| Summary: | 碩士 === 中國醫藥大學 === 臨床醫學研究所碩士班 === 98 === Background: Colorectal cancer is one of the most prevalent solid organ cancers in developed countries. Approximately 1 million cases are recorded every year worldwide, and over half a million patients die of this disease yearly. The natural phloroglucinol has been reported to exhibit multiple functions resulting in anti-tumoral effects. However, its effect on human colon carcinoma cells has never been scrutinized. In this study, the mechanisms underlying the anti-cancer effect of a new phloroglucinol derivative, 3-monochlorophenyl phloroglucinol (MCPP) in human colon carcinoma cells were studied.
Methods: Several derivatives of phloroglucinol were synthesized. MTT and SRB assays (for cell viability) were used to screen the synthesized phloroglucinol derivatives. MCPP was found to have the most obvious anti-tumor effect. The cell cycle and sub-G1 accumulation were evaluated by PI flowcytometry. The percentage of apoptotic cells was quantified by Annexin V-PI double labeling. Also, the percentage of apoptotic cells was determined by TUNEL method. The expression level of cleaved PARP was assessed by Western blotting. Bcl-2 family proteins and the associated endoplasmic recticulum stress response proteins (GRP78, GRP94, p-eIF2α, CHOP and GSK3α/β) were evaluated by Western blotting. With reverse transcription polymerase chain reaction (RT-PCR), the mRNA expression of target genes (GRP78 and CHOP) was quantified. Finally, with siRNA technology, HCT-116 cells were transfecetd with indicated siRNA (siRNA against GRP78 or CHOP) for further evaluation.
Results: MCPP inhibited HCT-116 cell growth in a concentration- and time-dependent manner. The inhibitory effects of HCT-116 cells were correlated with the arresting cell cycle at G0/G1 phase. Annexin V-PI double labeling also demonstrated the induction of cell apoptosis by MCPP treatment. With MCPP treatment, TUNEL method also revealed the induction of cell apoptosis. Cleaved-PARP level was also elevated after MCPP treatment. Treatment of HCT-116 human colon carcinoma cells with MCPP was found to induce pro-apoptotic member of Bcl-2 family proteins: Bax and Bak. Treatment of HCT-116 human colon carcinoma cells with MCPP was found to decrease the anti-apoptotic member of Bcl-2 family proteins: Bcl-xL and Bcl-2. The changed ratio of Bcl-2 family of proteins shifted the cells from pro-survival to pro-apoptotic pathway. Treatment of HCT-116 human colon carcinoma cells with MCPP was also found to induce a number of signature ER stress markers: phosphorylation of eukaryotic initiation factor-2α(eIF-2α), up-regulation of glucose-regulated protein (GRP)-78, and up-regulation of CCAAT / enhancer-binding protein homologous protein (CHOP). MCPP also induced activation of caspase-3, caspase-7, and caspase-9. Furthermore, transfection of cells with GRP78 or CHOP small interfering RNA (siRNA) attenuated MCPP-mediated cell death. GSK3βinhibitor also reduced MCPP-mediated cell apoptosis.
Conclusion: Taken together, the present study thus provides evidence to support an important role of ER stress response in mediating the MCPP-induced human colon cancer cell apoptosis. Thus, our findings reveal that MCPP might be a potential, new chemopreventive agent targeting human colon cancer cells.
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