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bisindo;ylmaleimide. Blood 2000; 95:3478-82. 6. Meng XW, Helderbrant MP, Kaufmann SH. Phorbol 12-myristate 13-acetate inhibits death receptor-mediated ...
[Cancer Biology & Therapy 2:3, 266-270, May/June 2003]; ©2003 Landes Bioscience

Research Paper

Bisindolylmaleimide IX Induces Reversible and Time-Dependent Tumor Necrosis Factor Receptor Family-Mediated Caspase Activation and Cell Death

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*Correspondence to: Oskar W Rokhlin, Ph.D.; Department of Pathology, 145 MRC; University of Iowa; Iowa City, Iowa 52242 USA; Tel.: 319.335.8214; Fax: 319.335.8453; Email: [email protected] Received 02/26/03; Accepted 03/24/03

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INTRODUCTION

Bisindolylmaleimide (Bis) derivatives were originally produced as protein kinase C (PKC) inhibitors.1-3 However, subsequent studies showed that Bis VIII induced Fas- and TNF-αmediated apoptosis in an astrocytoma cell line and in lymphoid cells independently of the inhibition of PKC.4 Several Bis derivatives (I, II, III, IV, and VIII) have been found to facilitate Fas- and TRAIL-mediated apoptosis in human dendritic cells.5 Bis VIII has also been described to potentiate TNF-α-, Fas-, and TRAIL-mediated apoptosis in Jurkat cells.6 Recently, it has been reported that Bis VIII in combination with agonistic Abs to TRAIL-R2 enhanced apoptosis through the MKK4/JNK/p38 kinase and mitochondrial pathways.7 Our own work has shown that Bis IX is a potent inducer of apoptosis in human prostatic carcinoma cell lines under simultaneous treatment with TNF-α, agonistic anti-Fas mAb and TRAIL. In addition, Bis IX was found to become fluorescent inside cells, binds to the mitochondria, and inhibits transcription by binding with DNA to actinomycin D-related sites.8 In summary, these data indicate that Bis derivatives are agents with multiple effects with potential utility in tumor therapy. In this study, we determined that BisIX induces reversible, time-dependent tumor necrosis factor receptor family-mediated cell death and caspase activation. Using Bis IX as a reversible inducer of apoptosis we have shown that there are different inhibitory factor(s) in prostatic carcinoma cell lines that are responsible for the resistance to TRAIL-, Fas-, and TNF-α-mediated apoptosis.

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acetyl actinomycin D aminomethylcoumarin bisindolylmaleimids Asp-Glu-Val-Asp protein kinase C; rhodamine 123 tumor necrosis factor TNF-related apoptosis-inducing ligand Val-Glu-Ile-Asp

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ABBREVIATIONS

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bisindolylmaleimides, apoptosis, prostatic neoplasms

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KEY WORDS

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Previously published online as a CB&T Paper in Press at: http://www.landesbioscience.com/journals/cbt/toc.php?volume=2&issue=3

Ac Act D AMC Bis’s DEVD PKC R123 TNF TRAIL VEID

We have previously shown that Bisindolylmaleimide (Bis) IX is localized in mitochondria but also acts as an inhibitor of transcription and facilitates tumor necrosis factor receptor family-mediated apoptosis (Rokhlin et al. J Biol Chem 2002; 277: 33213-9). In this study, we found that Bis IX is freely distributed both within cells and extracellular medium and acts as a reversible apoptosis-inducing agent. Bis IX was found to induce time-dependent apoptosis in combination with TNF-α, TRAIL, and anti-Fas Ab. Using human prostatic carcinoma cell lines DU145 and LNCaP that are resistant to treatment with TNF family death-inducing ligands, we have shown that different, albeit still unidentified, inhibitory factors are responsible for the resistance to TRAIL-, Fas-, and TNF-α-mediated apoptosis. Our data also suggest that the turnover of apoptosis suppressor factors is much faster in DU145 compared to LNCaP. Lastly, we have found that Bis IX can override the apoptosisinhibitory effects of Bcl-2 overexpression. In conclusion, Bis IX could be used as a drug to facilitate apoptosis of cancer cells that are resistant to treatment with death-inducing ligands as well as a valuable tool to discern the factors that mediate resistance to different death-inducing ligands.

