c-FLIP Expression Research Articles

Expression of c-FLIP in peripheral blood mononuclear cells of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway

To investigate the expression of apoptosis related protein cellular Fas associated death domain like interleukin 1 converting enzyme inhibitory protein (c-FLIP) in peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway. Sixty patients with rheumatoid arthritis were collected from Zhangzhou Affiliated Hospital of Fujian Medical University during January 2014 and June 2015, including 22 patients with low activities (DAS28<3.2), 20 patients with middle activities (3.2 ≤ DAS28 ≤ 5.1), and 18 patients with high activities (DAS28>5.1). And 25 healthy controls were also collected. The mRNA and protein expression levels of c-FLIP and the extrinsic apoptotic pathway related proteins Fas-associated protein with death domain (FADD), caspase-8 in PBMCs were detected by real-time RT-PCR and Western blot, respectively. Correlations between c-FLIP and FADD, caspase-8 in PBMCs were analyzed by pearson test. mRNA expression levels of c-FLIP, FADD and caspase-8 in PBMCs of patients with rheumatoid arthritis were all higher than those of healthy controls (all P<0.05). mRNA expression levels of FADD and caspase-8 in patients with middle activities were significantly higher than those in patients with low activities (all P<0.05), but the mRNA expression level of c-FLIP was not significantly higher than that in patients with low activities. mRNA expression level of c-FLIP in patients with high activities was higher than those in patients with middle or low activities (all P<0.05), while the mRNA expression level of caspase-8 was lower than those in patients with middle or low activities (all P<0.05). mRNA expression level of FADD in patients with high activities was higher than those in patients with low activities (P<0.05). Pearson analysis showed that there was a positive correlation between c-FLIP and FADD mRNA expression (r=0.323, P<0.05), and negative correlation between c-FLIP and caspase-8 mRNA expression (r=-1.104, P<0.05). The protein expression levels of c-FLIP and FADD in patients with middle activities were significantly higher than those in control group and patients with low or high activities (P<0.05 or 0.01). The protein expression levels of caspase-8 in patients with middle and high activities were significantly higher than those in control group and patients with low activities (P<0.05 or P<0.01), and the protein expression level of caspase-8 in patients with high activities was higher than that in patients with middle activities (P<0.05). c-FLIP may be involved in the extrinsic apoptotic pathway in rheumatoid arthritis, and can provide reference for the evaluation of disease activities.

Cutaneous expression of antiapoptotic protein c-FLIP in psoriasis

Cutaneous expression of antiapoptotic protein c-FLIP in psoriasis

WITHDRAWN: Overexpression of miR-382 sensitizes hepatocellular carcinoma cells to γδ T cells through inhibiting the expression of c-FLIP

// Benxin Hou 1 , Zhong Chen 1 , Zhiang Zheng 1 , Lan Feng 1 , Zhenqing Huo 1 , Liju Huang 1 , Mingchang Fu 1 , Qiuzan Chen 1 , Keyan Zhuang 1 and Jinjun Yang 1 1 Department of General Surgery, The Third People's Hospital of Hainan Province, Sanya, Hainan, China Correspondence to: Benxin Hou, email: pi0283@126.com Keywords: hepatocellular carcinoma; &gamma;&delta; T cells; miR-382; c-FLIP; caspase 8 Received: July 28, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: August 28, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: January 04, 2018 ABSTRACT Immunotherapy is an important strategy for cancer treatment. Recently, human &gamma;&delta;T lymphocytes are reported to display anti-tumor effect against multiple cancers, including hepatocellular carcinoma (HCC). However, resistance to &gamma;&delta;T cells is a major obstacle in immunotherapy of HCC. Studies have demonstrated that microRNAs (miRNAs) are usually dysregulated in cancer cells. Aberrant expression of miRNAs leads to low response to immunotherapy. In the present study, we observed that ex vivo expanded human &gamma;&delta;T cells were able to induce cell lysis of HCC. Furthermore, as miR-382 was observed to be downregulated in HCC tissues and cell lines, we found that overexpression of miR-382 increased the sensitivity of HCC cells to &gamma;&delta;T cells. Mechanically, we proved that mRNA of c-FLIP was the target of miR-382. Inhibition of c-FLIP by miR-382 significantly promotes the cell lysis of HCC through strengthening the activation caspase 8 induced by &gamma;&delta;T cells treatment. These data strongly demonstrates that overexpression of miR-382 promotes HCC cell lysis induced by &gamma;&delta;T cells through inhibiting the expression of c-FLIP.

