Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression

Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. In this study we analyzed the expression and function of TRAIL and its agonistic and antagonistic receptors as well as expression of cellular FLICE-like inhibitory protein and caspase-2, -3, -8, -9, and -10 in 18 NB cell lines. Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cellular FLICE-like inhibitory protein. Surprisingly, caspase-8 and caspase-10 mRNA expression was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. Treatment with 5-aza-2'-deoxycytidine restored mRNA and protein expression of caspase-8 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. The TRAIL system seems to be functional in NB cells expressing caspase-8 and/or caspase-10. Because many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.     

Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and has recently been shown to exert tumoricidal activity in vivo in the absence of any observable toxicity. The signaling pathways triggered by TRAIL stimulation and the mechanisms involved in resistance against TRAIL-mediated apoptosis are still poorly defined. We show here that TRAIL-induced apoptosis involves late dissipation of mitochondrial membrane potential (delta psi(m)) and cytochrome c release. These events follow activation of caspase-8 and caspase-3 and induction of DNA fragmentation. In addition, caspase-8-deficient cells are resistant against TRAIL-induced apoptosis, and inhibition of caspase-8 but not caspase-9 prevents mitochondrial permeability transition and apoptosis. In contrast, various Bcl-2- or Bcl-xL-overexpressing tumor cell lines are sensitive to TRAIL-induced apoptosis; however, they show a delay in TRAIL-induced mitochondrial permeability transition compared with control transfectants. This indicates that TRAIL-induced apoptosis depends on caspase-8 activation rather than on the disruption of mitochondrial integrity. Because most chemotherapeutic drugs used in the treatment of malignancies lead to apoptosis primarily by engagement of the mitochondrial proapoptotic machinery, we tested whether drug-resistant tumor cells retain sensitivity for TRAIL-induced apoptosis. Tumor cells overexpressing Bcl-2 or Bcl-xL become resistant to apoptosis induced by the chemotherapeutic drug etoposide. However, these cells are not protected or are only marginally protected against TRAIL-induced apoptosis. Thus, TRAIL may still kill tumors that have acquired resistance to chemotherapeutic drugs by overexpression of Bcl-2 or Bcl-xL. These data will influence future treatment strategies involving TRAIL.     

Anticancer agents sensitize osteosarcoma cells to TNF-related apoptosis-inducing ligand downmodulating IAP family proteins

Although TNF-related apoptosis-inducing ligand (TRAIL) usually induces cell death in tumor cells, there are some tumor cell types that are resistant to its apoptogenic effects. Some chemotherapeutic drugs, however, can sensitize resistant cancer cells to TRAIL by either upregulating surface TRAIL death receptor expression or by modulating intracellular signalling pathways emanating from TRAIL receptors. U2OS human osteosarcoma cells express TRAIL-R2 but are resistant to TRAIL-induced apoptosis. however, the genotoxic drugs, Doxorubicin and Cisplatin, are able to sensitize U2OS cells to TRAIL, without affecting their surface expression of either death or decoy TRAIL receptors. We demonstrate that Doxorubicin and Cisplatin downmodulate X-IAP, while not affecting FLIP levels in U2OS cells. Selective downmodulation of X-IAP protein synthesis by specific small interference RNA transfection induced a sensitization of U2OS cells to TRAIL comparable to that induced by pharmacological treatment with genotoxic drugs. TRAIL-R2 downmodulation by siRNAs completely abolished the TRAIL-induced apoptosis of genotoxin-treated U2OS cells. Our findings demonstrate that Doxorubicin and Cisplatin do not sensitize U2OS osteosarcoma cells to TRAIL by surface receptor modulation but rather by the removal of the intracellular signalling inhibition generated by X-IAP, suggesting a foreseeable relevant advantage to the therapy of these tumors by the combined regimen of genotoxin-based chemotherapy and TRAIL.     

ROS and CHOP are critical for dibenzylideneacetone to sensitize tumor cells to TRAIL through induction of death receptors and downregulation of cell survival proteins

