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

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) 1 -related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with strong apoptosis-inducing activity in most tumor cells (1,2). 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 (3)(4)(5). 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 (6 -11).
It has been demonstrated that while TRAIL induces apoptosis, it also activates NF-B, c-Jun N-terminal protein kinase (JNK, also referred to as stress-activated protein kinase (SAPK)), and p38 mitogen-activated protein kinase (MAPK) (7,(12)(13)(14). 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 (15) and overexpression of caspase-8 stimulated the JNK activity (16). The caspase inhibitors such as N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) can block activation of JNK and p38 by many apoptotic stimuli (14,17,18). 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 (19); MAPK kinase (MKK) 6b, an activator of p38 (20); and apoptosis signal-regulating kinase 1 (21) and mammalian STE20-like kinase 1 (MstI) (22), 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 (23). 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 (24). 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 (25), 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 (26,27), substantially facilitates Fas-mediated apoptosis and inhibits T cell-mediated autoimmune disease (28). * This work was supported in part by Sankyo Co., Ltd. of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  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 receptormediated 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.

EXPERIMENTAL PROCEDURES
Reagents-An agonistic anti-human DR5 monoclonal antibody, TRA-8, was prepared as described (25). Bis VIII and SB203580 were A, time dependence of Bis VIII and TRA-8 on enhancement of cell death. 1321N1 cells were incubated with indicated concentrations of Bis VIII and/or TRA-8 for indicated periods, and cell viability was determined by the ATPLite assay. The results are the mean Ϯ S.D. of a representative of at least two independent experiments performed in triplicate. B, Bis VIII enhances nuclear fragmentation induced by TRA-8. 1321N1 cells were incubated with the indicated concentrations of Bis VIII and/or TRA-8 for 15 h, stained with Hoechst 33342, and then examined under ultraviolet fluorescence. C, detection of DNA ladder formation induced by Bis VIII and/or TRA-8. 1321N1 cells were incubated with indicated concentrations of Bis VIII and/or TRA-8 for 15 h. After the incubation, cellular DNA was extracted and analyzed by electrophoresis on 2% agarose gel to detect DNA fragmentation. M, size marker DNA (1-kb Plus DNA Ladder; Invitrogen). D, enhancement of tumoricidal activity of TRA-8 by Bis VIII. SCID mice were inoculated with 1321N1 cells. Mice were injected with three doses of 100 g of TRA-8 and/or 100 g of Bis VIII on the 7 days after tumor inoculation (n ϭ 10, each group). Tumor growth was determined by weight.  Cell Culture and Cell Viability Assays-Human 1321N1 astrocytoma cells were kindly provided by Dr. Richard Jope (University of Alabama at Birmingham) and maintained at 37°C in a humidified atmosphere of 5% CO 2 in air in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated (56°C for 30 min) fetal calf serum (FCS), 50 g/ml streptomycin, and 50 units/ml penicillin (all from Cellgro, Mediatec, Inc., Herndon, VA). Human ovarian cancer cell line UL-3C was kindly provided by Dr. Cicek Gercel Taylor (University of Louisville) and maintained in RPMI1640 medium supplemented with 10% heat- For cell viability assay, cells (1-2 ϫ 10 3 cells/well) were seeded onto a 96-well plate in a volume of 50 l. The caspase inhibitors were added 1 h before the addition of stimulants. After incubating the cells for the indicated periods, cell viability was determined using the ATPLite kit according to the manufacturer's instructions (Packard Instrument Co.). For trypan blue dye exclusion assays, the medium was removed, and the adherent cells were detached with 10 mM EDTA in phosphate-buffered saline for 2 min at room temperature. Both the medium and adherent cells were placed in a tube, and the cells were collected by centrifugation. An aliquot of the cells resuspended in the medium was mixed with 0.4% dye at a 1:1 ratio. Live and dead cells were quantitated with a hemocytometer.
