Cisplatin Inactivation of Caspases Inhibits Death Ligand-induced Cell Death in Vitro and Fulminant Liver Damage in Mice

Abstract

Cisplatin is a platinum-containing chemotherapeutic drug that has been widely used to treat various human cancers. It acts by forming inter- and intracross-links of DNA, which is believed to be a major cause for its therapeutic efficacy. However, little attention has been paid to the effect of cisplatin on death ligand-induced cell death. Here we demonstrate that cisplatin inhibits death ligand-induced cell death in cell lines in a p53-independent manner. This inhibitory effect of cisplatin on cell death is direct, whereby cisplatin forms a complex with caspases leading to their inactivation. The cisplatin-caspase complex is reversed by the addition of reducing agent dithiothreitol, and caspase activity is regained. In addition, cisplatin shows a death-inhibition effect in in vivo animal models of fulminant liver damage induced by Fas activation and lipopolysaccharide-induced liver shock mediated by tumor necrosis factor-α. Together, we demonstrate that cisplatin inhibits cell death induced by death ligands in cell lines and in mice through caspase inactivation. Cisplatin is a platinum-containing chemotherapeutic drug that has been widely used to treat various human cancers. It acts by forming inter- and intracross-links of DNA, which is believed to be a major cause for its therapeutic efficacy. However, little attention has been paid to the effect of cisplatin on death ligand-induced cell death. Here we demonstrate that cisplatin inhibits death ligand-induced cell death in cell lines in a p53-independent manner. This inhibitory effect of cisplatin on cell death is direct, whereby cisplatin forms a complex with caspases leading to their inactivation. The cisplatin-caspase complex is reversed by the addition of reducing agent dithiothreitol, and caspase activity is regained. In addition, cisplatin shows a death-inhibition effect in in vivo animal models of fulminant liver damage induced by Fas activation and lipopolysaccharide-induced liver shock mediated by tumor necrosis factor-α. Together, we demonstrate that cisplatin inhibits cell death induced by death ligands in cell lines and in mice through caspase inactivation. Cell death induced by death ligands, such as TNF-α, 1The abbreviations used are: TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; DTT, dithiothreitol; TRAIL, TNF-related apoptosis-inducing ligand; z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; pNA, p-nitroanilide; d-gal, d-galactosamine. TRAIL, and FasL, has been shown to play a pivotal role in maintaining a homeostasis like the immune response (1Wang S. El-Deiry W.S. Oncogene. 2003; 22: 8628-8633Crossref PubMed Scopus (708) Google Scholar, 2Nagata S. Annu. Rev. Genet. 1999; 33: 29-55Crossref PubMed Scopus (676) Google Scholar). However, death ligands have been shown to participate in many pathological processes. For instance, FasL, TNF-α, or TRAIL contributes to acute hepatic failure such that blocking of these death ligands ameliorates the hepatic cell death in various experimental animal models (3Zheng S.J. Wang P. Tsabary G. Chen Y.H. J. Clin. Investig. 2004; 113: 58-64Crossref PubMed Scopus (142) Google Scholar, 4Schmaltz C. Alpdogan O. Kappel B.J. Muriglan S.J. Rotolo J.A. Ongchin J. Willis L.M. Greenberg A.S. Eng J.M. Crawford J.M. Murphy G.F. Yagita H. Walczak H. Peschon J.J. van den Brink M.R. Nat. Med. 2002; 8: 1433-1437Crossref PubMed Scopus (135) Google Scholar, 5Kondo T. Suda T. Fukuyama H. Adachi M. Nagata S. Nat. Med. 1997; 3: 409-413Crossref PubMed Scopus (461) Google Scholar). Upon engagement of death receptors by death ligands, the death signaling pathway is initiated by formation of death-inducing signaling complex at the trimerized cytoplasmic domain of death receptors (Apo-1/Fas, DR4, or DR5) (1Wang S. El-Deiry W.S. Oncogene. 2003; 22: 8628-8633Crossref PubMed Scopus (708) Google Scholar, 2Nagata S. Annu. Rev. Genet. 