Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460

Chemotherapy-induced apoptosis is generally thought to be dependent on a pathway headed by caspase-9. This model is primarily based on studies performed in leukemia cells; however, little is known about caspase cascades in relatively resistant solid tumor cells, including non-small cell lung cancer (NSCLC) cells. Using the NSCLC cell line NCI-H460 (H460), here, we studied the effect of stable expression of various caspase inhibitors on apoptosis induced by the anticancer drugs cisplatin, topotecan, and gemcitabine. Interestingly, overexpression of caspase-9S and X-linked inhibitor of apoptosis (XIAP), both able to inhibit caspase-9 activity, failed to block apoptosis. In contrast, stable expression of caspase-8 inhibitors, such as cytokine response modifier A (CrmA) and dominant-negative caspase-8, almost completely abrogated apoptosis and also enhanced clonogenic survival. Caspase-8 activation in H460 cells was not mediated by death receptors, inasmuch as overexpression of dominant-negative Fas-associated death domain (FADD-DN) did not prevent procaspase-8 cleavage and subsequent apoptosis. However, stable expression of Bcl-2 and Bcl-xL did suppress these apoptotic events, including the release of cytochrome c from mitochondria, which was observed in drug-treated H460 cells. In the NSCLC cell line H460, we, thus, provide evidence for the existence of a novel drug-inducible apoptotic pathway in which activation of caspase-8, and not of caspase-9, forms the apical and mitochondria-dependent step that subsequently activates the downstream caspases.     

EGFR inhibitors sensitize non-small cell lung cancer cells to TRAIL-induced apoptosis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.     

In vitro efficacy of AdTRAIL gene therapy of bladder cancer is enhanced by trichostatin A-mediated restoration of CAR expression and downregulation of cFLIP and Bcl-XL

Current therapies for bladder cancer are suboptimal and adenoviral gene therapy has been explored as an alternative treatment. In this study, we evaluated the in vitro efficacy of an adenovirus expressing TNF-related apoptosis-inducing ligand (AdTRAIL). At low concentrations of virus, T24 cells were more resistant to AdTRAIL-induced apoptosis than 5637 bladder carcinoma cells. Resistance in T24 cells correlated with poor infectivity and lack of surface expression of coxsackie and adenovirus receptor (CAR). Pretreatment with low concentrations of the histone deacetylase inhibitor trichostatin A, restored CAR expression in T24 cells, which facilitated viral infection and resulted in apoptosis at low concentrations of AdTRAIL. In addition, trichostatin A reduced the expression of Bcl-X(L) and cFLIP resulting in increased sensitivity to recombinant TRAIL. Overexpression of cFLIP inhibited TRAIL-mediated killing in trichostatin A pretreated cells, indicating that downregulation of this antiapoptotic protein is required for sensitization. Therefore, trichostatin A can enhance the efficacy of AdTRAIL by restoring CAR expression and by generating a more pro-apoptotic phenotype that would facilitate bystander activity of TRAIL. Combination of histone deacetylase inhibitors with intravesical AdTRAIL gene therapy may be a novel treatment strategy for bladder cancer.     

Recombinant adenoviruses expressing TRAIL demonstrate antitumor effects on non-small cell lung cancer (NSCLC)

INTRODUCTION: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of malignant cells, but not in normal cells. This preferential toxicity to the abnormal cells renders TRAIL potentially a very powerful therapeutic weapon against cancer. However, a requirement for large quantities of TRAIL to suppress tumor growth in vivo is one of the major factors that has hindered it from being widely applied clinically. To overcome this, we constructed a replication-deficient adenovirus that carries a human full-length TRAIL gene (Ad-TRAIL) and tested its efficacy against a lung cancer model system in comparison to that of the recombinant soluble TRAIL protein. METHODS: To investigate the antitumor activity and therapeutic value of the Ad-TRAIL on the non-small cell lung cancer (NSCLC), four NSCLC cell lines, namely, YTMLC, GLC, A549, and H460 cells, were used. TRAIL protein expression was determined by Western blotting and flow cytometry. Cell viability was analyzed by proliferation assay, and DNA ladder and cell-cycle analysis were used to identify apoptosis. To further evaluate the effect of Ad-TRAIL in vivo, YTMLC cells were inoculated to the subcutis of nude mice. The Ad-TRAIL was subsequently administered into the established tumors. Tumor growth and the TRAIL toxicity were evaluated after treatment. RESULTS: YTMLC cells infected with Ad-TRAIL showed decreased cell viability and a higher percentage of apoptosis. Similar, Ad-TRAIL treatment also significantly suppressed tumor growth in vivo. CONCLUSIONS: TRAIL gene therapy provides a promising therapy for the treatment of NSCLC.     

