肿瘤坏死因子相关的凋亡诱导配体对胰腺癌细胞抗肿瘤作用的研究

Objective To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL).Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry.Procaspase-8 and procaspase-3 were determined by Western blot. Results The stable overexpression of TRAIL was detected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore,co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P ＜ 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and themechanisms involved in this effect may be proapoptosis and bystander effect.     

Lung-cancer chemoprevention by induction of synthetic lethality in mutant KRAS premalignant cells in vitro and in vivo

Lung cancer is the leading cause of cancer death in both men and women in the United States, with a low 5-year survival rate despite improved treatment strategies. These data underscore the great need for effective chemoprevention of this cancer. Mutations and activation of KRAS occur frequently in, and are thought to be a primary driver of the development of, non-small cell lung cancers (NSCLC) of the adenocarcinoma subtype. In this study, we developed a new approach for the chemoprevention of NSCLC involving specific targeting of apoptosis in mutant KRAS cells. This approach is based on a synthetic lethal interaction among TNF-related apoptosis-inducing ligand (TRAIL), the second mitochondria-derived activator of caspase Smac/DIABLO (Smac), and KRAS. Mutational activation of KRAS modulated the expression of TRAIL receptors by upregulating death receptors and downregulating decoy receptors. Furthermore, oncogenic KRAS repressed cellular FADD-like interleukin 1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) expression through activation of Erk/mitogen-activated protein kinase (MAPK)-mediated activation of c-Myc. Smac overcame KRAS-induced cell-survival signaling by antagonizing X-linked inhibitor of apoptosis protein (XIAP). Therefore, the combination of TRAIL and a small molecule mimic of Smac induced apoptosis specifically in mutant KRAS cells without harming normal cells. We further showed that short-term, intermittent in vivo treatment with TRAIL and Smac mimic induced apoptosis in tumor cells and reduced tumor burden in a murine model of KRAS-induced lung cancer. These results reflect the potential benefit of a selective therapeutic approach for the chemoprevention of NSCLC.     

Adenovirus-TRAIL can overcome TRAIL resistance and induce a bystander effect

TRAIL is a cytokine with a unique ability to induce apoptosis selectively in many transformed cell lines. The instability of TRAIL and the resistance of some cancer cells to TRAIL present the main obstacles for clinical experimentation. We generated an adenovirus expressing full-length TRAIL and tested its efficacy in several cancer cell lines. Ad-TRAIL-infected cancer cells localized full-length TRAIL protein to the cytoplasm and released same-sized TRAIL in the media. Ad-TRAIL was found to induce apoptotic cell death in several cancer cell lines resistant to soluble TRAIL (A549, SKOV3, HT-29 and LNCap) and in TRAIL-sensitive cell lines. Ad-TRAIL, but not soluble TRAIL, induced apoptotic cell death in TRAIL-resistant cell lines, manifested by an increased sub-G1 proportion, caspase-3 activation and PARP cleavage. Ad-TRAIL also induced a media-transferable bystander effect, but only in soluble TRAIL-sensitive cell lines. In conclusion, two novel characteristics of ad-TRAIL were found during this study. First, that ad-TRAIL can induce apoptotic cell death in several cancer cell lines resistant to sTRAIL. Second, that ad-TRAIL induces a media-transferable bystander effect, which is expected to increase its therapeutic value by allowing TRAIL to overcome the locally acting nature and low transduction rate commonly encountered in clinical situation.     

Cotylenin A,a differentiation-inducing agent,and IFN-alpha cooperatively induce apoptosis and have an antitumor effect on human non-small cell lung carcinoma cells in nude mice

Cotylenin A, a novel inducer of the differentiation of leukemia cells, and IFN-alpha synergistically inhibited the growth of and induced apoptosis in several human non-small cell lung carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5 were the early genes induced by the combination of cotylenin A and IFN alpha in lung carcinoma cells. Neutralizing antibody to TRAIL inhibited apoptosis, suggesting that cotylenin A and IFN alpha cooperatively induced apoptosis through the TRAIL signaling system. This combined treatment preferentially induced apoptosis in human lung cancer cells while sparing normal lung epithelial cells and significantly inhibited the growth of human lung cancer cells as xenografts without apparent adverse effects, suggesting that this combination may have therapeutic value in treating lung cancer.     

