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Bisindolylmaleimide VIII Enhances DR5-mediated Apoptosis through the MKK4/JNK/p38 Kinase and the Mitochondrial Pathways

2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês

10.1074/jbc.m203342200

ISSN

1083-351X

Autores

Toshiaki Ohtsuka, Tong Zhou,

Tópico(s)

NF-κB Signaling Pathways

Resumo

Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism. In the present study, we examined the effect of Bis VIII on apoptosis induced by DR5 (TRAIL-R2), using an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our results demonstrated that Bis VIII was able to enhance the apoptosis-inducing activity of TRA-8 both in vitro and in vivo. The combination of TRA-8 and Bis VIII led to a synergistic and sustained activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase, which was mediated by MAPK kinase 4 and was caspase-8-dependent. The mitochondrial pathway is involved in the synergistic induction of apoptosis by Bis VIII and TRA-8. Bis VIII alone induced the loss of mitochondrial membrane potential in a caspase-independent fashion without subsequent release of cytochrome c. However, in the presence of Bis VIII, TRA-8 induced more profound loss of mitochondrial membrane potential and release of cytochrome c. These results suggest that the enhanced and persistent activation of the JNK/p38 and the decreased mitochondrial membrane potential play a crucial role in synergistic induction of the death receptor-mediated apoptosis by Bis VIII. The unique ability of Bis VIII to enhance DR5-mediated apoptosis signal transduction discloses a potential utility of this compound in combination with anti-DR5 antibody in cancer therapy. Bisindolylmaleimide VIII (Bis VIII) has been previously shown to enhance Fas-mediated apoptosis through a protein kinase C-independent mechanism. In the present study, we examined the effect of Bis VIII on apoptosis induced by DR5 (TRAIL-R2), using an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our results demonstrated that Bis VIII was able to enhance the apoptosis-inducing activity of TRA-8 both in vitro and in vivo. The combination of TRA-8 and Bis VIII led to a synergistic and sustained activation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase, which was mediated by MAPK kinase 4 and was caspase-8-dependent. The mitochondrial pathway is involved in the synergistic induction of apoptosis by Bis VIII and TRA-8. Bis VIII alone induced the loss of mitochondrial membrane potential in a caspase-independent fashion without subsequent release of cytochrome c. However, in the presence of Bis VIII, TRA-8 induced more profound loss of mitochondrial membrane potential and release of cytochrome c. These results suggest that the enhanced and persistent activation of the JNK/p38 and the decreased mitochondrial membrane potential play a crucial role in synergistic induction of the death receptor-mediated apoptosis by Bis VIII. The unique ability of Bis VIII to enhance DR5-mediated apoptosis signal transduction discloses a potential utility of this compound in combination with anti-DR5 antibody in cancer therapy. tumor necrosis factor TNF-related apoptosis-inducing ligand c-Jun N-terminal protein kinase stress-activated protein kinase mitogen-activated protein kinase N-benzyloxycarbonyl- fluoromethyl ketone MAPK kinase bisindolylmaleimide VIII 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide MAPK phosphatase-1 extracellular signal-regulated kinase fetal calf serum 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate Tumor necrosis factor (TNF)1-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with strong apoptosis-inducing activity in most tumor cells (1Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.-P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2635) Google Scholar, 2Griffith T.S. Lynch D.H. Curr. Opin. Immunol. 1998; 10: 559-563Crossref PubMed Scopus (440) Google Scholar). Several receptors for TRAIL in humans have been identified, including DR4 (TRAIL-R1) and DR5 (TRAIL-R2), both of which are able to induce cell death, and decoy receptors DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4), as well as osteoprotegerin, which serve as an inhibitor of TRAIL-mediated apoptosis (3Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1544) Google Scholar, 4Pan G. Ni J. Wei Y.F. Yu G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1372) Google Scholar, 5Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar). Similar to TNF-R1 and Fas, DR4 and DR5 contain a death domain in their cytoplasmic region and are able to transduce a death signal in a Fas-associated death domain and caspase-8-dependent fashion (6Chaudhary P.M. Eby M. Jasmin A. Bookwalter A. Murray J. Hood L. Immunity. 