Potentiation of TRAIL-Induced Apoptosis in TRAIL-Resistant Cholangiocarcinoma Cells by Curcumin through the Induction of DR5 Membrane Localization and Disruption of the Anti-Apoptotic Complex DR5/DDX3/GSK3β

Objective: Cholangiocarcinoma (CCA) is a cancer of the bile duct with a poor prognosis. The present study examined the ability of curcumin to sensitize apoptosis in the TNF-related apoptosis-inducing ligand (TRAIL)-resistant CCA cell lines of HuCCA-1 and KKU-213A. Methods: Apoptosis was measured using a TUNEL assay. Protein expression was determined by immunoblotting. Membrane death receptor 5 (DR5) was detected by flow cytometry. Protein complex was examined by co-immunoprecipitation. Result: Curcumin potentiated TRAIL-induced apoptosis in both cell lines, indicating the sensitization to TRAIL-induced apoptosis by curcumin. Additionally, curcumin increased DR5 expression and membrane localization; however, the curcumin/TRAIL combination did not result in further increases in DR5 expression and membrane localization in either cell line. Moreover, the curcumin/TRAIL combination reduced DR5/decoy receptor 2 (DcR2) complexes in both cell lines, suggesting that curcumin may enhance TRAIL-induced apoptosis by disrupting DR5/DcR2 interaction. In addition, levels of the anti-apoptotic complex DR5/ DDX3/GSK3β were reduced by the curcumin/TRAIL combination in HuCCA-1 but not in KKU-213A cells. This study also demonstrated that the DR5/DcR2 and DR5/DDX3/GSK3β complexes could be observed under basal conditions, suggesting that these anti-apoptotic complexes may contribute to TRAIL-resistant phenotypes in both cell lines. Pretreatment with the antioxidant N-acetylcysteine attenuated curcumin-enhanced apoptosis by TRAIL, indicating that curcumin sensitized TRAIL-induced apoptosis through an oxidative stress–dependent mechanism. Conclusion: The present study demonstrates the potential of using curcumin in combination with TRAIL to yield better TRAIL therapy outcomes in TRAIL-resistant CCA.     

Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels

We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.     

Doxorubicin potentiates TRAIL cytotoxicity and apoptosis and can overcome TRAIL-resistance in rhabdomyosarcoma cells

Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Tumour necrosis factor-alpha (TNF-alpha) is used for STS in the setting of isolated limb perfusions. Like TNF-alpha, TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis. In contrast to TNF-alpha preliminary studies suggest that TRAIL lacks systemic side effects. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO were evaluated in the TNF-alpha sensitive rhabdomyosarcoma cell line KYM-1, its 5-fold TNF-alpha sensitive subline KD4 and its >150-fold TNF-alpha resistant subline 37B8R. Membrane expression of TRAIL-receptors DR4 (death receptor 4), DR5 (pro-apoptotic), DcR1 (decoy receptor 1), DcR2 (anti-apoptotic) was assessed by flow cytometry. Cytotoxicity was determined by microculture tetrazolium assays. Apoptosis assays were performed with acridine orange. DOX (doxorubicin) and 4-OH-IFO decreased survival in all cell lines; a 2-fold resistance was observed for both drugs in 37B8R. All cell lines expressed DR4 and DR5, but hardly any DcR1 or DcR2. TRAIL was cytotoxic in KYM-1, even more in KD4 and induced massive apoptosis; 37B8R was >500-fold resistant to TRAIL and little apoptosis could be observed. TRAIL plus DOX showed synergistic cytotoxicity in KYM-1 and 37B8R. TRAIL plus 4-OH-IFO showed addition in all three cell lines. DOX plus TRAIL-induced more cytotoxicity and apoptosis in all cell lines compared to TRAIL alone. In 37B8R, DOX overcame resistance to TRAIL. In KYM-1, KD4 and 37B8R, sensitivity and resistance to TNF-alpha and TRAIL parallels. TRAIL-resistance was independent from expression of TRAIL-receptors. DOX with TRAIL could overcome TRAIL-resistance in 37B8R cells.     

Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand

Fas (APO-1/CD95) is a transmembrane protein of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily that induces apoptosis in susceptible normal and neoplastic cells upon cross-linking by its ligand (FasL). TNF-related apoptosis-inducing ligand (TRAIL) is a more recently identified member of the TNF superfamily that has been shown to selectively kill neoplastic cells by engaging two cell-surface receptors, DR4 and DR5. Two additional TRAIL receptors (DcR1 and DcR2) do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis. We addressed the expression of Fas, DR4, and DR5 in thyroid carcinoma cell lines and in 31 thyroid carcinoma specimens by Western blot analysis and immunohistochemistry, respectively, and tested the sensitivity of thyroid carcinoma cell lines to Fas- and TRAIL-induced apoptosis. Fas was found to be expressed in most thyroid carcinoma cell lines and tissue specimens. Although cross-linking of Fas did not induce apoptosis in thyroid carcinoma cell lines, Fas-mediated apoptosis did occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting the presence of a short-lived inhibitor of the Fas pathway in these cells. Cross-linking of Fas failed to induce recruitment and activation of caspase 8, whereas transfection of a constitutively active caspase 8 construct effectively killed the SW579 papillary carcinoma cell line, arguing that the action of the putative inhibitor occurs upstream of caspase 8. By contrast, recombinant TRAIL induced apoptosis in 10 of 12 thyroid carcinoma cell lines tested, by activating caspase-10 at the receptor level and triggering a caspase-mediated apoptotic cascade. Resistance to TRAIL did not correlate with DcR1 or DcR2 protein expression and was overcome by protein synthesis inhibition in 50% of the resistant cell lines. One medullary carcinoma cell line was resistant to Fas-and TRAIL-induced apoptosis, even in the presence of cycloheximide, and to transfection of constitutively active caspase-8, suggesting a different regulation of the apoptotic pathway. Our observations indicate that TRAIL effectively kills carcinomas that originate from the follicular epithelium of the thyroid gland, by inducing caspase-mediated apoptosis, and may provide a potentially potent therapeutic reagent against thyroid cancer.     

Pharmacological inhibition of Bcl-2 family members reactivates TRAIL-induced apoptosis in malignant glioma

The major aim of this study was to develop novel therapeutic approaches to potentiate and reactivate apoptosis induced by TNF-Related Apoptosis Inducing Ligand (TRAIL) in malignant glioma. Analysis of five glioma cell lines (U87, U251, U373, MZ-54 and MZ-18) indicated that only two of the cell lines were sensitive to apoptosis induced by TRAIL alone. TRAIL resistance was not correlated to expression levels of the death receptors DR4 and DR5 or the decoy receptors DcR1 and DcR2, suggesting that it was mediated by inactivation of TRAIL-induced downstream signalling. Activation of the BH3 only protein Bid and subsequent activation of the mitochondrial apoptosis pathway are known to play a pivotal role in TRAIL-induced apoptosis. Since this process is blocked by overexpression of anti-apoptotic Bcl-2 family members, we analyzed the therapeutic potential of BH3 mimetics in potentiating TRAIL-induced apoptosis. Treatment with TRAIL in combination with the specific Bcl-2 inhibitor HA14-1 and the Bcl-2/Bcl-xL inhibitor BH3I-2′ potently enhanced apoptosis in TRAIL-sensitive U87 cells in a dose-dependent fashion. TRAIL-induced apoptosis was significantly reactivated by HA14-1 and BH3I-2′ in one (U343) and two (MZ-54 and MZ-18) of three investigated TRAIL-insensitive cell lines, respectively. Knockdown of the anti-apoptotic Bcl-2 family member Mcl-1 by RNA interference had no additional effect on apoptosis induced by TRAIL and HA14-1 in U87 and U343 cells. Our data indicate that Bcl-2 and Bcl-xL play fundamental roles in TRAIL resistance of malignant glioma and suggest that using TRAIL or agonistic TRAIL receptor antibodies in combination with BH3 mimetics may represent a promising approach to reactivate apoptosis in therapy-resistant high grade gliomas.     

Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.     

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.     

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.     

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression

Background  

TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression.     

Doxorubicin pretreatment sensitizes prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of c-FLIP expression

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) can induce receptor-mediated apoptosis in prostate cancer cell lines that have been co-treated with the chemotherapeutic agent doxorubicin (Voelkel-Johnson C, et al. Cancer Gene Therapy 2002; 9:164-172). In this study, we report that pretreatment with doxorubicin is sufficient to sensitize cells to TRAIL. To identify possible targets of doxorubicin, we analyzed levels of several Bcl-2 family members, TRAIL receptors and the anti-apoptotic protein c-FLIP. Doxorubicin did not affect steady state levels of Bax, Bcl-2 and Bcl-X(L) in the majority of the prostate cancer cell lines. TRAIL receptor mRNAs (DR4, DR5, and DcR2) were induced by doxorubicin but these changes were not reflected at the protein level. In contrast, in response to doxorubicin, levels of c-FLIP, particularly FLIP(S), decreased in all cell lines tested. The decrease in c-FLIP(S) correlated with onset and magnitude of caspase-8 and PARP cleavage in PC3 cells. In two TRAIL resistant cell lines, DU145 and LNCaP, treatment with TRAIL alone resulted in processing of c-FLIP(L) and initiated abortive caspase-8 proteolysis. TRAIL treatment did not affect levels of c-FLIP(S) in Du145 and LNCaP cells and did not result in PARP cleavage. Therefore, our results suggest that doxorubicin- mediated down regulation of c-FLIP(S) predisposes cells to TRAIL-induced apoptosis.     

