DcR2 (TRAIL-R4) siRNA and adenovirus delivery of TRAIL (Ad5hTRAIL) break down in vitro tumorigenic potential of prostate carcinoma cells

High levels of decoy receptor 2 (DcR2; TRAIL-R4) expression are correlated with TRAIL resistance in prostate cancer cells. In addition, upregulation of TRAIL death receptor (DR4 and DR5) expression, either by ionizing radiation or chemotherapy, can sensitize cancer cells to TRAIL. Considering more than half of human cancers are TRAIL resistant, modulation of surface TRAIL receptor expression appears to be an attractive treatment modality to counteract TRAIL resistance. In this study, three siRNA duplexes targeting DcR2 receptor were tested. Ad5hTRAIL infections were performed to overexpress human full-length TRAIL to induce cell death, and the in vitro tumorigenic potential of prostate cancer cells was assessed using colony-forming assays on soft agar. The DU145 and LNCaP prostate cancer cell lines, which express high levels of DcR2, were resistant to Ad5hTRAIL-induced death. Downregulation of surface DcR2 expression by siRNA sensitized these prostate cancer cell lines to Ad5hTRAIL. In addition, DcR2 siRNA-mediated knockdown of DcR2, followed by Ad5hTRAIL infection, dramatically reduced the in vitro tumorigenic potential of prostate cancer cells. Collectively, our results suggest the potential for combining receptor-specific siRNA with TRAIL in the treatment of certain cancers.     

Anti-tumor Effects of IL-1β Induced TRAIL-Expressing hUCMSCs on Embelin Treated Breast Cancer Cell Lines

Background: Human umbilical cord mesenchymal stem cells (hUCMSCs) have high therapeutic value in cancer treatment. We have found that pre-activating hUCMSCs with IL-1β promotes tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression and facilitates anti-tumor effect. Furthermore, embelin has been found to induce apoptosis of different cancer cell lines by upregulating the expression of TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). This study investigated whether IL-1β induced TRAIL-expressing hUCMSCs, in combination with low-dose embelin, could further induce apoptosis in breast cancer cell lines. Materials and Methods: MTT assay was used to examine the cytotoxicity of embelin in MDA-MB-231 and MCF-7. To detect the interested protein expression in cells, Western blot and cell immunofluorescence were used to double-confirm the observed results. Annexin V/PI apoptosis assay was detected by flow cytometry to analyze the apoptosis rate of embelin treated breast cancer cell lines and the effect of co-culturing with breast cancer cells and hUCMSCs. Results: Using Western blot and immunofluorescence, we found that breast cancer cell lines treated with low-dose embelin (2.5-5 μM) increased the expression of apoptosis-related receptor DR4, DR5 and the cleaved caspase 8, 9 and 3. Moreover, TRAIL expression was enhanced in IL-1β induced hUCMSCs. Combining these observations, we expected that coculturing IL-1β induced hUCMSCs with low dose embelin treated MDA-MB-231 and MCF-7 cells might enhance the apoptosis of breast cancer cells. We confirmed via flow cytometry that coculture of IL-1β induced TRAIL-expressing hUCMSCs and embelin treated MDA-MB-231 and MCF-7 cells enhances the apoptosis rate of these breast cancer cells. Conclusion: We found that embelin upregulated the expression of DR4 and DR5 to increase the TRAIL-mediated apoptosis in breast cancer cell lines. Low dose embelin treated breast cancer cell lines in combination with IL-1β induced TRAIL-expressing hUCMSCs may become a potential anti-tumor therapy.     

Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells, thus providing a therapeutic potential. In this study, we examined a large panel of human malignant glioma cell lines and primary cultures of normal human astrocytes for their sensitivity to TRAIL. Of 13 glioma cell lines, 3 were sensitive (80-100% death), 4 were partially resistant (30-79% death), and 6 were resistant (< 30% death). Normal astrocytes were also resistant. TRAIL-induced cell death was characterized by activation of caspase-8 and -3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation. Decoy receptor (DcR1 and DcR2) expression was limited in the glioma cell lines and did not correlate with TRAIL sensitivity. Both sensitive and resistant cell lines expressed TRAIL death receptor (DR5), adapter protein Fas-associated death domain (FADD), and caspase-8; but resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). In contrast, cellular FADD-like IL-1beta-converting enzyme-like inhibitory protein (cFLIP) expression was similar in sensitive and resistant cells. Transfection of sense PED/PEA-15 cDNA in sensitive cells resulted in cell resistance, whereas transfection of antisense in resistant cells rendered them sensitive. Inhibition of protein kinase C (PKC) activity restored TRAIL sensitivity in resistant cells, suggesting that PED/ PEA-15 function might be dependent on PKC-mediated phosphorylation. In summary, TRAIL induces apoptosis in > 50% of glioma cell lines, and this killing occurs through activation of the DR pathway. This caspase-8-induced apoptotic cascade is regulated by intracellular PED/PEA-15, but not by cFLIP or decoy receptors. This pathway may be exploitable for glioma and possibly for other cancer therapies.     

Expression of TRAIL (Apo2L), DR4 (TRAIL receptor 1), DR5 (TRAIL receptor 2) and TRID (TRAIL receptor 3) genes in multidrug resistant human acute myeloid leukemia cell lines that overexpress MDR 1 (HL60/Tax) or MRP (HL60/AR)

Previously we have reported a differential expression of CD95/CD95L and Bcl-2 family of genes in multidrug resistant tumor cells. TRAIL, a member of the TNF receptor family, induces apoptosis in many tumor cells by binding to DR4 (TRAIL receptor 1) and DR5 (TRAIL receptor 2). In contrast, TRAIL-induced apoptosis is prevented by a decoy receptor (DcR1, TRID or TRAIL receptor 3). In the present study, we compared the expression of TRAIL, DR4, DR5, and TRID between a drug sensitive HL60, a myeloid leukemia cell line, and its multidrug resistant (MDR) sublines that either overexpressed MDR 1 gene (HL60/Tax) or MRP gene (HL60/AR), using RT-PCR. TRAIL mRNA was expressed in HL60 cells but was present in low levels in HL60/AR cells and was completely lacking in HL60/Tax cells. Both DR4 and DR5 were undetectable in HL60/Tax but were present at comparable levels in HL60/AR and drug sensitive HL60 cells. TRID were absent in HL60 and HL60/Tax cells, but was present in low but comparable levels in peripheral blood mononuclear cells and HL60/AR cells. These data suggest that the multidrug resistance in MDR HL60 cell lines, regardless of overexpression of MDR 1 or MRP, may be due to different mechanisms. In HL60/AR cells it appears that MDR may be due to decreased expression of TRAIL and constitutive expression of TRID, whereas in HL60/Tax cells, MDR could be due to the absence of TRAIL and/or DR4 and DR5.     

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL

Object Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Methods Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Results Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Conclusion Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death. This work was supported in part by Award P50CA127001 from the National Cancer Institutes and Grants from the National Brain Tumor Foundation and the American Cancer Society-IRG to VG.     

Role of the TRAIL/APO2-L death receptors in chlorambucil- and fludarabine-induced apoptosis in chronic lymphocytic leukemia

The standard treatments for chronic lymphocytic leukemia (CLL) include the alkylating agent chlorambucil (CLB) and the nucleoside analog fludarabine (F-ara-AMP, Flu). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death receptor ligand that induces apoptosis preferentially in tumors. However, CLL cells seem to be resistant to TRAIL-induced apoptosis. The TRAIL apoptotic signaling pathway has also been implicated in genotoxin-induced apoptosis through upregulation of TRAIL death receptors DR4 and DR5. In the present study, we demonstrate that the treatment of primary CLL cells with CLB or Flu increases the mRNA, protein and cell surface expression levels of DR4 and DR5 in a dose-dependent manner. In contrast to CLL cells, drug treatment fails to increase significantly the expression of DR4 or DR5 in normal lymphocytes. CLL cells are, however, resistant to TRAIL-induced apoptosis compared to B-cell lines. In contrast, combinational treatment using CLB or Flu with TRAIL (100 ng/ml) gave a synergistic apoptotic response. Furthermore, TRAIL is readily detectable on the cell surface of CLL cells, but TRAIL expression fails to increase following drug treatment. Preventing TRAIL from interacting with DR4 and DR5 decreases CLB-induced apoptosis in CLL cells. A similar, but less marked effect is observed with Flu. These findings indicate the involvement of the TRAIL apoptotic pathway in the mechanism of action of chemotherapy, and this mechanism could be utilized to sensitize CLL cells to TRAIL-induced apoptosis.     

