Combination Of TRAIL Research Articles

Perifosine Induces DR4/DR5 Expression Leading to Apoptosis That Can Be Enhanced with Exogenous TRAIL, and Blocked with Strategies Directed at DR4/DR5 Inhibition.

Background: Perifosine is an oral AKT inhibitor which exerts a marked cytotoxic effect on human tumor cell lines. It is currently being tested in several phase II trials for the treatment of major cancers including multiple myeloma. While the proposed mechanism of action relates to downregulation of AKT expression, overepxression of constitutively active AKT does not abrogate perifosine induced cell death suggesting alternative mechanisms.Hypothesis: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma cells in vitro after binding to their membrane specific receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). It is our hypothesis that DR4/DR5 upregulation occurs in response to perifosine treatment, and thus may be additive with exogenous TRAIL.Materials and Methods: TRAIL-sensitive myeloma cell lines (MM.1S, RPMI8226, MM.1R) and TRAIL- resistant myeloma cell lines (U266) were used in this study. Apoptosis was assessed by annexin-V binding assay using flow-cytometry and cell death was assessed by MTT assay. Recombinant human TRAIL, chimeras of DR4 and DR5 were obtained from R&D systems.Results: Perifosine alone(5μM and 10μM) induced apoptosis of MM.1S in 40% and 50% of the treated cells as measured by flow cytometry, that increased to 81% and 91% when 50ng/ml of TRAIL was added to 5μM and 10 μM of perifosine. TRAIL alone induced only nominal apoptosis. Use of the TRAIL resistant U266 cell line showed only minimal apoptosis in response to perifosine, TRAIL, or the combination of both agents. Perifosine also induced DR4 and DR5 expression in less than 2hrs upon the Perifosine exposure in MM.1S as shown by RT-PCR. The combination of perifosine and TRAIL was not sequence specific. Furthermore, we observed that the enhanced apoptosis induced by perifosine and TRAIL in combination was almost or partially blocked by the administration of the DR4 and DR5 blocking antibodies only in the case of MM.1S, MM.1R, RPMI8226 TRAIL sensitive cells lines. Apoptosis was completely blocked in the case of U266 TRAIL resistant cell line when the chimera antibodies were used with perifosine alone or in combination with TRAIL.Conclusion and future directions: Perifosine, an agent proposed to function via inhibition of p-AKT and PDK-1, may have other effects on cell cycle regulation and it pro-apoptotic effects may be partially related to the TRAIL pathway. Our data suggests that an additional mechanism of action relates to the effect perifosine has on DR4 and DR5 expression thus directly effecting apoptosis via the TRAIL mediated effects. The limited response the trail resistant cell line U266 cells suggest that the TRAIL resistant myeloma cells have less DR4 or DR5 surface receptors as compared to the TRAIL sensitive cell lines, MM.1S, MM.1R, and RPMI8226 further validating this alternative mechanism. Further experiments such as inhibition of DR4, DR5, and FADD by small interfering RNAs, RT-PCR, the response in primary myeloma cells and also using more TRAIL resistant cell lines to support our preliminary observations are currently in progress.

Triptolide Sensitizes Leukemic Cells to TRAIL Induced Apoptosis Via Decrease of XIAP and p53-Mediated Increase of DR5.