The proteasomal and apoptotic phenotype determine bortezomib sensitivity of non-small cell lung cancer cells

Bortezomib is a novel anti-cancer agent which has shown promising activity in non-small lung cancer (NSCLC) patients. However, only a subset of patients respond to this treatment. We show that NSCLC cell lines are differentially sensitive to bortezomib, IC₅₀ values ranging from 5 to 83 nM. The apoptosis-inducing potential of bortezomib in NSCLC cells was found to be dependent not only on the apoptotic phenotype but also on the proteasomal phenotype of individual cell lines. Upon effective proteasome inhibition, H460 cells were more susceptible to apoptosis induction by bortezomib than SW1573 cells, indicating a different apoptotic phenotype. However, exposure to a low dose of bortezomib did only result in SW1573 cells, and not in H460 cells, in inhibition of proteasome activity and subsequent apoptosis. This suggests a different proteasomal phenotype as well. Additionally, overexpression of anti-apoptotic protein Bcl-2 in H460 cells did not affect the proteasomal phenotype of H460 cells but did result in decreased bortezomib-induced apoptosis. In conclusion, successful proteasome-inhibitor based treatment strategies in NSCLC face the challenge of having to overcome apoptosis resistance as well as proteasomal resistance of individual lung cancer cells. Further studies in NSCLC are warranted to elucidate underlying mechanisms.     

The novel thymidylate synthase inhibitor trifluorothymidine (TFT) and TRAIL synergistically eradicate non-small cell lung cancer cells

Purpose

TRAIL, a tumor selective anticancer agent, may be used for the treatment of non-small cell lung cancer (NSCLC). However, TRAIL resistance is frequently encountered. Here, the combined use of TRAIL with trifluorothymidine (TFT), a thymidylate synthase inhibitor, was examined for sensitizing NSCLC cells to TRAIL.

Methods

Interactions between TRAIL and TFT were studied in NSCLC cells using growth inhibition and apoptosis assays. Western blotting and flow cytometry were used to investigate underlying mechanisms.

Results

The combined treatment of TFT and TRAIL showed synergistic cytotoxicity in A549, H292, H322 and H460 cells. For synergistic activity, the sequence of administration was important; TFT treatment followed by TRAIL exposure did not show sensitization. Combined TFT and TRAIL treatment for 24 h followed by 48 h of TFT alone was synergistic in all cell lines, with combination index values below 0.9. The treatments affected cell cycle progression, with TRAIL inducing a G1 arrest and TFT, a G2/M arrest. TFT activated Chk2 and reduced Cdc25c levels known to cause G2/M arrest. TRAIL-induced caspase-dependent apoptosis was enhanced by TFT, whereas TFT alone mainly induced caspase-independent death. TFT increased the expression of p53 and p21/WAF1, and p53 was involved in the increase of TRAIL-R2 surface expression. TFT also caused downregulation of cFLIP and XIAP and increased Bax expression.

Conclusions

TFT enhances TRAIL-induced apoptosis in NSCLC cells by sensitizing the apoptotic machinery at different levels in the TRAIL pathway. Our findings suggest a possible therapeutic benefit of the combined use of TFT and TRAIL in NSCLC.     

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.     

Apoptosis induction in renal cell carcinoma by TRAIL and gamma-radiation is impaired by deficient caspase-9 cleavage

TNF-related apoptosis-inducing ligand (TRAIL APO-2L) is a member of the TNF family and induces apoptosis in cancer cells without affecting most non-neoplastic cells. The present investigation is focused on apoptosis induction by combined exposure to TRAIL and ionising radiation (IR) in human renal cell carcinoma (RCC) cell lines. Here, we demonstrate that all RCC cell lines coexpress TRAIL and the death-inducing receptors, TRAIL-R1 and TRAIL-R2. Exposure to TRAIL alone induced marked apoptosis in three out of eight RCC cell lines. Combined exposure to TRAIL and IR resulted in a sensitisation to TRAIL-induced apoptosis in one RCC cell line only. Enhanced apoptosis induction by TRAIL in combination with IR was paralleled by an increase in PARP cleavage and activation of executioner caspase-3, whereas caspases-6 and -7 were not involved. Moreover, exposure to TRAIL and/or IR resulted in a marked activation of initiator caspase-8, possibly augmented by the observed reduction of inhibitory c-FLIP expression. In contrast to other tumour types, activation of initiator caspase-9 was not detectable in our RCC model system after exposure to TRAIL and/or IR. This lack of caspase-9 activation might be related to an impaired 'crosstalk' with the caspase-8 pathway as suggested by the missing Bid cleavage and to the appearance of an XIAP cleavage product known to inhibit caspase-9 activation. Deficient activation of caspase-9, therefore, might contribute to the clinically known resistance of human RCC against IR and also argues against an effective combination therapy with TRAIL and IR in this tumour type.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with down-regulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.     

