Efficacy of TRAIL treatment against HPV16 infected cervical cancer cells undergoing senescence following siRNA knockdown of E6/E7 genes

In this study we investigated E6 and E7 oncogenes from the Human Papilloma Virus as targets for siRNA knockdown in order to boost the efficacy of the anti-cancer drug ‘tumor necrosis factor-related apoptosis inducing ligand’ (TRAIL). SiHa cells were treated with TRAIL following transfection with E6/E7 siRNA and the expression of death receptors DR4 and DR5, cell viability, apoptosis, senescence and cell cycle analysis were undertaken using flow cytometry, MTT viability assay and cellular β-galactosidase activity assays. E6/E7 siRNA resulted in significant upregulation of death receptors DR4 and DR5 but did not result in an enhanced sensitivity to TRAIL. Our results indicate that E6/E7-siRNA induces senescence rather than apoptosis in SiHa cells. The occurrence of senescence in drug resistant cervical cancer cells such as the SiHa cell line by E6/E7 siRNA, among other factors, may prevent TRAIL induced activation of extrinsic and intrinsic pathways that lead to apoptotic cell death. Our findings are significant for combinatorial strategies for cancer therapy since the induction of senescence can preclude apoptosis rendering cells to be recalcitrant to TRAIL treatment.     

Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G₁ cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-x_L, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Snake venom toxin from Vipera lebetina turanica sensitizes cancer cells to TRAIL through ROS- and JNK-mediated upregulation of death receptors and downregulation of survival proteins

We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.     

An anthraquinone derivative, emodin sensitizes hepatocellular carcinoma cells to TRAIL induced apoptosis through the induction of death receptors and downregulation of cell survival proteins

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical trials for cancer, however many tumor cells, including hepatocellular carcinoma (HCC) develop resistance to TRAIL-induced apoptosis. Hence, novel agents that can alleviate TRAIL-induced resistance are urgently needed. In the present report, we investigated the potential of emodin to enhance apoptosis induced by TRAIL in HCC cells. As observed by MTT cytotoxicity assay and the externalization of the membrane phospholipid phosphatidylserine, we found that emodin can significantly potentiate TRAIL-induced apoptosis in HCC cells. When investigated for the mechanism(s), we observed that emodin can downregulate the expression of various cell survival proteins, and induce the cell surface expression of both TRAIL receptors, death receptors (DR) 4 as well as 5. In addition, emodin increased the expression of C/EBP homologous protein (CHOP) in a time-dependent manner. Knockdown of CHOP by siRNA decreased the induction of emodin-induced DR5 expression and apoptosis. Emodin-induced induction of DR5 was mediated through the generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of DR5 and the induction of apoptosis. Also, the knockdown of X-linked inhibitor of apoptosis protein by siRNA significantly reduced the sensitization effect of emodin on TRAIL-induced apoptosis. Overall, our experimental results clearly indicate that emodin can indeed potentiate TRAIL-induced apoptosis through the downregulation of antiapoptotic proteins, increased expression of apoptotic proteins, and ROS mediated upregulation of DR in HCC cells.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential

Background

We have previously shown that prostate cancer LNCaP cells are resistant to TRAIL, and downregulation of PI-3K/Akt pathway by molecular and pharmacological means sensitizes cells to undergo apoptosis by TRAIL and curcumin. The purpose of this study was to examine the molecular mechanisms by which resveratrol sensitized TRAIL-resistant LNCaP cells.

Results

Resveratrol inhibited growth and induced apoptosis in androgen-dependent LNCaP cells, but had no effect on normal human prostate epithelial cells. Resveratrol upregulated the expression of Bax, Bak, PUMA, Noxa, Bim, TRAIL-R1/DR4 and TRAIL-R2/DR5, and downregulated the expression of Bcl-2, Bcl-X_L, survivin and XIAP. Treatment of LNCaP cells with resveratrol resulted in generation of reactive oxygen species, translocation of Bax and p53 to mitochondria, subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2), activation of caspase-3 and caspase-9 and induction of apoptosis. The ability of resveratrol to sensitize TRAIL-resistant LNCaP cells was inhibited by dominant negative FADD, caspase-8 siRNA or N-acetyl cysteine. Smac siRNA inhibited resveratrol-induced apoptosis, whereas Smac N7 peptide induced apoptosis and enhanced the effectiveness of resveratrol.

Conclusion

Resveratrol either alone or in combination with TRAIL or Smac can be used for the prevention and/or treatment of human prostate cancer.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53?/?) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: Evidence for the role of c-Jun N-terminal kinase activation

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Ligand nanovectorization using graphene to target cellular death receptors of cancer cell

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is nowadays envisaged as a natural cytokine useful in nanomedicine to eradicate the cancer cells and not the healthy surrounding ones. However, it suffers from cell resistance and strong dispersion in body to prove its efficiency. The understanding at the molecular level of the TRAIL interaction with death receptors (DRs) on cancer cells is thus of fundamental importance to improve its action. We demonstrate here via molecular simulations that TRAIL can bind to its both agonistic DRs (ie, DR4 and DR5) with a preference for DR4. In this study, the role of a graphene nanoflake as a potential cargo for TRAIL is examined. Furthermore, both TRAIL self-assembling and TRAIL affinity when adsorbed on graphene are considered to enhance efficacy toward the targeted cancer cell. Our modelization results show that TRAIL can bind to DR4 and DR5 when transported by graphene nanoflake, as a proof of concept.     