Triptolide, a recently identified anticancer agent from a Chinese herb, has been shown to synergistically enhance TRAIL-induced cell death in various solid tumor cell lines. We have found that triptolide potently induces apoptosis in leukemic cell lines and blasts from AML patients at least in part by decreasing XIAP levels. XIAP is known to be a resistance factor in TRAIL-induced cell death. XIAP is highly expressed in AML blasts and primary AML cells generally are insensitive to TRAIL. We therefore hypothesize that triptolide will sensitize AML cells to TRAIL-induced apoptosis. We treated OCI-AML3, U937, and Jurkat cells with sub-optimal concentrations of triptolide, TRAIL, and their combination. At concentrations showing no or minimal effects by each agent alone, their combinations significantly promoted cell death with combination indices (CI) <1.0 for all three leukemia cell lines. To ensure that XIAP contributes to TRAIL resistance, we treated U937 cells overexpressing XIAP (U937XIAP) and the control cells (U937neo) with TRAIL. U937XIAP cells were 6-fold more resistant to TRAIL (IC50=455.8 ng/ml) than U937neo cells (IC50=74.3 ng/ml). We then treated these cells with TRAIL and 1396–11, a small molecule XIAP antagonist that binds to BIR2 of XIAP and promotes caspase-dependent apoptosis. At 3μM, 1396–11 had no significant effect on survival of either U937neo or U939XIAP cells. When combined with TRAIL, 1396–11 augmented TRAIL-induced cell death of both U937neo (IC50=45.0 ng/ml with 1396-11 vs. IC50=74.3 ng/ml without) and U939XIAP cells (IC50=318.9 ng/ml with 1396–11 vs. IC50=455.8 ng/ml without). Furthermore, we observed that triptolide decreased MDM2 and increased p53 protein levels in p53 wild type OCI-AML3 but not in p53 null U937 cells. TRAIL receptor DR5 has been shown to be regulated by p53. To elucidate the role of p53 in TRAIL-induced cell death sensitized by triptolide, we treated OCI-AML3 and U937 cells with triptolide and examined DR5 expression. We found that triptolide induced DR5 protein levels in OCI-AML3, but much less so in U937 cells. Treatment of OCI-AML3 cells with Nutlin3a, a MDM2 inhibitor that binds to MDM2 and stabilizes p53, increased DR5 protein levels and sensitized to TRAIL-induced cell death. Knockdown of p53 with retrovirus expressing p53siRNA in OCI-AML3 cells abolished cellular responses to nutlin3a and significantly decreased the sensitization to TRAIL by nutlin3a. Finally, we treated OCI-AML3 cells with 1396–11, nutlin3a, and TRAIL and our results showed that the triple combination (CI=0.045 at 24 hours, average of ED50, ED75, and ED90) was more effective in inducing cell death than either 1396–11 and TRAIL (CI=0.066) or nutlin3a and TRAIL (CI=0.190) combinations supporting our notion that triptolide sensitizes to TRAIL-induced cell death by modulating both XIAP expression and p53 signaling. Collectively, our studies suggest that inhibition of XIAP and induction of DR5 mediated by p53 activation both independently sensitize leukemic cells to TRAIL-induced apoptosis. Triptolide not only inhibits XIAP, which is overexpressed in AML, but also activates p53 signaling, which is intact in the majority of AMLs. Thus, combinations of triptolide and TRAIL may provide a novel strategy for treating AML patients by overcoming critical mechanisms of apoptosis resistance.

Induction of RKIP and Inhibition of the Transcription Repressor YY1 in B-NHL by the Novel Proteasome Inhibitor NPI-0052 and Sensitization to TRAIL-Induced Apoptosis.

Proteasome inhibitors have been shown to exert multiple effects including direct cytotoxic activity in sensitive cancer cells. In addition, proteasome inhibitors have recently been used therapeutically for certain cancers such as multiple myeloma and mantle cell lymphoma. We have recently reported that the novel proteasome inhibitor, NPI-0052 (Nereus Pharmaceuticals), can sensitize drug-resistant B-NHL tumor cells to apoptosis by various chemotherapeutic drugs. Also, NPI-0052 inhibits the NF-κB survival pathway and inhibition of NF-κB sensitizes tumor cells to TRAIL-induced apoptosis. We hypothesized that inhibition of NF-κB by NPI-0052 may also lead to sensitization of TRAIL-resistant B-NHL cells to TRAIL-mediated apoptosis. Human Burkitt's lymphoma,Ramos cells, were treated with various concentrations of NPI-0052 (0–10ng/ml for 24h) and were then treated with recombinant human TRAIL (0–100ng/ml for 24h). The cells were examined for apoptosis by Annexin V/PI. The combination treatment resulted in significant potentiation of cytotoxicity and synergy in apoptosis was achieved. We have then examined a potential underlying mechanism of NPI-0052-mediated sensitization. The transcription repressor YY1 was recently shown to negatively regulate the transcription of the TRAIL death receptor DR5 and thus overexpression of YY1 is a resistant factor for TRAIL-induced apoptosis. Hence, treatment of Ramos cells with NPI-0052 resulted in significant inhibition of YY1 expression and upregulation of surface DR5 expression. We have also examined the effect of NPI-0052 on RKIP (Raf-kinase inhibitor protein) (Yeung et al., Molecular Cellular Biology; 21:7207, 2001) expression. Since both NPI-0052 and RKIP inhibit the NF-κB pathway, we hypothesized that treatment of Ramos cells with NPI-0052 may result in significant upregulation of RKIP expression. The findings, indeed, demonstrate that NPI-0052 significantly induced RKIP expression in Ramos cells. Overall, the findings show, for the first time, that NPI-0052 sensitizes B-NHL tumor cells to TRAIL-mediated apoptosis. Sensitization by NPI-0052 was correlated with both the induction of RKIP expression and inhibition of YY1 and upregulation of DR5 expression. Currently, TRAIL or agonist monoclonal antibodies against DR4 or DR5 are being examined clinically for anti-tumor activity. Therefore, our findings suggest the potential application of the combination of NPI-0052 and TRAIL or agonistic monoclonal antibodies against DR4 or DR5 in the treatment of tumor cells that are resistant to drugs and TRAIL.

