TRAIL-treated Cells Research Articles

Anti-apoptotic hPEBP4 silencing promotes TRAIL-induced apoptosis of human ovarian cancer cells by activating ERK and JNK pathways

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cell lines but not typically in normal or nontransformed cells, which makes TRAIL a desirable therapeutic agent to fight cancer. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a recently identified anti-apoptotic molecule and has been shown to be highly expressed in breast and ovarian cancer cells. We demonstrate that silencing of hPEBP4 in CaoV-3 ovarian cancer cells potentiates TRAIL-induced apoptosis. We found that endogenous hPEBP4 interacts with Raf-1 and MEK1 in TRAIL-treated CaoV-3 cells by co-immunoprecipitation analysis. Simultaneously, silencing of hPEBP4 in CaoV-3 cells enhances TRAIL-induced ERK and JNK activation. Moreover, the inhibitors of MEK1 or JNK can reduce hPEBP4-silence-induced TRAIL sensitivity. Therefore, silencing of hPEBP4 in CaoV-3 ovarian cancer promotes TRAIL-induced apoptosis, and the increased MAPK activation is required for the apoptosis sensitization. All these data indicate that silencing of hPEBP4, an important potential target, may be a promising approach for the treatment of ovarian cancer.

Proteins Made for Dying

It has long been understood that programmed cell death is an active process that requires protein synthesis. Although overall rates of translation are decreased in apoptosis, a subset of proteins bucks the trend and continues to be made, or is even more abundant, in cells destined to die. Bushell et al. explored how the mRNAs encoding these proteins are differentially regulated. They screened polysomal RNAs on microarrays to identify those whose translation was maintained or increased in cells of the human breast cancer-derived cell line MCF7 that were induced to undergo apoptosis by treatment with the cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). About 3% of all mRNAs were maintained or recruited onto polysomes in the TRAIL-treated cells. Of these, about a third encoded proteins known to function in organization of chromatin or regulation of transcription. Also overrepresented were mRNAs encoding proteins of the Notch signaling pathway. Of 11 mRNAs examined in detail, 8 contained internal ribosomal entry sites (IRESs) that allow internal recruitment of ribosomes for initiation of translation. There is specificity within the IRES sequences, however, as not all mRNAs with functional IRESs were maintained on polysomes in the apoptotic cells. The function of the IRESs that promoted synthesis in the apoptotic cells appeared to require PTB (polypyrimidine tract-binding protein), a factor known to enhance IRES function. Expression of PTB was increased in TRAIL-treated cells. Furthermore, reduced expression of PTB after treatment of cells with siRNA inhibited apoptosis in response to TRAIL. Thus IRESs, which are often activated in conditions of cell stress, provide one mechanism by which translation is maintained of proteins that accumulate during cell death. M. Bushell, M. Stoneley, Y. W. Kong, T. L. Hamilton, K. A. Spriggs, H. C. Dobbyn, X. Qin, P. Sarnow, A. E. Willis, Polypyrimidine tract binding protein regulates IRES-mediated gene expression during apoptosis. Mol. Cell 23 , 401-412 (2006). [PubMed]

TRAIL-induced apoptosis proceeding from caspase-3-dependent and -independent pathways in distinct HeLa cells

The apoptotic pathway in higher eukaryotes remains controversial with respect to the necessity of activation of caspase-3 in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated cells. In this study, a fluorescence resonance energy transfer (FRET) probe was developed to image the activation of caspase-3 and the related apoptotic pathway in TRAIL-treated cells in real time. Both kinds of apoptotic pathways were observed simultaneously in the same experiment proceeding from activation and non-activation of caspase-3. The total apoptotic rate was 56.08%, the apoptotic rates for activation and non-activation of caspase-3 pathways were 21.5% and 34.58%, respectively, which were examined later for Hoechst 33258 staining and morphological characteristics. The apoptotic rate due to the activation of caspase-3 pathways in TRAIL-treated cells has been independently measured to be around 25.11% by capillary electrophoresis (CE) analysis, which confirmed the apoptotic rate due to activation of caspase-3 pathways as found by FRET analysis. This result also suggests that rest apoptosis is preceded by caspase-3-independent pathways, as CE has the ability to quantitatively detect caspase-dependent apoptosis. The observation of the coexistence of caspase-3-dependent and caspase-3-independent apoptotic pathways in the TRAIL-treated cells was unusual in comparison with the previous reports.