1997; 7: 821-830Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 7Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 8Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (581) Google Scholar, 9Sprick M.R. Weigand M.A. Rieser E. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 10Kuang A.A. Diehl G.E. Zhang J. Winoto A. J. Biol. Chem. 2000; 275: 25065-25068Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar).It has been demonstrated that while TRAIL induces apoptosis, it also activates NF-κB, c-Jun N-terminal protein kinase (JNK, also referred to as stress-activated protein kinase (SAPK)), and p38 mitogen-activated protein kinase (MAPK) (7Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 12Muhlenbeck F. Haas E. Schwenzer R. Schubert G. Grell M. Smith C. Scheurich P. Wajant H. J. Biol. Chem. 1998; 273: 33091-33098Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar). The activation of JNK/p38 through death receptors requires caspase-8, since Fas antibody did not activate JNK and p38 in caspase-8-deficient cells (15Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and overexpression of caspase-8 stimulated the JNK activity (16Chaudhary P.M. Eby M.T. Jasmin A. Hood L. J. Biol. Chem. 1999; 274: 19211-19219Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The caspase inhibitors such asN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) can block activation of JNK and p38 by many apoptotic stimuli (14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar, 17Cahill M.A. Peter M.E. Kischkel F.C. Chinnaiyan A.M. Dixit V.M. Krammer P.H. Nordheim A. Oncogene. 1996; 13: 2087-2096PubMed Google Scholar, 18Juo P. Kuo C.J. Reynolds S.E. Konz R.F. Raingeaud J. Davis R.J. Biemann H.P. Blenis J. Mol. Cell. Biol. 1997; 17: 24-35Crossref PubMed Scopus (280) Google Scholar). Thus, the JNK and p38 pathways may function downstream of caspase activation in apoptotic signaling. However, it seems that JNK and p38 activation is upstream of caspases in the apoptotic signal transduction, because overexpression of upstream components of the JNK and p38 pathway such as MAPK kinase kinase 1, which functions in the JNK pathway (19Cardone M.H. Salvesen G.S. Widmann C. Johnson G. Frisch S.M. Cell. 1997; 90: 315-323Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar); MAPK kinase (MKK) 6b, an activator of p38 (20Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar); and apoptosis signal-regulating kinase 1 (21Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1999) Google Scholar) and mammalian STE20-like kinase 1 (MstI) (22Ura S. Masuyama N. Graves J.D. Gotoh Y. Genes Cells. 2001; 6: 519-530Crossref PubMed Scopus (100) Google Scholar), which activate JNK and p38 pathways, can also induce caspase activation and cell death.Numerous proapoptotic signal transduction and damage pathways converge on mitochondrial membranes to induce their permeabilization (23Ferri K.F. Kroemer G. Nat Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1291) Google Scholar). Changes in mitochondrial membrane potential are believed to be associated with the release of cytochrome c from the mitochondrial intermembrane space together with procaspase-9 and -2 and apoptosis-inducing factor (24Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). These studies indicate cross-talk between three signaling pathways including mitochondrial, JNK/p38 kinase, and caspases. However, the mechanism by which the JNK/p38 and mitochondrial pathways might influence the caspase pathway is still not clear.Recently, we generated a novel agonistic anti-human DR5 monoclonal antibody, TRA-8, which strongly induces apoptosis of most TRAIL-sensitive tumor cells both in vitro and in vivo (25Ichikawa K. Liu W. Zhao L. Wang Z. Liu D. Ohtsuka T. Zhang H. Mountz J.D. Koopman W.J. Kimberly R.P. Zhou T. Nat. Med. 2001; 7: 954-960Crossref PubMed Scopus (497) Google Scholar), which