Rituximab-Mediated Sensitization of B-NHL Cells Lines to TRAIL-Induced Apoptosis: Involvement of YY1 Suppression and DR5 Upregulation.

There has been considerable interest in the treatment of drug-resistant tumor cells with TRAIL or agonist monoclonal antibody directed against the TRAIL receptors DR4 and DR5. TRAIL has been shown to be largely non-toxic to normal tissues and cytotoxic to transformed tumor cells. However, many tumors, including B-NHL, are resistant to TRAIL-induced apoptosis. We have reported that rituximab signals B-NHL cells and inhibits several cell survival signaling pathways leading to chemosensitization (Jazirehi and Bonavida, Oncogene ; 2004: 2121, 2005). In addition, we have recently reported that rituximab sensitizes B-NHL cells to Fas-ligand-induced apoptosis, via inhibition of the transcription repressor Yin Yang 1 (YY1) (Vega et al. The Journal of Immunology;175:2174 2005). We have found that YY1 negatively regulates DR5 transcription and expression and thus, we hypothesized that rituximab-mediated inhibition of YY1 may sensitize TRAIL-resistant B-NHL cells lines to TRAIL-induced apoptosis. The B-NHL cells lines, Ramos and Daudi, were treated with rituximab (20μg/ml for 6h) and were then exposed to various concentrations of recombinant TRAIL (2.5–10 ng/ml for 24h). Following incubation, the cells were examined for apoptosis by assessing activation of caspase-3 and by Annexin V/PI. The findings revealed that the cell lines were relatively resistant to TRAIL but following treatment with rituximab significant potentiation of apoptosis and synergy were achieved. Optimal apoptosis was observed with a concentration of TRAIL of 10ng/ml. The rituximab-treated cells showed a 2 fold upregulation of cell surface DR5 expression as compared to untreated cells. In addition, rituximab treated cells showed significant inhibition of YY1 expression as determined by Western and EMSA. We have also examined the expression of YY1 in tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma, obtained from the Aids and Cancer Specimen Resource of the NCI. These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma and 6 Small Cell Lymphoma and were examined for YY1 by immunhistochemistry. The findings revealed that YY1 was overexpressed as compared to normal tissues. Currently, we are examining the effect of rituximab-mediated sensitization of patients derived B-NHL cells to TRAIL-induced apoptosis. The present findings demonstrate that drug-resistant and TRAIL-resistant B-NHL cells can be sensitized by rituximab to TRAIL-induced apoptosis. Further, the studies revealed a potential novel mechanism of rituximab-mediated effect in vivo by recruiting host cells expressing/secreting TRAIL to exert a cytotoxic activity on the rituximab-treated cells. The findings also suggest the potential therapeutic efficacy, in vivo, of combination of rituximab and either recombinant TRAIL or agonist monoclonal antibodies against DR4 or DR5 in the treatment of resistant cells. We propose that inhibitors of YY1 can serve as a sensitizing agent for TRAIL-induced apoptosis in rituximab-resistant B-NHL cells.

Adenovirus-mediated transfection of caspase-8 sensitizes hepatocellular carcinoma to TRAIL- and chemotherapeutic agent-induced cell death

Caspase-8 belongs to the cysteine protease family and is known to be activated at the initial step in the cascade of TRAIL-induced apoptosis. The activation of procaspase-8 can be blocked by a relatively large amount of c-FLIP, which renders resistance to death receptor-mediated apoptosis in many types of cancer cells. To ask if extrinsic over-expression of caspase-8 contributes to the induction of apoptosis, we introduced the caspase-8 gene into HCC cells using an adenoviral (Adv) vector (Adv-Casp8). We demonstrated that Adv-Casp8 increased expression of active forms of caspase-8 in MOI-dependent manner. A large amount of Adv-Casp8 (MOI of 50) induced apoptosis significantly in HCC cells and resulted in downregulation of c-FLIP (in SK-Hep1, HLE, and HepG2 cells), XIAP, survivin, and Bcl-xL (in HLE cells) and dynamic release of cytochrome c and Smac from the mitochondria into the cytosol. On the other hand, a small amount of Adv-Casp8 (MOI of 10) causes a slight but detectable increase in the level of apoptosis with only a small effect on anti-apoptotic proteins and mitochondrial activation. However, small amounts of Adv-Casp8 augmented TRAIL- or chemotherapeutic agent-induced cell death (with an MOI of 10 or 20, respectively). These results suggest both that exogenous over-expression of caspase-8 by Adv-Casp8 may be essential for induction of HCC cell death and that the combination of Adv-Casp8 and TRAIL or chemotherapeutic agents could provide a useful strategy for treatment of HCC.

