关键词: 一叶秋碱, 结肠肿瘤, 细胞凋亡, 细胞增殖, 活性氧

Abstract:

Objective To investigate the effect of securinine （SEC） on apoptosis of the human colon cancer cell line SW620， and to elucidate its possible mechanism. Methods The nude mice with subcutaneously transplanted tumor were divided into control group （n=6）， oxaliplatin （OXA） group （n=7）， and SEC group（n=7）. The volume and mass of subcutaneous tumors in the nude mice were measured in various groups， and the tumor inhibitory rates in various groups were calculated. The SW620 cells were treated with different doses （5-120 μmol·L^-1） of SEC for 12， 24， 48， and 72 h， respectively. Cell counting kit-8 （CCK-8） assay was used to assess the survival rates of cells in various groups， and the optimal doses of SEC were confirmed. The SW620 cells were divided into control group， 20 μmol·L^-1 SEC group， 40 μmol·L^-1 SEC group， and 40 μmol·L^-1 OXA group. TUNEL staining method and flow cytometry were used to detect the apoptotic rates of cells in various groups. JC-1 staining was used to detect the mitochondrial membrane potentials of cells in various groups， and 2'，7'-dichlorodi-hydrofluorescin diacetate （DCFH-DA） fluorescence staining and flow cytometry were used to measure the reactive oxygen species （ROS） levels in the cells in various groups. Western blotting method was used to detect the expression levels of cytochrome C， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， c-Jun N-terminal kinase （JNK）， phosphorylated JNK （p-JNK）， mitogen-activated protein kinase p38， phosphorylated p38 （p-p38）， extracellular signal-regulated kinase （ERK） and phosphorylated ERK （p-ERK） proteins in the cells in various groups. Results Compared with control group， the volume and mass of subcutaneously transplanted tumors in the nude mice in SEC group were significantly decreased （P<0.05 or P<0.01 or P<0.001）； the inhibitory rates of tumor in SEC group and OXA group were 20.42% and 6.50%. The CCK-8 assay results showed that compared with 0 μmol·L^-1 SEC， when the SEC dose exceeded 20 μmol·L^-1， the survival rates of SW620 cells were significantly decreased （P<0.001）. The optimal condition for subsequent experiments was set as doses of 20 μmol·L^-1 SEC and 40 μmol·L^-1 SEC， and duration of 24 h. The TUNEL results showed that compared with control group， the apoptotic rates of cells in 20 and 40 μmol·L^-1 SEC groups were significantly increased （P<0.05 or P<0.001）. The results of flow cytometry showed that compared with control group， the apoptotic rate in 40 μmol·L^-1 SEC group was significantly increased （P<0.001）. The JC-1 staining results showed that compared with control group， the mitochondrial membrane potentials of cells in 20 and 40 μmol·L^-1 SEC groups were significantly decreased （P<0.001）. Compared with control group， the levels of ROS detected by DCFH-DA fluorescence staining in the cells of 20 and 40 μmol·L^-1 SEC groups and 40 μmol·L^-1 OXA group were significantly increased （P<0.001）， while the level of ROS detected by flow cytometry in 40 μmol·L^-1 SEC group was significantly increased （P<0.05）. Compared with control group， the expression levels of Bcl-2 protein in the cells in 20 and 40 μmol·L^-1 SEC groups and 40 μmol·L^-1 OXA group were decreased （P<0.01）， while the expression levels of cytochrome C and Bax proteins were increased （P<0.001）.Compared with control group， the ratios of p-JNK/JNK， p-p38/p38 and p-ERK/ERK in 20 and 40 μmol·L^-1 SEC groups were significantly increased （P<0.05 or P<0.01 or P<0.001）. Conclusion SEC can inhibit the proliferation of SW620 cells， increase the cellular ROS levels， reduce the mitochondrial membrane potential， and induce the mitochondrial apoptosis； its mechanism may be related to the regulation of the mitogen-activated protein kinase（MAPK） signaling pathway.

Key words: Securinine, Colon neoplasms, Apoptosis, Cell proliferation, Reactive oxygen species