Co-treatment of birinapant with TRAIL synergistically induces apoptosis by downregulating cFLIP(L) in MDA-MB-453 cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received much attention owing to its ability to specifically induce cell death in cancer. However, several types of cancer, including some forms of breast cancer, are resistant to TRAIL. Various chemotherapeutic agents, phytochemicals, and TRAIL combination therapies have been proposed to resolve TRAIL resistance. Here, we explored the sensitization effect of birinapant on TRAIL-induced apoptosis in the MDA-MB-453 cell line. Although neither birinapant nor TRAIL showed any cytotoxic effect when used alone, apoptosis was induced when birinapant and TRAIL were used together. Our data suggest that the combination of birinapant and TRAIL induces downregulation of FLICE-like inhibitory protein (cFLIP) (L) protein expression. Interestingly, cFLIP(L) overexpression reversed apoptosis caused by co-treatment with TRAIL. Taken together, our results indicate that a combination of birinapant and TRAIL may be a promising treatment for TRAIL-resistant breast cancer.

Metformin induces caspase-dependent and caspase-independent apoptosis in human bladder cancer T24 cells.

Bladder cancer (BC) is the sixth most common cancer in men. Moreover, chemotherapy for BC leads to various side effects. Metformin is known to induce apoptosis in vitro in many types of cancer. Furthermore, it has feasibility as a drug repositioning used for the treatment of cancer. The molecular mechanism of metformin mediating apoptosis in BC is still unclear. In this study, we showed that metformin stimulated the caspase-dependent apoptotic signaling pathway in T24 cells, a human BC cell line. Moreover, the induced apoptosis was partially inhibited by a general caspase inhibitor, z-VAD-fmk, which suggested that metformin-induced apoptosis in T24 cells is partially caspase-independent. Notably, we observed the nuclear translocation of apoptosis-inducing factors (AIFs) in metformin-promoted apoptosis, which is a typical characteristic of the caspase-independent apoptotic pathway. In addition, we found that metformin-mediated apoptosis occurred via degradation of the cellular FADD-like interleukin-1ß-converting enzyme inhibitory protein (c-FLIP) by facilitating ubiquitin/proteasome-mediated c-FLIPL degradation. Furthermore, treatment with the reactive oxygen species scavenger N-acetylcysteine, failed to suppress metformin-induced apoptosis and c-FLIPL protein degradation in metformin-treated T24 cells. In conclusion, these results indicate that metformin-induced apoptosis was mediated through AIF-promoted caspase-independent pathways as well as caspase-dependent pathways in T24 cells. As such, metformin could be used as a possible apoptotic agent for the treatment of BC.

Mathematical Modeling Reveals the Importance of the DED Filament Composition in the Effects of Small Molecules Targeting Caspase-8/c-FLIPL Heterodimer.

Procaspase-8 activation at the death-inducing signaling complex (DISC) triggers extrinsic apoptotic pathway. Procaspase-8 activation takes place in the death effector domain (DED) filaments and is regulated by c-FLIP proteins, in particular, by the long isoform c-FLIPL. Recently, the first-in-class chemical probe targeting the caspase-8/c-FLIPL heterodimer was reported. This rationally designed small molecule, FLIPin, enhances caspase-8 activity after initial heterodimer processing. Here, we used a kinetic mathematical model to gain an insight into the mechanisms of FLIPin action in a complex with DISC, in particular, to unravel the effects of FLIPin at different stoichiometry and composition of the DED filament. Analysis of this model has identified the optimal c-FLIPL to procaspase-8 ratios in different cellular landscapes favoring the activity of FLIPin. We predicted that the activity FLIPin is regulated via different mechanisms upon c-FLIPL downregulation or upregulation. Our study demonstrates that a combination of mathematical modeling with system pharmacology allows development of more efficient therapeutic approaches and prediction of optimal treatment strategies.

Codium fragile F2 sensitize colorectal cancer cells to TRAIL-induced apoptosis via c-FLIP ubiquitination.