ERK1/2 activation attenuates TRAIL-induced apoptosis through the regulation of mitochondria-dependent pathway

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as an extracellular signal, which triggers apoptosis in tumor cells. In order to characterize the molecular events involved in TRAIL cytotoxic signaling, we attempted to determine the role of extracellular signal-regulated kinase 1/2 (ERK1/2), as well as its downstream targets in TRAIL-treated HeLa cells. Here we demonstrate that TRAIL exposure resulted in the activation of ERK1/2, and the elevation of anti-apoptotic Bcl-2 protein levels. ERK1/2 inhibition with PD98059 promoted cell death via the down-regulation of Bcl-2 protein levels, together with increasing mitochondrial damage, including the collapse of mitochondrial membrane potential, the release of cytochrome c from mitochondria to cytoplasm and caspase activity. These results suggest that the ERK1/2 activation is a kind of survival mechanism to struggle against TRAIL-induced stress condition in early stage, via activating cellular defense mechanisms like as the up-regulation of the Bcl-2/Bax ratio, as well as several mitochondrial events.

Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily that selectively induces apoptosis in malignant cells. However, not all cancer cells are susceptible to TRAIL and mechanisms of resistance and new strategies to enhance sensitivity are an area of intense investigation. Glucose withdrawal or paclitaxel increase intracellular ceramide, down-regulate cellular FLICE inhibitory protein (cFLIP), and sensitize cells to TRAIL. Therefore, we investigated whether TRAIL resistance is due to ceramide levels and/or defects in ceramide generation following ligand binding. Colon cancer cells isolated from the primary tumor (SW480) and a subsequent metastasis (SW620) of the same patient have different sensitivities to TRAIL. Mass spectrometry was used to compare ceramide content in untreated and TRAIL-treated cells. Overall levels of ceramide were comparable in the cell lines but TRAIL-sensitive SW480 cells contained a higher percentage of C(16)-, and C(18)-ceramide and lower C(24)-ceramides than TRAIL-resistant SW620 cells. Upon TRAIL treatment, ceramide (primarily C(16)-ceramide) increased in SW480 but not SW620 cells. The increase in ceramide occurred with slow kinetics, paralleling caspase-3/7 activation. Combination of C(6)-ceramide with TRAIL resulted in apoptosis of SW620 cells. However, exogenous C(6)-ceramide did not affect levels of cFLIP nor did pretreatment sensitize cells to TRAIL. Exposure to TRAIL prior to ceramide was required to induce apoptosis, suggesting that ceramide plays a role in enhancing or amplifying TRAIL-mediated signaling. Our results suggest that ceramide plays a role in promoting TRAIL-mediated apoptosis and that TRAIL-resistant cancers may benefit from combination therapy with ceramide or agents that enhance ceramide accumulation.

Caspase-Mediated p65 Cleavage Promotes TRAIL-Induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is cytotoxic to a wide variety of transformed cells, but not to most normal cells, implying potential therapeutic value against advanced cancer. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood compared with other TNF family members. Specifically, it is not yet understood how TRAIL controls nuclear factor kappaB (NF-kappaB) activation and overcomes its anti-apoptotic effect. We explored the regulation of NF-kappaB activity by TRAIL and its role in apoptosis. TRAIL combined with IkappaBalpha-"superrepressor" induced potent apoptosis of SK-Hep1 hepatoma cells at low concentrations of TRAIL that do not independently induce apoptosis. Apoptosis by high concentrations of TRAIL was not affected by IkappaBalpha-superrepressor. Although TRAIL alone did not induce NF-kappaB activity, TRAIL combined with z-VAD significantly increased NF-kappaB activation. Analysis of the NF-kappaB activation pathway indicated that TRAIL unexpectedly induced cleavage of p65 at Asp97, which was blocked by z-VAD, accounting for all of these findings. p65 expression abrogated apoptosis and increased NF-kappaB activity in TRAIL-treated cells. Cleavage-resistant p65D97A further increased NF-kappaB activity in TRAIL-treated cells, whereas the COOH-terminal p65 fragment acted as a dominant-negative inhibitor. XIAP levels were increased by TRAIL in combination with z-VAD, whereas XIAP levels were decreased by TRAIL alone. Cleavage of p65 was also detected after FRO thyroid cancer cells were treated with TRAIL. These results suggest that TRAIL induces NF-kappaB activation, but simultaneously abrogates NF-kappaB activation by cleaving p65, and thereby inhibits the induction of anti-apoptotic proteins such as XIAP, which contributes to the strong apoptotic activity of TRAIL compared with other TNF family members.