Preclinical Differentiation between Apparently Safe and Potentially Hepatotoxic Applications of TRAIL Either Alone or in Combination with Chemotherapeutic Drugs

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) exhibits potent antitumor activity on systemic administration in nonhuman primates without deleterious side effects for normal tissue. However, there is a controversy about the potential toxicity of TRAIL on human hepatocytes. The use of different recombinant TRAIL forms only partially explains the contradicting reports on TRAIL sensitivity in primary human hepatocytes (PHH). To clarify this issue, we comprehensively tested four different recombinant forms of TRAIL for their apoptosis-inducing capacity on PHH obtained from a total of 55 human livers between day 1 and day 8 of in vitro culture. One day after single-cell isolation, all but one recombinant form of TRAIL [i.e., an untagged form of TRAIL (TRAIL.0)] induced apoptosis in PHH. Apoptosis induction by TRAIL in these cells could only be fully inhibited by concomitant blockade of TRAIL receptor 1 and TRAIL receptor 2. At day 4 of in vitro culture, when surrogate markers indicated optimal hepatocyte in vitro function, only high doses of cross-linked FLAG-TRAIL killed PHH whereas the other three recombinant TRAIL forms did not. Strikingly, cotreatment of day 4 PHH with cisplatin sensitized for TRAIL-induced apoptosis whereas 5-fluorouracil, etoposide, gemcitabine, irinotecan, or oxaliplatin, which are commonly used in the treatment of gastrointestinal cancers, did not. Our data show that whereas TRAIL alone or together with selected chemotherapeutic drugs seems to be safe, the combination of TRAIL with cisplatin is toxic to PHH.

Synergistic cytotoxic effects of TRAIL and chemotherapeutic agents on pancreatic carcinoma cells

目的 研究肿瘤坏死因子相关凋亡诱导配体以及联合应用亚毒性剂量的化疗药物对胰腺癌的治疗作用.方法半定量RT-PCR检测TRAILR mRNA在胰腺癌细胞株Canpan-2中的表达.DNA琼脂糖凝胶电泳检测细胞凋亡情况.应用不同浓度的TRAIL及联合亚毒性剂量的氟尿嘧啶(5-fluorouracil)、吉西他滨(gemcitabin)处理胰腺癌细胞,通过MTT法检测细胞毒性作用,流式细胞仪分析细胞凋亡率.结果死亡受体DR4、DR5及诱骗受体DcR1、DcR2在胰腺癌细胞株Canpan-2中均有表达.DNA琼脂糖凝胶电泳可见到典型的凋亡梯形带.TRAIL100 ng/ml作用胰腺癌细胞24 h后,细胞杀伤率为(29.5±1.2)%,且其作用存在浓度依赖性;联合应用亚毒性剂量的氟尿嘧啶、吉西化滨能够大大提高TRAIL的细胞毒活性,杀伤率分别为TRAIL+氟尿嘧啶(43.7±1.4)%,TRAIL+吉西化滨(49.8±1.2)%,联合用药前后差异有显著性(P＜0.01).结论TRAIL受体在胰腺癌细胞普遍表达,并存在受体类型的表达差异;TRAIL与化疗药物氟尿嘧啶、吉西他滨有协同杀伤胰腺癌细胞的作用。

Role of Protein Kinase CK2 in the Regulation of Tumor Necrosis Factor–Related Apoptosis Inducing Ligand–Induced Apoptosis in Prostate Cancer Cells

