CHOP down-regulates cFLIP(L) expression by promoting ubiquitin/proteasome-mediated cFLIP(L) degradation.

The transcription factor CHOP/GADD153 is induced during the unfolded protein response and is related to the induction of ER stress-mediated apoptosis. However, how CHOP is organized between the pro-survival and pro-apoptotic roles of ER stress remains largely undefined. In this study, we identified the apoptosis regulating protein suppressed by CHOP. We found that treatment of Caki cells with CHOP-inducing drugs including withaferin A, thapsigargin, brefeldin A, and silybin led to a strong reduction in cFLIP(L) protein levels together with a concomitant increase in the CHOP protein. Interestingly, Wit A down-regulated cFLIP(L) expression via both suppressing mRNA transcription and increasing cFLIPL protein instability. We also found that forced expression of CHOP dose-dependently led to a decrease of cFLIP(L) protein expression but did not alter cFLIP(L) mRNA levels. Additionally, we observed that siRNA-mediated CHOP silencing recovered the cFLIP(L) expression decreased by CHOP-inducing agents in Caki cells. Finally, we showed that CHOP facilitates ubiquitin/proteasome-mediated cFLIP(L) degradation, leading to down-regulation of cFLIP(L). Finally, cFLIP(L) over-expression reduced cell death induced by treatment with brefeldin A, thapsigargin, and silybin. Taken together, our results provide novel evidence that cFLIP(L) is a CHOP control target and that CHOP-induced down-regulation of cFLIP(L) is due to activation of the ubiquitin/proteasome pathways.

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.

Suppression of phorbol-12-myristate-13-acetate-induced tumor cell invasion by apigenin via the inhibition of p38 mitogen-activated protein kinase-dependent matrix metalloproteinase-9 expression.

Apigenin has special interest for the development of chemopreventive agents against cancer because it is a widely distributed plant flavonoid that has antitumor properties. In this study, we investigated the apigenin effects on the protease-mediated invasiveness in human metastatic cancer cell lines Caski, SK-Hep1, and MDA-231. We found that apigenin markedly inhibits the phorbol-12-myristate-13-acetate (PMA)-induced increase in MMP-9 expression and activity in several cancer cell lines. These effects of apigenin are dose-dependent and correlate with the suppression of MMP-9 mRNA expression levels. PMA caused about a 5-fold induction in MMP-9 promoter activity, which was also suppressed by apigenin treatment in Caski cells. We found that apigenin could inhibit PMA-induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was involved in the down-regulation of the expression of matrix metalloproteinase-9 (MMP-9) at mRNA levels. Furthermore, the treatment of inhibitors specific for p38 MAPK (SB203580) to Caski cells caused the reduction of MMP-9 expression. Restoration of p38 expression partly increased PMA-induced MMP-9 secretion blocked by apigenin treatment in Caski cells. These results showed apigenin might inhibit the invasion and migration abilities of Caski cells by reducing the MMP-9 expression through suppressing the p38 MAPK signaling pathway. These findings indicate that apigenin might be a useful strategy for controlling metastasis and the invasiveness of tumors.

Overexpression of Par-4 sensitizes TRAIL-induced apoptosis via inactivation of NF-kappaB and Akt signaling pathways in renal cancer cells.

The prostate-apoptosis-response-gene-4 (Par-4) is up-regulated in prostate cells undergoing programmed cell death. Furthermore, Par-4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. In this study, we investigated how Par-4 modulates TRAIL-mediated apoptosis in TRAIL-resistant Caki cells. Par-4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par-4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase-8 and the effector caspase-3, together with an enforced cleavage of XIAP and c-FLIP. TRAIL-induced reduction of XIAP and c-FLIP protein levels in Par-4 overexpressing cells was prevented by z-VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL-treated Par-4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par-4 recovered Bcl-2 level to basal level induced by wild type Par-4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par-4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par-4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl-2, Akt, and NF-kappaB.