ABSTRACT
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.
Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Gene Expression Regulation, Neoplastic , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Transcription Factor CHOP/metabolism , Ubiquitin/metabolism , Apoptosis , Blotting, Western , Brefeldin A/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Immunoprecipitation , Leupeptins/pharmacology , Proteasome Inhibitors/pharmacology , Protein Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Silybin , Silymarin/pharmacology , Thapsigargin/pharmacology , Transcription Factor CHOP/genetics , Transcription, Genetic , Transfection , Ubiquitination , Unfolded Protein Response , Withanolides/pharmacologyABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Berberine/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Kidney Neoplasms/pathology , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Blotting, Western , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation , Flow Cytometry , Humans , Kidney Neoplasms/drug therapy , Kidney Neoplasms/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Proteasome Inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand/geneticsABSTRACT
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.
Subject(s)
Apigenin/pharmacology , Matrix Metalloproteinase 9/genetics , Neoplasms/pathology , Tetradecanoylphorbol Acetate/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Cell Movement/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Evaluation, Preclinical , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/metabolism , Phosphorylation/drug effects , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
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.