Prediction of proapoptotic anticancer therapeutic response in vivo based on cell death visualization and TRAIL death ligand-receptor interaction

Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5 agonistic monoclonal antibodies are being developed as targeted therapeutics because they kill cancer cells while sparing normal cells. A challenge to targeted therapeutics is the selection of patients who are most likely to benefit from targeted drugs because of the heterogeneity of cancer. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and for predicting therapeutic response. We hypothesized that the cell surface expression level of DR4/5 may predict the proapoptotic targeted therapeutic response if the signaling pathway downstream is intact. The goal of this proof-of-concept study was to develop a molecular imaging strategy to predict proapoptotic anti-cancer therapy response at an early stage of treatment. TRAIL and the DR5 agonistic monoclonal antibody HGS-ETR2 (Lexatumumab, TRM-2) were labeled with a near-infrared dye and these were used to image the TRAIL receptors on cultured TRAIL sensitive and TRAIL resistant human tumor cells as well as tumor xenografts. Imaging of cells and tumor-bearing animals was conducted with near infrared fluorescence imagers and apoptosis in cells was assessed by western blots of PARP-cleavage and flow cytometry of sub-G1 content. Apoptosis in tumors was evaluated by imaging near-infrared dye-labeled Annexin V and tumor tissue activated caspase-3 staining. Both in vitro and in vivo studies showed that imaging of death inducing ligand-receptor interaction was consistent with the apoptosis readout. Thus TRAIL sensitive tumors that express TRAIL receptors underwent cell death following treatment whereas tumors lacking TRAIL receptor expression were shown to be TRAIL resistant. In vivo molecular imaging of TRAIL receptor expression correlated with response to TRAIL therapy and an apoptotic response in vivo.     

Cisplatin-enhanced sensitivity of glioblastoma multiforme U251 cells to adenovirus-delivered TRAIL in vitro

TRAIL is a novel therapeutic agent for potential use in glioblastoma multiforme therapy; however, glioblastoma multiforme cells exhibit resistance to TRAIL-induced apoptosis. To evaluate the effects of cisplatin on sensitivity of human glioma cell line U251 to Ad-TRAIL and to investigate the potential mechanism, U251 cells were transfected with Ad-TRAIL and then exposed to cisplatin. The proliferation inhibition of the treated cells was studied by the method of MTT. The cell apoptosis was analyzed by Hoechst33342 staining and by flow cytometry with propidium iodide staining. Semi-quantitative RT-PCR was introduced to detect the mRNA expression of TRAIL, DR4, DR5, Caspase 3, and survivin. Protein expression of DR5 and cleaved Caspase 3 was detected by Western blot assay. The results showed that the combination treatment of cisplatin and Ad-TRAIL could inhibit the proliferation of U251 cells significantly compared with the alone treatment (P < 0.01), which was chiefly attributed to the induction of obvious apoptosis. The enhancement of Ad-TRAIL by cisplatin was due to the up-regulation of DR5 but not DR4 expression, and followed by the down-regulation of survivin and activation of Caspase 3. In conclusion, cisplatin could enhance the apoptosis induction of U251 cells to adenovirous vector carried TRAIL.     

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.     

Therapeutic strategy using phenotypic modulation of cancer cells by differentiation-inducing agents

A low concentration of differentiation inducers greatly enhances the in vitro and in vivo antiproliferative effects of interferon (IFN)α in several human cancer cells. Among the differentiation inducers tested, the sensitivity of cancer cells to IFNα was most strongly affected by cotylenin A. Cotylenin A, which is a novel fusicoccane diterpene glycoside with a complex sugar moiety, affected the differentiation of leukemia cells that were freshly isolated from acute myelogenous leukemia patients in primary culture. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5 were early genes induced by the combination of cotylenin A and IFNα in carcinoma cells. Neutralizing antibody to TRAIL inhibited apoptosis, suggesting that cotylenin A and IFNα cooperatively induced apoptosis through the TRAIL signaling system. Combined treatment preferentially induced apoptosis in human lung cancer cells while sparing normal lung epithelial cells. In an analysis of various cancer cell lines, ovarian cancer cells were highly sensitive to combined treatment with cotylenin A and IFNα in terms of the inhibition of cell growth. This treatment was also effective toward ovarian cancer cells that were refractory to cisplatin, and significantly inhibited the growth of ovarian cancer cells as xenografts without apparent adverse effects. Ovarian cancer cells from patients were also sensitive to the combined treatment in primary cultures. Combined treatment with cotylenin A and IFNα may have therapeutic value in treating human cancers including ovarian cancer. ( Cancer Sci 2007; 98: 1643–1651)     

Overexpression of Bcl2 abrogates chemo- and radiotherapy-induced sensitisation of NCI-H460 non-small-cell lung cancer cells to adenovirus-mediated expression of full-length TRAIL

TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo-2L) is a promising novel anticancer agent that selectively induces apoptosis in tumour cells and the activity of which can be enhanced by combined treatment with chemo- or radiotherapy. For therapeutic purposes, the use of full-length TRAIL may be favourable to recombinant TRAIL based on its increased tumour cell killing potential, and the delivery of TRAIL at the tumour site by adenovirus vectors may provide an approach to overcome the short half-life of recombinant TRAIL and hepatocyte toxicity in vivo. Here, we constructed an adenoviral vector expressing full-length TRAIL (AdTRAIL) and studied the potential of chemo- and radiotherapy in enhancing AdTRAIL-induced apoptosis in non-small cell lung cancer (NSCLC) H460 cells and normal cells and, in addition, investigated the mechanism of AdTRAIL-induced apoptosis. AdTRAIL effectively killed H460 cells, which we previously showed to have a deficiency in mitochondria-dependent apoptosis by downstream activation of caspase-8 rather than caspase-9. Further analyses revealed that AdTRAIL induces death receptor- and mitochondria-dependent apoptosis that could be partially suppressed by Bcl2 overexpression. Combined treatment with doxorubicin (DOX), cisplatin (CDDP), paclitaxel (PTX) and radiation strongly enhanced AdTRAIL-induced cytotoxicity in a synergistic way. Synergy was accompanied by the cleavage of Bid and an increase in caspase-8 processing that was abolished by Bcl2 overexpression, indicating that the Bid-mitochondrial amplification loop is functional in H460 cells. Moreover, combination treatment did not alter the tumour selectivity of AdTRAIL since normal human fibroblasts (NHFs) remained resistant under these conditions. These findings further indicate that the combined use of chemo/radiotherapy and adenovirus-produced full-length TRAIL may provide a valuable treatment option for NSCLC.     

Photochemically mediated delivery of AdhCMV-TRAIL augments the TRAIL-induced apoptosis in colorectal cancer cell lines

Tumor targeting is an important issue in cancer gene therapy. We have developed a light-specific transduction method, named photochemical internalization (PCI), to enhance gene expression from adenoviral vectors selectively in illuminated areas. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in cancer cells, and the aim of this study was to investigate the potential of PCI to enhance transgene expression from AdhCMV-TRAIL and evaluate its impact on apoptotic induction in the two human colorectal cancer cell lines HCT116 and WiDr. PCI-mediated delivery of AdhCMV-TRAIL enabled an increased expression of TRAIL, induced a synergistic reduction in cell viability compared to the individual action of AdhCMV-TRAIL and photochemical treatment, and enhanced the induction of apoptosis demonstrated by an increase in cytoplasmic histone-associated DNA fragments, caspase-8 and caspase-3 activation, PARP cleavage and a decrease in the mitochondrial membrane potential. The synergistic effect could be related to the enhanced TRAIL expression in PCI-treated samples and a modest sensitization of the cancer cells to TRAIL induced apoptosis due to the photochemical treatment. Furthermore, an increased cleavage of Bid and a cell line dependent reduction in the expression levels of anti-apoptotic Bcl-2 family members were observed and could possibly contribute to the enhanced apoptotic level in samples exposed to the combined treatment. The presented results indicate that photochemically mediated delivery of AdhCMV-TRAIL allows a selective enhancement in cell killing, and suggest that PCI may be relevant and advantageous for therapeutic gene delivery in vivo.     

Apoptosis mediated by lentiviral TRAIL transfer involves transduction-dependent and -independent effects

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, which selectively induces apoptosis in many transformed cells without apparent toxic side effects in normal tissue. We recently described the construction and characterization of a lentiviral vector for expression of TRAIL. In this report, we evaluate its suitability for therapeutic application. In vitro, we observed specific induction of apoptosis upon transduction in human lung cancer cells. Cell death was partially dependent on successful integration and TRAIL expression by the vectors, but was to some extent mediated by protein carryover, as we found TRAIL protein associated with virus particles. Transduction of subcutaneously growing lung tumors on nude mice with lentiviral TRAIL mediated a transient suppression of tumor growth. Analysis of tumor sections revealed that transduction efficiency of lentiviral control vector but not of lentiviral TRAIL vector was high. This was because of the direct cytotoxic activity of recombinant TRAIL present in viral particles, which prevented efficient tumor transduction. These data therefore suggest that enveloped viral vectors constitutively expressing TRAIL are well suited for ex vivo applications, such as the transduction of tumor-homing cells, but may have a lower effect when used directly for the transduction of tumor cells in vivo.     

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.     

