Cell proliferation was inhibited obviously when c-FLIP expression

Cell proliferation was inhibited obviously when c-FLIP expression was knocked down by siRNA. Our data showed that si-526-siRNA significantly decreased the growth rate of 7721 cells, with a >50% decrease after 3 days repeatedly in three separate experiments (Figure.

4). Figure 4 Cell viability was accessed by cell counting. The study showed that 7721 cell viability was reduced by the transfetion CYC202 chemical structure with recombinant iRNA vectors. pSuper-Si1 had more significant effect on the reduction of the cell viability. Then, the cells were assayed by the TUNEL method to assess the drug-LB-100 chemical structure induced apoptosis. Positive TUNEL staining would be indicative of the DNA fragmentation that was characteristic of apoptosis. Without c-FLIP RNAi, the fewer 7721 cells were TUNEL positive. In contrast, in cells transfected with the specific siRNA vector, pSuper-Si1, the apoptosis induced by treatment with doxorubicin was significantly elevated (Figure. 5).

Figure 5 Cells were assayed Alisertib for apoptosis by the TUNEL method and photographed by fluorescence microscopy at ×100. Green cells are positive for DNA fragmentation, consistent with apoptosis. A: 7721/pSuper-Neg; B: 7721/pSuper-Si1. Discussion Tumor cells have developed different ways to escape apoptosis induced by DR-triggering such as surface DR down-regulation, loss or mutation. Other mechanisms elaborated by tumor cells to develop cell death resistance include aberrant expression of anti-apoptotic molecules such as c-FLIP, Bcl-2, Bcl-xL, survivin and Livin. The current belief holds that perturbations in apoptotic death regulation

constitute a vital step in cancer evolution [17]. Each step in DR-mediated apoptosis is well regulated. c-FLIP is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs). Furthermore, c-FLIP over-expression can activate nuclear factor (NF)-κB activation induced by TNF-α or TRAIL. c-FLIP has a more Cobimetinib ic50 central role in the antiapoptotic NF-kB response than the TRAF/IAP complex. On the other hand, c-FLIP expression is regulated by NF-κB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. So, c-FLIP plays an important role in cell survival not simply by inhibiting DR-mediated apoptosis but also by regulating NF-κB activation in human HCCs [10, 18]. Moreover, c-FLIP has recently been shown to be associated with the generation of positive signals for cell proliferation by activation of the Erk pathway through Raf-1 binding [19, 20]. There is increasing evidence that in regard to its anti-apoptotic functions, c-FLIP can be considered as a tumor-progression factor. At present, the role of c-FLIP, as an anti-apoptotic protein involved in the regulation of the DR extrinsic apoptotic pathway, remains unclear.

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