IFN-γ联合阿霉素或依托泊苷增强TRAIL对神经母细胞瘤细胞的诱导凋亡作用

  目的  探讨半胱-天冬氨酸蛋白酶8(Caspase 8)和死亡受体(DR)在肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导神经母细胞瘤(NB)细胞凋亡中的作用。  方法  应用RT-PCR方法检测γ干扰素(IFN-γ)作用前后NB细胞Caspase 8 mRNA的表达; 应用Western blot方法检测Caspase 8、DR4和DR5蛋白表达; 应用四甲基偶氮唑蓝(MTT)比色法及流式细胞术检测IFN-γ, TRAIL, IFN-γ+TRAIL, Caspase 8抑制剂+TRAIL及IFN-γ+阿霉素/依托泊苷+TRAIL对NB细胞株生长及凋亡的影响。  结果  IFN-γ在NB细胞株SKNDZ中诱导了Caspase 8mRNA及蛋白表达。SY5Y细胞对TRAIL不敏感, 而IFN-γ与TRAIL或阿霉素, 依托泊苷联用对SY5Y细胞有明显诱导凋亡作用。IFN-γ+TRAIL组SY5Y细胞早期凋亡率(23.09+2.35)%高于TRAIL组[(6.15±0.54)%(P< 0.01)], 但低于IFN-γ+阿霉素/依托泊苷+TRAIL组[(43.41±6.46)%/(38.86±7.29)%, P< 0.01]。阿霉素或依托泊苷可以诱导NB细胞株DR5蛋白表达, 但未诱导DR4蛋白表达。IFNγ诱导后表达Caspase 8的SKNDZ细胞对TRAIL的诱导凋亡作用仍不敏感, 而阿霉素或依托泊苷处理后同时表达DR5的SKNDZ细胞对TRAIL的诱导凋亡作用相对敏感。IFN-γ+阿霉素/依托泊苷+TRAIL组SKNDZ细胞早期凋亡率(11.54±2.49)%/(13.38±1.65)%高于IFN-γ+TRAIL组(P< 0.01)。  结论  IFN-γ上调Caspase 8表达及化疗药阿霉素或依托泊苷诱导DR5表达可以恢复NB细胞对TRAIL的敏感性, Caspase 8和DR5在TRAIL诱导NB细胞凋亡中起着十分关键的作用。      

Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection.

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis in susceptible cells by binding to death receptors 4 (DR4) and 5 (DR5). TRAIL preferentially induces apoptosis in transformed cells and the identification of mechanisms by which TRAIL-induced apoptosis can be enhanced may lead to novel cancer chemotherapeutic strategies. Here we show that reovirus infection induces apoptosis in cancer cell lines derived from human breast, lung and cervical cancers. Reovirus-induced apoptosis is mediated by TRAIL and is associated with the release of TRAIL from infected cells. Reovirus infection synergistically and specifically sensitizes cancer cell lines to killing by exogenous TRAIL. This sensitization both enhances the susceptibility of previously resistant cell lines to TRAIL-induced apoptosis and reduces the amount of TRAIL needed to kill already sensitive lines. Sensitization is not associated with a detectable change in the expression of TRAIL receptors in reovirus-infected cells. Sensitization is associated with an increase in the activity of the death receptor-associated initiator caspase, caspase 8, and is inhibited by the peptide IETD-fmk, suggesting that reovirus sensitizes cancer cells to TRAIL-induced apoptosis in a caspase 8-dependent manner. Reovirus-induced sensitization of cells to TRAIL is also associated with increased cleavage of PARP, a substrate of the effector caspases 3 and 7.     

Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells.