1999; 33: 29-55Crossref PubMed Scopus (676) Google Scholar). The activation of procaspase-8 at the death-inducing signaling complex stimulates the downstream signaling cascades transmitted through two major pathways, mitochondria-dependent and -independent pathways. In the mitochondria-independent pathway, caspase-8 directly activates procaspase-3 to active caspase-3, which cleaves target substrates and eventually leads to cell death. In the mitochondria-dependent pathway, caspase-8 cleaves Bid into tBid, which translocates to the mitochondria and causes mitochondrial damage culminating in an efflux of death-promoting proteins such as cytochrome c. This in turn leads to activation of caspase-9 and then caspase-3 (6Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar, 7Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). Despite the existence of these two pathways, the mitochondria-dependent pathway is thought to be critical in hepatic cell death, because effective death induction of hepatocytes does not occur unless the mitochondria-dependent signaling pathway is actively engaged (8Yin X.M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (867) Google Scholar). Cisplatin was first identified as a growth suppressant of bacterial cells and later found to have a tumor-killing activity in mice, leading to its use as an anti-cancer agent in humans. It has been widely prescribed to treat the various human tumors including germ-cell tumors, advanced bladder carcinoma, breast cancer, and lung carcinoma. Cisplatin is believed to cause cross-linking in intra- and interstrands of DNA, eventually leading to cell death mediated through the activation of caspases (9Siddik Z.H. Oncogene. 2003; 22: 7265-7279Crossref PubMed Scopus (2672) Google Scholar, 10Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Many studies have demonstrated that chemotherapeutic agents including cisplatin significantly increase the death-inducing activity of death ligands. Even at a subtherapeutic doses, which show no induction of cell death, chemotherapeutic agents are able to promote death ligand-induced killing (11Arizono Y. Yoshikawa H. Naganuma H. Hamada Y. Nakajima Y. Tasaka K. Br. J. Cancer. 2003; 88: 298-306Crossref PubMed Scopus (48) Google Scholar, 12Munshi A. McDonnell T.J. Meyn R.E. Cancer Chemother. Pharmacol. 2002; 50: 46-52Crossref PubMed Scopus (62) Google Scholar). Here, using in vitro and in vivo models, we show that cisplatin has the opposite effect. Rather than promoting death ligand-mediated cell killing, cisplatin inhibits cell death induced by death ligands, and this effect is achieved through direct association of cisplatin with caspases. Cell Culture and Measurement of Cell Viability—HeLa, PC-3, and HCT116 cells were cultured in Dulbecco's modified Eagle's medium or McCoy's 5A medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were plated on 48-well plates for cell death assay. Cell viability was determined by crystal violet staining as described previously (23Kim Y.M. Kim T.H. Seol D.W. Talanian R.V. Billiar T.R. J. Biol. Chem. 1998; 273: 31437-31441Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In brief, cells were stained with 0.4% crystal violet in methanol for 10 min at room temperature and then washed with tap water. Stained cells were extracted with 4% methanol, and dye extracts were measured at a 540 nm wavelength using a micro-plate reader. Western Blotting—Cell lysates or recombinant proteins were separated by a 15% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The membranes were hybridized with anticaspase-8 antibody (Cell Signaling, Beverly, MA), anti-Bid antibody (a generous gift from Dr. Xiao-Ming Yin at the University of Pittsburgh), or anti-caspase-3 antibody (Santa Cruz, San Diego, CA). Analysis of Bid Cleavage—Active recombinant caspase-8 protein (100 ng) was pretreated with cisplatin, etoposide, z-VAD-fmk, or cisplatin plus DTT in the reaction buffer (10 mm PIPES, pH 7.4, 100 mm NaCl, 0.1 mm EDTA, 10 mm DTT, 10% sucrose, 0.1% CHAPS), and then recombinant Bid protein (100 ng) or cytosolic lysate (200 μg) prepared from mouse liver was added to the reaction mixtures. The cleavage of Bid was analyzed by Western blotting with anti-Bid antibody. In Vitro Analysis of Caspase Activities—Caspase-8 (Calbiochem) and caspase-3 assay kits (R&D System) were purchased, and the assays were carried out according to the manufacturer's instructions. Ac-IETD-pNA and Ac-DETD-pNA were used as the substrates for caspase-8 and caspase-3, respectively. Recombinant caspase-8 or caspase-3 protein was preincubated with cisplatin, etoposide, z-VAD-fmk, or cisplatin plus DTT for 10 min at 25 °C prior to the addition of substrates and then further incubated for1hat37 °C upon the addition of substrates. Caspase activities were measured at a 405 nm wavelength using a micro-plate reader. Binding of Cisplatin to Caspases—Active recombinant caspase-8 (50 ng) or caspase-3 protein (50 ng) was pretreated with cisplatin (25 μg) or etoposide (25 μg) in 10 μl of phosphate-buffered saline buffer and then further incubated on ice for 1 h. Reaction mixtures were subjected to SDS-PAGE in the presence or absence of 2-mercaptoethanol, and caspase-3 and -8 were visualized by Western blotting with anti-caspase-3 antibody