Low-dose 12-O-tetradecanoylphorbol-13-acetate enhances tumor necrosis factor related apoptosis-inducing ligand induced apoptosis in prostate cancer cells.

PURPOSE: Previously, we have shown that c-Fos/activator protein-1 (AP-1) promotes tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by repressing the antiapoptotic molecule c-FLIP(L). In this study, we investigated whether synthetic induction of c-Fos/AP-1 by 12-O-tetradecanoylphorbol-13-acetate (TPA) converts the phenotype of TRAIL-resistant prostate cancer cells to a TRAIL-sensitive phenotype in vitro and in vivo. EXPERIMENTAL DESIGN: Low-dose TPA was used to determine whether LNCaP prostate cancer cells could be converted to a TRAIL-sensitive phenotype in in vitro and in vivo studies. We also assessed whether TPA enhancement of TRAIL-induced apoptosis varies between androgen-sensitive and androgen-insensitive prostate cancer cells and evaluated the role of TRAIL receptors, DR4 and DR5, in TPA-enhanced TRAIL-induced apoptosis. RESULTS: We show that the combination of TRAIL with low-dose TPA has no effect on nonmalignant prostate epithelial cells; however, TPA up-regulates most AP-1 proteins and AP-1 activity, reduces c-FLIP(L), and potentiates TRAIL-induced apoptosis. We show that the combination of TPA + TRAIL is effective in promoting apoptosis in both hormone-sensitive LNCaP and hormone-insensitive LNCaP-C4-2 prostate cancer cells. Although TPA enhances the TRAIL-receptor 1 (DR4) level, sensitization of prostate cancer cells seems to be more dependent on TRAIL-receptor 2 (DR5) than TRAIL-receptor 1 levels. In vivo xenograft experiments suggest that TPA elevates the expression of c-Fos and reduces c-FLIP(L). Combination of TPA with TRAIL-receptor 2 agonist antibody, lexatumumab, effectively increases apoptosis and reduces LNCaP xenograft tumor burden. CONCLUSIONS: TPA, when combined with the proapoptotic agent TRAIL, is effective in changing the phenotype of some TRAIL-resistant prostate cancer cells to a TRAIL-sensitive phenotype.     

Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in a variety of tumorigenic and transformed cell lines but not in many normal cells. Hence, TRAIL has the potential to be an ideal cancer therapeutic agent with minimal cytotoxicity. FLICE inhibitory protein (c-FLIP) is an important regulator of TRAIL-induced apoptosis. Here, we show that persistent expression of c-FLIP(Long) [c-FLIP(L)] is inversely correlated with the ability of TRAIL to induce apoptosis in prostate cancer cells. In contrast to TRAIL-sensitive cells, TRAIL-resistant LNCaP and PC3-TR (a TRAIL-resistant subpopulation of PC3) cells showed increased c-FLIP(L) mRNA levels and maintained steady protein expression of c-FLIP(L) after treatment with TRAIL. Ectopic expression of c-FLIP(L) in TRAIL-sensitive PC3 cells changed their phenotype from TRAIL sensitive to TRAIL resistant. Conversely, silencing of c-FLIP(L) expression by small interfering RNA in PC3-TR cells reversed their phenotype from TRAIL resistant to TRAIL sensitive. Therefore, persistent expression of c-FLIP(L) is necessary and sufficient to regulate sensitivity to TRAIL-mediated apoptosis in prostate cancer cells.     