Resistance of Human Melanoma Cells Against the Death Ligand TRAIL Is Reversed by Ultraviolet-B Radiation via Downregulation of FLIP

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug since it induces apoptosis preferentially in malignant but not in normal cells. Not all cancer cells, however, are TRAIL susceptible. Chemotherapeutic drugs and ionizing radiation have been found to be able to sensitize resistant tumor cells for TRAIL-induced apoptosis. Since ultraviolet B radiation (UVB) is a potent inducer of apoptosis but exhibits much less adverse effects, we studied whether UVB sensitizes TRAIL-resistant melanoma cells. Therefore, we analyzed the TRAIL-sensitive human cell line A-375 in comparison to the resistant cell line IGR-37. Both cell lines showed expression of the long form of the antiapoptotic FLICE inhibitory protein FLIP(L) which, however, was partially cleaved into the 43 kDa form in A-375 cells. In addition, only IGR-37 cells expressed the short splicing variant FLIP(S), which exerts high antiapoptotic activity. Accordingly, transient overexpression of FLIP(S) rendered A-375 cells resistant to TRAIL. Upon exposure to low UVB doses, TRAIL-treated IGR-37 cells underwent pronounced apoptosis and TRAIL sensitivity of A-375 cells was dramatically increased. In both cases, UVB caused an inhibition of flip expression. This study indicates that (i) the expression level and the processing status of FLIP may play a crucial role in determining the sensitivity of melanoma cells towards TRAIL and (ii) expression of FLIP is regulated by UVB.

Elevated Cystatin expression in metastatic oral cancer cells confers greater resistance to TRAIL-induced apoptosis

Background TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immunosurveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by inhibitors known as cystatins (CY). Objective To relate the TRAIL sensitivity of clonally related primary and metastatic oral cancer (OC) cells with their CB and CY levels. Study design Two pairs of primary (686Tu and 101Tu) and metastatic (686Ln and 101Ln) OC cell lines were treated with various concentrations (10-1000 ng/mL) recombinant human TRAIL protein for 14 hours. Cell viability was quantified by MTT assay. Apoptosis rate among TRAIL-treated cells was measured using the TUNEL and M30 CytoDEATH immunodetection assays. CB and CY (A, B, C, and M) levels in these cells lines were analyzed by RT-PCR and Western blots. Results Primary cells revealed greater susceptibility to TRAIL-induced killing (ED 50 of 50 and 250 ng/mL for 686Tu and 101Tu, respectively) than their metastatic clones (ED 50 of 250 and 1000 ng/mL for 686Ln and 101Ln, respectively). Primary cells in the presence of CB-specific chemical inhibitor CA074 revealed markedly increased resistance to TRAIL (ED 50 > 1000 ng/mL). Expression levels of CY were markedly higher in metastatic OC cells than in their respective primary cells whereas CB levels remain unchanged. Conclusion CB is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells correlate with their greater resistance to TRAIL-induced apoptosis. Our data suggest that overexpression of CY in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.

Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo.

