not in of cell or increased caspase activity, as compared with control transfected with In this we have that the molecular chaperone Hsp60 (12Deocaris C.C. Kaul S.C. Wadhwa R. Cell Stress Chaperones. 2006; 11: 116-128Crossref PubMed Scopus (157) Google Scholar) is in human in and orchestrates a centered on stabilization of survivin and of p53 function ablation of Hsp60 results in of the mitochondrial pool of survivin, which is to apoptosis T. Xia F. Altieri D.C. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), increased expression of and activation of apoptosis in tumor cells This mechanism of Hsp60 is selectively exploited in tumors in vivo, Hsp60 is differentially as compared with normal tissues, and of Hsp60 in normal cells is not associated with mitochondrial or cell survivin is recognized as a cancer gene with roles in cell and of apoptosis D.C. Cell Biol. 2006; 18: PubMed Scopus Google Scholar, G. Cell Biol. 2006; 18: PubMed Scopus Google Scholar), the molecular of how survivin contributes to have not been However, a critical of this process is the of a pool of survivin compartmentalized in in tumor and in the in response to cell death T. Beltrami E. Plescia J. Altieri D.C. J. 2004; PubMed Scopus Google Scholar). There is now evidence that mitochondrial survivin for a pool of the to apoptosis T. Beltrami E. Plescia J. Altieri D.C. J. 2004; PubMed Scopus Google Scholar, H. Y. J. C. 2005; PubMed Scopus Google Scholar), tumor in T. Beltrami E. Plescia J. Altieri D.C. J. 2004; PubMed Scopus Google Scholar), and that this is by compartmentalized and binding to the anti-apoptotic inhibitor of T. Xia F. Altieri D.C. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the of a mitochondrial we have that survivin is imported in This may by molecular chaperones to with survivin in the including Hsp90 (22Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar) (23Kang B.H. Altieri D.C. J. Biol. Chem. 2006; 281: 24721-24727Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), that have been in mitochondrial (3Young J.C. Hoogenraad N.J. Hartl F.U. Cell. 2003; 112: 41-50Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, M. K. M. J. Cell Biol. 2003; PubMed Scopus Google Scholar). in survivin may the of a with Hsp60 (12Deocaris C.C. Kaul S.C. Wadhwa R. Cell Stress Chaperones. 2006; 11: 116-128Crossref PubMed Scopus (157) Google Scholar) to after the mitochondrial a that involves of imported proteins F.U. J. W. Annu. Rev. 21: PubMed Scopus Google Scholar). with this we have that ablation of Hsp60 results in of survivin and and of the mitochondrial pool of survivin T. Beltrami E. Plescia J. Altieri D.C. J. 2004; PubMed Scopus Google Scholar, H. Y. J. C. 2005; PubMed Scopus Google Scholar), this anti-apoptotic response T. Xia F. Altieri D.C. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In to stabilization of mitochondrial survivin, a mechanism of Hsp60 identified was the of Hsp60-p53 which p53 function in tumor cells. There is for the role of molecular chaperones in restraining p53 of the mitochondrial also has been to and p53 in the R. T. Mitsui Y. Reddel R.R. Kaul S.C. Cell Res. 2002; PubMed Scopus Google Scholar, C. S. B. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), its and in H. M. Y. K. 2006; PubMed Scopus Google Scholar), this a of Hsp60 of p53 does not to in the p53 regulator Mdm-2 and is from the role of in Cancer Res. PubMed Scopus Google Scholar). a mitochondrial pool of p53 has been that apoptosis via of family proteins P. M. Nat. Genet. 2003; PubMed Scopus Google Scholar), and of Hsp60 in increased expression of p53 in the data that Bax is for apoptosis by Hsp60 with of tumor maintenance (8Whitesell L. Lindquist S.L. Nat. Rev. Cancer. 2005; 5: 761-772Crossref PubMed Scopus (1985) Google Scholar) and overexpression in cancer in vivo, molecular chaperones have been for novel cancer (9Isaacs J.S. Xu W. Neckers L. Cancer Cell. 2003; 3: 213-217Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). together (4Beere H.M. J. Cell Sci. 2004; 117: 2641-2651Crossref PubMed Scopus (519) Google Scholar), the data that may a general of molecular chaperones, including Hsp60, at an anti-apoptotic threshold in tumor cells in vivo. this process to exploited selectively in transformed cells but not in normal In to a expression of chaperones, Hsp60, in cancer as to normal in vivo, other may contribute to the of this in tumor cells. may in chaperone activity, as has been for Hsp90 ATPase function A. L. J. Zhang L. 2003; PubMed Scopus Google Scholar), or with cancer genes differentially expressed in cancer, as is the for complexes (22Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar, 23Kang B.H. Altieri D.C. J. Biol. Chem. 2006; 281: 24721-24727Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). may promote tumor cell survival and the expression of this in tumor cells may for anticancer this molecular or of complexes survivin and Hsp90 (24Plescia J. Salz W. Xia F. Pennati M. Zaffaroni N. Daidone M.G. Meli M. Dohi T. Fortugno P. Nefedova Y. Gabrilovich D.I. Colombo G. Altieri D.C. Cancer Cell. 2005; 7: 457-468Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), and p53 R. T. Yoshida A. Nomura H. Reddel R.R. R. H. Kaul S.C. Cancer Res. 2000; Google Scholar), and Hsp60 and has been associated with of mitochondrial cell death in tumor cells normal cell including cells (24Plescia J. Salz W. Xia F. Pennati M. Zaffaroni N. Daidone M.G. Meli M. Dohi T. Fortugno P. Nefedova Y. Gabrilovich D.I. Colombo G. Altieri D.C. Cancer Cell. 2005; 7: 457-468Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). We Dr. Vogelstein for HCT116 cells.