This study demonstrates that combined treatment with subtoxic doses of Codium extracts (CE), a flavonoid found in many fruits and vegetables, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces apoptosis in TRAIL-resistant colorectal cancer (CRC) cells. Effective induction of apoptosis by combined treatment with CE and TRAIL was not blocked by Bcl-xL overexpression, which is known to confer resistance to various chemotherapeutic agents. While TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in various CRC cells treated with TRAIL alone, co-treatment with CE efficiently recovered TRAIL-induced caspase activation. We observed that CE treatment of CRC cells did not change the expression of anti-apoptotic proteins and pro-apoptotic proteins, including death receptors (DR4 and DR5). However, CE treatment markedly reduced the protein level of the short form of the cellular FLICE-inhibitory protein (c-FLIPS), an inhibitor of caspase-8, via proteasome-mediated degradation. Collectively, these observations show that CE recovers TRAIL sensitivity in various CRC cells via down-regulation of c-FLIPS.

Overcoming cell death resistance in skin cancer therapy: Novel translational perspectives.

In the last decade, significant progress has been made in understanding skin cancer cell death resistance mechanisms, and a number of new treatment strategies have been developed. Systematic approach genomic studies of various cancer types have opened new possibilities for the development of anticancer therapies. However, there are still fundamental gaps in the challenging biomedical puzzle, which will form a complete picture for curing cancer. Thus, herein, we describe some of the current cancer treatment strategies and discuss additional cell signalling pathways that could be potential targets for skin cancer treatment.

Genome-wide association analysis identified splicing single nucleotide polymorphism in CFLAR predictive of triptolide chemo-sensitivity.

BACKGROUND: Triptolide is a therapeutic diterpenoid derived from the Chinese herb Tripterygium wilfordii Hook f. Triptolide has been shown to induce apoptosis by activation of pro-apoptotic proteins, inhibiting NFkB and c-KIT pathways, suppressing the Jak2 transcription, activating MAPK8/JNK signaling and modulating the heat shock responses. RESULTS: In the present study, we used lymphoblast cell lines (LCLs) derived from 55 unrelated Caucasian subjects to identify genetic markers predictive of cellular sensitivity to triptolide using genome wide association study. Our results identified SNPs on chromosome 2 associated with triptolide IC50 (p < 0.0001). This region included biologically interesting genes as CFLAR, PPIl3, Caspase 8/10, NFkB and STAT6. Identification of a splicing-SNP rs10190751, which regulates CFLAR alternatively spliced isoforms predictive of the triptolide cytotoxicity suggests its role in triptolides action. Our results from functional studies in Panc-1 cell lines further demonstrate potential role of CFLAR in triptolide toxicity. Analysis of gene-expression with cytotoxicity identified JAK1 expression to be a significant predictor of triptolide sensitivity. CONCLUSIONS: Overall out results identified genetic factors associated with triptolide chemo-sensitivity thereby opening up opportunities to better understand its mechanism of action as well as utilize these biomarkers to predict therapeutic response in patients.

FLIP: molecular switch between apoptosis and necroptosis.

Cancerous growth is one of the most difficult diseases to target as there is no one clear cause, and targeting only one pathway does not generally produce quantifiable improvement. For a truly effective cancer therapy, multiple pathways must be targeted at the same time. One way to do this is to find a gene that is associated with several pathways; this approach expands the possibilities for disease targeting and enables multiple points of attack rather than one fixed point, which does not allow treatment to evolve over time as cancer does. Inducing programmed cell death (PCD) is a promising method to prevent or inhibit the progression of tumor cells. Intricate cross talk among various programmed cell death pathways including cell death by apoptosis, necroptosis or autophagy plays a critical role in the regulation of PCD. In addition, the complex and overlapping patterns of signaling and lack of understanding of such networks between these pathways generate hurdles for developing effective therapeutic approaches. This review article focuses on targeting FLIP (Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein) signaling as a bridge between various PCD processes as an effective approach for cancer management.

Synthesis of methyl 2-cyano-3,12-dioxo-18ß-olean-1,9(11)-dien-30-oate analogues to determine the active groups for inhibiting cell growth and inducing apoptosis in leukemia cells.

Fourteen of the methyl 2-cyano-3,12-dioxo-18ß-olean-1,9(11)-dien-30-oate (CDODO-Me-12, 10d) analogues with different structures of ring C were synthesized to determine the active groups for inhibiting cell growth and inducing apoptosis in human leukemia HL-60 cells. An unsaturated group in ring C was required to maintain the ability to inhibit cell growth and induce apoptosis. Compound 10e with 9(11),12-dien in ring C displayed comparable apoptosis induction ability to 10d associated with decreased levels of c-FLIP, but not Mcl-1 and XIAP. Compound 10e had decreased ability to deplete GSH compared to compound 10d. Compound 10e represents a new active compound acting through a different mechanism from that of compound 10d.