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.     

Splicing reprogramming of TRAIL/DISC-components sensitizes lung cancer cells to TRAIL-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selective killing of cancer cells underlines its anticancer potential. However, poor tolerability and resistance underscores the need to identify cancer-selective TRAIL-sensitizing agents. Apigenin, a dietary flavonoid, sensitizes lung cancer cell lines to TRAIL. It remains unknown, however, whether apigenin sensitizes primary lung cancer cells to TRAIL and its underlying mechanisms. Here we show that apigenin reprograms alternative splicing of key TRAIL/death-inducing-signaling-complex (DISC) components: TRAIL Death Receptor 5 (DR5) and cellular-FLICE-inhibitory-protein (c-FLIP) by interacting with the RNA-binding proteins hnRNPA2 and MSI2, resulting in increased DR5 and decreased c-FLIP_S protein levels, enhancing TRAIL-induced apoptosis of primary lung cancer cells. In addition, apigenin directly bound heat shock protein 70 (Hsp70), promoting TRAIL/DISC assembly and triggering apoptosis. Our findings reveal that apigenin directs alternative splicing and inhibits Hsp70 enhancing TRAIL anticancer activity. These findings underscore impactful synergies between diet and cancer treatments opening new avenues for improved cancer treatments.Subject terms: Cancer, Molecular biology     

Casticin Potentiates TRAIL-Induced Apoptosis of Gastric Cancer Cells through Endoplasmic Reticulum Stress

Background

Casticin is one of the main active components obtained from Fructus Viticis and has been reported to exert anti-carcinogenic activity on a variety of cancer cells but the precise mechanism underlying this activity remains unclear.

Materials and Methods

Apoptotic activities of casticin (1.0 µmol/l) and TRAIL (25, 50 ng/ml) alone or in combination in the gastric cancer cell lines BGC-823, SGC-7901 and MGC-803 were detected by the use of a cell apoptosis ELISA detection kit, flow cytometry (FCM) with propidium iodide (PI) staining and activities of caspase-3, -8 and -9 by ELISA and cleavage of polyADP-ribose polymerase (PARP) protein using western blot analysis. Death receptors (DR) expression levels were evaluated using FCM analysis and western blotting. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to measure the increase in reactive oxygen species (ROS) levels in cells. Multiple interventions, such as siRNA transfection and pharmacological inhibitors were used to explore the mechanisms of these actions.

Results

Subtoxic concentrations of casticin significantly potentiated TRAIL-induced cytotoxicity and apoptosis in BGC-823, SGC-7901 and MGC-803 cells. Casticin dramatically upregulated DR5 receptor expression but had no effects on DR4 or decoy receptors. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by the co-application of TRAIL and casticin. Gene silencing of the CCAAT/enhancer binding protein homologous protein (CHOP) and pretreatment with salubrinal, an endoplasmic reticulum (ER) stress inhibitor, attenuated casticin-induced DR5 receptor expression, and apoptosis and ROS production. Casticin downregulated the expression levels of the cell survival proteins cFLIP, Bcl-2, XIAP, and survivin. In addition, casticin also induced the expressions of DR5 protein in other gastric cancer cells (SGC-7901 and MGC-803).

Conclusion/Significance

Casticin enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-ER stress-CHOP pathway.     

Encorafenib enhances TRAIL-induced apoptosis of colorectal cancer cells dependent on p53/PUMA signaling

TRAIL has been demonstrated to play a critical role in the apoptosis of colorectal cancer (CRC) cells, but drug resistance markedly restricts its therapeutic effects. Objectives: This study aims to investigate whether encorafenib can enhance TRAIL-induced apoptosis of colorectal cancer cells and the underlying mechanism. TRAIL was first used to induce CRC cells. CCK-8 assays were conducted for detecting cell viability of TRAIL-induced CRC cells with encorafenib treatment. Flow cytometry was used to detect the cell apoptosis of CRC cells and western blot was used to measure the expressions of apoptosis-related proteins. The expressions of DR4, DR5, p53, and PUMA were then evaluated by qPCR and western blot. After transfecting the interference plasmid of p53 into CRC cells, the expressions of PUMA and DR5 were further explored. TRAIL reduced the cell viability of CRC cells, and the inhibition was further reinforced under co-treatment of TRAIL and encorafenib. Encorafenib also triggered the promotion of CRC cell apoptosis induced by TRAIL. It was also found that encorafenib exerted its promoting effects on cell apoptosis of CRC cells via the elevation of DR5. Besides, encorafenib administration promoted the expression levels of p53 and PUMA in TRAIL-induced CRC cells. Furthermore, p53 knockdown attenuated the expression of PUMA and DR5 in TRAIL-induced CRC cells treated with encorafenib. This study indicates that encorafenib stimulates TRAIL-induced apoptosis of CRC cells dependent on p53/PUMA signaling, which may provide instructions for the treatment of CRC.     