or anti-caspase-8 antibody. Animal Models—All animal studies followed the institutional guidelines. Adult BALB/c mice were purchased from Damool science (Daejeon, Korea) and injected intravenously with 1.5 μg of LPS/mouse (Sigma) and 20 mg of d-galactosamine (d-gal)/mouse (Sigma) or with 7.5 μg of Jo-2 antibody/mouse (Pharmingen). Cisplatin (0.6 mg/mouse) was injected intravenously 2 h before LPS/d-gal injection. After treatment, the mice were sacrificed by cervical dislocation. The photographs of the gross liver were taken with an Olympus digital camera, and the liver tissues were collected and processed for hematoxylin and eosin staining. Protective Effect of Cisplatin on Death Ligand-induced Cell Death in Cell Lines—Chemotherapeutic agents substantially increase cell death induced by TRAIL, FasL, or TNF-α and even induce cell death in cells resistant to the individual agent alone (12Munshi A. McDonnell T.J. Meyn R.E. Cancer Chemother. Pharmacol. 2002; 50: 46-52Crossref PubMed Scopus (62) Google Scholar, 13Jazirehi A.R. Ng C.P. Gan X.H. Schiller G. Bonavida B. Clin. Cancer Res. 2001; 7: 3874-3883PubMed Google Scholar, 14Evdokiou A. Bouralexis S. Atkins G.J. Chai F. Hay S. Clayer M. Findlay D.M. Int. J. Cancer. 2002; 99: 491-504Crossref PubMed Scopus (132) Google Scholar, 15Kelly M.M. Hoel B.D. Voelkel-Johnson C. Cancer Biol. Ther. 2002; 1: 520-527Crossref PubMed Scopus (32) Google Scholar). Thus, we first evaluated the death-enhancing activity of various chemotherapeutic agents on TRAIL-induced cell death. HeLa cells were treated with TRAIL for 6 h in the presence of cisplatin or etoposide and analyzed for cell death. Cisplatin or etoposide alone did not show any significant cell death-inducing activity at 6 h of treatment, whereas TRAIL effectively induced cell death in HeLa cells (Fig. 1A). When co-treated with TRAIL, etoposide increased TRAIL-induced cell death as previously observed (Fig. 1A). Similarly, other chemotherapeutic agents, such as actinomycin D, doxorubicin, cycloheximide, and MG132, also promoted TRAIL-induced cell death (data not shown). However, cisplatin inhibited TRAIL-induced cell death in HeLa cells. Moreover, cisplatin attenuated the death-enhancing activity of etoposide in TRAIL-induced cell death (Fig. 1, C-E), indicating that the death-inhibitory activity of cisplatin is dominant over the death-enhancing activity of etoposide. These findings prompted us to investigate the detailed mechanism of the death-inhibitory activity of cisplatin. First of all, we asked the question whether the death-inhibitory activity of cisplatin is only for TRAIL or whether it extends to other death ligands. To answer the question, we examined the effects of cisplatin and etoposide on FasL-induced cell death in HeLa cells. Similar to the results observed with TRAIL, cisplatin prevents agonistic anti-Fas (CH11)-induced cell death in HeLa cells (Fig. 1B). Our data showed that cisplatin effectively inhibits the cell death-inducing activity of both TRAIL and FasL, indicating that cisplatin may block a common signaling pathway activated by TRAIL and FasL. The death-induction activity of cisplatin has been thought to be mainly because of the formation of intra- and interstrand DNA cross-links, which leads to the stabilization of the p53 tumor suppressor protein and subsequently the induction of either cell cycle arrest or apoptosis (16Caelles C. Helmberg A. Karin M. Nature. 1994; 370: 220-223Crossref PubMed Scopus (835) Google Scholar, 17Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (837) Google Scholar). Thus, we examined whether or not the inhibitory effect of cisplatin on TRAILinduced cell death is associated with p53 function. We first utilized a PC-3 prostate tumor cell line that has a p53-null mutation and is susceptible to TRAIL-induced cell death. As shown in Fig. 1C, cisplatin inhibited TRAIL-induced cell death and the death-enhancing activity of etoposide in PC-3 cells, as observed in HeLa cells. This indicates that p53 is not associated with the death-inhibitory activity of cisplatin. To further confirm that p53 is not associated with the inhibitory effect of cisplatin on TRAIL-induced cell death, we next utilized a human colon cancer HCT116 p53-deficient cell line in which the p53 gene was specifically deleted by homologous recombination. HCT116 p53+/+ or HCT116 p53-/- cells were treated with cisplatin or etoposide in the presence or absence of TRAIL and analyzed for cell death. As shown in Fig. 2, D and E, cisplatin inhibited TRAIL-induced cell death regardless of p53 status, whereas etoposide did not show any inhibitory effect. Our data confirm that the