Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer

TRAIL/Apo-2L is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis in cancer cells, but not in normal cells. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without side effects. Akt promotes cell survival and block apoptosis. Some prostate cancer cells express high levels of Akt due to lack of active lipid phosphatase PTEN, a negative regulator of PI-3 kinase pathway, which may be responsible for drug resistance. The objective of this paper is to investigate the intracellular molecules that regulate TRAIL resistance. We have examined caspase-8 activity, BID cleavage, Akt activity, mitochondrial membrane potential (DeltaPsi(m)) and apoptosis in prostate cancer (LNCap, PC-3, PC-3M and DU145) cells treated with or without TRAIL. PC-3, PC-3M and DU145 cells are sensitive to TRAIL, whereas LNCap cells are resistant. LNCap cells express the highest level of constitutively active Akt, which is directly correlated with TRAIL resistance. TRAIL activates caspase-8 in all the cell lines. Downregulation of constitutively active Akt by PI-3 kinase inhibitors (wortmannin and LY-294002), dominant negative Akt or PTEN, renders LNCap cells sensitive to TRAIL. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage. Inhibition of protein synthesis by cycloheximide also causes LNCap cells sensitive to TRAIL. Overexpression of Bcl-2 or Bcl-X(L) inhibits TRAIL-induced DeltaPsi(m) and apoptosis. Overexpression of constitutively active Akt in PC-3M cells (express very low levels of constitutively active Akt) restores TRAIL resistance. These data suggest that elevated Akt activity protects LNCap cells from TRAIL-induced apoptosis, and the PI-3 kinase/Akt pathway may inhibit apoptotic signals by inhibiting processing of BID. Thus, constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer.     

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

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

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.     

CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release.

Death ligands such as CD95 ligand (CD95L) or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) induce apoptosis in radiochemotherapy-resistant human malignant glioma cell lines. The death-signaling TRAIL receptors 2 (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/DR4 were expressed more abundantly than the non-death-inducing (decoy) receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the absence of a protein synthesis inhibitor, cycloheximide (CHX). Three of the 12 cell lines were still TRAIL/Apo2L-resistant in the presence of CHX. TRAIL-R2 expression predicted sensitivity to apoptosis. Coexposure to TRAIL/Apo2L and cytotoxic drugs such as topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, CCNU) or temozolomide resulted in synergistic killing. Synergistic killing was more often observed in cell lines retaining wild-type p53 activity (U87MG, LN-229) than in p53 mutant cell lines (LN-18, T98G, U373MG). Drug exposure resulted in enhanced TRAIL-R2 expression, but decreased TRAIL-R4 expression in U87MG cells. Ectopic expression of dominant-negative p53(V135A) abrogated the drug-induced changes in TRAIL-R2 and TRAIL-R4 expression, but had no effect on synergy. Thus, neither wild-type p53 function nor changes in TRAIL receptor expression were required for synergy. In contrast, synergy resulted possibly from drug-induced cytochrome c release from mitochondria, serving as an amplifier of the TRAIL/Apo2L-mediated cascade of caspase activation. These data provide novel insights into the role of the TRAIL/Apo2L system in malignant gliomas and illustrate that TRAIL/Apo2L-based immunochemotherapy may be an effective therapeutic strategy for these lethal neoplasms.     

Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.     

Genistein synergizes with RNA interference inhibiting survivin for inducing DU-145 of prostate cancer cells to apoptosis

To further investigate the effect of a combination of genistein with survivin of RNA interference on the proliferation and apoptosis of DU-145 cells, the effect of genistein on the proliferation of DU-145 cells was detected by the MTT method and cytometry, and the apoptosis of cells was observed with fluorescence microscopy. In order to test combined genistein with transfection of small interfering RNA (siRNA) against survivin, a survivin siRNA plasmid was constructed and transfected into DU-145 cells. Genistein inhibited proliferation and induced apoptosis of cancerous DU-145 and Hela cells, whereas genistein had minimal effects for normal L-O2 cells. The stable transfected cell lines of DU-145, knockdown survivin by siRNA, displayed stronger apoptotic than untransfected DU-145, the transfected cell of DU-145 treated with genistein demonstrated the inhibition of proliferation and induction of apoptosis significantly; it showed genistein synergistic effect with RNAi in survivin for inhibition of prostate cancer cells.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of topotecan and ionizing radiation on TRAIL/Apo2L-mediated apoptosis in malignant glioma