TRAIL enhances efficacy of radiotherapy in a p53 mutant, Bcl-2 overexpressing lymphoid malignancy.

BACKGROUND AND PURPOSE: Resistance to apoptosis is a contributing factor in the response to radiotherapy. Aim of this study was to evaluate whether TRAIL--in a soluble isoleucine zippered form--enhances the cytotoxic effect of irradiation on tumour cells with a blockade in the mitochondrial apoptosis route and/or a dysfunctional p53 pathway. MATERIALS AND METHODS: The p53 mutant human T acute lymphoblastic leukemia line Jurkat transduced with the Bcl-2 gene was used as model system in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by bioluminescence and palpation in vivo. RESULTS: Jurkat cells overexpressing Bcl-2 did not undergo apoptosis after irradiation, but the combination with TRAIL synergistically induced apoptosis without breaking mitochondrial resistance. TRAIL also reduced clonogenic survival after irradiation. In vivo, radiotherapy or TRAIL alone delayed tumour outgrowth, but combination treatment had the most profound effect. CONCLUSIONS: Isoleucine zippered TRAIL can strongly enhance the efficacy of tumour therapy with ionising radiation in an unfavourable setting of p53 mutation and Bcl-2 overexpression.     

TRAIL and apoptosis induction by TNF-family death receptors

Tumor necrosis factor-related apoptosis-inducing ligand or Apo 2 ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) family of ligands capable of initiating apoptosis through engagement of its death receptors. TRAIL selectively induces apoptosis of a variety of tumor cells and transformed cells, but not most normal cells, and therefore has garnered intense interest as a promising agent for cancer therapy. TRAIL is expressed on different cells of the immune system and plays a role in both T-cell- and natural killer cell-mediated tumor surveillance and suppression of suppressing tumor metastasis. Some mismatch-repair-deficient tumors evade TRAIL-induced apoptosis and acquire TRAIL resistance through different mechanisms. Death receptors, members of the TNF receptor family, signal apoptosis independently of the p53 tumor-suppressor gene. TRAIL treatment in combination with chemo- or radiotherapy enhances TRAIL sensitivity or reverses TRAIL resistance by regulating the downstream effectors. Efforts to identify agents that activate death receptors or block specific effectors may improve therapeutic design. In this review, we summarize recent insights into the apoptosis-signaling pathways stimulated by TRAIL, present our current understanding of the physiological role of this ligand and the potential of its application for cancer therapy and prevention.     

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.     

The proteasome inhibitor PS-341 (bortezomib) up-regulates DR5 expression leading to induction of apoptosis and enhancement of TRAIL-induced apoptosis despite up-regulation of c-FLIP and survivin expression in human NSCLC cells

The proteasome inhibitor PS-341 (bortezomib or Velcade), an approved drug for treatment of patients with multiple myeloma, is currently being tested in clinical trials against various malignancies, including lung cancer. Preclinical studies have shown that PS-341 induces apoptosis and enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells with undefined mechanisms. In the present study, we show that PS-341 induced caspase-8-dependent apoptosis, cooperated with TRAIL to induce apoptosis, and up-regulated death receptor 5 (DR5) expression in human non-small cell lung cancer (NSCLC) cells. DR5 induction correlated with the ability of PS-341 to induce apoptosis. Blockage of PS-341-induced DR5 up-regulation using DR5 small interfering RNA (siRNA) rendered cells less sensitive to apoptosis induced by either PS-341 or its combination with TRAIL, indicating that DR5 up-regulation mediates PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells. We exclude the involvement of c-FLIP and survivin in mediating these events because c-FLIP (i.e., FLIP(S)) and survivin protein levels were actually elevated on exposure to PS-341. Reduction of c-FLIP with c-FLIP siRNA sensitized cells to PS-341-induced apoptosis, suggesting that c-FLIP elevation protects cells from PS-341-induced apoptosis. Thus, the present study highlights the important role of DR5 up-regulation in PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells.     