Quinacrine Inhibits ICAM-1 Transcription by Blocking DNA Binding of the NF-κB Subunit p65 and Sensitizes Human Lung Adenocarcinoma A549 Cells to TNF-α and the Fas Ligand.

Quinacrine has been used for therapeutic drugs in some clinical settings. In the present study, we demonstrated that quinacrine decreased the expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor (TNF)-α and interleukin-1 (IL-1) α in human lung adenocarcinoma A549 cells. Quinacrine inhibited ICAM-1 mRNA expression and nuclear factor κB (NF-κB)-responsive luciferase reporter activity following a treatment with TNF-α and IL-1α. In the NF-κB signaling pathway, quinacrine did not markedly affect the TNF-α-induced degradation of the inhibitor of NF-κB or the TNF-α-induced phosphorylation of the NF-κB subunit, p65, at Ser-536 and its subsequent translocation to the nucleus. In contrast, a chromatin immunoprecipitation assay showed that quinacrine prevented the binding of p65 to the ICAM-1 promoter following TNF-α stimulation. Moreover, TNF-α and the Fas ligand effectively reduced the viability of A549 cells in the presence of quinacrine only. Quinacrine down-regulated the constitutive and TNF-α-induced expression of c-FLIP and Mcl-1 in A549 cells. These results revealed that quinacrine inhibits ICAM-1 transcription by blocking the DNA binding of p65 and sensitizes A549 cells to TNF-α and the Fas ligand.

Relationship between High Expression of c-FLIP and Drug Resistance of Leukemia Cells

To explore the effect of c-FLIP expression on drug resistance of Kasumi-1 leukemia cells and its mechanisms. Tet-on inducible system was used to construct the conditional expression vector of c-FLIP by cloning the c-FLIP gene into lentivirus vector pLVX-Tight-Puro, then the Kasumi-1 cells were transfected with lentivirus pLVX-Tight-Puro-c-FLIP. The expression of c-FLIP was induced by doxycycline(Dox) for different time and doses, and verified by qRT-PCR and Western blot. On the basis of the overexpression of c-FLIP, the Kasumi-1-c-FLIP cells were treated with CH11 and PB in order to induce apoptosis, and the Giemsa staining was used to show the apoptotic cell morphology. qRT-PCR and Western blot showed the overexpression of c-FLIP, the CH11 and PB can induce Kasumi-1 cell apoptosis, while the c-FLIP overexpression weakened this effects. Western blot showed that the c-FLIP blocked the caspase-8 activation. The overexpression of c-FLIP inhibits the apoptosis caused by CH11 and PB, and leads to drug resistance in leukemia cells.

Caspase-1 Engagement and TLR-Induced c-FLIP Expression Suppress ASC/Caspase-8-Dependent Apoptosis by Inflammasome Sensors NLRP1b and NLRC4

SummaryThe caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1−/− macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1β (IL-1β) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.

Volasertib Enhances Sensitivity to TRAIL in Renal Carcinoma Caki Cells through Downregulation of c-FLIP Expression.

Polo-like kinase 1 (PLK1) plays major roles in cell cycle control and DNA damage response. Therefore, PLK1 has been investigated as a target for cancer therapy. Volasertib is the second-in class dihydropteridinone derivate that is a specific PLK1 inhibitor. In this study, we examined that combining PLK1 inhibitor with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) would have an additive and synergistic effect on induction of apoptosis in cancer cells. We found that volasertib alone and TRAIL alone had no effect on apoptosis, but the combined treatment of volasertib and TRAIL markedly induced apoptosis in Caki (renal carcinoma), A498 (renal carcinoma) and A549 (lung carcinoma) cells, but not in normal cells (human skin fibroblast cells and mesangial cells). Combined treatment induced accumulation of sub-G1 phase, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and activation of caspase 3 activity in Caki cells. Interestingly, combined treatment induced downregulation of cellular-FLICE-inhibitory protein (c-FLIP) expression and ectopic expression of c-FLIP markedly blocked combined treatment-induced apoptosis. Therefore, this study demonstrates that volasertib may sensitize TRAIL-induced apoptosis in Caki cells via downregulation of c-FLIP.