The survival of patients with malignant gliomas is still unsatisfactory despite multimodality treatment, therefore new therapeutic strategies are required. Tumor necrosis factor apoptosis related ligand (TRAIL/Apo2L), a member of the tumor necrosis factor superfamily, may induce apoptotic cell death in several tumors, but not in normal cells, upon binding with specific receptors. In the present study, the expression and function of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) has been investigated in five human glioma cell lines (U87, U138, U373, A172, SW1783) in ex vivo tumors and in primary cultures obtained from the tumors. Our data show that gliomas preferentially express TRAIL R2 and that treatment with topotecan, a topoisomerase I inhibitor, significantly up-regulates its expression as detected by flow cytometry and western blotting. Moreover, in most cases, treatment with topotecan resulted in an increased sensitivity to TRAIL-dependent apoptosis, although cyclohexymide had to be added to induce apoptosis. On glioma cell lines, the effects of irradiation on TRAIL receptors were also analysed. In our experimental conditions, irradiation with 2Gy had a modest additive effect on TRAIL-dependent apoptosis and was not able to modulate TRAIL receptor expression.     

Adriamycin sensitizes the adriamycin-resistant 8226/Dox40 human multiple myeloma cells to Apo2L/tumor necrosis factor-related apoptosis-inducing ligand-mediated (TRAIL) apoptosis.

The newly discovered member of the tumor necrosis factor superfamily, Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), has been identified as an apoptosis-inducing agent in sensitive tumor cells but not in the majority of normal cells, and hence it is of potential therapeutic application. However, many tumor cells are resistant to Apo2L/TRAIL-mediated apoptosis. Various chemotherapeutic drugs have been shown to sensitize tumor cells to members of the tumor necrosis factor family. However, it is not clear whether sensitization by drugs and sensitivity to drugs are related or distinct events. This study examined whether an Adriamycin-resistant multiple myeloma (MM) cell line (8226/Dox40) can be sensitized by Adriamycin (ADR) to Apo2L/TRAIL-mediated apoptosis. Treatment with the combination of Apo2L/TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental 8226/S and the 8226/Dox40 tumor cells. Adriamycin treatment modestly up-regulated Apo2L/TRAIL-R2 (DR5) and had no effect on the expression of Fas-associated death domain, c-FLIP, Bcl-2, Bcl(xL), Bax, and IAP family members (cIAP-1, cIAP-2, XIAP, and survivin). The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR, whereas pro-caspase-9 and Apaf-1 were up-regulated. Combination treatment with Apo2L/TRAIL and ADR resulted in significant mitochondrial membrane depolarization and activation of caspase-9 and caspase-3 and apoptosis. Because ADR is shown to sensitize ADR-resistant tumor cells to Apo2L/TRAIL, these findings reveal that ADR can still signal ADR-resistant tumor cells, resulting in the modification of the Apo2L/TRAIL-mediated signaling pathway and apoptosis. These in vitro findings suggest the potential application of combination therapy of Apo2L/TRAIL and subtoxic concentrations of sensitizing chemotherapeutic drugs in the clinical treatment of drug-resistant/Apo2L/TRAIL-resistant multiple myeloma.     

Sensitization of tumor cells to Apo2 ligand/TRAIL-induced apoptosis by inhibition of casein kinase II

Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.     