Protein kinase CK2 (formerly casein kinase 2 or II) is a ubiquitous and highly conserved protein Ser/Thr kinase that plays diverse roles such as in cell proliferation and apoptosis. With respect to the latter, we originally showed that elevated CK2 could suppress various types of apoptosis in prostate cancer cells; however, the downstream pathways that respond to CK2 for mediating the suppression of apoptosis have not been fully elucidated. Here, we report studies on the role of CK2 in influencing activities associated with tumor necrosis factor-related ligand (TRAIL/Apo2-L)-mediated apoptosis in prostate carcinoma cells. To that end, we show that both androgen-insensitive (PC-3) and androgen-sensitive (ALVA-41) prostate cancer cells are sensitized to TRAIL by chemical inhibition of CK2 using its specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). Furthermore, we have shown that overexpression of CK2alpha using pcDNA6-CK2alpha protected prostatic cancer cells from TRAIL-mediated apoptosis by affecting various activities associated with this process. Thus, overexpression of CK2 resulted in the suppression of TRAIL-induced apoptosis via its effects on the activation of caspases, DNA fragmentation, and downstream cleavage of lamin A. In addition, the overexpression of CK2 blocked the mitochondrial apoptosis machinery engaged by TRAIL. These findings define the important role of CK2 in TRAIL signaling in androgen-sensitive and -insensitive prostatic carcinoma cells. Our data support the potential usefulness of anticancer strategies that may involve the combination of TRAIL and down-regulation of CK2.

Sensitization to gimatecan-induced apoptosis by tumor necrosis factor-related apoptosis inducing ligand in prostate carcinoma cells

Since the intrinsic resistance of prostate carcinoma likely reflects a low susceptibility to drug-induced apoptosis, in this study we explored the possibility of sensitizing prostate carcinoma cells to apoptosis by combination of TRAIL with camptothecins. Indeed, these agents are known to activate different pathways of apoptosis. Topotecan- and gimatecan induced moderate up-regulation of TRAIL-R1 and -R2 which resulted in a different cell response to the combination in androgen-independent cells (DU-145 and PC-3). In DU-145 cells apoptosis was increased by lower TRAIL concentrations and was earlier than in PC-3 cells, as shown using Annexin V-binding assay. The relative resistance of PC-3 cells to drug-induced apoptosis was associated with constitutive Akt activation, higher levels of cFLIP-L and Bcl-2, and lower levels of Bax. The different expression/activation of apoptosis-related factors appears to influence the sensitization of prostate carcinoma cells by TRAIL. Potentiation of camptothecin-induced apoptosis by TRAIL appears dependent on cooperation between extrinsic and intrinsic pathways, as documented by loss of the sensitization to apoptosis following reduction of caspase 8 after small interfering RNA transfection. The efficacy of the approach may be critically dependent on the intrinsic susceptibility to apoptosis of different tumors. These observations support that the activation of multiple signals could enhance apoptotic response and suggest the therapeutic interest of the TRAIL/camptothecin combination.

FLIPLong(L) and FLIPShort(S) Overexpression in the Human Myeloid Leukemia Cell Line ML-1 and Its Role in TRAIL [Tumor Necrosis Factor(TNF)-Related Apoptosis-Inducing Ligand] and TNFa Induced Apoptosis.

TRAIL initiates activation of Caspase-8, which is blocked by the FLICE inhibitory protein (FLIP), resulting in resistance to apoptosis. Here we show that overexpression of FLIPL and FLIPS in ML1 cells, with low constitutive levels of FLIP, protects these cells against apoptosis induced by TRAIL, not only via Caspase-8 inhibition, but also via upregulation of anti-apoptotic molecules.Methods: 1) Apoptosis was determined by Annexin V/ 7-AAD following treatment with TRAIL (100–500ng/ml), or TNFa (20–100ng/ml) in ML1 cells transduced with FLIPL.GFP (green fluorescent protein), FLIPS.GFP or Neo.GFP (control). 2) Caspase-8, Caspase-3, Bid, Bcl-xL, XIAP, phosphorylated (P)-IKBa and P-Akt were determined by western blots. 3) Active Caspase-3 was determined using EnzChek Caspase-3 assay kit.Results: Both FLIPL and FLIPS transduction protected ML1 cells against apoptosis induced by TRAIL (300ng/ml), while no protection was observed in Neo.GFP cells. FLIPL had a more profound protective effect than FLIPS (Fig.1A). Both FLIPL and FLIPS, but not Neo.GFP, blocked Caspase-8 and Caspase-3 activation (Fig.1B); FLIPS cells showed two-fold higher levels of active Caspase-3 than FLIPL cells, consistent with higher apoptosis in FLIPS cells. Caspase-3 can be activated through Caspase-8 (extrinsic pathway) or via Caspase-8/Bid (intrinsic pathway). The latter was responsible for high active Caspase-3 levels in FLIPS cells as shown by the presence of cleaved Bid (t-Bid) (Fig.1B); cleavage of Bid was inhibited by combination of TRAIL and Z-IETD-FMK (Caspase-8 inhibitor). Anti-apoptotic molecules, including Bcl-xL, XIAP and FLIP are regulated by NF-kB and FLIP participates in an NF-kB auto-amplification loop. While Neo.GFP cells showed little Bcl-xL after 4h of TRAIL exposure and there was a twofold reduction in FLIPS cells, only a slight reduction of Bcl-xL was noted in FLIPL cells. FLIPL cells showed the lowest rates of apoptosis when exposed to TNFa and BMS543541, a specific inhibitor of IkB kinase (Fig. 1C). In the presence of BMS543541, phosphorylation of IkBa and levels of Bcl-xL and XIAP decreased in Neo.GFP and FLIPS but not in FLIPL cells. Additional data suggest that the PI3-kinase/Akt pathway is involved in constitutive NF-kB activation and differentially affected by FLIPL and FLIPS (Fig. 1D). Preliminary results in immunodeficient mice transplanted with transduced ML1 cells indicated the in vivo relevance of the differences between FLIPL and FLIPS with FLIPL cells engrafting earlier and showing earlier signs of sickness.Conclusions: FLIPL and FLIPS conferred resistance to TRAIL induced apoptosis but showed differential effects: Caspase-8/Bid was involved in the apoptosis pathway in FLIPS, but not in FLIPL cells. FLIPL cells' resistance was due not only to caspase inhibition but to the recruitment of downstream anti-apoptotic pathways such as NF-kB and PI3K/Akt. In vivo data further substantiated the antiapoptotic/pro-survival behavior of FLIPL cells. [Display omitted]