Inhibitory effect of riccardin D on growth of human non-small cell lung cancer: in vitro and in vivo studies

Riccardin D is a macrocyclic bisbibenzyl compound extracted from liverwort plant Dumortiera hirsuta. Our previous study showed that riccardin D induced apoptosis of human leukemia cells by targeting DNA topoisomerase II (topo II). Riccardin D has been considered as a novel DNA topo II inhibitor and potential chemotherapeutic agent for treatment of cancers. In this study, we evaluated the inhibitory effects of riccardin D on growth of human non-small cell lung cancer (NSCLC) both in vitro and in vivo. Riccardin D effectively inhibited the proliferation of NSCLC cells as estimated by the MTT assay. Further examination showed that the ability of invasion and migration of NSCLC cells was suppressed on exposure to riccardin D as estimated by the assays of scratch and transwell chamber. The anticancer activity of riccardin D was verified in mice bearing human NSCLC H460 xenografts. Riccardin D injection produced a 44.5% inhibition of cancer growth without apparent signs of toxicity to animals. Further, riccardin D induced apoptosis of NSCLC cells as evidenced by the increases of cells with externalization of phosphatidylserine and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive in H460 xenografts. The analysis of apoptotic proteins showed that riccardin D activated the caspases cascade signaling pathway as demonstrated by the increases of cleaved caspase-3 and cleaved PARP in NSCLC cells in vitro and in H460 xenografts in mice. The pBR322 DNA relaxation assay indicated that riccardin D inhibited the activity of DNA topo II in H460 and A549 cells, suggesting the mechanism of riccardin D in induction of NSCLC apoptosis. In addition, we studied the activity and expression of matrix metalloproteinases (MMPs) in NSCLC cells. The activities of MMP-2 and MMP-9 in supernatants of NSCLC cells were suppressed on exposure to riccardin D as estimated by gelatin zymography assay. The inhibitory effects of riccardin D on expressions of MMP-2 and MMP-9 were verified in H460 xenografts in mice and the decreases of vascular endothelial growth factor (VEGF) and Erk1/2 might associate with the inhibition of MMPs and NSCLC growth. Together, our results suggest that riccardin D has a high inhibitory effect on human NSCLC growth through induction of apoptosis.     

Molecular requirements for the combined effects of TRAIL and ionising radiation.

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

Cisplatin enhances the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand gene therapy via recruitment of the mitochondria-dependent death signaling pathway

Despite adequately expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4/DR5, malignant cells are frequently refractory to the cytotoxic effect of this apoptosis-inducing ligand. The susceptibility of cancer cells to TRAIL can be potentiated by cisplatin (CDDP). This study was designed to evaluate the ability of cisplatin to enhance the cytotoxic effect of TRAIL gene therapy using the recombinant adenovirus-mediated tumor-selective expression of membrane-bound green fluorescence protein (GFP)-TRAIL fusion protein (AdVgTRAIL) on thoracic cancer cells and to elucidate the putative mechanisms responsible for this synergistic combination effect. While causing little death of cultured thoracic cancer cells by itself, AdVgTRAIL in combination with CDDP, on the other hand, mediated profound supra-additive cytotoxicity and apoptosis via a strong bystander effect. CDDP/AdVgTRAIL-induced cytotoxicity was completely abrogated either by the pancaspase inhibitor zVAD-fmk or by the selective caspase 9 inhibitor or by transient knockdown of caspase 9 by siRNA, indicating that this process was caspase-mediated and mitochondria-dependent. This was confirmed by the observation that Bcl2 overexpression protected the cells from combination-induced cytotoxicity. Robust activation of caspase 8 activity in combination-treated cells was blocked by overexpression of Bcl2, indicating that caspase 8 activation was secondary to the mitochondria-mediated amplification feedback loop. Combining CDDP with AdVgTRAIL greatly enhances its tumoricidal efficacy in cultured thoracic cancer cells in vitro. The two agents interact to mediate profound activation of caspase cascade via recruitment of the mitochondria and positive feedback loop. The CDDP/AdVgTRAIL combination also exhibits a strong antitumor effect in in vivo animal model of human cancer xenografts.