The dietary flavonol fisetin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous agent that induces apoptosis selectively in cancer cells. Soluble or expressed in immune cells, TRAIL plays an important role in the defense against tumour cells. The resistance of cancer cells to TRAIL immune surveillance is implicated in tumour development. Naturally occurring flavonoids can sensitize TRAIL-resistant cancer cells and augment their apoptotic activity. Fisetin, a dietary flavonol has cancer preventive properties. This study was designed to investigate the effect of fisetin on the TRAIL-induced apoptosis potential in prostate cancer cells. Prostate cancer cell lines represent an ideal model for research in chemoprevention. Cytotoxicity was measured by MTT and LDH assays. Apoptosis was detected using Αnnexin?V-FITC by flow cytometry and fluorescence microscopy. Mito-chondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence microscopy. Death receptor (TRAIL-R1 and TRAIL-R2) expression was analysed by flow cytometry. Inhibition of NF-κB (p65) activation was confirmed with an ELISA-based TransAM NF-κB kit. Caspase-8 and caspase-3 activities were determined by colorimetric protease assays. Our study demonstrates that fisetin sensitizes the TRAIL-resistant androgen-dependent LNCaP and the androgen-independent DU145 and PC3 prostate cancer cells to TRAIL-induced death. Fisetin augmented TRAIL-mediated cytotoxicity and apoptosis in prostate cancer LNCaP cells by engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways. Fisetin increased the expression of TRAIL-R1 and decreased the activity of NF-κB. Co-treatment of cancer cells with TRAIL and fisetin caused significant activation of caspase-8 and caspase-3 and disruption of ΔΨm. Our data indicate the usefulness of fisetin in prostate cancer chemoprevention through enhancement of TRAIL-mediated apoptosis.     

Cotreatment with histone deacetylase inhibitor LAQ824 enhances Apo-2L/tumor necrosis factor-related apoptosis inducing ligand-induced death inducing signaling complex activity and apoptosis of human acute leukemia cells

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21(WAF1) in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G(1) phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-x(L), XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.     

Enhancement of Ad5-TRAIL cytotoxicity against renal cell carcinoma with histone deacetylase inhibitors

Renal cell carcinoma (RCC) will cause greater than 12,000 deaths in the United States this year. The lack of effective therapy for disseminated RCC has stimulated the search for novel treatments including immunotherapeutic strategies, but poor therapeutic responses and marked toxicity have limited their use. The tumor necrosis factor (TNF) family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L induces apoptosis in various tumor cell types, while having little cytotoxicity against normal cells. In this study, we investigated the tumoricidal potential of a recombinant adenovirus encoding human TNFSF10 (Ad5-TRAIL), alone and in combination with a panel of histone deacetylase inhibitors (HDACi), against the TRAIL/Apo-2L-resistant RCC line 786-O and normal human renal proximal tubule epithelial cells (RPTEC). Ad5-TRAIL was unable to induce apoptosis in either 786-O or RPTEC alone; however, tumor cell apoptosis occurred when Ad5-TRAIL was combined with HDAC inhibition. Except when combined with trichostatin A, RPTEC were not sensitized to Ad5-TRAIL by HDACi. In 786-O, HDAC inhibition induced CAR expression, permitting increased adenoviral infection and transgene expression. It also induced TRAIL-R2 expression, accelerated the death-inducing signaling complex formation and enhanced caspase-8 activation. Our results demonstrate the utility of combining Ad5-TRAIL with HDACi against RCC, and mechanistically define how this combination modulates RCC sensitivity to TRAIL/Apo-2L and adenoviral infection.     

Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert potent cytotoxic activity against many tumor cell lines but not against normal cells. It has been hypothesized that this difference in TRAIL sensitivity between normal and transformed cells might be due to the expression of the non-death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and TRAIL-R4, presumably by competition for limited amounts of TRAIL. To assess the regulation of resistance versus sensitivity to TRAIL in primary as well as transformed keratinocytes, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. Although TRAIL induced apoptosis in primary as well as transformed keratinocytes, a marked difference in sensitivity could be observed with primary keratinocytes (PK) being 5-fold less sensitive to TRAIL than transformed keratinocytes (TK). Yet both cell types exhibited similar TRAIL receptor surface expression, suggesting that expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of sensitivity to TRAIL. Biochemical analysis of the signaling events induced by TRAIL revealed that PK could be sensitized for TRAIL and, similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide (CHX). This sensitization concomitantly resulted in processing of caspase-8, which did not occur in TRAIL-resistant PK. These data indicate that an early block of TRAIL-induced apoptosis was present in PK compared with TK or PK treated with CHX. Interestingly, cellular FLICE inhibitory protein (cFLIP) levels, high in PK and low in TK and several other squamous cell carcinoma cell lines, decreased rapidly after treatment of PK with CHX, correlating with the increase in TRAIL sensitivity and caspase-8 processing. Furthermore, ectopic expression of cFLIP long (cFLIP(L)) in TK by transfection with a cFLIP(L) expression vector resulted in resistance to TRAIL-mediated apoptosis of these cells. Thus, our results demonstrate that TRAIL sensitivity in PK is primarily regulated at the intracellular level rather than at the receptor level.     

In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs

TRAIL/Apo-2L has shown promise as an anti-glioma drug, based on investigations of TRAIL sensitivity in established glioma cell lines, but it is not known how accurately TRAIL signalling pathways of glioma cells in vivo are reproduced in these cell lines in vitro. To replicate as closely as possible the in vivo behaviour of malignant glioma cells, 17 early passage glioma cell lines and 5 freshly resected gliomas were exposed to TRAIL-based agents and/or chemotherapeutic drugs. Normal human hepatocytes and astrocytes and established glioma cell lines were also tested. Cross-linked TRAIL, but not soluble TRAIL, killed both normal cell types and cells from three tumours. Cells from only one glioma were killed by soluble TRAIL, although only inefficiently. High concentrations of cisplatin were lethal to glioma cells, hepatocytes and astrocytes. Isolated combinations of TRAIL and chemotherapy drugs were more toxic to particular gliomas than normal cells, but no combination was generally selective for glioma cells. This study highlights the widespread resistance of glioma cells to TRAIL-based agents, but suggests that a minority of high-grade glioma patients may benefit from particular combinations of TRAIL and chemotherapy drugs. In vitro sensitivity assays may help identify effective drug combinations for individual glioma patients.     

Resistance of prostate cancer cells to soluble TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) can be overcome by doxorubicin or adenoviral delivery of full-length TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to induce apoptosis in malignant cells without harming normal cells. To determine the antitumor potential of TRAIL against prostate cells, we undertook a comprehensive study that included eight prostate cancer cells lines (CWR22Rv1, Du145, DuPro, JCA-1, LNCaP, PC-3, PPC-1, and TsuPr1) and primary cultures of normal prostate epithelial cells (PrEC). Cells were tested for susceptibility to soluble TRAIL in the presence or absence of the chemotherapeutic agent doxorubicin. TRAIL was also delivered by an adenoviral vector. Our results reveal that Du145, DuPro, LNCap, TsuPr1, and PrEC were resistant to 100 ng/mL TRAIL. JCA-1 and PPC-1 were slightly sensitive (20% killing) and PC-3 and CWR22Rv1 exhibited the highest sensitivity to TRAIL (30% and 50% killing, respectively). The combination of 10 ng/mL TRAIL with doxorubicin resulted in 60-80% cytotoxicity in seven of eight prostate cancer cells. TRAIL-mediated apoptosis involved cleavage of Bid, caspase-3, and PARP, and required caspase-8 and -9 activity. Full-length TRAIL delivered by an adenoviral vector (AdTRAIL-IRES-GFP) killed prostate cancer cell lines and PrEC without requisite doxorubicin cotreatment. Therefore, expression of the transgene from a tissue-specific promotor would make gene therapy with AdTRAIL-IRES-GFP a possibility.     