Allogeneic transplantation, Fas signaling, and dysregulation of hepcidin.

Hepatic iron overload is common in patients undergoing hematopoietic cell transplantation. We showed previously in a murine model that transplantation of allogeneic T cells induced iron deposition and down-regulation of hepcidin (Hamp) in hepatocytes. We hypothesized that hepatic injury was related to disrupted iron homeostasis triggered by the interaction of Fas-ligand, expressed on activated T cells, with Fas on hepatocytes. In the current study, we determined the effects of modified expression of the Flice inhibitory protein (FLIP long [FLIPL]), which interferes with Fas signaling, on the impact of Fas-initiated signals on the expression of IL-6 and Stat3 and their downstream target, Hamp. To exclude a possible contribution by other pathways, we used agonistic anti-Fas antibodies (rather than allogeneic T cells) to trigger Fas signaling. Inhibition of FLIPL by RNA interference resulted, as expected, not only in enhanced hepatocyte apoptosis in response to Fas signals, but also in decreased levels of IL-6, Stat3, and Hamp. In contrast, overexpression of FLIPL protected hepatocytes against agonistic anti-Fas antibody-mediated apoptosis and increased the levels of IL-6 and Stat3, thereby maintaining the expression of Hamp in an NF-κB-dependent fashion. In vivo overexpression of FLIPL in the liver via hydrodynamic transfection, similarly, interfered with Fas-initiated apoptosis and prevented down-regulation of IL-6, Stat3, and Hamp. These data indicate that Fas-dependent signals alter the regulation of iron homeostasis and suggest that signals initiated by Fas may contribute to peritransplantation iron accumulation.

Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins.

Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with other agents is a promising strategy to overcome TRAIL resistance in malignant cells. Wogonin, a flavonoid originated from Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL's antitumor activity in vivo has never been studied. In this study, the effect of combination of TRAIL and wogonin was tested in a non-small-cell lung cancer xenografted tumor model in nude mice. Consistent with the in vitro study showing that wogonin sensitized A549 cells to TRAIL-induced apoptosis, wogonin greatly enhanced TRAIL-induced suppression of tumor growth, accompanied with increased apoptosis in tumor tissues as determined by TUNEL assay. The expression levels of antiapoptotic proteins including long form of cellular FLICE-like inhibitory protein (cFLIPL), X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues after co-treatment with wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated by proteasomal degradation that involved intracellular reactive oxygen species (ROS), because wogonin robustly induced ROS accumulation and ROS scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins in cells co-treated with wogonin and TRAIL. These results show for the first time that wogonin enhances TRAIL's antitumor activity in vivo, suggesting this strategy has an application potential for clinical anticancer therapy.

Cellular FLIP(L) plays a survival role and regulates morphogenesis in breast epithelial cells.

Strong evidences support the inhibitory activity of cellular FLICE-inhibitory protein (FLIP) in the apoptotic signalling by death receptors in tumor cells. However, little is known about the role of FLIP in the regulation of apoptosis in non-transformed cells. In this report, we demonstrate that FLIP(L) plays an important role as a survival protein in non-transformed breast epithelial cells. Silencing of FLIP(L) by siRNA methodology enhances TRAIL-R2 expression and activates a caspase-dependent cell death process in breast epithelial cells. This cell death requires the expression of TRAIL, TRAIL-R2, FADD and procaspase-8 proteins. A mitochondria-operated apoptotic pathway is partially required for FLIP(L) siRNA-induced apoptosis. Interestingly, FLIP(L) silencing markedly abrogates formation of acinus-like structures in a three-dimensional basement membrane culture model (3D) of the human mammary MCF-10A cell line through a caspase-8 dependent process. Furthermore, over-expression of FLIP(L) in MCF-10A cells delayed lumen formation in 3D cultures. Our results highlight the central role of FLIP in maintaining breast epithelial cell viability and suggest that the mechanisms regulating FLIP levels should be finely controlled to prevent unwanted cell demise.