Cisplatin triggers cancer stem cell enrichment in platinum-resistant cells through NF-\u03baB-TNF\u03b1-PIK3CA loop

BackgroundParallel to complex alteration in molecular and cellular events, enrichment of cancer stem cells (CSC) contributes significantly in deliberation and maintenance of cisplatin resistance. Cisplatin mediated CSC enrichment is well established in various cancers, yet the underlying mechanism is largely unknown. Cisplatin also promotes transcriptional upregulation of PIK3CA, hence activating PI3K/AKT signaling in resistant cells. However, such cisplatin-induced transcriptional regulators of PIK3CA and their impact on cancer stem cell population in resistant cells are largely unknown.MethodsDNA-binding protein pulldown using PIK3CA promoter as bait followed by nLCMS was used to identify, cisplatin-induced potential transcriptional regulators of PIK3CA promoter. PIK3CA promoter activity was estimated by luciferase based reporter assay. ChIP was used to assess interaction of NF-κB with PIK3CA promoter. CSC-enriched side-population was sorted using DCV-dye exclusion methods. All the gene expression levels were assessed using qPCR.ResultsUsing a transcription factor pull-down assay with PIK3CA promoter, we identified NF-κB as a prime regulator, which escalates both TNFα and PIK3CA expression only in CSC enriched side-population (SP) but not in non side-population (NSP) in platinum resistant ovarian cancer cells upon cisplatin treatment. This SP-specific NF-κB-TNFα-PIK3CA bi-modal loop, on one hand, maintains persistent activation of NF-κB through TNFα- NF-κB autocrine loop, while NF-κB-PIK3CA loop nurture CSC population under cisplatin treatment. Activation of PI3K/AKT signalling drives SP’s into an undifferentiated, anti-apoptotic stage through upregulating P21, P27,cFLIP expression. Contrarily, lack of active NF-κB-TNFα-PIK3CA loop makes NSPs vulnerable towards cisplatin and undergoes apoptosis. Altogether, cisplatin enriches cancer stem cells properties in SP fraction, which is evident from increased levels of pluripotency gene OCT4/SOX2/NANOG expression. Disruption of PIK3CA-NF-κB loop by Wortamannin reduces SP fraction by 1.4–1.6 fold in control and treated cells.ConclusionTogether, our study signifies an active role of NF-κB-TNFα-PIK3CA bi-modal loop in cisplatin-mediated promotion and maintenance of CSC-like population in platinum-resistant cells.

Anti-leukemia activity of NSC-743380 in SULT1A1-expressing acute myeloid leukemia cells is associated with inhibitions of cFLIP expression and PI3K/AKT/mTOR activities.

Our recent study showed that acute myeloid leukemia (AML) cells expressing SULT1A1 are highly sensitive to NSC-743380, a small molecule that inhibits STAT3 activity and induces SULT1A1-dependent apoptosis of various cancer cell lines. In this study, we characterized the molecular mechanisms of NSC-743380–mediated anti-leukemia activity in AML cell lines and antileukemia activity of NSC-743380 in patient-derived primary leukemia cells from AML patients. Our results showed that treatment with NSC-743380 triggered robust apoptosis in SULT1A1-positive AML cells. Treatment with NSC-743380 did not increase intracellular reactive oxygen species or change of STAT3 activity in AML cells, but did dramatically and rapidly decrease cFLIP expression. Proteomic analysis with reverse phase protein microarray revealed that treatment of U937 and THP-1 AML cells with NSC-743380 led to drastic and time-dependent suppression of phosphorylation of several key nodes in the PI3K/AKT/mTOR pathway, including AKT and mTOR. Moreover, primary AML cells expressed SULT1A1 were highly sensitive to treatment with NSC-743380, which was not affected by co-culture with bone marrow mesenchymal stem cells. Thus, our results provide proof-of-concept evidence that AML cells expressing SULT1A1 can be targeted by small molecules that induce apoptosis through inhibiting the expression or activities of multiple targets.

Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4+, CD8+ T-cells and in CD56+ NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.

PELI1 functions as a dual modulator of necroptosis and apoptosis by regulating ubiquitination of RIPK1 and mRNA levels of c-FLIP

