Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Inhibition of RIP and c-FLIP enhances TRAIL-induced apoptosis in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it is capable of preferentially inducing apoptosis in human cancer over normal cells. The majority of human pancreatic cancers, unfortunately, are resistant to TRAIL treatment. Here, we show that the inhibition of caspase-8 cleavage is the most upstream event in TRAIL resistance in pancreatic cancers. TRAIL treatment led to the cleavage of caspase-8 and downstream caspase-9, caspase-3, and DNA fragmentation factor 45 (DFF45) in TRAIL-sensitive pancreatic cancer cell lines (BXPC-3, PACA-2). This caspase-8-initiated caspase cascade, however, was inhibited in TRAIL-resistant pancreatic cancer cell lines (PANC-1, ASPC-1, CAPAN-1, CAPAN-2). The long and short forms of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP(L), c-FLIP(S)) were highly expressed in the TRAIL-resistant as compared to the sensitive cells; knockdown of c-FLIP(L) and c-FLIP(S) by a short hairpin RNA (shRNA) rendered the resistant cells sensitive to TRAIL-induced apoptosis through the cleavage of caspase-8 and activation of the mitochondrial pathway. Receptor-interacting protein (RIP) has been reported in TRAIL-induced activation of NF-kappaB and we show here that knockdown of RIP sensitized the resistant cells to TRAIL-induced apoptosis. These results indicate the role of c-FLIP and RIP in caspase-8 inhibition and thus TRAIL resistance. Treatment of the resistant cells with camptothecin, celecoxib and cisplatin resulted in the downregulation of c-FLIP and caused a synergistic apoptotic effect with TRAIL. These studies therefore suggest that combination treatment with chemotherapy can overcome TRAIL resistance and enhance TRAIL therapeutic efficacy in treating pancreatic cancers.     

Progesterone induces apoptosis in TRAIL-resistant ovarian cancer cells by circumventing c-FLIPL overexpression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds great potential as an anticancer drug, since it induces selective cell death in cancer cells but not in normal ones. However, cancer cells often acquire resistance to TRAIL, which hinders its clinical efficacy. We previously demonstrated that progesterone triggers apoptosis in human ovarian cancer (OCa) cells. In the present study, we evaluated the prospect of utilizing progestins in combination with TRAIL to enhance cell death in TRAIL-sensitive (OVCA 420, OVCA 429, and OVCA 433) and -resistant (OVCA 432) OCa cell lines. TRAIL sensitivity (60-80% cell kill) bore no correlation with expression of the TRAIL receptors (DR4, DR5) or their decoys (DcR1 and DcR2), but was associated with activation of caspase-8 and -3, and downregulation of the long isoform of FLICE-like inhibitory protein (c-FLIP(L)), an anti-apoptosis mediator. Small interfering RNA-mediated knockdown of c-FLIP(L) expression restored TRAIL sensitivity in OVCA 432 cells. Induction of c-FLIP(L) overexpression increased TRAIL resistance in TRAIL-sensitive lines. Thus, persistent high level of c-FLIP(L) expression likely mediates TRAIL resistance in OCa cells. Treatment of OCa cells with progesterone enhanced TRAIL-induced cell death (>85%), but only in TRAIL-sensitive cell lines. Combined treatment with two progestins was superior to single progestin treatment, with progesterone plus medroxyprogesterone acetate (MPA) achieving over 85% cell kill in both TRAIL-sensitive and -resistant OCa cell lines. Significantly, unlike TRAIL, progestin-induced cell death did not involve c-FLIP(L) downregulation. Hence, combined progestin regimens, with or without TRAIL, may serve as an effective therapy for OCa by circumventing the anti-apoptotic action of c-FLIP(L).     