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Departments of 1Pathology; 2Urology; and 3Epidemiology, The University of Iowa; Iowa City, Iowa USA

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ABSTRACT

Oskar W. Rokhlin1,* Michael B. Cohen1,2,3

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Supported by NIH grant CA 87717.

MATERIALS AND METHODS Cell Lines and Estimation of Cells Viability. The human prostatic carcinoma cell lines LNCaP, PC3, and DU145 were cultured in RPMI 1640 as has been previously described.9 Human TRAIL was purchased from PeptoTech (Rocky Hill, NJ), TNF-α was purchased from R&D Systems (Minneapolis, MN), and agonistic anti-Fas (IgM) monAb was previously described.9 Bisindolylmaleimid IX was purchased from Alexis Biochemicals (San Diego, CA). Actinomycin D (act D) was purchased from Sigma. To measure cell viability, we used calcein AM assay (Molecular Probes, Eugene, OR) as previously described.10 Con A cell line expressing MDR gene was kindly provided by Dr. A. Gudkov (Cleveland Clinic Foundation, Cleveland, OH). Assessment of Caspase Activity. Caspase activity was estimated using fluorogenic substrates as previously described.8 Briefly, cytosolic extracts were prepared in 1% Triton X100 buffer, pH 7.2 containing 0.1 mM PMSF, and 2 µg/ml pepstatin, leupeptin, and antipain. 40 µg of protein lysate

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was incubated for 60–120 min in assay buffer (20mM PIPES, pH 7.2, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS and 10% sucrose) with 40 µM fluorescent substrates (BioMol): Ac-DEVD-AMC as a substrate that is cleaved by caspase-3, -7, and, to some extent, -8; Ac-VEID-AMC as a substrate for caspase-6.

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RESULTS Bis IX Is a Reversible Death-Inducing Agent. We compared the death effects of Bis IX and act D during treatment of DU145, LNCaP, and PC3 for 48 h (Fig. 1A). Surprisingly, we have found a dramatic difference between act D and Bis IX in a dose-dependent manner in the range of concentrations used: more than 70% of PC3, 50% of DU145, and 50% of LNCaP were killed by 4 nM of act D. The same effect with Bis IX was achieved only at concentration of 4 µM. The next question we addressed was whether the killing effects of Bis IX and act D were time-dependent. To answer this, we treated DU145, LNCaP, and PC3 cells with 4 µM of Bis IX and act D, then washed the cells after 1, 2, 4, 8, and 24 h of treatment, and estimated cell death after 48 h of culturing. Figure 1B shows that the killing effect of Bis IX is clearly timedependent. Cells were not killed after 1, 2, and 4 h of exposure to Bis IX; approximately 30% were killed after 24 h, and 60–70% after 48 h of treatment. In contrast, 70–75% of cells were killed by act D after 1 h of exposure with no significant change up to 48 h. One possible explanation for these differences is that the binding of act D to its targets is irreversible. Binding of Bis IX to its targets however, might be reversible and Bis IX could be distributed between the cells and culture medium outside the cells. To test this hypothesis, we capitalized on the fluorescent properties of Bis IX. We have previously shown that Bis IX becomes fluorescent in cells and this fluorescence can be quantitatively measured with excitation at 488 nm and emission at 645 nm.8 In this experiment, DU145 was treated with Bis IX (10 µM) for 1 h, the cells were then washed with medium, harvested and fluorescence measured either just after washing (0 time point) or after culturing in fresh medium for 1, 2, 4, and 8 Figure 1. Assessment of cell death of PC3, DU145, and LNCaP cell lines hours. As can be seen from Table 1, more than 80% of Bis IX was found following treatment with Bis IX and act D. (A) Cells were treated with different outside of the cells after 1 hour and 97% after 3 h. These data concentrations of Bis IX and act D at the time of plating and cell death was suggest that during treatment of cells with Bis IX it is predominantly localized estimated 48 h later. (B) Cells were treated with 4 µM of Bis IX and actD, outside of the cells. The replacement of medium resulted in the sharp then washed after 1, 2, 4, 8, and 24 h of treatment and cell death was estidecrease of the effective concentrations of Bis IX. mated after 48 h of culturing. Each point represents mean values of four We next examined whether the efflux of Bis IX is mediated by the replicates in one of three separate experiments. multidrug resistance (MDR) mechanism. To answer this question, we compared the efflux values for rhodamine 123 (R123) and Bis IX using the con- Table 1 FLUORESCENCE IN DU145 CELLS AFTER TREATMENT WITH BIS IX AND trol cell line ConA, which expresses the MDR-assoSUBSEQUENT CCULTURING OF CELLS IN FRESH MEDIUM FOR DIFFERENT TIMES ciated p-glycoprotein, and DU145 and PC3. As shown in Table 2, Bis IX efflux occurred at the Culturing in fresh medium after treatment with Bis IX (hours) same level in ConA cells and in the prostate cell 0 1 2 4 8 lines, whereas R123 efflux was not observed in prostate cells but was pumped out of ConA cells. Fluorescence 9540 ± 1020 1720 ± 310 700 ± 160 300 ± 120 420 ± 110 These data indicate that Bis IX efflux from the cells % fluorescence 100 18 7.3 3.1 4.4 occurs independent of p-glycoprotein. retained inside cells Bis IX Induces Time-Dependent Tumor Necrosis Factor Receptor Family-Mediated Cell Death and Caspase Activation. Since the killing effects of Bis IX Table 2 FLUORESCENCE OF R123 AND BIS IX IN CELL LINES AFTER TREATMENT AND itself were found to be time-dependent, we next SUBSEQUENT CULTURING OF CELLS IN FRESH MEDIUM investigated the time dependence of Bis IX effects in combination with different ligands (TNF-α, Fas, ConA DU145 PC3 and TRAIL). For these experiments we chose Cell line R123 Bis IX R123 Bis IX R123 Bis IX DU145 because it is resistant to treatment with all these ligands in the asbsence of Bis IX.8,9 We first Fluorescence treated the cells with Bis IX for different times, Time(hours) 0 19260 13500 22780 15060 27110 14530 then washed the cells after the indicated times, and 2 2710 2180 21360 1890 24860 1280 then ligands were added to the cells. Cell death was % fluorescence estimated after 24 h of treatment with ligands. As 14 16 94 12.5 92 9 can be seen from Figure 2, pretreatment with Bis retained in the cells