Malignant ascites protect against TRAIL-induced apoptosis by activating the PI3K/Akt pathway in human ovarian carcinoma cells

Ascites are commonly found in ovarian cancer patients with advanced disease and are rich in cellular components and growth-promoting factors. The purpose of this study was to assess the effect of malignant ascites on TRAIL-induced apoptosis. We demonstrate that malignant ascites obtained from women with advanced ovarian cancer protect tumor cells from TRAIL- and FasL-induced apoptosis but not against cisplatin-induced apoptosis. This antiapoptotic effect was consistently found among different malignant ascites while nonmalignant peritoneal fluids or conditioned medium from TRAIL-resistant cells failed to protect tumor cells against TRAIL killing. Malignant ascites strongly inhibits TRAIL-induced caspase-3 activation and PARP cleavage. Furthermore, ascites activate PI3K and its downstream target Akt and increases c-FLIP(S) protein levels without affecting ERK phosphorylation status. The antiapoptotic effect of malignant ascites is abrogated by the inhibition of PI3K with LY294002, by a specific inhibitor of Akt and by Akt siRNA. We further show that the pro-survival effect of ascites can be suppressed by down-regulation of c-FLIP(S). Our data indicate that malignant effusions protect against TRAIL-induced apoptosis by activating the PI3K/Akt pathway. These findings demonstrate that the tumor microenvironment may contribute to the resistance of ovarian cancer cells to death receptor-induced apoptosis.     

Flavopiridol induces cellular FLICE-inhibitory protein degradation by the proteasome and promotes TRAIL-induced early signaling and apoptosis in breast tumor cells

The cyclin-dependent kinase inhibitor flavopiridol is undergoing clinical trials as an antitumor drug. We show here that pretreatment of different human breast cancer cell lines with flavopiridol facilitates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In breast tumor cells, apoptosis induction by TRAIL is blocked at the level of apical caspase-8 activation. Flavopiridol treatment enhances TRAIL-induced formation of death-inducing signaling complex and early processing of procaspase-8. Subsequently, a TRAIL-induced, mitochondria-operated pathway of apoptosis is activated in cells treated with flavopiridol. Down-regulation of cellular FLICE-inhibitory proteins (c-FLIP; c-FLIP(L) and c-FLIP(S)) is observed on flavopiridol treatment. c-FLIP loss and apoptosis sensitization by flavopiridol are both prevented in cells treated with an inhibitor of the ubiquitin-proteasome system. Furthermore, targeting c-FLIP directly with small interfering RNA oligonucleotides also sensitizes various human breast tumor cell lines to TRAIL-induced apoptosis. Our results indicate that flavopiridol sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating early events in the apoptotic pathway, and this combination treatment could be regarded as a potential therapeutic tool against breast tumors.     

c-FLIP downregulation contributes to apoptosis induction by the novel synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate (CDDO-Me) in human lung cancer cells

The novel synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate (CDDO-Me) induces apoptosis of cancer cells, enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and exhibits potent anticancer activity in animal models with a favorable pharmacokinetic profile. Thus, CDDO-Me is being tested in Phase I clinical trials. In an effort to understand the mechanism by which CDDO-Me induces apoptosis, particularly in human lung cancer cells, we previously demonstrated that CDDO-Me induces apoptosis involving c-Jun N-terminal kinase (JNK)-dependent upregulation of death receptor 5 (DR5) expression. In the current work, we determined the modulatory effects of CDDO-Me on the levels of c-FLIP, a major inhibitor of death receptor-mediated caspase-8 activation, and its impact on CDDO-Me-induced apoptosis and enhancement of TRAIL-induced apoptosis in human lung cancer cells. CDDO-Me rapidly and potently decreased c-FLIP levels including both long (FLIP(L)) and short (FLIP(S)) forms of c-FLIP in multiple human lung cancer cell lines. The presence of the proteasome inhibitor MG132, but not the JNK inhibitor SP600125, prevented CDDO-Me-induced c-FLIP reduction. Moreover, CDDO-Me increased ubiquitination of c-FLIP. Thus, CDDO-Me induces ubiquitin/proteasome-dependent c-FLIP degradation independently of JNK activation. Importantly, overexpression of c-FLIP (e.g., FLIP(L)) protected cells not only from CDDO-Me-induced apoptosis, but also from induction of apoptosis by the combination of CDDO-Me and TRAIL. Accordingly, silencing of c-FLIP with c-FLIP siRNA sensitized cancer cells to CDDO-Me. Collectively, these results indicate that c-FLIP downregulation contributes to CDDO-Me-initiated apoptosis and also to enhancement of TRAIL-induced apoptosis by CDDO-Me.     

Delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis,by inducing DR5 and causing caspase-mediated HDAC3 cleavage

TRAIL can induce apoptosis in some cancer cells and is an immune effector in the surveillance and elimination of developing tumors. Yes, some cancers are resistant to TRAIL. Delphinidin, a polyphenolic compound contained in brightly colored fruits and vegetables, has anti-inflammatory, anti-oxidant, and anti-tumorigenic activities. Here we showed that delphinidin sensitized TRAIL-resistant human prostate cancer cells to undergo apoptosis. Cells treated with delphinidin and TRAIL activated the extrinsic and intrinsic pathways of caspase activation. TRAIL-induced apoptosis in prostate cancer cells pretreated with delphinidin was dependent on death receptor 5 (DR5) and downstream cleavage of histone deacetylase 3 (HDAC3). In conclusion, delphinidin sensitizes prostate cancer cells to TRAIL-induced apoptosis by inducing DR5, thus causing caspase-mediated HDAC3 cleavage. Our data reveal a potential way of chemoprevention of prostate cancer by enabling TRAIL-mediated apoptosis.     

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.     

Berberine sensitizes TRAIL-induced apoptosis through proteasome-mediated downregulation of c-FLIP and Mcl-1 proteins

Berberine (BBR) is an isoquinoline alkaloid which has a wide spectrum of clinical applications including anti-tumor, anti-microbial and anti-inflammatory activities. In this study, we showed that co-treatment with subtoxic doses of BBR and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis in human renal cancer cells, Caki cells, but not in normal tubular kidney cells. Treatment of Caki cells with BBR resulted in downregulation of c-FLIP and Mcl-1 proteins in a dose-dependent manner. The BBR-induced downregulation of c-FLIP and Mcl-1 proteins were involved in proteasome dependent pathways, which was confirmed by the result that pre-treatment with the proteasome inhibitor MG132 inhibited berberine-induced downregulation of the c-FLIP and Mcl-1 proteins. Pretreatment with N-acetyl-L-cysteine (NAC) significantly inhibited the cell death induced by the combined treatment with BBR and TRAIL as well as recovered the expression levels of c-FLIP and Mcl-1 downregulated by treatment with BBR. These results suggested that BBR-stimulated TRAIL-induced apoptosis is dependent on the generation of reactive oxygen species through the downregulation of c-FLIP and Mcl-1 proteins. In conclusion, this study demonstrates that BBR enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated c-FLIP and Mcl-1 down-regulation.     

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.     

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition

API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity.     

Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.     

Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: Relationship to Bcl-2 family and caspase activation

In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.     

Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.     

Ethanolic extract of Brazilian green propolis sensitizes prostate cancer cells to TRAIL-induced apoptosis