allows us to examine the signal transduction of DR5 in a more precise way. Moreover, we have previously shown that bisindolylmaleimide VIII (Bis VIII), an inhibitor of protein kinase C (26Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 27Bit R.A. Davis P.D. Elliott L.H. Harris W. Hill C.H. Keech E. Kumar H. Lawton G. Maw A. Nixon J.S. Vesey D.R. Wadsworth J. Wilkinson S.E. J. Med. Chem. 1993; 36: 21-29Crossref PubMed Scopus (160) Google Scholar), substantially facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune disease (28Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar). Although the molecular mechanism(s) by which Bis VIII enhances the death receptor-mediated apoptosis is unknown, our study suggest that the apoptosis-enhancing effect of Bis VIII might be associated with signal transduction of death receptor-mediated apoptosis and independent of its protein kinase C inhibition function. In the present study, we examined the effect of Bis VIII on DR5-mediated apoptosis and the signaling mechanisms of DR5-mediated apoptosis. Our results demonstrate that Bis VIII enhances DR5-mediated apoptosis through the JNK/p38 kinase pathway and a mitochondria-dependent pathway.DISCUSSIONMany chemotherapy agents have been shown to be able to enhance TRAIL-mediated apoptosis of tumor cells (42Keane M.M. Ettenberg S.A. Nau M.M. Russell E.K. Lipkowitz S. Cancer Res. 1999; 59: 734-741PubMed Google Scholar, 43Bonavida B. Ng C.P. Jazirehi A. Schiller G. Mizutani Y. Int. J. Oncol. 1999; 15: 793-802PubMed Google Scholar, 44Gibson S.B. Oyer R. Spalding A.C. Anderson S.M. Johnson G.L. Mol. Cell. Biol. 2000; 20: 205-212Crossref PubMed Scopus (228) Google Scholar, 45Mizutani Y. Yoshida O. Miki T. Bonavida B. Clin. Cancer Res. 1999; 5: 2605-2612PubMed Google Scholar, 46Lacour S. Hammann A. Wotawa A. Corcos L. Solary E. Dimanche-Boitrel M.T. Cancer Res. 2001; 61: 1645-1651PubMed Google Scholar). Therefore, the combined apoptosis-inducing molecules with chemotherapy agents have been proposed as a more effective strategy for cancer therapy. However, the molecular mechanisms by which chemotherapy agents sensitize tumor cells to apoptosis stimuli are poorly understood. In the present study, we utilized a previously identified leading compound, Bis VIII, and a newly generated novel agonistic anti-human DR5 antibody to examine the molecular signaling mechanisms of DR5-mediated apoptosis. Our results demonstrate that the JNK/p38 kinase plays a central role in the enhancement of DR5-mediated apoptosis with Bis VIII. Although many studies have shown that TRAIL induces activation of JNK/p38 (12Muhlenbeck F. Haas E. Schwenzer R. Schubert G. Grell M. Smith C. Scheurich P. Wajant H. J. Biol. Chem. 1998; 273: 33091-33098Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar), the role of activation of JNK/p38 in TRAIL-mediated apoptosis is not clear. Our results suggest that in the presence of DR5 signaling, the activation of JNK/p38 controls the process of apoptosis. TRA-8 induced a moderate and caspase-8-dependent activation of the JNK/p38, which was mediated by an upstream kinase, MKK4 (Figs. 3, 4, and 6). The moderate activation of the JNK/p38 was correlated with a weak apoptosis response (Fig. 3). However, in the presence of Bis VIII, the activation of the JNK/p38 induced by TRA-8 was significantly enhanced (Fig. 3). Importantly, Bis VIII was able to enhance the activation of not only JNK/p38 but also caspase-8 (Figs. 2 and 3). These results suggest that there might be a positive feedback between the JNK/p38 and caspase-8, in which the DR5 signaling initiates a weak activation of MKK4/JNK/p38 through caspase-8, and in this event, enhanced activation of JNK/p38 by Bis VIII further leads to activation of caspase-8 and enhancement of apoptosis. MKK4 is likely to be a key upstream kinase responsible for JNK and p38 activation by TRA-8 (Fig.4). Both inhibitor and activator of the JNK/p38 pathway also prove the central role of the JNK/p38 in DR5-mediated apoptosis. The inhibition of MKK4/JNK/p38 activation by curcumin antagonizes both the enhancing effect of Bis VIII on TRA-8-induced caspase activation and cell death, and the activation of the JNK/p38 pathway by anisomycin increases DR5-induced cell death (Fig. 5). Since the JNK/p38 pathway is crucially involved in stress