Apoptosis and necroptosis are two distinct cell death mechanisms that may be activated in cells on stimulation by TNFα. It is still unclear, however, how apoptosis and necroptosis may be differentially regulated. Here we screened for E3 ubiquitin ligases that could mediate necroptosis. We found that deficiency of Pellino 1 (PELI1), an E3 ubiquitin ligase, blocked necroptosis. We show that PELI1 mediates K63 ubiquitination on K115 of RIPK1 in a kinase-dependent manner during necroptosis. Ubiquitination of RIPK1 by PELI1 promotes the formation of necrosome and execution of necroptosis. Although PELI1 is not directly involved in mediating the activation of RIPK1, it is indispensable for promoting the binding of activated RIPK1 with its downstream mediator RIPK3 to promote the activation of RIPK3 and MLKL. Inhibition of RIPK1 kinase activity blocks PELI1-mediated ubiquitination of RIPK1 in necroptosis. However, we show that PELI1 deficiency sensitizes cells to both RIPK1-dependent and RIPK1-independent apoptosis as a result of down-regulated expression of c-FLIP, an inhibitor of caspase-8. Finally, we show that Peli1-/- mice are sensitized to TNFα-induced apoptosis. Thus, PELI1 is a key modulator of RIPK1 that differentially controls the activation of necroptosis and apoptosis.

Angelicin potentiates TRAIL-induced apoptosis in renal carcinoma Caki cells through activation of caspase 3 and down-regulation of c-FLIP expression.

Preclinical Research & Development Angelicin is a furocoumarin derived from Psoralea corylifolia L. fruit that has anti-inflammatory and anti-tumor activity. In the present study, the effect of angelicin in enhancing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic cell death was studied in Caki (renal carcinoma) cells. Angelicin alone and TRAIL alone had no effect on apoptosis, but in combination these compounds markedly induced apoptosis in the cancer cell lines while not inducing apoptosis in normal cells. The combination treatment induced accumulation of the sub-G1 population, DNA fragmentation, and activated caspase 3 activity in Caki cells, induced down-regulation of c-FLIP expression post-translationally, and over-expression of c-FLIP markedly blocked apoptosis induced by combined treatment with angelicin plus TRAIL. This study provides evidence that angelicin might be a TRAIL sensitizer.

TRAF2 is a Valuable Prognostic Biomarker in Patients with Prostate Cancer.

BackgroundTRAF2 exerts important functions in regulating the development and progression of cancer. The aim of this study is to investigate whether TRAF2 is a valuable prognostic biomarker and to determine if it regulates TRAIL-induced apoptosis in prostate cancer.Material/MethodsMicroarray gene expression data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to determine TRAF2 expression in prostate cancer. TRAF2 expression in prostate cancer was further investigated by immunohistochemistry assay. Kaplan–Meier curves and log-rank test were used to assess the recurrence-free rate. Cox regression was used to analyze prognostic factors. Effects of TRAF2 on regulating TRAIL-induced apoptosis in DU-145 cells were further investigated.ResultsWe found that TRAF2 was significantly upregulated in prostate cancer compared with normal prostate samples (P<0.001). In addition, compared with primary prostate cancer, TRAF2 was upregulated in metastatic prostate cancer (P=0.006). Furthermore, our results showed that high expression of TRAF2 was significantly associated with tumor stage of prostate cancer (P=0.035). TRAF2 high expression was associated with poorer recurrence-free survival in prostate cancer patients (P=0.013). TRAF2 was found to be a valuable independent prognostic factor for predicting recurrence-free survival (P=0.026). In addition, the present results indicate that TRAF2 affects TRAIL-induced apoptosis in prostate cancer DU-145 cells via regulating cleaved Caspase-8 and c-Flip expression.ConclusionsTRAF2 could be a novel prognostic biomarker for predicting recurrence-free survival in patients with prostate cancer, which might be associated with the effects of TRAF2 in regulating TRAIL-induced apoptosis in prostate cancer cells via c-Flip/Caspase-8 signalling.

The coffee diterpene kahweol enhances sensitivity to sorafenib in human renal carcinoma Caki cells through down-regulation of Mcl-1 and c-FLIP expression.

Sorafenib is approved for the treatment of hepatocellular carcinoma (HCC) and advanced renal cell carcinoma (RCC). However, low tumor response and side effects have been widely reported. Therefore, to improve the efficacy of sorafenib, we investigated whether combined treatment with sorafenib and kahweol, the coffee-specific diterpene, has a synergistic effect on apoptotic cell death. Combined treatment with sorafenib and kahweol markedly induced caspase-mediated apoptosis in renal carcinoma Caki cells. Combined treatment with sorafenib and kahweol induced down-regulation of Mcl-1 and c-FLIP expression. We found down-regulation of Mcl-1 and c-FLIP expression was modulated by the ubiquitin-proteasome pathway. Ectopic expression of Mcl-1 inhibited sorafenib plus kahweol-induced apoptosis. Interestingly, combined treatment with sorafenib and kahweol induced apoptotic cell death in c-FLIP overexpressed cells. In addition, combined treatment with sorafenib and kahweol markedly induced apoptosis in human lung carcinoma (A549) and breast carcinoma (MDA-MB-361) cells, but not in human normal mesangial cells and human skin fibroblast cells (HSF). Collectively, our study demonstrates that combined treatment with sorafenib and kahweol induces apoptotic cell death through down-regulation of Mcl-1 expression.