Identification of inhibitors of TRAIL-induced death (ITIDs) in the TRAIL-sensitive colon carcinoma cell line SW480 using a genetic approach

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cell lines, whereas normal cells appear to be protected from its cytotoxic effects. Therefore TRAIL holds promise as a potential therapeutic agent against cancer. To elucidate some of the critical factors that contribute to TRAIL resistance, we performed a genetic screen in the human colon carcinoma cell line SW480 by infecting this TRAIL-sensitive cell line with a human placental cDNA retroviral library and isolating TRAIL-resistant clones. Characterization of the resulting clones for inhibitors of TRAIL-induced death (ITIDs) led to the isolation of c-FLIP(S), Bax inhibitor 1, and Bcl-XL as candidate suppressors of TRAIL signaling. We have demonstrated that c-FLIP(S) and Bcl-XL are sufficient when overexpressed to convey resistance to TRAIL treatment in previously sensitive cell lines. Furthermore both c-FLIP(S) and Bcl-XL protected against overexpression of the TRAIL receptors DR4 and KILLER/DR5. When c-FLIP(S) and Bcl-XL were overexpressed together in SW480 and HCT 116, an additive inhibitory effect was observed after TRAIL treatment suggesting that these two molecules function in the same pathway in the cell lines tested. Furthermore, we have demonstrated for the first time that a proapoptotic member of the Bcl-2 family, Bax, is required for TRAIL-mediated apoptosis in HCT 116 cells. Surprisingly, we have found that the serine/threonine protein kinase Akt, which is an upstream regulator of both c-FLIP(S) and Bcl-XL, is not sufficient when overexpressed to protect against TRAIL in the cell lines tested. These results suggest a key role for c-FLIP(S), Bcl-XL, and Bax in determining tumor cell sensitivity to TRAIL.     

Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients' stroma-conditioned medium, we determined that soluble factor(s) produced by stroma-myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.     

Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1

We recently demonstrated that heme oxygenase (HO)-1 is constitutively expressed in human CD4+CD25+ regulatory T cells and induced by anti-CD28 or anti-CD28/anti-CD3 stimulation, even in CD4+CD25- responder T cells. To study the effects of HO-1 expression on lymphocyte survival, we transfected the HO-1 gene or induced the gene to express HO-1 protein with cobalt protoporphyrin (CoPP) in Jurkat T cells. Consistently, anti-Fas antibody triggered apoptotic cell death in wild-type Jurkat T cells. Surprisingly, however, HO-1-overexpressing Jurkat T cells showed strong resistance to Fas-mediated apoptosis. In contrast, abrogation of HO-1 expression by antisense oligomer against HO-1 gene from CoPP-treated cells or depletion of iron by desferrioxamine from HO-1-transfected cells abolished the resistance. In addition, exogenously added iron rendered wild-type Jurkat T cells resistant. The resistance involved IkappaB kinase (IKK) activation via iron-induced reactive oxygen species formation, NF-kappaB activation by activated IKK, and c-FLIP expression by activated NF-kappaB. Primary CD4+ T cells induced by CoPP to express HO-1 also showed more resistance to Fas-mediated apoptosis than untreated cells. Our findings suggest that HO-1 plays a critical and nonredundant role in Fas-mediated activation-induced cell death of T lymphocytes.     

Modulation of tumor necrosis factor apoptosis-inducing ligand- induced NF-kappa B activation by inhibition of apical caspases.

Tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), a member of the TNF family, induces apoptosis in many transformed cells. We report TRAIL-induced NF-kappaB activation, concomitant with production of the pro-inflammatory cytokine Interleukin-8 in the relatively TRAIL-insensitive cell line, HEK293. In contrast, TRAIL-induced NF-kappaB activation occurred in HeLa cells only upon pretreatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.fmk), indicating that this was due to a caspase-sensitive component of TRAIL-induced NF-kappaB activation. NF-kappaB activation was mediated by the death receptors, TRAIL-R1 and -R2, but not by TRAIL-R3 or -R4 and was only observed in HeLa cells in the presence of z-VAD.fmk. Receptor-interacting protein, an obligatory component of TNF-alpha-induced NF-kappaB activation, was cleaved during TRAIL-induced apoptosis. We show that receptor-interacting protein is recruited to the native TRAIL death-inducing signaling complex (DISC) and that recruitment is enhanced in the presence of z-VAD.fmk, thus providing an explanation for the potentiation of TRAIL-induced NF-kappaB activation by z-VAD.fmk in TRAIL-sensitive cell lines. Examination of the TRAIL DISC in sensitive and resistant cells suggests that a high ratio of c-FLIP to caspase-8 may partially explain cellular resistance to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis was also modulated by inhibition or activation of NF-kappaB. Thus, in some contexts, modulation of NF-kappaB activation possibly at the level of apical caspase activation at the DISC may be a key determinant of sensitivity to TRAIL-induced apoptosis.     

NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling

The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptor-induced apoptosis. We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-kappaB pathway, but NF-kappaB activation was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-kappaB activation, inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IkappaBalpha mutant (IkappaBalpha-SR) or lack of IkappaB kinase gamma expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data point to an important role for NF-kappaB in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide (CHX). However, overexpression of IkappaBalpha-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX, and cFLIP expression was able to reverse the proapoptotic effect of NF-kappaB inhibition. Western blot analysis further revealed that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data suggest a key role for cFLIP in the antiapoptotic response of NF-kappaB activation.     

Erythroid differentiation sensitizes K562 leukemia cells to TRAIL-induced apoptosis by downregulation of c-FLIP

Regulation of the apoptotic threshold is of great importance in the homeostasis of both differentiating and fully developed organ systems. Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells, in which the apoptotic threshold is often characteristically elevated. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied death receptor responses during erythroid differentiation of K562 erythroleukemia cells, which normally are highly resistant to tumor necrosis factor (TNF) alpha-, FasL-, and TRAIL-induced apoptosis. However, upon hemin-mediated erythroid differentiation, K562 cells specifically lost their resistance to TNF-related apoptosis-inducing ligand (TRAIL), which efficiently killed the differentiating cells independently of mitochondrial apoptotic signaling. Concomitantly with the increased sensitivity, the expression of both c-FLIP splicing variants, c-FLIP(L) and c-FLIP(S), was downregulated, resulting in an altered caspase 8 recruitment and cleavage in the death-inducing signaling complex (DISC). Stable overexpression of both c-FLIP(L) and c-FLIP(S) rescued the cells from TRAIL-mediated apoptosis with isoform-specific effects on DISC-recruited caspase 8. Our results show that c-FLIP(L) and c-FLIP(S) potently control TRAIL responses, both by distinct regulatory features, and further imply that the differentiation state of malignant cells determines their sensitivity to death receptor signals.     

Transrepression of NF-kappaB is not required for glucocorticoid-mediated protection of TNF-alpha-induced apoptosis on fibroblasts

The cellular resistance to tumor necrosis factor (TNF) of most cell types has been attributed to both a protective pathway induced by this cytokine and the preexistence of protective factors in the target cell. NF-kappaB has been postulated as one of the principal factors involved in antiapoptotic gene expression control on TNF-resistant cells. We have previously shown that glucocorticoids protect the naturally TNF-sensitive L-929 cells from apoptosis. Here we analyze the role of NF-kappaB and glucocorticoids on TNF-induced apoptosis in L-929 cells. We found that inhibition of NF-kappaB enhanced the sensitivity to TNF-induced apoptosis. Glucocorticoids inhibited NF-kappaB transactivation via IkappaB induction. Moreover, glucocorticoids protected from TNF-induced apoptosis even when NF-kappaB activity was inhibited by stable or transient expression of the superrepressor IkappaB. These results demonstrate that although glucocorticoids inhibit NF-kappaB transactivation in these cells, this is not required for their protection from TNF-induced apoptosis.     

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.     

NF-kappaB signaling pathway governs TRAIL gene expression and human T-cell leukemia virus-I Tax-induced T-cell death.

The Tax oncoprotein encoded by human T-cell leukemia virus induces both T-cell activation and apoptosis. The mechanism by which Tax induces apoptosis has remained unclear. Using genetically manipulated T-cell lines, we demonstrate that Tax-induced T-cell death is dependent on NF-kappaB signaling. Tax fails to induce apoptosis in T cells lacking IkappaB kinase gamma (IKKgamma), an essential component of the NF-kappaB signaling pathway. This defect was rescued when the mutant cells were reconstituted with exogenous IKKgamma. We further demonstrate that the Tax-induced T-cell death is mediated by TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), because this event can be effectively inhibited by a TRAIL-blocking antibody. Consistent with this functional aspect, Tax stimulates the expression of TRAIL mRNA. Finally, we provide genetic evidence demonstrating that the NF-kappaB signaling pathway is essential for TRAIL gene induction by both Tax and T-cell activation signals. These studies reveal a novel function of the NF-kappaB signaling pathway and suggest a key mechanism by which Tax induces T-cell death.     

Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL

Chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. The death receptor ligand TRAIL also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. We show here that the treatment of four different epithelial cell lines with the topoisomerase II inhibitor etoposide in combination with TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. The mechanism of the synergistic effect results from the etoposide-mediated increase in the expression of the death receptors 4 (DR4) and 5 (DR5). Inhibition of NF-kappaB activation by expression of kinase-inactive MEK kinase 1(MEKK1) or dominant-negative IkappaB (DeltaIkappaB) blocked the increase in DR4 and DR5 expression following etoposide treatment. Addition of a soluble decoy DR4 fusion protein (DR4:Fc) to cell cultures reduced the amount of etoposide-induced apoptosis in a dose-dependent manner. The addition of a soluble TNF decoy receptor (TNFR:Fc) was without effect, demonstrating the specificity of DR4 binding ligands in the etoposide-induced apoptosis response. Thus, genotoxin treatment in combination with TRAIL is an effective inducer of epithelial-cell-derived tumor cell apoptosis relative to either treatment alone.     

Hypermethylation of the death-associated protein kinase promoter attenuates the sensitivity to TRAIL-induced apoptosis in human non-small cell lung cancer cells

Death-associated protein (DAP) kinase plays an important role in IFN-gamma, tumor necrosis factor (TNF)-alpha, or Fas-ligand induced apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family and can induce caspase-dependent apoptosis in cancer cells while sparing most of the normal cells. However, some of the cancer cell lines are insensitive to TRAIL, and such resistance cannot be explained by the dysfunction of TRAIL receptors or their known downstream targets. We reported previously that DAP kinase promoter is frequently methylated in non-small cell lung cancer (NSCLC), and such methylation is associated with a poor clinical outcome. To determine whether DAP kinase promoter methylation contributes to TRAIL resistance in NSCLC cells, we measured DAP kinase promoter methylation and its gene expression status in 11 NSCLC cell lines and correlated the methylation/expression status with the sensitivity of cells to TRAIL. Of the 11 cell lines, 1 had a completely methylated DAP kinase promoter and no detectable DAP kinase expression, 4 exhibited partial promoter methylation and substantially decreased gene expression, and the other 6 cell lines showed no methylation in the promoter and normal DAP kinase expression. Therefore, the amount of DAP kinase expression amount was negatively correlated to its promoter methylation (r = -0.77; P = 0.003). Interestingly, the cell lines without the DAP kinase promoter methylation underwent substantial apoptosis even in the low doses of TRAIL, whereas those with DAP kinase promoter methylation were resistant to the treatment. The resistance to TRAIL was reciprocally correlated to DAP kinase expression in 10 of the 11 cell lines at 10 ng/mL concentration (r = 0.91; P = 0.001). We treated cells resistant to TRAIL with 5-aza-2'-deoxycytidine, a demethylating reagent, and found that these cells expressed DAP kinase and became sensitive to TRAIL. These results suggest that DAP kinase is involved in TRAIL-mediated cell apoptosis and that a demethylating agent may have a role in enhancing TRAIL-mediated apoptosis in some NSCLC cells by reactivation of DAP kinase.     

Mechanisms of resistance of human glioma cells to Apo2 ligand/TNF-related apoptosis-inducing ligand.

BACKGROUND: Many tumor cells are resistant to Apo2L/TRAIL-induced apoptosis in the absence of inhibitors of protein synthesis. Apo2L/TRAIL, in addition to induction of apoptosis, may therefore also activate survival pathways. METHODS: Here we investigated whether such survival pathways mediate resistance to Apo2L.0-induced apoptosis in human glioma cells. RESULTS: Apo2L.0 induced the phosphorylation of ERK1/2, but not of Akt. This effect was unaffected by caspase inhibition. Inhibitors of protein synthesis, PI3 kinase, ERK kinase, NF-kappaB or casein kinase 2 sensitized for Apo2L.0-induced apoptosis to a different extent in a panel of human malignant glioma cell lines. However, none of the sensitizers overcame resistance mediated by ectopic expression of the viral caspase 8 inhibitor, crm-A. Primary glioma cultures were almost completely resistant to Apo2L.0-induced cell death even in the presence of the inhibitors. Caspase-8 was expressed in these cells whereas only weak expression of DR5 was detected. Transient expression of DR5 conferred sensitivity to Apo2L.0. CONCLUSION: These data challenge the view that specific cell lines harbour specific mechanisms of resistance to Apo2L/TRAIL. Weak expression of DR5 in primary glioma might limit the therapeutic application of Apo2L/TRAIL in human glioblastoma patients.