抑制PI3K信号通路促进TRAIL诱导人乳腺癌细胞MCF-7凋亡

肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand, TRAIL）对癌细胞有独特的细胞毒性作用,而对正常细胞没有影响. 但乳腺癌细胞耐受TRAIL诱导凋亡.本研究探索磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase,PI3K）信号通路对人乳腺癌MCF-7细胞耐受TRAIL的影响. 采用MTT法、显微照相以及DAPI染色观察TRAIL对MCF-7细胞生长的抑制作用以及诱导细胞凋亡状况;流式细胞分析细胞凋亡的情况;激光共聚焦显微镜观察多聚ADP核糖多聚酶-1（poly(ADP-ribose) polymerase -1,PARP-1）的迁移和定位;Western印迹分析死亡受体、caspase-3/8、磷酸化的AKT［pAKT(Ser473)］、Src和PARP-1等蛋白质表达. 结果显示,小剂量TRAIL（< 80 nmol/L）和Ly294002（< 40μmol/L）对MCF-7细胞生长没有显著的抑制作用,但是大剂量TRAIL（160 nmol/L）和Ly294002（80 μmol/L）则能抑制MCF-7细胞生长;低剂量Ly294002协同TRAIL抑制MCF-7细胞生长,并诱导细胞凋亡;Ly294002和TRAIL共同作用能促进PARP-1从胞浆进入细胞核;蛋白质表达分析显示,MCF-7细胞均表达死亡受体DR4、DR5、诱骗受体DcR1和DcR2、以及caspase-8,但是不表达caspase-3;Ly294002和TRAIL共同作用也能抑制pAKT(Ser473)和Src的表达,并且导致PARP-1断裂. 本研究结果提示,抑制PI3K信号可增加MCF-7细胞对TRAIL诱导的敏感性;MCF-7细胞通过PI3K/AKT途径促进Src的表达耐受TRAIL的细胞毒性作用Ly294002联合TRAIL是一种新的药物组合方式治疗乳腺癌.     

Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis

Melanoma cells are relatively resistant to Apo2L/TRAIL (TNF-related apoptosis-inducing ligand). We postulated that resistance might result from higher expression of inhibitors of apoptosis including Bcl-2, FLIP (FLICE-like inhibitory protein) or IAPs such as XIAP (X-linked inhibitor of apoptosis) or survivin. Compared to scrambled or mismatch controls, targeting individual inhibitors with siRNA (si-Bcl-2, si-XIAP, si-FLIP or si-Surv), followed by Apo2L/TRAIL resulted in marked increase in apoptosis in melanoma cells. Compared to Bcl-2 or FLIP, siRNAs against XIAP and survivin were most potent in sensitizing melanoma cells. A similar substantial increase in apoptosis was seen in renal carcinoma cells (SKRC-45, Caki-2), following the inhibition of either XIAP or survivin by siRNAs. Apo2L/TRAIL treatment in IAP-targeted cells resulted in cleavage of Bid, activation of caspase-9 and cleavage of PARP (poly ADP-ribose polymerase). Thus, Apo2L/TRAIL resistance can be overcome by interfering with expression of inhibitors of apoptosis regulating both extrinsic (death receptor) or intrinsic (mitochondrial) pathways of apoptosis in melanoma cells.     

DR4-selective tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) variants obtained by structure-based design

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. Different tumor types respond either to DR4 or to DR5 activation, and chemotherapeutic drugs can increase the expression of DR4 or DR5 in cancer cells. Thus, DR4 or DR5 receptor-specific TRAIL variants would permit new and tumor-selective therapies. Previous success in generating a DR5-selective TRAIL mutant using computer-assisted protein design prompted us to make a DR4-selective TRAIL variant. Technically, the design of DR4 receptor-selective TRAIL variants is considerably more challenging compared with DR5 receptor-selective variants, because of the lack of a crystal structure of the TRAIL-DR4 complex. A single amino acid substitution of Asp at residue position 218 of TRAIL to His or Tyr was predicted to have a favorable effect on DR4 binding specificity. Surface plasmon resonance-based receptor binding tests showed a lowered DR5 affinity in concert with increased DR4 specificity for the designed variants, D218H and D218Y. Binding to DcR1, DcR2, and osteoprotegerin was also decreased. Cell line assays confirmed that the variants could not induce apoptosis in DR5-responsive Jurkat and A2780 cells but were able to induce apoptosis in DR4-responsive EM-2 and ML-1 cells.