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Figure 2. Assessment of cell death of DU145 to treatment with Bis IX (4 µM) in the absence/presence of death-inducing ligands. Cell death was estimated by calcein assay. Cells were plated at a density of 7000 cells/well in 96 well flat-bottom plates. Bis IX was added 24 h after plating, then cells were washed after the indicated times and then TNF-α (20 ng/ml), agonistic antiFas mAb (1000 ng/ml), and TRAIL (400 ng/ml) were added to the cells. Cell death was estimated after 24 h of treatment with ligands. Each point represents mean values of six replicates in one of two separate experiments, which gave similar results.

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IX for 8 h was sufficient to make DU145 sensitive to treatment with TRAIL. However, to make cells sensitive to treatment with anti-Fas Ab, 24 h of Bis IX treatment was necessary; DU145 cells remained resistant with TNF-α treatment. We subsequently investigated whether the activation of caspases is timedependent under Bis IX treatment in combination with TNF-α, anti-Fas mAb and TRAIL. In these experiments cells were treated either simultaneously for 4 and 8 hours with Bis IX and ligands, or cells were first treated for 4 hours with Bis IX, then washed, and finally ligands were added for 4 hours. As shown in Figure 3A, simultaneous treatment for 8 h with Bis IX and TNF-α or anti Fas mAb sharply increased caspase activity compared to simultaneous treatment for 4 h. Importantly, there were no differences between DU145 and LNCaP in response to this treatment. However, a dramatic difference between these cell lines was found when cells were treated sequentially: first with Bis IX for 4 hours and then with TNF-α or anti-Fas for 4 h. Under this treatment, caspase activity increased in LNCaP but strongly decreased in DU145. These differences were also found after treatment with Bis IX and TRAIL despite the fact that after simultaneous treatment with Bis IX and TRAIL we did not find increased caspase activity (after 8 h compared to 4 h of treatment) in DU145 and only a modest increase in LNCaP (Fig. 4). One explanation for these findings is that four hours of treatment with Bis IX and TRAIL in DU145 resulted in increased caspase activity that was 2-5 times higher compared to the levels induced by Bis IX in the presence of TNF-α and anti-Fas Abs. Apparently, TRAIL in the presence of Bis IX induced maximal caspase activity for these cells after 4 h of treatment and therefore further treatment of DU145 did not increase caspase activity. Bis IX Overrides Bcl-2-Dependent Resistance to Fas- and TRAILMediated Apoptosis. We have previously described that human prostatic carcinoma cell line PC3 was sensitive to treatment with anti-Fas agonistic Ab (IPO-4) and TRAIL9,10 and overexpression of Bcl-2 in PC3 converted these cells from sensitive to resistant.10 Since Bis IX binds to mitochondria and acts as inhibitor of transcription we examined whether Bis IX could overcome the Bcl-2-dependent resistance to Fasand TRAIL-mediated apoptosis. As can be seen from Figure 5, Bis IX synergistically increased cell death of PC3-Bcl-2 under simultaneous treatment with Bis IX in dose-dependent manner, and different concentrations of anti-Fas mAb or TRAIL. By western blot analysis, we examined the level of Bcl-2 after Bis IX treatment and found that the Bcl-2 expression was decreased three fold after 24 h of treatment (data not shown).