PURPOSE: Many melanoma cell lines and primary cultures are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In this study, we investigated the molecular mechanisms that control melanoma cell resistance and searched for chemotherapeutic drugs that could overcome the TRAIL resistance in melanoma cells. EXPERIMENTAL DESIGN: We examined 21 melanoma cell lines and 3 primary melanoma cultures for their sensitivity to TRAIL-induced apoptosis, and then tested cisplatin, chemptothecin, and etoposide for their synergistic effects on TRAIL sensitivity in resistant melanoma cells. RESULTS: Of 21 melanoma cell lines, 11 showed various degrees of sensitivity to TRAIL-induced apoptosis through caspase-8-initiated cleavage of caspase-3 and DNA fragmentation factor 45. The remaining cell lines and primary cultures were resistant to TRAIL, but cisplatin, chemptothecin, and etoposide sensitized the resistant cell lines and primary cultures to TRAIL-induced apoptosis, which also occurred through the caspase-8-initiated caspase cascade. Of the two TRAIL death receptors (DR4 and DR5), melanoma cells primarily expressed DR5 on cell surface. Cisplatin treatment had no effects on cell surface DR5 expression or intracellular expression of Fas-associated death domain and caspase-8. Instead, cisplatin treatment down-regulated intracellular expression of the short form of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory protein (c-FLIP) and inhibited phosphorylation of the long form of c-FLIP. CONCLUSIONS: The results presented here indicate that cisplatin inhibits c-FLIP protein expression and phosphorylation to restore TRAIL-induced caspase-8-initiated apoptosis in melanoma cells, thus providing a new combined therapeutic strategy for melanomas.     

H-Ras regulation of TRAIL death receptor mediated apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.     

Synergistic induction of apoptosis in neuroblastoma cells using a combination of cytostatic drugs with interferon-gamma and TRAIL

The majority of high-risk neuroblastomas lack the expression of caspase-8 due to gene silencing which suggest a mechanism for the selection of tumour cells that are refractory to multiple cytotoxic drugs including tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Inhibitors of DNA methyltransferases and IFN-gamma induce expression of caspase-8, and sensitise some neuroblastoma cells to TRAIL-mediated apoptosis. Here we demonstrate that a combination of cytostatic drugs with IFN-gamma and TRAIL synergistically induces neuroblastoma cell death, which may have implications for future therapy of children with neuroblastoma. Treatment of neuroblastoma cells with IFN-gamma induced caspase-8 expression in all cell lines investigated. In five of the neuroblastoma cell lines (SHEP-1, SK-N-AS, SK-N-FI, SH-SY-5Y and Kelly), IFN-gamma promoted TRAIL-mediated cleavage of caspase-8, initiating a caspase cascade involving caspase-7 and PARP followed by apoptosis. IFN-gamma-mediated facilitation of apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk and the caspase-8 specific inhibitor zIEDT-fmk, indicating an important role of caspase-8 in mediating sensitation by IFN-gamma in neuroblastoma cells. In three of the cell lines [SK-N-BE(2), SK-N-DZ and IMR-32] caspase-8 expression was induced by IFN-gamma, but the cells were still resistant to TRAIL-mediated apoptosis. The pattern of basal TRAIL receptor expression, decoy receptors, FLIP and FADD could not be correlated with resistance or sensitivity to TRAIL-induced apoptosis. Importantly, treatment of neuroblastoma cell lines with cytostatic drugs increased apoptosis in the TRAIL-sensitive cell lines whereas the resistant cell lines were susceptible to TRAIL-mediated apoptosis in the presence of the anticancer drugs. The mechanism of the increased susceptibility to apoptosis might results from drug-mediated up-regulation of the death receptors DR4 and DR5.     

An antibody against DR4 (TRAIL-R1) in combination with doxorubicin selectively kills malignant but not normal prostate cells

Prostate cancer is a major health problem among American men and new treatment approaches are needed. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL/Apo2L) is a death ligand that can induce apoptosis in some but not all cancer cells. Resistance to TRAIL-mediated apoptosis can be overcome by radiation or chemotherapy. The effect of doxorubicin/TRAIL combination therapy was compared among PC3, normal prostate epithelial (PrEC) and stromal (PrSC) cells and cell viability measured by MTS assay. Combination of doxorubicin and TRAIL caused cytotoxicity in all cells tested, although PrSC were more resistant. There was no correlation between TRAIL phenotype and expression of c-FLIP, caspases or TRAIL decoy receptors, although PrSC failed to express DR4. A DR4-specific antibody, which behaved as an agonist in combination with doxorubicin, selectively induced cell death in malignant but not normal prostate cells. Although normal PrEC expressed DR4 as determined by western blot, flow cytometry revealed that only maligant prostate cancer cells (PC3, JCA-1) and not PrEC's exhibited DR4 surface expression. Therefore, combination of doxorubicin and an antibody to DR4 might have therapeutic potential for the treatment of prostate cancer by selectively targeting malignant prostate cells.     

