Preliminary study on the role and mechanism of cells viability, invasive ability and induction of apoptosis of giant cell tumor cells in vitro by norcantharidin
Wang Jin, Chen Feiyan, Xia Jun, Wang Siqun, Wei Yibing, Huang Gangyong, Chen Jie, Shi Jingsheng
Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai 200040, China
Abstract：Objective To investigate the role of norcantharidin (NCTD) in cells viability, invasive ability and inducing apoptosis of giant cell tumor of bone (GCTB) in vitro and its related mechanisms.Methods Three cases of intraoperative tumor tissue specimens of GCTB (Campanacci Grade Ⅱ) patients in Huashan hospital were collected from January 2018 to March 2019, including 2 males (35 years old and 40 years old) and 1 female (27 years old). The primary GCTB cells were isolated, cultured and identified in vitro. The GCTB cells were divided into negative control group (0 μg/mL) and NCTD intervention group (200 μL of 5, 10, 20, 40 μg/mL NCTD solution was added in each well). After 24 h intervention, the cell morphology of each group was observed under microscope and the cells viability of each group were detected using CCK-8. Then GCTB cells were divided into control group, 5 μg/mL NCTF group and 20 μg/mL NCTD group. Transwell assay was used to detect cell invasive ability of each group. Flow cytometry was applied to evaluate the apoptosis rate of each group. The enzyme activity of caspase-3/cleaved-caspase-3 and caspase-9/cleaved caspase-9 were detected by commercial kit. JC-1 and DHE staining was used to assess the mitochondrial membrane potential and the level reactive oxygen species (ROS) in GCTB cells. Finally, the expression of apoptosis-related proteins cleaved caspase3, caspase-3, cleaved caspase9, caspase-9, Bcl-2-associatedx(Bax) and B-cell leukemia-lymphoma(Bcl-2) were measured by western blot.Results The GCTB tissue samples were isolated and cultured in vitro. Most of cells were irregular stromal cells after passaging. The expression of CD68 around the nucleus were positive, suggesting that the cells were giant cell tumor cells. Under microscopic observation, the cells shrank, suspended and gradually 25 lost their original morphology with the increase of NCTD concentration. CCK-8 results showed that the survival rates of GCTB cells in each group (5,10,20,40 μg/mL) were 100%,76.45%±9.86%, 62.34%±10.21%, 39.74%±8.36%, 25.54%±7.18%, respectively. The cell survival rate decreased gradually with the increase of the NCTD concentration, and the difference was statistically significant(F=75.716, P<0.05). Transwell test showed that the proportion of invasive cells in each group was 49.33%±9.84%, 32.84%±2.73%, 8.34%±1.41%, respectively (F=36.034, P＜0.01). There were significantly different apoptosis rates among the control group, 5 μg/mL and 20 μg/mL group (4.73%±0.05%, 14.23%±0.85%, 51.21%±10.47%, respectively, F=49.183, P＜0.01). The activities of cleaved-caspase-3/9 were gradually increased from the control group to 20 μg/mL NCTD group, and the difference was statistically significant (F=15.223, 30.841, all P values＜0.01). The level of ROS level in GCTB cells was significantly increased with the increase of NCTD concentration in the control group, 5μg/mL NCTD group and 20 μg/mL NCTD group. The mitochondrial membrane potential levels were detected in all of three groups. The values in control group, 5, 20 μg/mL NCTD group were 2.01±0.11, 1.27±0.07 and 0.79±0.14, respectively (F=128.641, P＜0.01). The results of Western blot suggested that the relative expression of cleaved-caspase-3/9 and Bax increased, while the expression of Bcl-2 was decreased with the increase of NCTD concentration. There was a statistically significant difference between three groups (all P values＜0.01). However, there was no significant change in the expression level of caspase-3 and caspase-9 (all P values>0.05).Conclusions NCTD can significantly inhibit the activity of GCTB cells in vitro, increase cell invasion and promote the apoptosis of GCTB cells. Its mechanism may be related to reducing the mitochondrial membrane potential, increasing the level of cellular reactive oxygen species, and regulating the expression of apoptosis-related proteins.
王进, 陈飞雁, 夏军, 王思群, 魏亦兵, 黄钢勇, 陈杰, 石晶晟. 去甲斑蝥素体外对骨巨细胞瘤细胞的活性、侵袭能力及凋亡的作用及其机制初探[J]. 中华解剖与临床杂志, 2020, 25(4): 411-420.
Wang Jin, Chen Feiyan, Xia Jun, Wang Siqun, Wei Yibing, Huang Gangyong, Chen Jie, Shi Jingsheng. Preliminary study on the role and mechanism of cells viability, invasive ability and induction of apoptosis of giant cell tumor cells in vitro by norcantharidin. Chinese Journal of Anatomy and Clinics, 2020, 25(4): 411-420.
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2020, Vol. 25 
