title: Protein Kinase RNA-Like Endoplasmic Reticulum Kinase-Mediated Bcl-2 Protein Phosphorylation Contributes to Evodiamine-Induced Apoptosis of Human Renal Cell Carcinoma Cells.
abstract: We investigated the anticancer mechanism of evodiamine (EVO) against the viability of human A498 renal cell carcinoma (RCC) cells in vitro and in vivo. The in vitro study showed that EVO decreased the viability of A498 cells with the occurrence of apoptotic characteristics such as hypodiploid cells, DNA ladders, chromatin-condensed cells, and cleaved caspase (Casp)-3/poly(ADP ribose) polymerase (PARP) proteins. Pharmacological studies using chemical inhibitors of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) indicated that phosphorylation of the c-Jun N-terminal kinase (JNK) protein participated in EVO-induced cell death of A498 cells, and application of the JNK inhibitor, SP600125 (SP), inhibited EVO-induced cleavage of the Casp-3/PARP proteins and chromatin condensation according to Giemsa staining. EVO disruption of the mitochondrial membrane potential (MMP) with increased protein levels of the phosphorylated Bcl-2 protein (p-Bcl-2) was prevented by JNK inhibitors in A498 cells. A structure-activity relationship study showed that a methyl group at position 14 in EVO was important for its apoptotic effects and increased p-Bcl-2 protein in A498 cells. Furthermore, significant increases in the phosphorylated endoplasmic reticular stress protein, protein kinase RNA-like endoplasmic reticulum kinase (p-PERK at Thr980), by EVO were detected in A498 cells, and the PERK inhibitor, GSK2606414, significantly suppressed EVO-induced apoptosis, p-JNK, p-PERK, and cleaved PARP proteins. The in vivo study showed that EVO significantly reduced RCC growth elicited by a subcutaneous injection of A498 cells, and an increased protein level of p-PERK was observed according to an immunohistochemical analysis. Apoptosis by EVO was also demonstrated in other RCC cells such as 786-O, ACHN, and Caki-1 cells. This is the first study to demonstrate the anti-RCC effect of EVO via apoptosis in vitro and in vivo, and activation of JNK and PERK to induce Bcl-2 protein phosphorylation, which led to disruption of the MMP.
authors.affiliation: Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, 110, Taiwan.|Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 110, Taiwan.
authors.forename: Wen-Shin
authors.initials: WS
authors.lastname: Wu
authors.affiliation: Department of Nephrology, Chi-Mei Medical Center, Tainan, Taiwan.|Department of Food Nutrition, Chung Hwa University of Medical Technology, Tainan, Taiwan.
authors.forename: Chih-Chiang
authors.initials: CC
authors.lastname: Chien
authors.affiliation: Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, 110, Taiwan.|Cancer Research Center and Orthopedics Research Center, Taipei Medical University Hospital, Taipei, 110, Taiwan.
authors.forename: Yen-Chou
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Neurosurgery, Taipei Municipal Wan-Fang Hospital and Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, 110, Taiwan.
authors.forename: Wen-Ta
authors.initials: WT
authors.lastname: Chiu
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008819:Mice, Nude; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays; D019892:eIF-2 Kinase / Q000235:genetics* / Q000378:metabolism
chemical_list: D000225:Adenine; D000873:Anthracenes; D000972:Antineoplastic Agents, Phytogenic; D053148:Caspase 3; D007211:Indoles; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D011065:Poly(ADP-ribose) Polymerases; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011799:Quinazolines; D019892:eIF-2 Kinase; C576403:7-methyl-5-(1-((3-(trifluoromethyl)phenyl)acetyl)-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo(2,3-d)pyrimidin-4-amine; C052617:Bcl2 protein, mouse; C432165:pyrazolanthrone; C049639:evodiamine; C505409:Casp3 protein, mouse