Analysis of Tumoricidal Activity in Vivo-6 -8-week-old female NOD/SCID mice were inoculated subcutaneously with 1321N1 cells (1 ϫ 10 7 ). Seven days after inoculation, mice were intraperitoneally injected with 100 g of TRA-8 and/or 100 g of Bis VIII every other day for three doses. Seven days after treatment, the growth of the 1321N1 cells was determined by the weight of the tumor mass.
Western Blot Analysis-After the required treatments, cells (1-3 ϫ 10 6 ) were washed once with phosphate-buffered saline and lysed in the sample buffer (100 -120 l) for SDS-polyacrylamide gel electrophoresis (PAGE) and immediately boiled for 4 min. To measure the release of cytochrome c from mitochondria, cytosolic and mitochondrial fractions were prepared as described in Ref. 29. Each sample was subjected to 7.5, 10, or 12.5% SDS-PAGE, and the proteins separated in the gel were subsequently electrotransferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris-HCl (pH 7.4), 8 g/liter NaCl, and 0.1% Tween 20) for 1-2 h at room temperature. The membrane was then incubated with the indicated primary antibodies in TBS-T containing either 5% nonfat dry milk or 5% bovine serum albumin at 4°C overnight. The membrane was washed three times with TBS-T and probed with peroxidase-conjugated secondary antibodies at room tem-  perature for 1.5-2 h. After washing four times with TBS-T, the protein was visualized using the ECL Plus Western blotting detection system (Amersham Biosciences) according to the manufacturer's instructions. Proteins were quantified by densitometric analysis using the Quantify One program (Bio-Rad). Kinase activities for JNK and p38 in cell extracts were determined using SAPK/JNK and p38 MAPK assay kits, respectively (both from Cell Signaling Technology, Inc., Beverly, MA).
DNA Fragmentation and Nuclear Staining Assay-After the required treatments of cells (1 ϫ 10 6 ), both adherent and detached cells were collected as described above, washed once with phosphate-buffered saline, and lysed in 100 l of TE-T buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100. Lysates were centrifuged at 14,000 rpm for 5 min at 4°C, and supernatants were then subjected to digestion with ribonuclease A (0.2 mg/ml) for 1 h at 37°C followed by incubation with proteinase K (0.2 mg/ml) for 1 h at 37°C. DNA in the sample was precipitated by centrifugation at 14,000 rpm for 15 min at 4°C after treatment with 50% isopropyl alcohol and 0.5 M NaCl overnight at Ϫ20°C. DNA was resuspended in 30 l of TE buffer and analyzed by electrophoresis on a 2% agarose gel in the presence of 0.2 g/ml ethidium bromide. For fluorescent microscopic analysis of apoptotic cells, Hoechst 33342 (300 ng/ml) was added in culture medium, and the cells were incubated for 20 min at room temperature before microscopic observation. Determination of Mitochondrial Membrane Potential-The mitochondrial membrane potential was assessed by using JC-1, a lipophilic cation that can selectively enter into mitochondria (30). JC-1 was dissolved in dimethyl sulfoxide to give a 1 mg/ml solution. This was further diluted to 20 g/ml in a fluorescence-activated cell sorting buffer containing 5% FCS and 0.1% NaN 3 in phosphate-buffered saline and filtered using a 0.45-m filter. After the required treatments of cells (2 ϫ 10 5 ), both adherent and detached cells were collected as described above and resuspended in 125 l of the fluorescence-activated cell sorting buffer. The cell suspension was incubated for 20 min at room temperature with 250 l of the filtered working solution of JC-1. Both red and green fluorescence emissions were analyzed with a flow cytometer (FACScan, Becton Dickinson, Sunnyvale, CA). A minimum of 10,000 cells per sample were acquired in list mode and analyzed using Winmdi software. The decrease in mitochondrial membrane potential was determined by a decrease in the ratio of red to green fluorescence intensities.