response, it would be reasonable to believe that the stress with radiation or chemotherapy agents would sensitize cancer cells to DR5-mediated apoptosis.Both caspase-dependent and -independent pathways of cell death are involved in cell death induced by a Bis VIII and TRA-8 combination. While TRA-8 transduces a caspase-dependent signal, Bis VIII also transduces an additional caspase-independent signal to facilitate cell death (Fig.6 A). Mitochondria are probably one of the targets of Bis VIII, since treatment of cells with Bis VIII alone resulted in a loss of mitochondria membrane potentials (Fig. 7 A), which was caspase-independent (Fig. 7 B). Whereas loss of mitochondria membrane potential with Bis VIII alone did not induce immediate cell death, it might be able to sensitize the cells to DR5-mediated apoptosis by enhancing utilization of the mitochondrial apoptosis pathway through cytochrome c release and caspase-9 activation (Fig. 7 C). Indeed, the several chemical agents that are able to induce the loss of mitochondrial membrane potential, such as carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (47Dispersyn G. Nuydens R. Connors R. Borgers M. Geerts H. Biochim. Biophys. Acta. 1999; 1428: 357-371Crossref PubMed Scopus (92) Google Scholar) andN,N,-dihexyl-2-(4-fluorophenyl)-indole-3-acetamide (48Pastorino J.G. Simbula G. Yamamoto K. Glascott Jr., P.A. Rothman R.J. Farber J.L. J. Biol. Chem. 1996; 271: 29792-29798Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), could enhance DR5-induced cell death by facilitating the caspase activation (data not shown).The signaling association between the JNK/p38 pathway and mitochondrial pathway is still not clear. Given the fact that activation of JNK/p38 after stimulation with TRA-8 or the combination with TRA-8 and Bis VIII (Fig. 3, A and B) occurred before the activation of caspase-9 (Figs. 2 A and 7 C) and release of cytochrome c into cytosol (Fig. 7 C), activated JNK/p38 might be able to regulate the mitochondrial apoptosis-inducing pathway through cleavage of Bid by caspase-8 and/or direct activation of mitochondrial death machinery as recently reported (49Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar).In conclusion, we propose a model in which a positive interaction between the JNK/p38 pathway and caspase-8 leads to enhanced apoptosis signal transduction. This finding suggests that any stress assaults against cancer cells through the JNK/p38 pathway might be able to enhance the tumoricidal activity of TRA-8 or TRAIL. The identification of the unique property of Bis VIII in enhancement of DR5-mediated apoptosis by targeting both the JNK/p38 pathway and the mitochondrial pathway might be helpful in the design and screening of better chemotherapy drugs to synergize cancer cells to death receptor-mediated apoptosis. Tumor necrosis factor (TNF)1-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with strong apoptosis-inducing activity in most tumor cells (1Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.-P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2635) Google Scholar, 2Griffith T.S. Lynch D.H. Curr. Opin. Immunol. 1998; 10: 559-563Crossref PubMed Scopus (440) Google Scholar). Several receptors for TRAIL in humans have been identified, including DR4 (TRAIL-R1) and DR5 (TRAIL-R2), both of which are able to induce cell death, and decoy receptors DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4), as well as osteoprotegerin, which serve as an inhibitor of TRAIL-mediated apoptosis (3Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1544) Google Scholar, 4Pan G. Ni J. Wei Y.F. Yu G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1372) Google Scholar, 5Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar). Similar to TNF-R1 and Fas, DR4 and DR5 contain a death domain in their cytoplasmic region and are able to transduce a death signal in a Fas-associated death domain and caspase-8-dependent fashion (6Chaudhary P.M. Eby M. Jasmin A. Bookwalter A. Murray J. Hood L. Immunity. 1997; 7: 821-830Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 7Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 8Bodmer J.L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Crossref PubMed Scopus (581) Google Scholar, 9Sprick M.R. Weigand M.A. Rieser E. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 10Kuang A.A. Diehl G.E. Zhang J. Winoto A. J. Biol. Chem. 2000; 275: 25065-25068Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 11Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar). It has been demonstrated that while TRAIL induces apoptosis, it also activates NF-κB, c-Jun N-terminal protein kinase (JNK, also referred to as stress-activated protein kinase (SAPK)), and p38 mitogen-activated protein kinase (MAPK) (7Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 12Muhlenbeck F. Haas E. Schwenzer R. Schubert G. Grell M. Smith C. Scheurich P. Wajant H. J. Biol. Chem. 1998; 273: 33091-33098Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar). The activation of JNK/p38 through death receptors requires caspase-8, since Fas antibody did not activate JNK and p38 in caspase-8-deficient cells (15Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and overexpression of caspase-8 stimulated the JNK activity (16Chaudhary P.M. Eby M.T. Jasmin A. Hood L. J. Biol. Chem. 1999; 274: 19211-19219Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The caspase inhibitors such asN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) can block activation of JNK and p38 by many apoptotic stimuli (14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar, 17Cahill M.A. Peter M.E. Kischkel F.C. Chinnaiyan A.M. Dixit V.M. Krammer P.H. Nordheim A. Oncogene. 1996; 13: 2087-2096PubMed Google Scholar, 18Juo P. Kuo C.J. Reynolds S.E. Konz R.F. Raingeaud J. Davis R.J. Biemann H.P. Blenis J. Mol. Cell. Biol. 1997; 17: 24-35Crossref PubMed Scopus (280) Google Scholar). Thus, the JNK and p38 pathways may function downstream of caspase activation in apoptotic signaling. However, it seems that JNK and p38 activation is upstream of caspases in the apoptotic signal transduction, because overexpression of upstream components of the JNK and p38 pathway such as MAPK kinase kinase 1, which functions in the JNK pathway (19Cardone M.H. Salvesen G.S. Widmann C. Johnson G. Frisch S.M. Cell. 1997; 90: 315-323Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar); MAPK kinase (MKK) 6b, an activator of p38 (20Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar); and apoptosis signal-regulating kinase 1 (21Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1999) Google Scholar) and mammalian STE20-like kinase 1 (MstI) (22Ura S. Masuyama N. Graves J.D. Gotoh Y. Genes Cells. 2001; 6: 519-530Crossref PubMed Scopus (100) Google Scholar), which activate JNK and p38 pathways, can also induce caspase activation and cell death. Numerous proapoptotic signal transduction and damage pathways converge on mitochondrial membranes to induce their permeabilization (23Ferri K.F. Kroemer G. Nat Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1291) Google Scholar). Changes in mitochondrial membrane potential are believed to be associated with the release of cytochrome c from the mitochondrial intermembrane space together with procaspase-9 and -2 and apoptosis-inducing factor (24Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). These studies indicate cross-talk between three signaling pathways including mitochondrial, JNK/p38 kinase, and caspases. However, the mechanism by which the JNK/p38 and mitochondrial pathways might influence the caspase pathway is still not clear. Recently, we generated a novel agonistic anti-human DR5 monoclonal antibody, TRA-8, which strongly induces apoptosis of most TRAIL-sensitive tumor cells both in vitro and in vivo (25Ichikawa K. Liu W. Zhao L. Wang Z. Liu D. Ohtsuka T. Zhang H. Mountz J.D. Koopman W.J. Kimberly R.P. Zhou T. Nat. Med. 2001; 7: 954-960Crossref PubMed Scopus (497) Google Scholar), which allows us to examine the signal transduction of DR5 in a more precise way. Moreover, we have previously shown that bisindolylmaleimide VIII (Bis VIII), an inhibitor of protein kinase C (26Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 27Bit R.A. Davis P.D. Elliott L.H. Harris W. Hill C.H. Keech E. Kumar H. Lawton G. Maw A. Nixon J.S. Vesey D.R. Wadsworth J. Wilkinson S.E. J. Med. Chem. 1993; 36: 21-29Crossref PubMed Scopus (160) Google Scholar), substantially facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune disease (28Zhou T. Song L. Yang P. Wang Z. Lui D. Jope R.S. Nat. Med. 1999; 5: 42-48Crossref PubMed Scopus (115) Google Scholar). Although the molecular mechanism(s) by which Bis VIII enhances the death receptor-mediated apoptosis is unknown, our study suggest that the apoptosis-enhancing effect of Bis VIII might be associated with signal transduction of death receptor-mediated apoptosis and independent of its protein kinase C inhibition function. In the present study, we examined the effect of Bis VIII on DR5-mediated apoptosis and the signaling mechanisms of DR5-mediated apoptosis. Our results demonstrate that Bis VIII enhances DR5-mediated apoptosis through the JNK/p38 kinase pathway and a mitochondria-dependent pathway. DISCUSSIONMany chemotherapy agents have been shown to be able to enhance TRAIL-mediated apoptosis of tumor cells (42Keane M.M. Ettenberg S.A. Nau M.M. Russell E.K. Lipkowitz S. Cancer Res. 1999; 59: 734-741PubMed Google Scholar, 43Bonavida B. Ng C.P. Jazirehi A. Schiller G. Mizutani Y. Int. J. Oncol. 1999; 15: 793-802PubMed Google Scholar, 44Gibson S.B. Oyer R. Spalding A.C. Anderson S.M. Johnson G.L. Mol. Cell. Biol. 2000; 20: 205-212Crossref PubMed Scopus (228) Google Scholar, 45Mizutani Y. Yoshida O. Miki T. Bonavida B. Clin. Cancer Res. 1999; 5: 2605-2612PubMed Google Scholar, 46Lacour S. Hammann A. Wotawa A. Corcos L. Solary E. Dimanche-Boitrel M.T. Cancer Res. 2001; 61: 1645-1651PubMed Google Scholar). Therefore, the combined apoptosis-inducing molecules with chemotherapy agents have been proposed as a more effective strategy for cancer therapy. However, the molecular mechanisms by which chemotherapy agents sensitize tumor cells to apoptosis stimuli are poorly understood. In the present study, we utilized a previously identified leading compound, Bis VIII, and a newly generated novel agonistic anti-human DR5 antibody to examine the molecular signaling mechanisms of DR5-mediated apoptosis. Our results demonstrate that the JNK/p38 kinase plays a central role in the enhancement of DR5-mediated apoptosis with Bis VIII. Although many studies have shown that TRAIL induces activation of JNK/p38 (12Muhlenbeck F. Haas E. Schwenzer R. Schubert G. Grell M. Smith C. Scheurich P. Wajant H. J. Biol. Chem. 1998; 273: 33091-33098Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar), the role of activation of JNK/p38 in TRAIL-mediated apoptosis is not clear. Our results suggest that in the presence of DR5 signaling, the activation of JNK/p38 controls the process of apoptosis. TRA-8 induced a moderate and caspase-8-dependent activation of the JNK/p38, which was mediated by an upstream kinase, MKK4 (Figs. 3, 4, and 6). The moderate activation of the JNK/p38 was correlated with a weak apoptosis response (Fig. 3). However, in the presence of Bis VIII, the activation of the JNK/p38 induced by TRA-8 was significantly enhanced (Fig. 3). Importantly, Bis VIII was able to enhance the activation of not only JNK/p38 but also caspase-8 (Figs. 2 and 3). These results suggest that there might be a positive feedback between the JNK/p38 and caspase-8, in which the DR5 signaling initiates a weak activation of MKK4/JNK/p38 through caspase-8, and in this event, enhanced activation of JNK/p38 by Bis VIII further leads to activation of caspase-8 and enhancement of apoptosis. MKK4 is likely to be a key upstream kinase responsible for JNK and p38 activation by TRA-8 (Fig.4). Both inhibitor and activator of the JNK/p38 pathway also prove the central role of the JNK/p38 in DR5-mediated apoptosis. The inhibition of MKK4/JNK/p38 activation by curcumin antagonizes both the enhancing effect of Bis VIII on TRA-8-induced caspase activation and cell death, and the activation of the JNK/p38 pathway by anisomycin increases DR5-induced cell death (Fig. 5). Since the JNK/p38 pathway is crucially involved in stress response, it would be reasonable to believe that the stress with radiation or chemotherapy agents would sensitize cancer cells to DR5-mediated apoptosis.Both caspase-dependent and -independent pathways of cell death are involved in cell death induced by a Bis VIII and TRA-8 combination. While TRA-8 transduces a caspase-dependent signal, Bis VIII also transduces an additional caspase-independent signal to facilitate cell death (Fig.6 A). Mitochondria are probably one of the targets of Bis VIII, since treatment of cells with Bis VIII alone resulted in a loss of mitochondria membrane