To investigate the synergistic cytotoxicity of TRAIL in combination with chemotherapeutic agents in A549 cell lines, we systematically evaluated the cytotoxicity of TRAIL alone and TRAIL in combination with cisplatin, paclitaxel (Taxol) or actinomycin D in A549 cell lines in vitro and in vivo, and whether the sensitivity was correlated with the expression level of TRAIL receptors. We investigated the cytotoxicity of TRAIL alone and the synergistic antitumor effects of TRAIL in combination with chemotherapeutic agents in A549 cells by crystal violet staining and FACS in vitro. The expression levels of DR4, DR5, DcR1 and DcR2 were measured in TRAIL-treated and chemotherapeutic agent-treated A549 cells by Western blotting. The growth inhibition of tumors was evaluated in terms of incidence, volume and weight in a A549-implanted nude mice model. Chemotherapeutic agents cisplatin (5.56 mug/ml), Taxol (10 and 30 mug/ml) or actinomycin D (9.26, 83.3 and 750 ng/ml) augmented the cytotoxicity of TRAIL in A549 cell lines within a range of concentrations of TRAIL (1.98-160 ng/ml) in vitro. The expression levels of DR4 and DR5 were not significantly different and the expression of DcR2 was slightly downregulated, but the expression of DcR1 was not detected in non-treated, TRAIL-treated and chemotherapeutic agent-treated A549 cells. The rates of tumor inhibition following treatment with TRAIL alone (15 mg/kg per day, daily for 10 days) and TRAIL/cisplatin (15 mg/kg per day TRAIL, daily for 10 days; 1.5 mg/kg per day cisplatin, daily for 10 days with 7-day intervals) were 28.3% and 76.8% by tumor weight ( P<0.05 for TRAIL alone versus control, P<0.05 for TRAIL/cisplatin versus cisplatin alone and TRAIL alone) on day 65 in vivo. TRAIL in combination with chemotherapeutic agents cisplatin, Taxol or actinomycin D exerted synergistic antitumor effects in A549 cell lines in vitro and TRAIL/cisplatin demonstrated synergistic antitumor effects in vivo. The expression levels of TRAIL receptors suggested that the synergistic effects of TRAIL in combination with chemotherapeutic agents are not at the receptor level in A549 cell lines.

Differential control of apoptosis by DJ-1 in prostate benign and cancer cells.

DJ-1 is a conserved protein reported to be involved in diverse cellular processes ranging from cellular transformation, control of protein-RNA interaction, oxidative stress response to control of male infertility, among several others. Mutations in the human gene have been shown to be associated with an autosomal recessive, early onset Parkinson's disease (PARK7). The present study examines the control of DJ-1 expression in prostatic benign hyperplasia (BPH-1) and cancer (PC-3) cell lines in which DJ-1 abundance differs significantly. We show that while BPH-1 cells exhibit low basal level of DJ-1 expression, stress-inducing agents such as H(2)O(2) and mitomycin C markedly increase the intracellular level of the polypeptide. In contrast, DJ-1 expression is relatively high in PC-3 cells, and incubation with the same cytotoxic drugs does not modulate further the level of the polypeptide. In correlation with the expression of DJ-1, both cytotoxic agents activate the apoptotic pathway in the prostatic benign cells but not in PC-3 cells, which are resistant to their action. We further demonstrate that incubation of BPH-1 cells with TNF-related-apoptosis-inducing-ligand/Apo-2L (TRAIL) also enhances DJ-1 expression and that TRAIL and H(2)O(2) act additively to stimulate DJ-1 accumulation but synergistically in the activation of the apoptotic pathway. Time-course analysis of DJ-1 stimulation shows that while DJ-1 level increases without significant lag in TRAIL-treated cells, there is a delay in H(2)O(2)-treated cells, and that the increase in DJ-1 abundance precedes the activation of apoptosis. Unexpectedly, over-expression of DJ-1 de-sensitizes BPH-1 cells to the action of apoptotic-inducing agents. However, RNA-interference-mediated silencing of DJ-1 expression results in sensitization of PC-3 cells to TRAIL action. These results are consistent with a model in which DJ-1 is involved in the control of cell death in prostate cell lines. DJ-1 appears to play a differential role between cells expressing a low but inducible level of DJ-1 (e.g., BPH-1 cells) and those expressing a high but constitutive level of the polypeptide (e.g., PC-3 cells).

Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia.

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP(L) but not c-FLIP(S). Both caspase-8 and c-FLIP(L) were cleaved to form two stable intermediates of approximately 43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP(L) to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.