TRAIL与卡铂联合诱导卵巢癌细胞凋亡的研究

目的探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）与卡铂（CBP）联合应用对体外培养的卵巢A2780细胞的生物学效应及其作用机制。方法采用MTT法、流式细胞术，检测体外培养的A2780细胞在卡铂和TRAIL共同作用下的细胞增殖抑制效应以及细胞凋亡程度，应用半定量逆转录多聚酶链反应（RT-PCR）法检测TRAIL受体的mRNA表达水平。结果A2780细胞对TRAIL敏感，TRAIL与卡铂联合用药对细胞的增殖抑制呈现高效协同作用，与单独用药组比较有显著性差异（P〈0．05）；流式细胞术分析协同性杀伤作用主要由于TRAIL和CBP联合诱导细胞凋亡引起；RT—PCR法检测结果显示A2780细胞在TRAIL与卡铂联合用药后均表现死亡受体DR4、DR5表达水平上调和诱骗受体DcR1、DcR2表达水平下调。结论在体外TRAIL与化疗药物联用能明显抑制肿瘤细胞增殖，诱导肿瘤细胞凋亡，卡铂能明显增强TRAIL对肿瘤细胞的敏感性，其诱导机制可能与死亡受体DR4、DR5表达水平上调和诱骗受体DcR1、DcR2表达水平下调相关。     

Response of normal and colon cancer epithelial cells to TNF-family apoptotic inducers

We compared the response of normal (FHC) and cancer (HT-29) human colon epithelial cells to the important apoptotic inducers TNF-alpha, anti-Fas antibody and TNF-related apoptosis inducing ligand (TRAIL). The two cell lines did not respond to TNF-alpha (15 ng/ml), expressed a limited sensitivity to anti-Fas antibody (200 ng/ml) and a different response to TRAIL (100 ng/ml). We studied apoptosis with regard to the changes at the receptor level (DR, DcR and FLIP) and at the level of mitochondria (Bid protein cleavage, Apo2.7 protein expression and caspase-9 activation). Two different approaches were used to sensitize the cells to TRAIL-induced apoptosis: inhibition of protein synthesis (cycloheximide, CHX) and inhibition of the pro-survival MEK/ERK pathway (U0126). While the two cell lines were markedly sensitized to all three TNF family members by CHX, a different degree of response (especially for TRAIL) was obtained when inhibition of the MEK/ERK pathway was achieved. TRAIL-induced apoptosis was significantly enhanced by U0126 co-treatment in the HT-29 cells, but not in the FHC cells. The most significant differences between the HT-29 and FHC cells co-treated with TRAIL and U0126 were demonstrated with regard to the involvement of the mitochondrial apoptotic pathway, suggesting its importance in the regulation of cell sensitivity to the TRAIL-induced apoptosis.     

Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo

Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL/Apo-2 ligand) induces apoptosis in the majority of cancer cells without appreciable effect in normal cells. Here, we report the effects of TRAIL on apoptosis in several human breast cancer cell lines, primary memory epithelial cells, and immortalized nontransformed cell lines, and we examine whether chemotherapeutic agents augment TRAIL-induced cytotoxicity in breast cancer cells in vitro and in vivo. TRAIL induced apoptosis with different sensitivities, and the majority of cancer cell lines were resistant to TRAIL. The chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, camptothecin, and Adriamycin) induced death receptors (DRs) TRAIL receptor 1/DR4 and TRAIL receptor 2/DR5, and successive treatment with TRAIL resulted in apoptosis of both TRAIL-sensitive and -resistant cells. Actinomycin D sensitized TRAIL-resistant cells through up-regulation of caspases (caspase-3, -9, and -8). TRAIL induces apoptosis in Adriamycin-resistant MCF7 cells already expressing high levels of death receptors DR4 and DR5. The pretreatment of breast cancer cells with chemotherapeutic drugs followed by TRAIL reversed their resistance by triggering caspase-3, -9, and -8 activation. The sequential treatment of nude mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity and apoptosis in xenografted tumors. Complete eradication of established tumors and survival of mice were achieved without detectable toxicity. Thus, the sequential administration of chemotherapeutic drugs followed by TRAIL may be used as a new therapeutic approach for cancer therapy.     

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.     