DISCUSSION The most important finding in this study is that Bis IX induces reversible, time-dependent tumor necrosis factor receptor family-mediated cell death and caspase activation. Bis IX was found to be freely distributed inside and outside of cells. Since Bis IX could be removed just by changing the medium, we have shown that the killing effect of Bis IX is clearly reversible and time-dependent. This observation, in turn, prompted us to use Bis IX as a tool to discern the factors responsible for the resistance to different deathinducing ligands. Eight hours of pretreatment with Bis IX was sufficient to sensitize DU145 to TRAIL treatment. In order to make DU145 sensitive to Figure 3. Caspase activity after simultaneous (A) or sequential (B) treatment with Bis IX (4 µM) and TNF-α or anti-Fas mAb. (A) Cells were treated simultaneously for 4 (unfilled columns) and 8 (stripped columns) hours with Bis IX and ligands. (B) Cells were treated for 4 hours with Bis IX (filled columns), or cells were first treated with Bis IX for 4 h, then washed and ligands were added for 4 hours. Ligands were used in the same concentrations as indicated in legend to Fig. 2. Caspase activity in cell lysates was estimated with two different substrates, Ac-DEVD-AMC and Ac-VEID-AMC, as described in “Materials and Methods”. Measurements were calibrated against a standard curve of 7-amino-4-methylcoumarin and data were expressed in nM of released AMC per µg of lysate protein. Each column represents mean values of three separate experiments.

