Role of Er Stress in Evodiamine-Induced Apoptosis and G2/M Arrest of Human Colorectal Carcinoma Cells

Colorectal carcinoma is one of the major leading causes of cancer mortality, as westernized diet, morbidity of colorectal carcinoma were increased. Evodiamin (EVO) derived from Chinese herb Evodiae fructus showed a significant cytotoxicity in cancer cells, however the anti-tumor mechanism of EVO is still unclear. Data from our preliminary study showed that EVO at the concentration of 1 ❍M effectively reduced the viability of colorectal carcinoma cells COLO205& HT-29 with an occurrence of apoptotic characteristics such as DNA ladders, chromatin condensation, and hypodiploid cells. Induction of caspase 3&9 protein cleavage, disruption of mitochondria membrane potential with increased cytosolic cytochrome c, AIF protein were detected in EVO-treated cells. Additionally, EVO stimulation significantly decreased the ratio of G1 phase and increased the ratio of G2/M phages via flow cytometry analysis in according with increases in Cyclin B1 and CDC25 protein expression in both colorectal carcinoma cells. Induction of ER stress-related proteins such as CHOP, PERK, eIF2α by EVO was detected in COLO205 and HT-29 cells. These data support the notion that EVO at the concentration of 1 mM induced ER stress, G2/M arrest, and apoptosis in colorectal carcinoma cells, however it is still unclear how ER stress contributes to EVO-induced G2/M arrest and apoptosis of colon carcinoma cells. In the present project, we will investigate the roles of ER stress and its related proteins including CHOP, PERK, eIF2α in EVO-induced G2/M arrest and apoptosis of colon carcinoma cells in vitro and in vivo. Specific Aim 1: To define the role of ER stress and its related proteins CHOP、 PERK、EIF2α in EVO-induced apoptosis of human colorectal carcinoma cells Hypothesis 1: ER stress participates in EVO-induced apoptosis of colorectal carcinoma cells 1.1. To identify the apoptotic mechanism of EVO against the viability of human colorectal carcinoma cells 1.2. To elucidate the role of reactive oxygen species (ROS) on EVO-induced ER stress and apoptosis in human colorectal carcinoma cells 1.3. To define the contribution of kinases cascade (MAPK&PI3K) to EVO-induced ER stress and apoptosis 1.4. Using MiRNA knockdown technique to investigate the role of ER stress genes such as CHOP、PERK、eIF2α on EVO-induced apoptosis in colorectal carcinoma cells 1.5. Overexpression of ER stress genes CHOP、PERK、EIF2α to verify their roles in EVO-induced apoptosis 1.6. Structure-activity relationship (SAR) study: to study the apoptotic effects of 12 structure-related chemicals of EVO (EVO1--EVO12) on human colorectal carcinoma cells Specific Aim 2 (1st and 2nd year): To define the role of ER stress and its related proteins CHOP、PERK、EIF2α on EVO-induced G2/M arrest in human colorectal carcinoma cells Hypothesis 2: Activation of ER stress contributes to EVO-induced G2/M arrest in human colorectal carcinoma cells 2.1. To define the mechanism of EVO-induced G2/M arrest in human colorectal carcinoma cells 2.2. To identify the role of ROS 與 kinases cascade (MAPK&PI3K) on EVO-induced G2/M arrest and cyclin B1/CDC25 protein expression in human colorectal carcinoma cells 2.3. Using miRNA knockdown or transfection of indicated expression vectors to investigate the role of ER stress genes CHOP、PERK、EIF2α on EVO-induced G2/M arrest and Cyclin B1/CDC25protein expression 2.4. To study if Ca+2 elevation contributes to EVO-induced G2/M arrest and apoptosis 2.5. SAR study: To examine the alternative activities of EVO-related chemicals (EVO1--EVO12) on the cell cycle progression of colorectal carcinoma cells Specific Aim 3 (3rd year): To confirm ER stress in EVO-inhibited colorectal carcinoma formation in a mouse model Hypothesis 3: EVO-inhibited colorectal carcinoma formation via ER stress in vivo 3.1. The in vivo animal model for studying colorectal carcinoma formation has been established in our lab. This model will be applied in the present project to study the in vivo role of ER stress on EVO-inhibited tumor growth。 3.2. To examine the growth of colorectal carcinoma cells in vivo in the presence or absence of EVO stimulation via IVIS 2000 systems. 3.3. To detect the protein expression of apoptosis (Caspases, PARP, Bcl-2 family)-, ER stress (CHOP, PERK, eRF2a)-, E2/M arrest (Cyclin B1, CDC25)-related genes in the tumor tissues by immunohistochemistry. 3.4.To establish stable ER stress genes (CHOP、PERK、eIF2α) knockdown COLO205 cells via miRNAs transfection, followed by in vivo study to verify the role of ER stress in EVO-inhibited tumor growth in vivo. 3.5. To examine the differential in vivo effects of 12 structure-related compounds of EVO (EVO1--EVO12) on the growth of colorectal carcinoma cells.