Prostate cancer represents an ideal disease for chemopreventive intervention. Propolis possesses immuno-modulatory, anti-tumour and chemopreventive properties. The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important endogenous anti-cancer agent that induces apoptosis selectively in tumour cells. However, some cancer cells are resistant to TRAIL-mediated apoptosis. Naturally occurring phenolic and polyphenolic compounds sensitize TRAIL-resistant cancer cells and augment the apoptotic activity of TRAIL. The ethanolic extract of Brazilian green propolis (EEP) is rich in phenolic components. Our in vitro results indicate the potential targets in the TRAIL-induced apoptotic pathway for the cancer chemopreventive activity of Brazilian propolis. We examined the cytotoxic and apoptotic effects of Brazilian EEP and its bioactive components in combination with TRAIL on LNCaP prostate cancer cells. The chemical composition of Brazilian green propolis was determined by high performance liquid chromatography-diode array detection. The cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium and lactate dehydrogenase assays. Apoptosis was detected using annexin V-FITC by flow cytometry and fluorescence microscopy. The mitochondrial membrane potential (?Ψm) was evaluated using DePsipher staining by fluorescence microscopy. Flow cytometry was used to analyse death receptor (TRAIL-R1 and TRAIL-R2) expression in LNCaP cells. The inhibition of nuclear factor-κB (NF-κB) (p65) activation in cancer cells was confirmed by the ELISA-based TransAM NF-κB kit. The LNCaP cells were shown to be resistant to TRAIL-induced apoptosis. Our study demonstrates that EEP sensitizes TRAIL-resistant prostate cancer cells. The main phenolic components detected in Brazilian green propolis are artepillin C, quercetin, kaempferol and p-coumaric acid. Brazilian propolis and its bioactive components markedly augmented TRAIL-mediated apoptosis and cytotoxicity in prostate cancer cells. Brazilian EEP enhanced the expression of TRAIL-R2 and the activity of NF-κB in LNCaP cells. The co-treatment of prostate cancer cells with 100 ng/ml TRAIL and 50 μg/ml EEP increased the percentage of apoptotic cells to 65.8 ± 1.2% and caused a significant disruption of ?Ψm in LNCaP cells. We show that Brazilian EEP helped cells overcome TRAIL resistance by engaging both intrinsic and extrinsic apoptotic pathways and regulating NF-κB activity. The data demonstrate the important role of Brazilian green propolis and its bioactive compounds in prostate cancer chemoprevention through the enhancement of TRAIL-mediated apoptosis.     

Protein kinase C inhibition and x-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an important member of the TNF superfamily with great potential in cancer therapy. Luteolin is a dietary flavonoid commonly found in some medicinal plants. Here we found that pretreatment with a noncytotoxic concentration of luteolin significantly sensitized TRAIL-induced apoptosis in both TRAIL-sensitive (HeLa) and TRAIL-resistant cancer cells (CNE1, HT29, and HepG2). Such sensitization is achieved through enhanced caspase-8 activation and caspase-3 maturation. Further, the protein level of X-linked inhibitor of apoptosis protein (XIAP) was markedly reduced in cells treated with luteolin and TRAIL, and ectopic expression of XIAP protected against cell death induced by luteolin and TRAIL, showing that luteolin sensitizes TRAIL-induced apoptosis through down-regulation of XIAP. In search of the molecular mechanism responsible for XIAP down-regulation, we found that luteolin and TRAIL promoted XIAP ubiquitination and proteasomal degradation. Next, we showed that protein kinase C (PKC) activation prevented cell death induced by luteolin and TRAIL via suppression of XIAP down-regulation. Moreover, luteolin inhibited PKC activity, and bisindolylmaleimide I, a general PKC inhibitor, simulated luteolin in sensitizing TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of luteolin and support its potential application in cancer therapy in combination with TRAIL. In addition, our data reveal a new function of PKC in cell death: PKC activation stabilizes XIAP and thus suppresses TRAIL-induced apoptosis.     

A double tyrosine phosphorylation of P68 RNA helicase confers resistance to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with the capability of inducing apoptosis specifically in tumor cells. However, cancer cells of many cancer types developed TRAIL resistance, limiting the applications of TRAIL in cancer therapies. We show here that p68 acquires a double tyrosine phosphorylation at Y593 and Y595 in TRAIL-resistant T98G glioblastoma cells. The double phosphorylations are induced by platelet-derived growth factor autocrine loop. The double phosphorylation mediates resistance to TRAIL-induced apoptosis. Our data suggest that the phosphorylated p68 protects the cells from programmed cell death by preventing procaspase-8 from proteolytic cleavage. The double-phosphorylated p68 may also confer apoptosis resistance by upregulation of X-chromosome-linked inhibitor apoptosis protein-associated factor 1. In addition, exogenous expression of p68 mutant that carries mutations at the phosphorylation sites (Y593/595F) dramatically sensitizes TRAIL-resistant cells to TRAIL-induced apoptosis, suggesting a potential therapeutic strategy to overcome TRAIL resistance.