Pharmacologic downregulation of c-FLIP(L) restores juxtacrine death receptor-mediated apoptosis in cancer cells in a peritoneal carcinomatosis model.

Metastasis occurs when circulating cancer cells implant in normal secondary tissues. Paradoxically, many cancer cells express death receptors while many normal tissues express the cognate death receptor ligands, suggesting that cancer cells possess mechanisms to inhibit death receptor signaling. Pharmacological restoration of juxtacrine-mediated death receptor signaling could prevent cancer cells from implanting in normal tissues such as the peritoneum. The results showed that BAY 11-7085 significantly inhibited peritoneal carcinomatosis in mice following the introduction of colon and pancreatic cancer cell lines into the intra-abdominal cavity. Treatment with BAY 11-7085 restored juxtacrine death receptor signaling during the adhesion of the cancer cells to mesothelial cells, which line the peritoneum. BAY 11-7085 rapidly inhibited c-FLIP(L) expression in colon and pancreatic cancer cell lines during adhesion to mesothelial cells. Pancreatic cancer cells sorted for high c-FLIP(L) expression formed peritoneal implants much more readily than cells with low c-FLIP(L) expression, and RNAi inhibition of c-FLIP(L) in colon cancer cells dramatically reduced peritoneal implantation. This is a novel demonstration that the restoration of death receptor-mediated apoptotic signaling in cancer cells through the pharmacological inhibition of c-FLIP(L) can inhibit tumor implantation in a clinically relevant model of peritoneal carcinomatosis, a fatal disease. Pharmacological inhibitors of FLIP hold promise as a way to curtail cancer cell colonization of secondary tissues.

Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α and death receptor stimulation in resistant variants of the human U937 cell line.

Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

Induction of CCL20 production by Kaposi sarcoma-associated herpesvirus: role of viral FLICE inhibitory protein K13-induced NF-kappaB activation.

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is the etiologic agent of Kaposi sarcoma (KS), an angioproliferative lesion characterized by dramatic angiogenesis and inflammatory infiltration. In this study, we report that expression of chemokine CCL20, a potent chemoattractant of dendritic cells and lymphocytes, is strongly induced in cultured cells either by KSHV infection or on ectopic expression of viral FLICE inhibitory protein K13. This induction is caused by transcriptional activation of CCL20 gene, which is mediated by binding of the p65, p50, and c-Rel subunits of the transcription factor nuclear factor-kappaB (NF-kappaB) to an atypical NF-kappaB-binding site present in the CCL20 gene promoter. The CCL20 gene induction is defective in K13 mutants that lack NF-kappaB activity, and can be blocked by specific genetic and pharmacologic inhibitors of the NF-kappaB pathway. CCR6, the specific receptor for CCL20, is also induced in cultured cells either by KSHV infection or on K13 expression. Finally, expression of CCL20 and CCR6 is increased in clinical samples of KS. These results suggest that KSHV and K13-mediated induction of CCL20 and CCR6 may contribute to the recruitment of dendritic cells and lymphocytes into the KS lesions, and to tumor growth and metastases.

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP.

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting.

Pre- and Post-Transcriptional Regulation of cFLIP for Effective Cancer Therapy Using pH-Ultrasensitive Nanoparticles.

Cellular FLIP (cFLIP) is a crucial player of apoptosis-regulated pathways that is frequently overexpressed in solid cancers. To inhibit c-FLIP, pre- and post-transcriptionally, a multifunctional nanoparticle (NP) was created to deliver cFLIP-specific small interfering RNA (siRNA) into cancer cells. Specifically, Vorinostat (Vor)-loaded mesoporous silica nanoparticles (MSN) were conjugated with polyethylenimine-biotin (PB), followed by electrostatically binding with cFLIP siRNA (Vor/siR@MSN-PB). To stabilize and prolong the circulation time of nanoparticles, a bialdehyde-modified poly(ethylene glycol) (PEG) was cross-linked onto the polyethylenimine (PEI) backbone via the formation of the imine linkage (Schiff base) (Vor/siR@MSN-PB-PEG). The Schiff base is highly stable at physiological pH 7.4 but labile under slightly acidic pH conditions. In the acidic tumor microenvironment (TME), the PEG outer layer could be rapidly cleaved, resulting in the switching of the nanoparticle surface charge to positive, which specifically enhances internalization of the NPs to the biotin-positive tumor cells. Our results demonstrated the successful preparation of Vor/siR@MSN-PB-PEG NPs, in which the siRNA was effectively protected in serum and regulated the expression of cFlip, post-transcriptionally. The presence of the PEG layer resulted in high tumor accumulation and high efficacy in tumor inhibition, which was a result of the efficient cFLIP suppression. Furthermore, in the low-dose regimen of Vorinostat-the pre-transcriptional cFLIP suppressor, treatment with Vor/siR@MSN-PB-PEG NPs was found to be safe with the treated mice, indicating a promising combination regimen for cancer therapy.