Because tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells, it is being tested in cancer patients. Unfortunately, patients develop resistance to the cytokine, therefore, agents that can sensitize cells to TRAIL are urgently needed. In this study, we investigated whether dibenzylideneacetone (DBA) can sensitize cancer cells to TRAIL and potentiates TRAIL-induced apoptosis. As indicated by accumulation of the membrane phospholipid phosphatidylserine, DNA breaks, intracellular esterase activity, and activation of caspase-8, -9, and -3, we concluded that DBA potentiated TRAIL-induced apoptosis in colon cancer cells. DBA also converted TRAIL resistant-cells to TRAIL-sensitive. When examined for the mechanism, we found that DBA decreased the expression of antiapoptotic proteins and decoy receptor-2 and increased proapoptotic proteins. DBA also induced both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 by small interfering RNA (SiRNA) reduced the sensitizing effect of DBA on TRAIL-induced apoptosis. In addition, DBA increased the expression of CHOP proteins. Knockdown of CHOP by siRNA decreased the induction of DBA-induced DR5 expression and apoptosis. Induction of receptors by DBA, however, was p53-independent, as deletion of p53 had no effect on receptor induction. We observed that DBA-induced induction of DR5 and DR4 was mediated through generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of death receptors and suppression of cell survival proteins by DBA. Overall, our results show that DBA potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of ROS and CHOP mediated pathways.     

Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells.

PURPOSE: Many melanoma cell lines and primary cultures are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In this study, we investigated the molecular mechanisms that control melanoma cell resistance and searched for chemotherapeutic drugs that could overcome the TRAIL resistance in melanoma cells. EXPERIMENTAL DESIGN: We examined 21 melanoma cell lines and 3 primary melanoma cultures for their sensitivity to TRAIL-induced apoptosis, and then tested cisplatin, chemptothecin, and etoposide for their synergistic effects on TRAIL sensitivity in resistant melanoma cells. RESULTS: Of 21 melanoma cell lines, 11 showed various degrees of sensitivity to TRAIL-induced apoptosis through caspase-8-initiated cleavage of caspase-3 and DNA fragmentation factor 45. The remaining cell lines and primary cultures were resistant to TRAIL, but cisplatin, chemptothecin, and etoposide sensitized the resistant cell lines and primary cultures to TRAIL-induced apoptosis, which also occurred through the caspase-8-initiated caspase cascade. Of the two TRAIL death receptors (DR4 and DR5), melanoma cells primarily expressed DR5 on cell surface. Cisplatin treatment had no effects on cell surface DR5 expression or intracellular expression of Fas-associated death domain and caspase-8. Instead, cisplatin treatment down-regulated intracellular expression of the short form of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory protein (c-FLIP) and inhibited phosphorylation of the long form of c-FLIP. CONCLUSIONS: The results presented here indicate that cisplatin inhibits c-FLIP protein expression and phosphorylation to restore TRAIL-induced caspase-8-initiated apoptosis in melanoma cells, thus providing a new combined therapeutic strategy for melanomas.     

Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo

Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL/Apo-2 ligand) induces apoptosis in the majority of cancer cells without appreciable effect in normal cells. Here, we report the effects of TRAIL on apoptosis in several human breast cancer cell lines, primary memory epithelial cells, and immortalized nontransformed cell lines, and we examine whether chemotherapeutic agents augment TRAIL-induced cytotoxicity in breast cancer cells in vitro and in vivo. TRAIL induced apoptosis with different sensitivities, and the majority of cancer cell lines were resistant to TRAIL. The chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, camptothecin, and Adriamycin) induced death receptors (DRs) TRAIL receptor 1/DR4 and TRAIL receptor 2/DR5, and successive treatment with TRAIL resulted in apoptosis of both TRAIL-sensitive and -resistant cells. Actinomycin D sensitized TRAIL-resistant cells through up-regulation of caspases (caspase-3, -9, and -8). TRAIL induces apoptosis in Adriamycin-resistant MCF7 cells already expressing high levels of death receptors DR4 and DR5. The pretreatment of breast cancer cells with chemotherapeutic drugs followed by TRAIL reversed their resistance by triggering caspase-3, -9, and -8 activation. The sequential treatment of nude mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity and apoptosis in xenografted tumors. Complete eradication of established tumors and survival of mice were achieved without detectable toxicity. Thus, the sequential administration of chemotherapeutic drugs followed by TRAIL may be used as a new therapeutic approach for cancer therapy.     

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

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

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.     

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.     

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.     

Cisplatin and a potent platinum(IV) complex-mediated enhancement of TRAIL-induced cancer cells killing is associated with modulation of upstream events in the extrinsic apoptotic pathway

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.     