death inhibition of cisplatin does not depend on the p53 status. Cisplatin Inhibition of Caspase Activation and Bid Cleavage—Caspase activation is a key mechanism for the induction of cell death by various death stimuli, including death ligands and chemotherapeutic agents. Studies have shown that upon engagement of TRAIL and FasL, caspase-8 activation is required for their induction of cell death, because caspase-8-deficient cells are resistant to these death ligands (18Seol D.W. Li J. Seol M.H. Park S.Y. Talanian R.V. Billiar T.R. Cancer Res. 2001; 61: Google Scholar). Thus, to the mechanism of the death-inhibitory activity of cisplatin, we the and downstream of the death pathway caspase-8 Bid and caspase-3 HeLa cells were pretreated with cisplatin or etoposide for min and incubated for 2 TRAIL Cisplatin inhibited TRAIL-induced caspase-8 activation and Bid cleavage in a etoposide TRAIL-induced caspase-8 activation (Fig. These results with of the death-inhibitory effect of cisplatin and the death-enhancing effect of etoposide on TRAIL caspase-8 has been to caspase-3 both directly and we examined whether TRAIL-induced caspase-3 activation was inhibited by cisplatin. activation was inhibited by cisplatin not by etoposide in a as caspase-8 activation (Fig. Bid is as a key for the of the mitochondria-dependent death pathway, leading to the of mitochondrial death from the mitochondria to the X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar, T.H. Zhao Y. Yin X.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we observed the cisplatin inhibition of caspase-8 activation and Bid we next examined whether cisplatin inhibits the of cytochrome in response to HeLa cells were pretreated with cisplatin or etoposide for h and treated with we the of cytochrome to the The of the cytochrome was by cisplatin not etoposide in with the attenuated caspase-8 activation and the Bid cleavage with cisplatin (Fig. Our data that cisplatin inhibits TRAIL-induced cell death by the activation activity of caspases including caspase-3 and the inhibition of which both the mitochondria-dependent and the mitochondria-independent pathways. Cisplatin it has been that cisplatin forms DNA studies have that cisplatin is also able to a complex with as observed in protein 1, and (9Siddik Z.H. Oncogene. 2003; 22: 7265-7279Crossref PubMed Scopus (2672) Google Scholar, M. J.M. Res. 2001; PubMed Scopus Google Scholar, M. J.M. Res. 2001; PubMed Scopus Google Scholar). It is that cisplatin has been shown to with the of the forming a complex J. J.A. A. J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). caspases are with a at the active and or of the to their Thus, we examined whether cisplatin directly caspases by with the of their recombinant caspase-8 was preincubated with cisplatin, etoposide, or a caspase in the presence or absence of a reducing DTT, and then recombinant Bid protein or cytosolic lysates from mouse liver were Bid cleavage was analyzed by Western Bid cleavage by caspase-8 was inhibited by cisplatin and not by etoposide (Fig. 2, and DTT the inhibitory effect of cisplatin on the Bid cleavage activity of caspase-8 (Fig. Our data that cisplatin directly which is reversed by To further confirm that caspases are by cisplatin, active recombinant caspases were preincubated with cisplatin, etoposide, or their and their activities were subsequently measured using substrates. As shown in Fig. cisplatin inhibited the activity of and this inhibitory effect of cisplatin was by the addition of Cisplatin also inhibited the activity of caspase-3 in a to that of caspase-8 (Fig. Cisplatin to to Caspase has been shown to effectively the cell death of hepatocytes by caspases formation of with caspases at the of their active and a reducing DTT, caspase inhibition activities by from caspases (23Kim Y.M. Kim T.H. Seol D.W. Talanian R.V. Billiar T.R. J. Biol. Chem. 1998; 273: 31437-31441Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Y.M. Kim T.H. Talanian R.V. Yin X.M. Billiar T.R. PubMed Scopus Google Scholar). Moreover, cisplatin is to a complex with proteins by to the of as J. J.A. A. J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Together, the that cisplatin may caspases by to the of their To this active recombinant caspase-3 or -8 was incubated with cisplatin or etoposide and analyzed by SDS-PAGE in the presence or absence of the reducing agent not etoposide, of caspase-3 and -8 in the absence of These were by the addition of (Fig. and Our data that cisplatin caspases by directly forming a cisplatin-caspase complex at the of of caspases. Inhibition of Cell Death in To whether cisplatin inhibits