Bis VIII Facilitates DR5-mediated Apoptosis and Enhances
Tumoricidal Activity of TRA-8 -To determine whether Bis VIII has a similar apoptosis-enhancing effect on DR5-mediated apoptosis to that on Fas-mediated apoptosis as previously shown (28), we examined cell death of a human astrocytoma cell line (1321N1) mediated by TRA-8 in the absence or presence of Bis VIII. 1321N1 cells are relatively insensitive to DR5-mediated apoptosis as a short term treatment with TRA-8-induced moderate cell death in a dose-dependent manner with no more than 30% of cells being killed by a high concentration of TRA-8 (1000 ng/ml) within a 12-h incubation (Fig.  1A). As shown previously (28), the treatment of 1321N1 cells with 5 M Bis VIII did not induce significant cell death after 8 h, and slightly increased toxicity of Bis VIII was observed at the end of a 12-h culture. However, in the presence of 5 M Bis VIII, TRA-8-induced cell death was dramatically increased (Fig. 1A). The increased cell death was both time-dependent and TRA-8 dose-dependent. Whereas 100 ng/ml TRA-8 induced no more than 20% cell death during a 12-h time period in the absence of Bis VIII, the same concentration of TRA-8 was able to induce up to 50 and 80% of cell death in the presence of 5 M Bis VIII at 8 and 12 h, respectively. More complete cell death could occur with a higher dose of TRA-8 and in a longer time period in the presence of Bis VIII. 1321N1 cells treated with TRA-8 alone or TRA-8 and Bis VIII combination showed typical morphological characteristics of apoptosis: membrane blebbing and condensed nuclei as demonstrated by Hoechst staining (Fig. 1B). TRA-8-induced DNA fragmentation was apparent, which was further enhanced by Bis VIII (Fig. 1C). The synergistic induction of apoptosis by TRA-8 and Bis VIII was also observed in other types of tumor cells including breast, ovary, prostate, liver, and cervix (Table I). Although these cells exhibited a different susceptibility to TRA-8-mediated apoptosis, they all became very susceptible to TRA-8 when used in combination with Bis VIII. These results indicate that Bis VIII is able to sensitize various tumor cells to TRA-8-mediated apoptosis.
Since TRA-8 has a tumoricidal activity in 1321N1 cells in vivo (25), we further examined the in vivo effect of co-administration of Bis VIII with TRA-8 on tumor growth. Compared with control-treated mice, the treatment with Bis VIII alone did not induce significant tumor regression (Fig. 1D). Although TRA-8 alone significantly inhibited tumor growth, complete tumor regression was never achieved. However, the treatment with combined TRA-8 and Bis VIII resulted in nearly complete tumor regression. These results indicate that similar to its effect on Fas-induced cell death (28), Bis VIII has a synergistic effect on DR5-mediated cell death both in vitro and in vivo.
Bis VIII Enhances Caspase Activation Induced by TRA-8 -TRA-8 is able to activate caspase-8, -9, and -3 (25). To deter-mine whether the potentiation of TRA-8-induced apoptosis by Bis VIII is through enhanced caspase activation, the time-dependent activation of caspases was determined by Western blotting (Fig. 2A) and caspase activity assay (Fig. 2B). The activation of caspase-8 is demonstrated by Western blot analysis with decreased intensity of the proform of caspase-8 as a result of cleavage. TRA-8 alone did not induce significant cleavage of procaspase-8 until 6 h after the treatment. However, a significantly decreased procaspase-8 was observed at 4 h after the combined treatment with TRA-8 and Bis VIII. At 6 h after the treatment with TRA-8 and Bis VIII, the procaspase-8 had completely disappeared (Fig. 2A). Similarly, the activation of caspase-9 and caspase-3 as indicated by the cleaved products was also increased by the combined treatment of TRA-8 and Bis VIII. As the substrates of caspases, Bid and poly(ADPribose) polymerase were cleaved by the treatment with TRA-8, which was further enhanced by Bis VIII (Fig. 2A). FLIP, an inhibitor for caspase-8 activation and death receptor-mediated apoptosis, was not affected by Bis VIII and TRA-8, although the down-modulation of FLIP by Bis VIII has been found to be a mechanism for increased susceptibility of dendritic cells to Fas-mediated apoptosis (31). A time course analysis of caspase-3 activity during the treatment showed that there was at least a 1-fold increase in caspase-3 activity induced by TRA-8 and Bis VIII compared with TRA-8 alone, although Bis VIII alone was unable to increase caspase-3 activity (Fig. 2B). These results indicate that Bis VIII is able to enhance DR5-mediated caspase activation.