α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway.

Vitamin E derivative RRR-α-tocopherol ether-linked acetic acid analog (α-TEA) induces apoptosis in MCF-7 and HCC-1954 human breast cancer cells in a dose- and time-dependent manner. α-TEA induces increased levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor-5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE-like inhibitory protein (c-FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c-FLIP (L), caspase-8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase-9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α-TEA to enhance DR5 protein levels, downregulate c-FLIP(L) protein levels and induce apoptosis. Combination of α-TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c-FLIP (L) protein levels. siRNA knockdown of c-FLIP produces a low level of spontaneous apoptosis and enhances α-TEA- and TRAIL-induced apoptosis. Taken together, these studies show that α-TEA induces TRAIL/DR5 mitochondria-dependent apoptosis in human breast cancer cells, and that TRAIL/DR5-dependent increases in DR5 and decreases in c-FLIP expression are triggered by TRAIL or α-TEA treatments.

Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.

BACKGROUND: Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. METHODS: We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. RESULTS: Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. CONCLUSIONS: Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.

4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells.

Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor for the tumor necrosis factor-related apoptosis-inducing ligand TRAIL and in drug resistance in human malignancies. c-FLIP is an antagonist of caspases-8 and -10, which inhibits apoptosis and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. c-FLIP is often overexpressed in various human cancers, including breast cancer. Several studies have shown that silencing c-FLIP by specific siRNAs sensitizes cancer cells to TRAIL and anticancer agents. However, systemic use of siRNA as a therapeutic agent is not practical at present. In order to reduce or inhibit c-FLIP expression, small molecules are needed to allow targeting c-FLIP without inhibiting caspases-8 and -10. We used a small molecule inhibitor of c-FLIP, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), and show that CMH, but not its inactive analog, downregulated c-FLIP(L) and c-FLIP(S) mRNA and protein levels, caused poly(ADP-ribose) polymerase (PARP) degradation, reduced cell survival, and induced apoptosis in MCF-7 breast cancer cells. These results revealed that c-FLIP is a critical apoptosis regulator that can serve as a target for small molecule inhibitors that downregulate its expression and serve as effective targeted therapeutics against breast cancer cells.

Bortezomib restores stroma-mediated APO2L/TRAIL apoptosis resistance in multiple myeloma.

OBJECTIVES: Hematopoietic stroma promotes resistance to immune control by APO2L/TRAIL in multiple myeloma (MM) cells in part by increasing synthesis of the anti-apoptotic protein c-FLIP. Here, we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistance (EM-IR). MATERIAL AND METHODS: MM cell lines (RPMI 8226 and U266) and CD138+ patient's MM cells were directly adhered to HS5 stroma exposed to HS5 or bone marrow stroma of patients with MM released soluble factors in a transwell system. Cells were treated with either APO2L/TRAIL (10 ng/mL), bortezomib (10 nm) or both. RESULTS: Pretreatment with bortezomib effectively overcomes APO2L/TRAIL apoptosis resistance in myeloma cell lines and in CD138+ cells while directly adhered or in transwell assay. Bortezomib was not cytotoxic to HS5 stroma cells and only altered monocyte chemotactic protein-2-3 and IL-10 levels in the stroma-myeloma milieu. Factors released by HS5 stroma increased expression of c-FLIP, induced STAT-3 and ERK phosphorylation and reduced DR4 receptor expression in MM cells. HS5 stroma-released factor(s) induced NF-kappaB activation after 20 h exposure in association with an enhanced c-FLIP transcription. Bortezomib effectively reduced c-FLIP protein expression without affecting other proteins. Bortezomib also increased DR4 and DR5 expression in the presence of stroma. CONCLUSIONS: These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on MM cells.