The Role of Histone Acetylation and Death Receptor 5 (DR5) Expression in the Treatment of Chronic Lymphocytic Leukemia (CLL).

Chronic lymphocytic leukemia (CLL) is a disease in which two-thirds of patients will require therapy, usually with the alkylating agent, chlorambucil (CLB) or the nucleoside analog, fludarabine (FLU). TRAIL is a death receptor ligand that has shown selective cytotoxicity towards a variety of malignancies. CLL cells, however, are relatively resistant to this agent. We have previously demonstrated that CLB- and Flu-induced apoptosis is partially mediated through activation of the TRAIL apoptotic pathway and this is related to the up-regulation of the TRAIL death receptors, DR4 and DR5 (Oncogene , 22:8356–8369, 2003). Combining CLB or Flu with TRAIL produced a synergistic apoptotic response in CLL cells. In contrast, the upregulation of DR5 and the synergistic apoptotic response was not observed in normal lymphocytes. We have subsequently demonstrated that the up-regulation of DR5 is mediated by transcription factors, nuclear factor κB (NFκB) and p53, and histone acetylation. Using chromatin immunoprecipitation assays, we have found that the p65 subunit of NFκB and p53 are bound to the DR5 gene in CLL cells following either CLB or Flu treatment. In addition, histone 3 is acetylated following CLB and Flu treatment corresponding to histone acetylase p300 binding to the DR5 gene. Histone deacetylase 1 (HDAC1) also binds to the DR5 gene following CLB or Flu treatment, but generally at later time points. Overall histone acetylation was also found to be increased in CLL cells, as compared to normal lymphocytes. Treatment with HDAC inhibitors, which are being evaluated in clinical trials, resulted in increased binding of p53 and NFκB, but not HDAC1, to the DR5 gene and increased DR5 mRNA levels in cells. Furthermore, enhanced apoptosis was also observed in CLL cells treated with combinations of SAHA (another HDAC inhibitor) and TRAIL. These findings suggest that histone acetylation is important in regulating DR5 expression and that HDAC inhibitors increase DR5 expression mediated by p53 and NFκB. This could provide a mechanism to sensitize CLL cells to TRAIL-induced apoptosis.

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation.

In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.

Transcription Factor NF-κB Differentially Regulates Death Receptor 5 Expression Involving Histone Deacetylase 1

The transcription factor nuclear factor kappaB (NF-kappaB) regulates the expression of both anti-apoptotic and proapoptotic genes. Death receptor 5 (DR5, TRAIL-R2) is a proapoptotic protein considered to be a potential target for cancer therapy, and its expression is mediated by NF-kappaB. The mechanism of NF-kappaB-induced DR5 expression is, however, unknown. Herein, we determined that etoposide-induced DR5 expression requires the first intronic region of the DR5 gene. Mutation of a putative NF-kappaB binding site in this intron eliminates DR5 promoter activity, as do mutations in the p53 binding site in this region. Reduction in p53 expression also blocks p65 binding to the intronic region of the DR5 gene, indicating cooperation between p53 and p65 in DR5 expression. In contrast, the anti-apoptotic stimulus, epidermal growth factor (EGF), fails to increase DR5 expression but effectively activates NF-kappaB and induces p65 binding to the DR5 gene. EGF, however, induces the association of histone deacetylase 1 (HDAC1) with the DR5 gene, whereas etoposide treatment fails to induce this association. Indeed, HDAC inhibitors activate NF-kappaB and p53 and upregulate DR5 expression. Blockage of DR5 activation decreased HDAC inhibitor-induced apoptosis, and a combination of HDAC inhibitors and TRAIL increased apoptosis. This provides a mechanism for regulating NF-kappaB-mediated DR5 expression and could explain the differential roles NF-kappaB plays in regulating apoptosis.