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Fas-mediated apoptosis, the cells required 24 h of B A pretreatment with Bis IX; the cells remained resistant with TNF-α treatment. These data indicate that there are different inhibitory factor(s) in DU145 that are responsible for the resistance to TRAIL-, Fas-, and TNF-α-mediated apoptosis. Interestingly, simultaneous treatment of DU145 with Bis IX and TNF-α resulted in synergistic induction of caspase activity but did not result in cell death. These data suggest that apoptosis is suppressed downstream of caspase activation after treatment with TNF-α in contrast to TRAIL- and Fas receptor-mediated apoptosis when Bis IX potentiated both caspase activation and cell death. We have also found that the differences between DU145 and LNCaP suggest that turnover of apoptosis suppressor factors in DU145 are much faster compared to LNCaP. This indicates Figure 4. Caspase activity after simultaneous (A) or sequential (B) treatment with Bis IX (4 µM) and TRAIL. Experiments were performed as described in legend to Figure 3. that short-lived proteins are responsible for resistance to apoptosis in DU145 but the proteins with longer half-lives are apparently responsible for the resistance of LNCaP. This hypothesis is currently being tested 24 hours 24 hours by analysis of gene expression after different time of treatment with Bis IX. It has been suggested in many studies that combined treatment with chemotherapy agents and apoptosis-inducing ligands is a more effective strategy for cancer treatment.11-13 This approach is especially promising in the case of TRAIL treatment since TRAIL does not damage normal cells. We have previously shown that Bis IX facilitates tumor necrosis factor receptor family-mediated apoptosis in three human prostatic carcinoma cell lines. Moreover, Bis IX was capable of potentiating TNF receptor familyinduced apoptosis in LNCaP after androgen with48 hours 48 hours drawal when cells stop proliferating.8 In this study, we have shown that Bis IX can overcome Bcl-2dependent resistance to Fas- and TRAIL-mediated apoptosis. Ohtsuka and Zhou have recently shown that another Bis derivative, Bis VIII, synergistically increased apoptosis induced by agonistic antibodies (TRA-8) to TRAIL-R2 in several tumor cell lines including DU145 and PC3.7 They have also detected that combined treatment with Bis VIII and TRA-8 resulted in nearly complete tumor regression upon treatment of human 1321N1 astrocytoma cells in SCID mice. The reversibility of Bis IX-mediated apoptosis could potentially be advantageous for combination therapy that includes TRAIL. Since the protein(s) that are responsible for mediating resistance to Figure 5. Bis IX potentiates cell death in PC3-Bcl-2. Cells were treated with indicated concenTRAIL-induced apoptosis have different half-lives in trations of Bis IX in the absence/presence of three concentrations of anti-Fas mAb or TRAIL. different tumor cells, any successful treatment will Cells were plated at a density of 7000 cells/well in 96 well flat-bottom plates. Reagents were require administration of multiple doses in order to added at the time of plating and celld eath was estimated 24 h or 48 h later. Each point repreconvert the vast majority of the tumor cells from sents mean values of six replicates in one of two separate experiments, which gave similar results. TRAIL-resistant to -sensitive. The reversible and time-dependent effect of Bis IX determines its low toxicity (see Fig. 1), ligands in different cell types.4-8 In addition to potential usage of Bis and should allow for the development of a flexible therapeutic regime derivatives as chemotherapeutic agents, we have shown in this study with, hopefully, minimal side effects. The published and our own that Bis IX may be used as a tool to discern the factors that mediate data indicate that Bis derivatives are multifunctional agents that are resistance to different death-inducing ligands. capable to overcome the resistance of tumor cells to TNF family death www.landesbioscience.com

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References 1. Toullec D, Pianetti P, Coste H, et al. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 1991; 266:15771-81. 2. Bit RA, Davis PD, Elliot LH, et al. Inhibitors of protein kinase C. 3. Potent and highly selective bisindolylmaleimides by conformational restriction. J Med Chem 1993; 36:21-9. 3. Jacobson PB, Kuchera SL, Metz A, Schachtele C, Imre K, Schrier DJ. Anti-inflammatory properties of Go 6850: a selective inhibitor of protein kinase C. J Pharmacol. Exp Ther 1995; 275:995-1002. 4. Zhou T, Song L, Yang P, Wang Z, Lui D, Jope RS. Bisindolylmeleimide VIII facilitates Fasmediated apoptosis and inhibits T cell-mediated autoimmune diseases. Nature Medicine 1999; 5:42-8. 5. Willerns F, Amraoui Z, Vanderheyde N, et al. Expression of c-FLIPL and resistance to CD95-mediated apoptosis of monocyte-derived dendritic cells: inhibition by bisindo;ylmaleimide. Blood 2000; 95:3478-82. 6. Meng XW, Helderbrant MP, Kaufmann SH. Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain. J Biol Chem 2002; 277:3776-83. 7. Ohtsuka T, Zhou T. Bisindolylmaleimide VIII enhances DR5-mediated apoptosis through the MKK4/JNK/p38 kinase and the mitochondrial pathways. J Biol Chem 2002; 277:29294-303. 8. Rokhlin OW, Glover RA, Taghiyev AF, et al. Bisindolylmaleimide IX facilitates tumor necrosis factor receptor family-mediated cell death and acts as an inhibitor of transcription. J Biol Chem 2002; 277:33213-9. 9. Rokhlin OW, Bishop GA, Hostager BS, et al. Fas-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res 1997; 57: 1758-68. 10. Rokhlin OW, Guseva N, Tagiyev A, Knudson MC, Cohen MB. Bcl-2 oncoprotein protects the human prostatic carcinoma cell line PC3 from TRAIL-mediated apoptosis. Oncogene 2001; 20:2836-43. 11. El-Deiry WS. Insights into cancer therapeutic design based on p53 and TRAIL receptor signaling. Cell Death Different 2001; 8:1066-75. 12. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nature Rev Cancer 2002; 2:420-443. 13. Reed JC. Apoptosis-based therapies. Nature Reviews Drug Discovery 2002; 1:111-21.

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