Cytotoxic drug-induced, p53-mediated upregulation of caspase-8 in tumor cells

Apoptosis resistance is crucially involved in cancer development and progression, represents the leading cause for failure of anticancer therapy and is caused, for example, by downregulation of proapoptotic intracellular signaling molecules such as caspase-8. We found that the cytotoxic drugs methotrexate (MTX) and 5-fluorouracil (5-FU) were both able to sensitize resistant tumor cells for induction of apoptosis by p53-mediated upregulation of caspase-8. Increase in caspase-8 messenger RNA and protein expression disabled tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced proliferation and restored sensitivity toward TRAIL-induced apoptosis which was inhibited by transfection of p53 decoy oligonucleotides, p53 shRNA and caspase-8 shRNA. Upregulation of caspase-8 and sensitization toward TRAIL-induced apoptosis was found both in a broad panel of tumor cell lines with downregulated caspase-8 and in TRAIL-resistant primary tumor cells of children with acute leukemia. Taken together, we have identified caspase-8 as an important p53 target gene regulated by cytotoxic drugs. These findings highlight a new drug-induced modulation of physiological apoptosis pathways, which may be involved in successful anticancer therapy using MTX and 5-FU in leukemia and solid tumors over decades.     

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.     

Androgens regulate TRAIL-induced cell death in prostate cancer cells via multiple mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising therapeutic agent for prostate cancer because it selectively induces apoptosis in cancer cells but not in normal cells. Previous reports have suggested that androgens regulate TRAIL-induced apoptosis in prostate cancer cells. However, there are discrepancies between these reports of how androgens affect TRAIL-induced cell death. To clarify the role of androgens on TRAIL-induced apoptosis in prostate cancer cells, we investigated the effects of androgen on TRAIL-induced cell death in a dose–response manner. Our results showed that although androgens sensitize LNCaP cells to TRAIL-induced apoptosis, this effect is dose-dependent and biphasic. We found that low levels of androgen are superior to high levels of androgen in term of sensitizing LNCaP cells to TRAIL. We also found that upregulation of DR5 (TRAIL-R2) expression by androgens is critical for sensitizing LNCaP cells to TRAIL. However, low levels of androgen are sufficient to induce DR5 expression and sensitize LNCaP cells to TRAIL-induced cell death. High levels of androgen alter the TRADD/RIP1 ratio, which may contribute to NF-κB activation and sequentially inhibit TRAIL-induced apoptosis.     

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.     

The topoisomerase I inhibitor topotecan increases the sensitivity of prostate tumor cells to TRAIL/Apo-2L-induced apoptosis

PURPOSE.:TRAIL/Apo-2L is cytotoxic against numerous prostate tumor cell lines; however, some lines are more resistant than others. Identification of an agent that increases prostate tumor cell sensitivity to TRAIL/Apo-2L would prove valuable for TRAIL/Apo-2L-mediated tumor therapy. Thus, we examined the effect of combining five clinically approved chemotherapeutic agents with TRAIL/Apo-2L for treating prostate tumor cells. METHODS: Four human prostate tumor cell lines were initially tested for TRAIL/Apo-2L sensitivity. Subsequent studies examined whether the TRAIL/Apo-2L-induced killing of DU-145 cells was augmented in the presence of the chemotherapeutic molecules, as measured by annexin V-FITC/propidium iodide staining. Furthermore, caspase 8 activation and BID cleavage were examined by immunoblotting. RT-PCR and flow cytometry were performed to monitor TRAIL-R1 and TRAIL-R2 levels after chemotherapeutic treatment. RESULTS: DU-145 cells were the least responsive of the prostate tumor cell lines tested to TRAIL/Apo-2L-induced death. Surprisingly, only topotecan, a topoisomerase I inhibitor, when used in combination with rTRAIL/Apo-2L led to significant apoptosis of DU-145 cells, as measured by caspase 8 activation, BID cleavage, and annexin V-FITC/PI staining. Topotecan alone had little to no toxicity on the DU-145 cells. Furthermore, the increase in TRAIL/Apo-2L sensitivity following topotecan treatment correlated with increased expression of TRAIL-R1 and TRAIL-R2 and decreased intracellular levels of the antiapoptotic protein survivin. CONCLUSIONS: Our results define a promising direction for alternative therapies against androgen-independent prostate cancers. The sensitivity of DU-145 cells to TRAIL/Apo-2L was dramatically increased when combined with topotecan, suggesting that low-dose topotecan treatment to upregulate TRAIL-R1 and TRAIL-R2 and downregulate survivin, followed by TRAIL/Apo-2L administration, may be a viable therapy for treating cancer of the prostate.     

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

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

Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer that is aggressively malignant or can undergo spontaneous regression that may involve apoptosis. NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Noninvasive S-type cell lines (NB cell lines of substrate adherent phenotype) are highly sensitive to TRAIL, whereas invasive N-type cell lines (NB cell lines of neuronal phenotype) are resistant. Whereas both S- and N-type cell lines express TRAIL-R2, FADD, and caspase-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a malignant stage IV NB tumor when compared with a benign ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage NB tumors. Caspase-8 expression can be induced by demethylation with 5-aza-2'deoxycytidine, which enhances sensitivity to TRAIL. Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.     

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.