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

Anti-Tumorigenic Effects of Resveratrol in Lung Cancer Cells Through Modulation of c-FLIP.

Resveratrol has been shown to have antioxidant and anti-proliferative properties in multiple cancer types. Here we demonstrate that H460 lung cancer cells are more susceptible to resveratrol treatment in comparison to human bronchial epithelial Beas-2B cells. Resveratrol decreases cell viability and proliferation, and induces significant apoptosis in H460 cells. The apoptosis observed was accompanied by an increase in hydrogen peroxide (H2O2) production, Bid, PARP and caspase 8 activation, and downregulation of pEGFR, pAkt, c-FLIP and NFkB protein expression. Furthermore, treatment with HH2O2 scavenger catalase significantly inhibited resveratrol-induced c-FLIP downregulation, caspase-8 activation and apoptosis. Overexpression of c-FLIP in H460 cells (FLIP cells) resulted in the inhibition of resveratrol-induced HH2O2 production, and a significant increase in resveratrolinduced apoptosis in comparison to H460 cells. In FLIP cells, catalase treatment did not rescue cells from a decrease in cell viability and apoptosis induction by resveratrol as compared to H460 cells. Resveratrol treatment also led to VEGF downregulation in FLIP cells. Furthermore, inhibition of pEGFR or pAkt using erlotinib and LY294002 respectively, enhanced the negative effect of resveratrol on FLIP cell viability and apoptosis. The reverse was observed when FLIP cells were supplemented with EGF, or transfected with WT-AKT plasmid; resulting in a 20% decrease in resveratrol-induced apoptosis. In addition, transfection with WT-AKT plasmid resulted in the inhibition of pro-apoptotic protein activation, and c-FLIP and pAkt downregulation. Overall, resveratrol induced apoptosis in H460 lung cancer cells by specifically targeting pAkt and c-FLIP dowregulation by proteasomal degradation in a EGFR-dependent manner.

MiR-126 reverses drug resistance to TRAIL through inhibiting the expression of c-FLIP in cervical cancer

TNF-related apoptosis-inducing ligand (TRAIL) represents one potential and ideal anti-tumor drug, because it kills cancer cells specifically without targeting normal cells. However, acquired drug resistance to TRAIL usually impedes the clinical use of TRAIL on cancer patients. In the present study, we established in vitro TRAIL-resistant cervical cancer cell lines through long-term exposure to TRAIL. Interestingly, we observed significant upregulation of c-FLIP in TRAIL-resistant Hela and SiHa cells (Hela-TR and SiHa-TR) compared to their parental Hela and SiHa cells. Although Hela-TR and SiHa-TR cells exhibited low-sensitivity to TRAIL treatment, knockdown of c-FLIP significantly increased the cytotoxicity of TRAIL to them. In contrast to high protein level of c-FLIP, expression of miR-126 was significantly downregulated in Hela-TR and SiHa-TR cells. Results of western blot analysis, luciferase assays and bioinformatics proved that c-FLIP was the target of miR-126. Furthermore, as c-FLIP is the cellular antagonist to caspase-8, transfection with miR-126 promoted the activation of caspase-8 induced by TRAIL. As a result, miR-126 increased the TRAIL-induced apoptosis in Hela-TR and SiHa-TR cells. In addition, miR-126 was also able to increase the cytotoxicity of TNF-α and FasL (caspase-8 inducers) to Hela-TR and SiHa-TR. We demonstrate that miR-126 impairs drug resistance to TRAIL, TNF-α and FasL through inhibiting the expression of c-FLIP in cervical cancer.

Plumbagin enhances TRAIL-induced apoptosis of human leukemic Kasumi‑1 cells through upregulation of TRAIL death receptor expression, activation of caspase-8 and inhibition of cFLIP.