Sensitization of human glioblastomas to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by NF-kappaB inhibitors

Glioblastoma is the most malignant form of primary brain tumor in adults, with no effective therapy and a low survival rate. TRAIL is a member of the TNF family, which selectively induces apoptosis in certain neoplastic cells, but not normal cells. In this study, we investigated the sensitivity of 7 human glioblastoma cell lines to TRAIL and the expression in them of TRAIL receptors. TRAIL exhibited significant cytotoxicity in 5 of 7 glioma cell lines. These glioblastoma cell lines expressed TRAIL-R2, but not TRAIL-R1, R3, or R4. However, no correlation was observed between the TRAIL sensitivity and the TRAIL-R2 expression level, suggesting that there is an additional determinant of TRAIL sensitivity. Treatments with NF-kappaB inhibitors, such as LLnL, MG132, and SN50, significantly increased the sensitivity of glioma cells to TRAIL. These results suggested that activation of NF-kappaB is a protective mechanism against TRAIL-induced cell death in some glioma cells, and thus NF-kappaB inhibitors may be useful to improve the clinical treatment of glioblastoma with TRAIL.     

Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma

The lack of effective therapy for disseminated renal cell carcinoma (RCC) has stimulated the search for novel treatments including immunotherapeutic strategies. However, poor therapeutic responses and marked toxicity associated with immunological agents has limited their use. The tumor necrosis factor family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand induces apoptosis in a variety of tumor cell types, while having little cytotoxic activity against normal cells. In this study the activation and regulation of TRAIL-induced apoptosis and TRAIL receptor expression in human RCC cell lines and pathologic specimens was examined. TRAIL induced caspase-mediated apoptotic death of RCC cells with variable sensitivities among the cell lines tested. Compared with TRAIL-sensitive RCC cell lines (A-498, ACHN, and 769-P), the TRAIL-resistant RCC cell line (786-O) expressed lesser amounts of the death-inducing TRAIL receptors, and greater amounts of survivin, an inhibitor of apoptosis. Incubation of 786-O with actinomycin D increased the expression of the death-inducing TRAIL receptors and, concomitantly, decreased the intracellular levels of survivin, resulting in TRAIL-induced apoptotic death. The link between survivin and TRAIL regulation was confirmed when an increase in TRAIL resistance was observed after overexpression of survivin in the TRAIL-sensitive, survivin-negative RCC line A-498. These findings, along with our observation that TRAIL receptors are expressed in RCC tumor tissue, suggest that TRAIL may be useful as a therapeutic agent for RCC and that survivin may partially regulate TRAIL-induced cell death.     

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

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

Inducible resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand receptor 2-mediated apoptosis by generation of a blockade at the death domain function

Induction of tumor cell resistance to therapeutics has been a major obstacle in cancer therapy. Targeting of the death receptors by a natural ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or agonistic monoclonal antibodies against TRAIL receptor 1 (TRAIL-R1) or TRAIL receptor 2 (TRAIL-R2) has been thought to be a promising cancer therapy. To determine whether tumor cells are able to generate a resistance to apoptosis induced by an anti-TRAIL-R2 antibody, TRA-8, we examined the apoptotic response of human breast and ovarian cancer cell lines after treatment with TRA-8. Our results show that tumor cell resistance to TRA-8 can be induced by repeated treatment of tumor cells with low, non-apoptosis-inducing doses of TRA-8. Interestingly, the induced resistance to apoptosis was not due to a global apoptotic defect in tumor cells but rather a selective defect in the TRAIL-R2 signaling pathway. Whereas TRA-8-treated tumor cells developed a selective resistance to TRAIL-R2-mediated apoptosis, the apoptotic responses induced by TRAIL, an anti-TRAIL-R1 antibody (2E12), and other apoptotic stimuli were not impaired. The expression levels of cell surface TRAIL-R2 were not altered and mutations of TRAIL-R2 were not found in the resistant cells. The induced TRA-8 resistance was due to a selective blockade at the level of the death domain and could be reversed by a wide array of chemotherapeutic agents. Proteomic analysis of death-inducing signaling complex formation during TRA-8 treatment shows that the translocation of TRAIL-R2-associated apoptotic proteins was significantly altered. Our results suggest that the prevention of tumor cell resistance to therapeutic agents that target the death receptors must be taken into consideration.