PG490-mediated sensitization of lung cancer cells to Apo2L/TRAIL-induced apoptosis requires activation of ERK2

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the family of programmed cell death-inducing cytokines. Apo2L/TRAIL induces apoptosis in a wide variety of tumor cells. Tumor cells that are resistant to Apo2L/TRAIL-induced apoptosis can be sensitized by chemotherapeutic drugs and other agents via an unknown mechanism. Here we report that PG490 (triptolide), a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii and used in traditional Chinese medicine, sensitizes lung cancer but not normal human bronchial epithelial cells to Apo2L/TRAIL-induced apoptosis. Sensitization was accompanied by caspase-3 and caspase-8 activation, whereas no cleavage of caspase-9 was observed. Determination of cell surface receptors by flow cytometry demonstrated no difference in Apo2L/TRAIL-R1 and -R2 expression, the two receptors with functional death domains, between resistant and sensitized cells. In cells treated with the combination of Apo2L/TRAIL and PG490, we observed activation of ERK2, a member of the mitogen-activated protein kinase family. Furthermore, sensitization could be blocked by the ERK inhibitor U0126 but not the p38 inhibitor SB203580, suggesting that activation of ERK2 is required for this effect. In addition, sensitization of lung cancer cells was also seen in ex vivo culture of lung cancer tissue from four patients who underwent surgery. Immunohistochemical staining showed a clear reduction in proliferation cell nuclear antigen (PCNA) in tissue treated with Apo2L/TRAIL and PG490. In conclusion, apoptosis induced by the combination of Apo2L/TRAIL and PG490 warrants further evaluation as a potential new strategy for the treatment of lung cancer.     

Adenoviral gene transfer of tumor necrosis factor-related apoptosis-inducing ligand overcomes an impaired response of hepatoma cells but causes severe apoptosis in primary human hepatocytes

Ligands of the tumor necrosis factor family play key roles in liver pathogenesis. The ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is unique, because it is thought to be nontoxic to normal cells while killing a broad range of tumor cells. However, hepatocellular carcinoma is considered resistant to soluble TRAIL treatment. Therefore, a direct gene transfer of TRAIL to malignant cells is part of an alternative delivery strategy. We show that an adenoviral gene transfer (Ad-TRAIL) overcomes an impaired response of hepatocellular carcinoma cell lines to soluble TRAIL, but the transduction of primary human hepatocytes revealed a high number of apoptotic cells. Our data imply that Ad-TRAIL administration in vivo must either be restricted to tumor tissue or controlled by a tumor-specific promoter to avoid severe liver damage in human trials.     

受体依赖性TRAIL重组腺病毒对人肺癌细胞凋亡的诱导作用

目的：观察重组TRAIL腺病毒（Ad5TRAIL及Ad5F35TRAIL）对人非小细胞肺癌（nonsmallcell lung cancer,NSCLC）原代培养细胞和细胞株的凋亡诱导作用,探讨两种AdTRAIL用于肺癌基因治疗的价值。方法：采用流式细胞术检测人肺癌细胞系A549、Z793、QG56和NCIH520及10例原代培养肺癌细胞中CAR和CD46的表达水平;分别以Ad5TRAIL及Ad5F35TRAIL重组腺病毒按MOI 10和50感染上述细胞,48 h后Annexin VFITC双标法流式细胞术检测细胞的早期凋亡。结果：A549、Z793、QG56和NCIH520 4株肺癌细胞中,CD46的表达均明显高于CAR表达。Z793、QG56细胞对Ad5TRAIL和Ad5F35TRAIL的作用较敏感, MOI=10感染后凋亡率分别为（11.76±2.10）%、（15.96±2.89）%和（6.05±158）%、（10.11±1.26）%,显著高于对照组\[（2.33±0.37）%和(5.95±1.89）%,P<0.05\];MOI=50感染时NCIH520细胞凋亡率分别为（12.89±3.2）%和（9.08±1.35）%,与对照组（7.04±2.17）%相比差异无统计学意义（P>0.05）;Ad5TRAIL和Ad5F35TRAIL均不能诱导A549细胞凋亡。10例原代肺癌细胞CD46表达也明显较CAR高;Ad5TRAIL或Ad5F35TRAIL 感染后,5例的原代肺癌细胞检测到凋亡;与Ad5TRAIL相比,Ad5F35TRAIL诱导的凋亡率更高。结论：两种TRAIL重组腺病毒对非小细胞肺癌细胞均有凋亡诱导作用,Ad5F35TRAIL的作用强于Ad5TRAIL,更适合于非小细胞肺癌的基因治疗。     

Chemosensitization of hormone-refractory prostate cancer cells by curcumin to TRAIL-induced apoptosis