Bis VIII and TRA-8 Synergistically Activate JNK and p38 -In addition to its ability to activate the caspase cascade, DR5 is also able to activate the JNK and p38 pathway. To determine the effect of Bis VIII on DR5-induced activation of JNK/p38, activation of three members of the MAPK family, JNK, p38, and ERK1/2, was examined by Western blot analysis during TRA-8-mediated apoptosis in the absence or presence of Bis VIII. Activation of these kinases was determined by detection of the phosphorylated form of the kinases with specific antibodies. Compared with TNF␣, which induced an earlier and transient activation of both JNK and p38 (within 1 h), TRA-8 alone induced the late but persistent activation of both kinases (2-12 h) (Fig. 3, A and B). Bis VIII alone also was able to induce moderate activation of both kinases in a pattern similar to that of TRA-8 (Fig. 3, A and B). However, the combined treatment with TRA-8 and Bis VIII resulted in a significant increase in activation of both kinases between 2 and 6 h after treatment (Fig. 3, A and B). This effect resulted from increased phosphorylation of JNK and p38 rather than an increased total amount of the protein (data not shown). The ability of TRA-8 and/or Bis VIII to induce JNK and p38 activation was confirmed in 1321N1 cells by in vitro kinase assay (data not shown). In addition, this effect appears to be selective for JNK and p38, since the activation of a third member of this kinase family, ERK1/2, was not affected at all (Fig. 3C).
The synergistic induction of JNK/p38 by TRA-8 and Bis VIII was also observed in other types of tumor cells including breast, T cell, ovary, colon, and cervix (Table II). Although these cells exhibited a different sensitivity to TRA-8, they all became very susceptible to TRA-8 when used in combination with Bis VIII (Table I and data not shown) in parallel with great increases in JNK/p38 activation. These results indicate that Bis VIII synergistically enhances the activation of JNK/ p38 in DR5 signal transduction in several different types of tumor cells.
MKK4 Mediates Activation of JNK and p38 Induced by TRA-8 and Bis VIII-It has been well established that activation of JNK/p38 kinase is modulated by either upstream ki-nases, MKKs, which phosphorylate and activate JNK/p38 kinase (32), or MKP-1, which dephosphorylates and inactivates JNK/p38 kinase (33). Thus, we examined the activation of three upstream kinases, MKK4, which activates both JNK and p38 (34), and MKK3 and -6 (MKK3/6), which only activate p38 (34,35), during the treatment with TRA-8 and Bis VIII. As shown in Fig. 4, only MKK4 and not MKK3/6 was activated by TRA-8, as demonstrated by a dramatic increase in the phosphorylated form of MKK4. Whereas Bis VIII alone did not induce significant activation of MKK4, TRA-8 alone led to a nearly 10-fold increase in the activated form of MKK4 after 4 h of the treatment. The combined treatment with both TRA-8 and Bis VIII resulted in a sharp increase (25-fold) in the activated MKK4 at 4 h after the treatment (Fig. 4A). The kinetic pattern of the activation of MKK4 was very similar to that of JNK/p38. In contrast, the activation of MKK3/6 was only slightly increased by the TRA-8 and Bis VIII combination (Fig.  4B), indicating that MKK4 is an upstream kinase responsible for DR5-induced activation of JNK/p38. Contrary to a previous report (36), which shows that down-modulation of MKP-1 by Ro318220, a similar compound to Bis VIII, contributes to enhanced apoptosis mediated by TNF-␣, MKP-1 is unlikely to be involved in DR5-and Bis VIII-induced sustained activation of JNK/p38, because the protein levels of expression of MKP-1 were not altered by either TRA-8 or Bis VIII alone or the combination as determined by Western blot analysis (data not shown). These results indicate that DR5 may activate JNK/p38 through the activation of MKK4, which can be synergistically enhanced by Bis VIII.