Augmentation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) through up-regulation of TRAIL receptors in human lung cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in different transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. The synthetic retinoid CD437 is a potent inducer of apoptosis in cancer cells through increased levels of death receptors. We demonstrate that treatment of human lung cancer cells with a combination of suboptimal concentrations of CD437 and TRAIL enhanced induction of apoptosis in tumor cell lines with wild-type p53 but not in normal lung epithelial cells. CD437 up-regulated DR4 and DR5 expression. The CD437 and TRAIL combination enhanced activation of caspase-3, caspase-7, caspase-8, and caspase-9 and the subsequent cleavage of poly(ADP-ribose) polymerase and DNA fragmentation factor 45. Caspase inhibitors blocked the induction of apoptosis by this combination. Moreover, this combination induced Bid cleavage and increased cytochrome c release from mitochondria. These results suggest that the mechanism of enhanced apoptosis by this combination involves p53-dependent increase of death receptors by CD437, activation of these receptors by TRAIL, enhanced Bid cleavage, release of cytochrome c, and activation of caspase-3, caspase-7, caspase-8, and caspase-9. These findings suggest a novel strategy for the prevention and treatment of human lung cancer with the CD437 and TRAIL combination.     

Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention as a novel anticancer agent. However, its efficiency may be diminished by occurring resistance in cancer cells. The mechanisms of TRAIL resistance in melanoma are still unsolved. Here we show for the first time that TRAIL-induced activation of NF-kappaB occurs in apoptosis-sensitive melanoma cell lines through TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4), whereas TRAIL failed to activate nuclear factor kappa B (NF-kappaB) in melanoma cells positive only for TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5). However, activation of NF-kappaB by TRAIL was not associated with enhanced expression of antiapoptotic factors: cellular FLICE-inhibitory protein (c-FLIP), Bcl-x(L), X-linked inhibitor of apoptosis protein (XIAP), Survivin, Livin. Rather in one of the cell lines, TRAIL induced the downregulation of DR4. In an established cell culture model for TRAIL resistance and regained TRAIL sensitivity, resistance was neither associated with increased NF-kappaB activity by TRAIL nor by an increased expression of antiapoptotic proteins. However, significant downregulation of caspase-8, caspase-10 and of DR4 was characteristic for TRAIL-resistant, DR4-positive melanoma cells, and regained TRAIL sensitivity coincided with re-expression of these factors. Sensitivity was also largely retained after their exogenous overexpression. Thus, initiator caspases and DR4 rather than NF-kappaB may control melanoma cell sensitivity to TRAIL, and strategies, which result in their upregulation, may be useful for enhancement of TRAIL sensitivity.     

Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-alpha2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a >3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-alpha2b and IFN-beta; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-beta or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-beta and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.     

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.     

Long-term tumor-free survival from treatment with the GFP-TRAIL fusion gene expressed from the hTERT promoter in breast cancer cells

We evaluated anti-tumor activity and toxic effect of an adenoviral vector expressing the GFP/TRAIL fusion gene from the hTERT promoter (designated Ad/gTRAIL) on human breast cancer cell lines and on normal human breast cells. Treatment with Ad/gTRAIL elicited high levels of transgene expression and apoptosis in a variety of breast cancer cell lines. Furthermore, treatment with Ad/gTRAIL was effective in killing breast cancer lines resistant to doxorubicin or soluble TRAIL protein. In contrast, only minimal transgene expression and toxicity was detected in normal human primary mammary epithelial cells after treatment with this vector. An in vivo study further showed that the intralesional administration of Ad/gTRAIL effectively suppressed the growth of human tumor xenografts derived from both doxorubicin-sensitive and doxorubicin-resistant breast cancer lines. Specifically, about 50% of animals bearing doxorubicin-sensitive and doxorubicin-resistant breast cancer xenografts showed complete tumor regression and remained tumor-free for over 5 months. These results suggest that the adenovirus encoding the GFP/TRAIL gene driven by the hTERT promoter has potential application in cancer therapy.     