potentials (Fig. 7 A), which was caspase-independent (Fig. 7 B). Whereas loss of mitochondria membrane potential with Bis VIII alone did not induce immediate cell death, it might be able to sensitize the cells to DR5-mediated apoptosis by enhancing utilization of the mitochondrial apoptosis pathway through cytochrome c release and caspase-9 activation (Fig. 7 C). Indeed, the several chemical agents that are able to induce the loss of mitochondrial membrane potential, such as carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (47Dispersyn G. Nuydens R. Connors R. Borgers M. Geerts H. Biochim. Biophys. Acta. 1999; 1428: 357-371Crossref PubMed Scopus (92) Google Scholar) andN,N,-dihexyl-2-(4-fluorophenyl)-indole-3-acetamide (48Pastorino J.G. Simbula G. Yamamoto K. Glascott Jr., P.A. Rothman R.J. Farber J.L. J. Biol. Chem. 1996; 271: 29792-29798Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), could enhance DR5-induced cell death by facilitating the caspase activation (data not shown).The signaling association between the JNK/p38 pathway and mitochondrial pathway is still not clear. Given the fact that activation of JNK/p38 after stimulation with TRA-8 or the combination with TRA-8 and Bis VIII (Fig. 3, A and B) occurred before the activation of caspase-9 (Figs. 2 A and 7 C) and release of cytochrome c into cytosol (Fig. 7 C), activated JNK/p38 might be able to regulate the mitochondrial apoptosis-inducing pathway through cleavage of Bid by caspase-8 and/or direct activation of mitochondrial death machinery as recently reported (49Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar).In conclusion, we propose a model in which a positive interaction between the JNK/p38 pathway and caspase-8 leads to enhanced apoptosis signal transduction. This finding suggests that any stress assaults against cancer cells through the JNK/p38 pathway might be able to enhance the tumoricidal activity of TRA-8 or TRAIL. The identification of the unique property of Bis VIII in enhancement of DR5-mediated apoptosis by targeting both the JNK/p38 pathway and the mitochondrial pathway might be helpful in the design and screening of better chemotherapy drugs to synergize cancer cells to death receptor-mediated apoptosis. Many chemotherapy agents have been shown to be able to enhance TRAIL-mediated apoptosis of tumor cells (42Keane M.M. Ettenberg S.A. Nau M.M. Russell E.K. Lipkowitz S. Cancer Res. 1999; 59: 734-741PubMed Google Scholar, 43Bonavida B. Ng C.P. Jazirehi A. Schiller G. Mizutani Y. Int. J. Oncol. 1999; 15: 793-802PubMed Google Scholar, 44Gibson S.B. Oyer R. Spalding A.C. Anderson S.M. Johnson G.L. Mol. Cell. Biol. 2000; 20: 205-212Crossref PubMed Scopus (228) Google Scholar, 45Mizutani Y. Yoshida O. Miki T. Bonavida B. Clin. Cancer Res. 1999; 5: 2605-2612PubMed Google Scholar, 46Lacour S. Hammann A. Wotawa A. Corcos L. Solary E. Dimanche-Boitrel M.T. Cancer Res. 2001; 61: 1645-1651PubMed Google Scholar). Therefore, the combined apoptosis-inducing molecules with chemotherapy agents have been proposed as a more effective strategy for cancer therapy. However, the molecular mechanisms by which chemotherapy agents sensitize tumor cells to apoptosis stimuli are poorly understood. In the present study, we utilized a previously identified leading compound, Bis VIII, and a newly generated novel agonistic anti-human DR5 antibody to examine the molecular signaling mechanisms of DR5-mediated apoptosis. Our results demonstrate that the JNK/p38 kinase plays a central role in the enhancement of DR5-mediated apoptosis with Bis VIII. Although many studies have shown that TRAIL induces activation of JNK/p38 (12Muhlenbeck F. Haas E. Schwenzer R. Schubert G. Grell M. Smith C. Scheurich P. Wajant H. J. Biol. Chem. 1998; 273: 33091-33098Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Crossref PubMed Scopus (189) Google Scholar, 14MacFarlane M. Cohen G.M. Dickens M. Biochem. J. 2000; 348: 93-101Crossref PubMed Scopus (62) Google Scholar), the role of activation of JNK/p38 in TRAIL-mediated apoptosis is not clear. Our results suggest that in the presence of DR5 signaling, the activation of JNK/p38 controls the process of apoptosis. TRA-8 induced a moderate and caspase-8-dependent activation of the JNK/p38, which was mediated by an upstream kinase, MKK4 (Figs. 3, 4, and 6). The