Intimate Relationship between Activation of the JNK/p38 Cascade and Induction of DR5-mediated Apoptosis-To assess the role of JNK and p38 in DR5-mediated and Bis VIII-enhanced apoptosis, we used several common inhibitors and activators for the JNK/p38 pathway. Curcumin acts as a potent inhibitor of JNK activation by interfering with the molecule(s) upstream of JNK in the signal transduction pathway (37, 38), whereas SB203580 is a highly specific inhibitor for p38 and has no inhibitory activity against ERK1/2 and JNK (39). Pretreatment with curcumin effectively inhibited activation of all three key kinases, MKK4, JNK, and p38, which were induced by either TRA-8 or Bis VIII alone or in combination (Fig. 5A). In contrast, SB203580 was able to partially inhibit activation of p38 but not MKK4 and JNK induced by TRA-8 or Bis VIII alone or in combination. Correlated with their inhibitory ability to the JNK/p38 pathway, only curcumin and not SB203580 was able to partially but significantly inhibit cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase after treatment with either TRA-8 alone or a TRA-8 and Bis VIII combination (Fig. 5B). Curcumin also significantly inhibited apoptosis induced by TRA-8 alone or a TRA-8 and Bis VIII combination. In contrast, selective inhibition of p38 by SB203580 had no effect on caspase activation and cell death induced by TRA-8 or a TRA-8 and Bis VIII combination. On the other hand, exposure to anisomycin, a well known activator for the JNK/p38 pathway (40), can induce activation of MKK4, JNK, and p38 in 1321N1 cells as expected (Fig. 5D). Similar to Bis VIII, anisomycin greatly enhanced TRA-8-induced apoptosis (Fig. 5F) and caspase activation (Fig. 5E) in parallel with synergistic activation of MKK4/JNK/p38 (Fig. 5D). Treatment with chelerythrine, another activator for the JNK/p38 pathway (41), in combination with TRA-8 also showed similar enhancement of apoptosis (data not shown). These results further support the notion that MKK4-mediated activation of both JNK and p38 is involved in DR5-mediated caspase activation and cell death.
DR5-mediated Activation of MKK4/JNK/p38 Is Caspase-8dependent-Caspase-8 is directly associated with the intracellular death domain of DR5, and the activation of caspase-8 plays a crucial role in the initiation of DR5 signal transduction (8,9,11). To determine whether caspase-8 is required for the activation of the MKK4/JNK/p38 pathway by DR5 signaling, we examined the effect of caspase inhibitors on cell death and the kinase activation induced by TRA-8 and Bis VIII. Among four caspase inhibitors tested, only the inhibitor for caspase-8 (IETD) and a general caspase inhibitor (Z-VAD) but not inhib- itors for caspase-3 and caspase-9 were able to significantly protect cells from TRA-8-induced cell death with or without Bis VIII (Fig. 6A). To further determine whether activation of caspase-8 is required for the activation of the MKK4/JNK/p38 pathway, 1321N1 cells were treated with TRA-8 and/or Bis VIII in the presence of caspase inhibitor, and the activation of MKK4, JNK, and p38 was examined. As shown above, TRA-8 induced a moderate activation of MKK4, JNK, and p38, which was greatly enhanced by Bis VIII (Fig. 6B). The inhibitor for caspase-8 but not for caspase-3 significantly inhibited the activation of all three kinases induced by either TRA-8 alone or by a TRA-8 and Bis VIII combination (Fig. 6B). Although Bis VIII alone induced a weak activation of MKK4, JNK, and p38, it was not caspase-dependent (Fig. 6B). These results suggest that activation of caspase-8 through DR5 plays a crucial role not only in apoptosis but also in activation of the MKK4/JNK/ p38 pathway.