肺腺癌细胞DR4基因启动子甲基化与TRAIL敏感性的相关性研究

目的 探讨DR4基因启动子甲基化水平与肺腺癌细胞株对肿瘤坏死因子相关凋亡诱导配体（TRAIL）敏感性之间的相关性。方法 选取未经5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理和10 μmol/L 5-Aza-CdR处理3 d后的肺腺癌细胞A549、LTEP-α-2,采用CCK-8法检测细胞增殖抑制率,倒置显微镜下观察细胞形态学改变,流式细胞仪检测细胞凋亡率;采用RT-PCR法、免疫印迹法和甲基化特异性PCR（MSP）法分别检测肺腺癌细胞株（A549、LTEP-α-2）DR4基因mRNA、蛋白表达和启动子区甲基化状态。结果 肺腺癌细胞（A549、LTEP-α-2）对低浓度TRAIL高度耐受,提高TRAIL浓度（15.625、31.25、62.5、125、250、500 μg／ml）作用细胞24、48 h后,细胞生长受到不同程度抑制,且呈剂量和时间依赖性;经5-Aza-CdR处理后,TRAIL对肺腺癌细胞的增殖抑制作用均较处理前显著增强（P＜0.05）,倒置显微镜下细胞形态表现出变圆、皱缩甚至脱落等特征。应用5-Aza-CdR处理后,125 μg／ml TRAIL诱导肺腺癌细胞的凋亡率较处理前明显升高（P＜0.05）。A549、LTEP-α-2细胞存在DR4 mRNA及蛋白的低表达,其基因启动子处于甲基化状态;经5-Aza-CdR处理后,肺腺癌细胞中DR4 mRNA及蛋白的表达均显著升高（P＜0.05）,其基因启动子处于非甲基化状态。结论 5-Aza-CdR可以逆转DR4基因启动子甲基化状态,上调基因表达,增强TRAIL诱导肺腺癌细胞凋亡的能力,从而逆转TRAIL耐药。5-Aza-CdR联合TRAIL可能是治疗肺腺癌的一种新策略。     

Ewing's sarcoma family tumors are sensitive to tumor necrosis factor-related apoptosis-inducing ligand and express death receptor 4 and death receptor 5

In this study, we investigated the sensitivity of Ewing's sarcoma family tumors (ESFTs) of children and adolescents to the tumor necrosis factor-related apoptosis-inducing Ligand (TRAIL). TRAIL binds to death receptors (DRs) DR4, DR5, DcR1, and DcR2. Either DR4 or DR5 can induce apoptosis, whereas DcR1 and DcR2 are considered inhibitory receptors. Nine of 10 ESFT cell lines, including several that were Fas resistant, underwent apoptosis with TRAIL through activation of caspase-10, capase-8 (FLICE), caspase-3, and caspase-9. In contrast to the Fas signaling pathway, caspase-10, but not caspase-8 or the Fas-associated death domain-containing molecule, was recruited to the TRAIL receptor-associated signaling complex. We found that 9 of 10 ESFT cell lines expressed both DR4 and DR5 by Western blotting, whereas the TRAIL-resistant line expressed only DR4. However, DR4 was absent from the cell surface in the resistant and two additional lines (three of five tested lines), suggesting that it may have been nonfunctional. On the contrary, DR5 was located on the cell surface in all four sensitive lines tested, being absent only from the cell surface of the resistant line that was also DR5-negative by Western blotting. In agreement with these findings, the resistance of the line was overcome by restoration of DR5 levels by transfection. Levels of DcR1 and DcR2 or levels of the FLICE-inhibitory protein (FLIP) did not correlate with TRAIL resistance, and protein synthesis inhibition did not sensitize the TRAIL-resistant line to TRAIL. Because these data suggested that sensitivity of ESFTs to TRAIL was mainly based on the presence of DR4/DR5, we investigated the presence of these receptors in 32 ESFT tissue sections by immunohistochemistry. We found that 23 of 32 tumor tissues (72%) expressed both receptors, 8 of 32 (25%) expressed one receptor only, and 1 was negative for both. Our finding of wide expression of DR4/DR5 in ESFT in vivo, in combination with their high sensitivity to TRAIL in vitro and the reported lack of toxicity of TRAIL in mice and monkeys, suggests that TRAIL may be a novel effective agent in the treatment of ESFTs.