Although the patients with t(8;21) acute myeloid leukemia (AML) have a favorable prognosis compared with other non-acute promyelocytic leukemia AML patients, only ~50% patients with this relatively favorable subtype can survive for 5years and refractory/relapse is common in clinical practice. So it is necessary to find novel agents to treat this type of AML. In this study, the effects and the mechanisms of plumbagin and recombinant soluble tumor necrosis factor‑α-related apoptosis-inducing ligand (rsTRAIL) on leukemic Kasumi‑1 cells were primarily investigated. Plumbagin and/or rsTRAIL could significantly inhibit the growth of Kasumi‑1 cells and induce apoptosis invitro and invivo. Plumbagin enhanced TRAIL-induced apoptosis of Kasumi‑1 cells in association with mitochondria damage, caspase activation, upregulation of death receptors (DRs) and decreased cFLIP expression. The effects of plumbagin on the expression of DR5, Bax and cFLIP could be partially abolished by the reactive oxygen species (ROS) scavenger NAC. Glutathione (GSH) depletion by plumbagin increased the production of ROS. Invivo, there was no obvious toxic pathologic change in the heart, liver and kidney tissues in any of the groups. Comparing with the control mice, a significantly increased number of apoptotic cells were observed in the combined treated mice by flow cytometry. Plumbagin also increased the expression of DR4 and DR5 in cells of xenograft tumors. Collectively, our results suggest that both plumbagin and rsTRAIL could be used as a single agent or synergistical agents to induce apoptosis of leukemic Kasumi‑1 cells invitro and in vivo.

Delayed neutrophil apoptosis is regulated by cancer associated a2 isoform vacuolar ATPase

Abstract Neutrophils are short life span cells but they can live for weeks in cancer tissues and promote tumor progression. Identifying cancer derived factors that enhance neutrophil survival will provide new targets for cancer therapy. A peptide from the N-terminal domain of a2- isoform vacuolar ATPase (a2NTD) is specifically secreted from cancer cells and promotes the neutrophil protumorigenic properties which in turn, enhance tumor progression. This gives a2NTD the propensity to regulate neutrophil survival. In fact the treatment of human neutrophils with recombinant a2NTD led to 2.1 fold increase in the percentage survival of neutrophils (P &amp;lt; 0.001). a2NTD treatment modulates different apoptosis pathways. Executive pathway; a2NTD treated neutrophils (a2Neuφ) showed a significantly decreased gene expression of caspase-3, -6, -7, that corresponded with decreased activity of these caspases. Intrinsic pathway; a2NTD treatment upregulates the gene expression of anti-apoptotic factors; Bcl2-A1, Bcl-xL and downregulates the pro-apoptotic factors; BAX and Apaf-1. Extrinsic pathway; a2NTD treatment dowenregulats the gene expression and the activity of caspase-8 and upregulates the gene expression of c-Flip. a2NTD treatment also stimulates reactive oxygen species generation that activate NF-kB p65 in neutrophils. Moreover, NF-kB inhibition negated that promoted neutrophil survival that was associated with downregulation of the gene expression of Bcl2-A1, Bcl-xL, G-CSF and upregulation of BAX. Thus, a2NTD enhances neutrophil survival by activating the NF-kB pathway in neutrophils. Together, these data demonstrate a novel role of the cancer associated a2-vacuolar ATPase on regulating neutrophil survival by the action of a2NTD.

Neem leaf glycoprotein prevents post-surgical sarcoma recurrence in Swiss mice by differentially regulating cytotoxic T and myeloid-derived suppressor cells.

Post-surgical tumor recurrence is a common problem in cancer treatment. In the present study, the role of neem leaf glycoprotein (NLGP), a novel immunomodulator, in prevention of post-surgical recurrence of solid sarcoma was examined. Data suggest that NLGP prevents tumor recurrence after surgical removal of sarcoma in Swiss mice and increases their tumor-free survival time. In NLGP-treated tumor-free mice, increased cytotoxic CD8+ T cells and a decreased population of suppressor cells, especially myeloid-derived suppressor cells (MDSCs) was observed. NLGP-treated CD8+ T cells showed greater cytotoxicity towards tumor-derived MDSCs and supernatants from the same CD8+ T cell culture caused upregulation of FasR and downregulation of cFLIP in MDSCs. To elucidate the role of CD8+ T cells, specifically in association with the downregulation in MDSCs, CD8+ T cells were depleted in vivo before NLGP immunization in surgically tumor removed mice and tumor recurrence was noted. These mice also exhibited increased MDSCs along with decreased levels of Caspase 3, Caspase 8 and increased cFLIP expression. In conclusion, it can be stated that NLGP, by activating CD8+ T cells, down regulates the proportion of MDSCs. Accordingly, suppressive effects of MDSCs on CD8+ T cells are minimized and optimum immune surveillance in tumor hosts is maintained to eliminate the residual tumor mass appearing during recurrence.