moderate activation of the JNK/p38 was correlated with a weak apoptosis response (Fig. 3). However, in the presence of Bis VIII, the activation of the JNK/p38 induced by TRA-8 was significantly enhanced (Fig. 3). Importantly, Bis VIII was able to enhance the activation of not only JNK/p38 but also caspase-8 (Figs. 2 and 3). These results suggest that there might be a positive feedback between the JNK/p38 and caspase-8, in which the DR5 signaling initiates a weak activation of MKK4/JNK/p38 through caspase-8, and in this event, enhanced activation of JNK/p38 by Bis VIII further leads to activation of caspase-8 and enhancement of apoptosis. MKK4 is likely to be a key upstream kinase responsible for JNK and p38 activation by TRA-8 (Fig.4). Both inhibitor and activator of the JNK/p38 pathway also prove the central role of the JNK/p38 in DR5-mediated apoptosis. The inhibition of MKK4/JNK/p38 activation by curcumin antagonizes both the enhancing effect of Bis VIII on TRA-8-induced caspase activation and cell death, and the activation of the JNK/p38 pathway by anisomycin increases DR5-induced cell death (Fig. 5). Since the JNK/p38 pathway is crucially involved in stress response, it would be reasonable to believe that the stress with radiation or chemotherapy agents would sensitize cancer cells to DR5-mediated apoptosis. Both caspase-dependent and -independent pathways of cell death are involved in cell death induced by a Bis VIII and TRA-8 combination. While TRA-8 transduces a caspase-dependent signal, Bis VIII also transduces an additional caspase-independent signal to facilitate cell death (Fig.6 A). Mitochondria are probably one of the targets of Bis VIII, since treatment of cells with Bis VIII alone resulted in a loss of mitochondria membrane potentials (Fig. 7 A), which was caspase-independent (Fig. 7 B). Whereas loss of mitochondria membrane potential with Bis VIII alone did not induce immediate cell death, it might be able to sensitize the cells to DR5-mediated apoptosis by enhancing utilization of the mitochondrial apoptosis pathway through cytochrome c release and caspase-9 activation (Fig. 7 C). Indeed, the several chemical agents that are able to induce the loss of mitochondrial membrane potential, such as carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (47Dispersyn G. Nuydens R. Connors R. Borgers M. Geerts H. Biochim. Biophys. Acta. 1999; 1428: 357-371Crossref PubMed Scopus (92) Google Scholar) andN,N,-dihexyl-2-(4-fluorophenyl)-indole-3-acetamide (48Pastorino J.G. Simbula G. Yamamoto K. Glascott Jr., P.A. Rothman R.J. Farber J.L. J. Biol. Chem. 1996; 271: 29792-29798Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), could enhance DR5-induced cell death by facilitating the caspase activation (data not shown). The signaling association between the JNK/p38 pathway and mitochondrial pathway is still not clear. Given the fact that activation of JNK/p38 after stimulation with TRA-8 or the combination with TRA-8 and Bis VIII (Fig. 3, A and B) occurred before the activation of caspase-9 (Figs. 2 A and 7 C) and release of cytochrome c into cytosol (Fig. 7 C), activated JNK/p38 might be able to regulate the mitochondrial apoptosis-inducing pathway through cleavage of Bid by caspase-8 and/or direct activation of mitochondrial death machinery as recently reported (49Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). In conclusion, we propose a model in which a positive interaction between the JNK/p38 pathway and caspase-8 leads to enhanced apoptosis signal transduction. This finding suggests that any stress assaults against cancer cells through the JNK/p38 pathway might be able to enhance the tumoricidal activity of TRA-8 or TRAIL. The identification of the unique property of Bis VIII in enhancement of DR5-mediated apoptosis by targeting both the JNK/p38 pathway and the mitochondrial pathway might be helpful in the design and screening of better chemotherapy drugs to synergize cancer cells to death receptor-mediated apoptosis. We thank Drs. W. J. Koopman and R. P. Kimberly for helpful discussion; Dr. F. Hunter for editing the manuscript; Drs. R. Jope and C. G. Taylor for providing 1321N1 and UL-3C cells, respectively; and Dr. D. Buchsbaum for providing MDA-MB-231-KS and MDA-MB-231-PO cells.

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