Tumor-specific down-regulation of the tumor necrosis factor-related apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in a large variety of cancer cells but not in most normal human cells. This feature makes TRAIL, a potential antitumor agent. TRAIL can bind to four different receptors, two pro-apoptotic death receptors (DRs), DR4 and DR5, and two antiapoptotic decoy receptors (DcRs), DcR1 and DcR2. Normal cells express all four of the receptors. The increased TRAIL sensitivity of tumor cells has been postulated to result from the lack of DcR expression. We studied the tumor-specific down-regulation of the TRAIL receptors DcR1 and DcR2, as well as DR4 and DR5, in a group of pediatric tumor cell lines [nine neuroblastoma and three peripheral primitive neuro-ectodermal tumors (PNETs)] and three cell lines from adult tumors. Lack of expression of DcR1 and DcR2 was widespread (13 of the 15 cell lines and 10 of 15, respectively), both in the adult tumor cell lines and in the pediatric tumor lines. DR4 and DR5 were expressed in 8 of 15 and 12 of 15 cell lines, respectively. To understand the tumor-specific down-regulation of the TRAIL receptors, the promoter regions were studied for possible methylation changes of their CpG islands. All normal tissues were completely unmethylated, whereas in the tumor cell lines, we found frequent hypermethylation of the promoter. For DcR1 and DcR2, we found dense hypermethylation in 9 (69%) of 13 and 9 (90%) of 10 of nonexpressing cell lines, respectively. DR4 and DR5 were methylated in 5 (71%) of 7 and 2 (67%) of 3 nonexpressing cell lines, respectively. Treatment with the demethylating agent 5-aza-2'deoxycytidine resulted in partial demethylation and restored mRNA expression. In addition, we performed mutation analysis of the death domains of DR4 and DR5 by sequencing exon 9. Mutations were not present in any of the neuroblastoma or PNET cell lines. A panel of 28 fresh neuroblastoma tumor samples also lacked expression of DcR1 and DcR2 in 85 and 74% of cases, respectively. Hypermethylation was observed in 6 (21%) of 28 for DcR1 and 7 (25%) of 28 for DcR2. DR4 and DR5 were both expressed in 22 of 28 tumors, and no promoter methylation was observed. These data suggest that hypermethylation of the promoters of DcR1 and DcR2 is important in the down-regulation of expression in neuroblastoma and other tumor types.     

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.     

Newly established tumourigenic primary human colon cancer cell lines are sensitive to TRAIL-induced apoptosis in vitro and in vivo

Most data on the therapeutic potential of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) as well as resistance to FAS ligand (FASL) in colorectal cancer have come from in vitro studies using cell lines. To gain a clearer understanding about the susceptibility of patient tumours to TRAIL and FASL, we derived primary human cancer epithelial cells from colon cancer patients. Characterisation of primary cultures PAP60 and MIH55 determined their highly proliferating advantage, transforming capability and tumorigenicity in vitro and in vivo. Although FASL treatment appeared to cause little apoptosis only in the PAP60 primary culture, increased apoptosis independent of p53 was observed in both primary PAP60 and MIH55 and control cell lines Caco-2, HT29 and DLD-1 after treatment with SuperKiller TRAIL. Expression analysis of death receptors (DR) in the original parental tumours, the primary cultures before and after engraftment as well as the mouse xenografts, revealed a significant upregulation of both DR4 and DR5, which correlated to differences in sensitivity of the cells to TRAIL-induced apoptosis. Treating patient tumour xenograft/SCID mouse models with Killer TRAIL in vivo suppressed tumour growth. This is the first demonstration of TRAIL-induced apoptosis in characterised tumorigenic primary human cultures (in vitro) and antitumour activity in xenograft models (in vivo).     

Androgens regulate TRAIL-induced cell death in prostate cancer cells via multiple mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent for prostate cancer because it selectively induces apoptosis in cancer cells but not in normal cells. Previous reports have suggested that androgens regulate TRAIL-induced apoptosis in prostate cancer cells. However, there are discrepancies between these reports of how androgens affect TRAIL-induced cell death. To clarify the role of androgens on TRAIL-induced apoptosis in prostate cancer cells, we investigated the effects of androgen on TRAIL-induced cell death in a dose–response manner. Our results showed that although androgens sensitize LNCaP cells to TRAIL-induced apoptosis, this effect is dose-dependent and biphasic. We found that low levels of androgen are superior to high levels of androgen in term of sensitizing LNCaP cells to TRAIL. We also found that upregulation of DR5 (TRAIL-R2) expression by androgens is critical for sensitizing LNCaP cells to TRAIL. However, low levels of androgen are sufficient to induce DR5 expression and sensitize LNCaP cells to TRAIL-induced cell death. High levels of androgen alter the TRADD/RIP1 ratio, which may contribute to NF-κB activation and sequentially inhibit TRAIL-induced apoptosis.