Synthetic Triterpenoids Cooperate with Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand to Induce Apoptosis of Breast Cancer Cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.

E1A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis through Enhancement of Caspase Activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis of cancer cells. Sensitization of cancer cells to TRAIL, particularly TRAIL-resistant cancer cells, could improve the effectiveness of TRAIL as an anticancer agent. The adenovirus type 5 E1A that associates with anticancer activities including sensitization to apoptosis induced by tumor necrosis factor is currently being tested in clinical trials. In this study, we investigated the sensitivity to TRAIL in the E1A transfectants ip1-E1A2 and 231-E1A cells and the parental TRAIL-resistant human ovarian cancer SKOV3.ip1 and TRAIL-sensitive human breast cancer MDA-MB-231 cells. The results indicated that the percentage of TRAIL-induced apoptotic cells was significantly higher in the E1A transfectants of both cell lines than it was in the parental cell lines. To further investigate the cellular mechanism of this effect, we found that E1A enhances TRAIL-induced activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity by a specific inhibitor, Z-DEVD-fmk, abolished TRAIL-induced apoptosis. In addition, E1A enhanced TRAIL expression in ip1-E1A2 cells, but not in 231-E1A cells, and the anti-TRAIL neutralizing antibody N2B2 blocked the E1A-mediated bystander effect in vitro. Taken together, these results suggest that E1A sensitizes both TRAIL-sensitive and TRAIL-resistant cancer cells to TRAIL-induced apoptosis, which occurs through the enhancement of caspase activation; activation of caspase-3 is required for TRAIL-induced apoptosis; and E1A-induced TRAIL expression is involved in the E1A-mediated bystander effect. Combination of E1A and TRAIL could be an effective treatment for cancer.

Hypoxia and low glucose differentially augments TRAIL-induced apoptotic death

Tumor microenvironment, which is characterized by hypoxia, low-glucose concentrations, high-lactate concentrations, low-extracellular pH, can alter the therapeutic response in tumors. In this study, we investigated whether hypoxia affects TRAIL-induced apoptotic death. When human prostate adenocarcinoma DU-145 cells were treated with 50 ng/mL TRAIL or hypoxia for 4 h, the survival was 45.7 and 32.5%, respectively. The combination of TRAIL and hypoxia synergistically increased cell death. Similar results were observed in human prostate adenocarcinoma LNCaP cells. Western blot analysis showed that the hypoxia augmented TRAIL-induced PARP cleavage as well as the activation of caspase-8 and caspase-3, but not caspase-9. Unlike hypoxia, low glucose promoted caspase-9 activation during TRAIL treatment. These results suggest that hypoxia or low glucose-augmented TRAIL cytotoxicity is mediated through the mitochondria-independent pathway or -dependent pathway, respectively.

Optimal selection of aspheric surfaces in optical design

Optical systems frequently use aspheric surfaces to improve performance. Typically the designer uses a combination of experience and trail and error to decide which surfaces to make aspheric, then the shape of the aspheric surface is optimized as part of the optical system. In this paper, a method of optimally choosing which surfaces are to be made aspheric is developed. This is implemented by allowing the surface number of the asphere to become a variable in the optimization. Imaginary surfaces are used as an intermediary to make the problem continuous for the optimization. The method of implementation and design examples are given.

HPC1/RNASEL Mediates Apoptosis of Prostate Cancer Cells Treated with 2′,5′-Oligoadenylates, Topoisomerase I Inhibitors, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2',5'-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH(2)-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH(2)-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.

Bortezomib Partially Overcomes TNF-Related Apoptosis Inducing ligand/Apo-2L (TRAIL/Apo-2L) Environment Mediated-Drug Resistance (EM-DR).