Failure to undergo apoptosis has been implicated in the resistance of tumor cells to anticancer therapies. Promotion of apoptosis in tumor cells could potentially increase the efficacy of conventional treatment regimens and improve prognosis. Prostate cancer cells are generally resistant to induction of apoptosis by anticancer agents and death ligands. We investigated the sensitization of prostate cancer cell lines by curcumin (diferuloyl-methane) to TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis. Prostate cancer cells treated with curcumin or TRAIL or curcumin and TRAIL together were assessed for induction of apoptosis and pathway of apoptosis was determined from the activation of procaspases and release of cytochrome c from mitochondria. Curcumin sensitized LNCaP, DU145 and PC3 tumor cell lines to TRAIL. Combined curcumin and TRAIL treatment produced the most loss of viable cells by inducing apoptosis as revealed by accumulation of hypodiploid cells in sub-G1 phase, enhanced annexin V binding, DNA fragmentation, cleavage of procaspases-3, -8, and 9, truncation of proapoptotic Bid, and release of cytochrome c from mitochondria. Tumor cells expressed constitutively active NF-kappaB and sensitization to TRAIL involved inhibition of NF-kappaB by curcumin. These findings suggest that combined curcumin/TRAIL chemo-immunotherapy may be a beneficial adjunct to the standard therapeutic regimens for prostate cancer.     

Expression of TRAIL and TRAIL death receptors in stage III non-small cell lung cancer tumors.

PURPOSE: Several in vitro studies have shown that non-small cell lung cancer (NSCLC) cell lines are sensitive to apoptosis induction by the recombinant human (rh) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death ligand, indicating that rhTRAIL might become an attractive molecule for treatment of NSCLCs. To investigate the therapeutic potential of rhTRAIL, the expression of TRAIL and its apoptosis-inducing receptors DR4 and DR5 was evaluated in tumors of stage III NSCLC patients. EXPERIMENTAL DESIGN: Before treatment, tumor biopsies from locally advanced NSCLC patients were obtained by bronchoscopy. DR4, DR5, and TRAIL expression were determined immunohistochemically in 87 tumors. Patients were randomized for treatment with 60 Gy radiotherapy with or without carboplatin as radiosensitizer. RESULTS: DR4, DR5, and TRAIL were expressed in 99%, 82%, and 91% of the tumors, respectively. Seventeen percent of the samples expressed only DR4 and no DR5. In NSCLCs with squamous cell differentiation, a typical staining pattern for DR4 and DR5 was observed. Cells from the basal layer were strongly positive, and the more mature cells were less positive or negative. An inverse staining pattern was observed for TRAIL. Poorly differentiated areas showed strong staining intensity for DR4, DR5, and TRAIL. DR5-positive staining was associated with increased risk of death (odds ratio, 5.76; 95% confidence interval, 1.04-31.93; P = 0.045). CONCLUSIONS: The majority of the locally irresectable stage III NSCLCs expressed at least one of the two death receptors for TRAIL. Therefore, these death receptors may provide a target for the use of rhTRAIL as a new adjunct in the treatment of stage III NSCLC.     

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.     

Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis-inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is capable of inducing apoptosis in non-small cell lung carcinoma (NSCLC). However, many of the human NSCLC cell lines are resistant to TRAIL, and TRAIL treatment of the resistant cells leads to the activation of nuclear factor-kappaB (NF-kappaB) and extracellular signal-regulated kinase 1/2 (ERK1/2). TRAIL can induce apoptosis in TRAIL-sensitive NSCLC cells through the induction of death-inducing signaling complex (DISC) assembly in lipid rafts of plasma membrane. In the DISC, caspase-8 is cleaved and initiates TRAIL-induced apoptosis. In contrast, TRAIL-DISC assembly in the nonraft phase of the plasma membrane leads to the inhibition of caspase-8 cleavage and NF-kappaB and ERK1/2 activation in TRAIL-resistant NSCLC cells. Receptor-interacting protein (RIP) and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP) mediates the DISC assembly in nonrafts and selective knockdown of either RIP or c-FLIP with interfering RNA redistributes the DISC from nonrafts to lipid rafts, thereby switching the DISC signals from NF-kappaB and ERK1/2 activation to caspase-8-initiated apoptosis. Chemotherapeutic agents inhibit c-FLIP expression, thereby enhancing the DISC assembly in lipid rafts for caspase-8-initiated apoptosis. These studies indicate that RIP and c-FLIP-mediated assembly of the DISC in nonrafts is a critical upstream event in TRAIL resistance and thus targeting of either RIP or c-FLIP may lead to the development of novel therapeutic strategies that can overcome TRAIL resistance in human NSCLC.