Loss of Mitochondrial Membrane Potential and Release of Cytochrome c Induced by TRA-8 and Bis VIII-Both loss of the mitochondrial membrane potential and the release of cytochrome c from mitochondrial intermembrane space are characteristic features of apoptosis, and regulations of the stability of mitochondrial membrane by the Bcl-2 family of proteins have been shown to play an important role in modulation of apoptosis (24). To determine whether the mitochondrial pathway is involved in DR5-and Bis VIII-induced apoptosis, we first examined the alteration of mitochondrial membrane potential during TRA-8-induced apoptosis using a fluorescent dye, JC-1. TRA-8 induced time-and dose-dependent decreases in mitochondrial membrane potential (Fig. 7A), which was completely blocked by a universal caspase inhibitor, Z-VAD (Fig. 7B), indicating that DR5 apoptosis signaling induces a caspase-dependent loss of the mitochondrial membrane potential. The induction of loss of mitochondrial membrane potential by TRA-8 took place in conjunction with the activation of caspases (Fig. 2) and JNK/p38 (Fig. 3). Although Bis VIII alone did not induce significant apoptosis, it was able to induce the loss of mitochondrial membrane potential in a similar time-dependent fashion to TRA-8 (Fig. 7A). However, while TRA-8 induced a caspase-dependent loss of mitochondrial potential, Bis VIII did not, indicating that the pathway leading to the loss of mitochondrial membrane potential by Bis VIII and TRA-8 is different (Fig. 7B). To further evaluate the effect of Bis VIII on the mitochondrial apoptosis pathway, we examined the release of cytochrome c from mitochondria in the presence or absence of DR5 signaling. Although Bis VIII alone induced a significant loss of the mitochondrial membrane potential, it was unable to induce the release of cytochrome c, the cleavage of Bid, and the activation of caspase-9 (Fig. 7C). In contrast, the loss of mitochondrial membrane potential induced by TRA-8 was associated with the release of cytochrome c and activation of caspase-9 (Fig. 7C). Importantly, the combined treatment of TRA-8 and Bis VIII resulted in an increase in cytochrome c release, Bid cleavage, and caspase-9 activation (Fig. 7C). These results suggest that the decreased mitochondrial membrane potential induced by Bis VIII might not be necessarily associated with induction of apoptosis but could lead to increased susceptibility of tumor cells to DR5-mediated apoptosis. DISCUSSION Many chemotherapy agents have been shown to be able to enhance TRAIL-mediated apoptosis of tumor cells (42)(43)(44)(45)(46). 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 (12)(13)(14), 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 mod- Alteration in mitochondrial membrane potential was measured by flow cytometry using JC-1 staining as described under "Experimental Procedures." One of three independent experiments is shown. B, caspasedependent and -independent loss of mitochondrial membrane potential induced by TRA-8 and Bis VIII, respectively. 1321N1 cells were preincubated with or without 10 M Z-VAD-FMK for 1 h and then treated with 5 M Bis VIII and/or 1 g/ml TRA-8 for 14 h. Alteration in mitochondrial membrane potential was measured by flow cytometry as in A. One of two independent experiments is shown. C, cytochrome c release in response to TRA-8 and/or Bis VIII. 1321N1 cells were treated with 5 M Bis VIII and/or 1 g/ml TRA-8 for 4 h and then harvested by two different methods depending on target proteins. Cytosolic fractions were subjected to Western blotting for cytosolic cytochrome c, whereas total cell lysates were used for detecting Bid and caspase-9. erate 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 caspaseindependent signal to facilitate cell death (Fig. 6A). 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. 7A), which was caspase-independent (Fig. 7B). 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. 7C). Indeed, the several chemical agents that are able to induce the loss of mitochondrial membrane potential, such as carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (47) and N,N,-dihexyl-2-(4-fluorophenyl)-indole-3-acetamide (48), 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. 2A and 7C) and release of cytochrome c into cytosol (Fig. 7C), 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 (49).
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.