Multiple Myeloma (MM) is an incurable disease that warrants novel treatments. MM survival is dependent on resistance conferred by interactions with the bone marrow (BM) microenvironment (stroma and/or extracellular matrix proteins): Environment Mediated-Drug Resistance (EM-DR). We have shown that adhesion to fibronectin inhibits CD95-induced caspase-8 activation resulting in apoptosis by regulating the cellular localization and availability of c- FLIPL (Shain K. et al. J Immunol . 2002; 168(5):2544–53). TNF-related apoptosis inducing ligand/Apo-2L (TRAIL/Apo-2L) is a member of the TNF superfamily of death ligands that induce apoptosis of resistant MM cell lines and patient's cells while cultured in suspension. TRAIL/Apo-2L also inhibits the growth of human plasmocytomas in xenografted NOD/SCID mice (Mitsiades C. et al. Blood 2001; 98(3):795–804). To explore if the tumor microenvironment confers resistance to TRAIL/Apo-2L mediated apoptosis, we treated RPMI 8226 cells with recombinant human killer TRAIL (Alexis Biochemicals). In a series of 3 experiments, we observed a 50.8 ± 10 % specific apoptosis, measured by annexin/PI staining, when cells were treated with TRAIL (10ng/mL) in suspension (Su) media. In contrast, a dramatic resistance to TRAIL mediated apoptosis (6.9 ± 1.2 %) was observed when RPMI 8226 cells were adhered (Ad) to stroma cells (HS-5 and HS-5 transfected with green fluorescent protein-GFP) suggesting the EM-DR phenotype. We hypothesized that EM-DR can potentially be reversed by Bortezomib, a proteosome inhibitor, via NF-κB inhibition resulting in reduced transcription of cellular adhesion proteins genes (Mitsiades et al. Blood 2002; 99(11): 4079–4086). Resistance to drug-induced apoptosis was also observed when RPMI 8226 cells were adhered to HS-5 and HS5-GFP and treated with Bortezomib alone at 5, 10 and 15nM (55.6 ± 11.6 % Su and 12.1 ± 1.6 % Ad). Microarray analysis of RPMI 8226 cells treated with Bortezomib showed that death receptor 4 (DR4) and DR5 expression was up-regulated suggesting a potential interaction between Bortezomib and TRAIL pathway. When RPMI 8226 cells were treated with Bortezomib (10nM) for 20 Hrs and subsequently treated with TRAIL for additional 4 Hrs, we observed an enhancement to 77.7 ± 3.64 % (Su) and 47.22 ± 13.6 % (Ad) specific apoptosis. In addition, in a series of 3 experiments using transwell inserts, RPMI 8226 cells (upper well) separated from HS-5 (lower well) were treated with Bortezomib (10nM), TRAIL (10ng/ML) or Bortezomib and TRAIL combined as described above. Results were comparable with direct contact experiments described, suggesting that cytokines and/or chemokines released by stroma may be responsible for EM-DR phenotype (Figure 1). Further mechanistic studies elucidating TRAIL and Bortezomib combination are currently under investigation. These preclinical studies provide the basis for clinical trial testing the combination of these agents. [Display omitted]

463 Translation targeting TRAIL receptors to the clinic

Background and purpose: Previously it was shown that combination of death ligand TRAIL and irradiation strongly increases cell kill in several human tumour cell lines. Since Bcl-2 overexpression did not strongly interfere with the efficacy, components of the mitochondrial death pathway are not required for an effective combined treatment. In the present study the minimal molecular prerequisites for the efficacy of a combined treatment were determined.Materials and methods: Apoptosis induction in control, caspase-8 and FADD negative Jurkat cells, BJAB control and FADD-DN cells was analysed by FACS. Activation of caspase-8, -10 and -3 and cleavage of PARP was determined by immunoblotting. TRAIL receptors were activated using recombinant human TRAIL. Surface expression of TRAIL receptors DR4 and DR5 was analysed by FACS.Results: Jurkat T-cells express the agonistic DR5 receptor but not DR4. Presence of FADD was found to be essential for TRAIL induced apoptosis. Caspase-8 negative cells show very low rates of apoptosis after prolonged stimulation with TRAIL. No combined effects of TRAIL with irradiation could be found in FADD-DN overexpressing and FADD deficient cells. However, the combination of TRAIL and irradiation clearly lead to a combined effect in caspase-8 negative Jurkat cells, albeit with reduced death rates. In these cells activation of the alternative initiator caspase-10 could be detected after combined treatment.Conclusion: Our data show that a combined therapy with TRAIL and irradiation will only be effective in cells expressing at least one agonistic TRAIL receptor, FADD and caspase-8 or caspase-10.