cell death induced by death ligands in animal models, we the of cell death of hepatocytes in mice induced by and d-galactosamine Y. Li S. Yin X.M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, X.M. J. Med. PubMed Scopus Google Scholar). BALB/c mice injected with 1.5 μg of LPS/mouse and 20 mg of which causes death of hepatic cells h. liver damage was observed (Fig. and (Fig. of mg of at 2 h before of LPS/d-gal hepatic cell death in the liver as by the gross of the liver (Fig. and and eosin staining (Fig. activity was significantly (Fig. and procaspase-3 and -8 were found to be to a (Fig. as with that observed in mice cisplatin of LPS/d-gal into mice cisplatin significantly the to (Fig. Together, these data that the of mouse was to a in liver damage to the caspase by cisplatin. Inhibition of FasL-induced in further confirm that cisplatin inhibits death ligand-induced cell death in mice, we utilized animal of FasL-induced fulminant liver damage in has been shown to induce fulminant liver damage in mice through a cell death of hepatocytes T. Suda T. Fukuyama H. Adachi M. Nagata S. Nat. Med. 1997; 3: 409-413Crossref PubMed Scopus (461) Google Scholar, X.M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (867) Google Scholar, X.M. J. Med. PubMed Scopus Google Scholar). BALB/c mice were intravenously injected with Jo-2 an agonistic antibody to mouse injected with 7.5 μg of Jo-2 antibody were of fulminant liver failure h. The liver damage was observed (Fig. and by staining (Fig. activation measured by a assay (Fig. and cleavage of procaspase-8 and (Fig. were with the mice, mice pretreated with cisplatin 2 h before Jo-2 were from the liver damage as by the in liver damage (Fig. and at the (Fig. by staining of activity (Fig. and the of caspase-8 and were substantially (Fig. as These data that cisplatin inhibits FasL-induced hepatic cell death in mice mediated through caspase inactivation. Cisplatin is of the widely used chemotherapeutic agents for of cancer because of its death-inducing activity various human from and and of studies have demonstrated that the tumor activity of cisplatin mainly upon DNA leading to cell death through p53 stabilization (9Siddik Z.H. Oncogene. 2003; 22: 7265-7279Crossref PubMed Scopus (2672) Google Scholar, 10Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (483) Google Scholar, M. J.M. Res. 2001; PubMed Scopus Google Scholar). we demonstrated that cisplatin inhibits death FasL, or cell death in cancer cells of p53 (Fig. and death ligand-induced hepatic in mice and Our studies that the mechanism by which cisplatin inhibits death ligand-induced cell death is through direct association of cisplatin with caspases. This association the in caspases and cisplatin leading to of caspase activity 2 and Many have demonstrated that cisplatin cell death in various tumor cells (11Arizono Y. Yoshikawa H. Naganuma H. Hamada Y. Nakajima Y. Tasaka K. Br. J. Cancer. 2003; 88: 298-306Crossref PubMed Scopus (48) Google Scholar, 12Munshi A. McDonnell T.J. Meyn R.E. Cancer Chemother. Pharmacol. 2002; 50: 46-52Crossref PubMed Scopus (62) Google Scholar, 14Evdokiou A. Bouralexis S. Atkins G.J. Chai F. Hay S. Clayer M. Findlay D.M. Int. J. Cancer. 2002; 99: 491-504Crossref PubMed Scopus (132) Google Scholar). This the and data on the of cisplatin leads us to that two for the of cisplatin which depend on the of The response to be associated with of caspases from the of cisplatin to caspases. This of the caspases by cisplatin is to be a major for death ligand-induced cell death. the other the initiated by are believed to like gene or of protein other which than h to be which is the cisplatin alone induce cell death in HeLa cells. These which may be to induce cell death. This of caspase activation to be a than a direct of cisplatin with caspases. Thus, the caspase activation by cisplatin observed in of studies results from the processes. In this in we have utilized both cisplatin and death ligands such that we a to the direct cisplatin and caspase activation by death ligands. This is mainly because of the activation of caspases induced by cisplatin and TRAIL in that caspase activation by TRAIL h in HeLa cells, the cisplatin alone the whereas caspase activation by cisplatin h. Thus, we are able to the caspase activation by death ligands or cisplatin This us to the of cisplatin that is of caspases. Together, we that cisplatin has the of caspase through direct association at the and caspase activation through at the We Dr. at University for the generous of HCT116 p53+/+ and p53-/- cells. We also Dr. G. C. Kim of the for and Dr. M. C. at the University of for critical of the