水飞蓟宾抑制缺氧状态下胃癌细胞mTOR磷酸化及HIF-1α表达

Silibinin inhibits mTOR phosphorylation and HIF-1α expression in gastric carcinoma cells under hypoxic condition

目的:探讨水飞蓟宾对胃癌细胞缺氧诱导因子1α(HIF-1α)表达的影响及其可能的分子机制。方法:低氧条件下体外培养胃癌细胞系MGC803,用不同浓度水飞蓟宾处理后,分别采用Real-time PCR和Western blotting法检测HIF-1αmRNA、蛋白的表达以及mTOR和Akt的磷酸化水平,MTT法检测水飞蓟宾对MGC803细胞增殖的影响。结果:细胞低氧状态下生长4 h后,与正常氧浓度组相比,细胞内HIF-1α蛋白表达水平显著增高[(0.94±0.16)vs(0.015±0.03),P<0.01)];经250μmol/L水飞蓟宾处理后,与处理前相比,HIF-1α蛋白表达水平明显减少[(0.24±0.09)vs(0.94±0.16),P<0.01]。与正常氧浓度组相比,低氧并不能增加HIF-1αmRNA的表达[(0.074±0.011)vs(0.07±0.02),P>0.05],即便加入250μmol/L水飞蓟宾处理对HIF-1αmRNA的表达也无明显影响[(0.081±0.011)vs(0.07±0.02),P>0.05],但水飞蓟宾能显著增加泛素化HIF-1α蛋白的表达[(0.94±0.16)vs(0.24±0.09),P<0.01];水飞蓟宾处理后能明显抑制mTOR磷酸化水平[(0.17±0.06)vs(0.53±0.14),P<0.05)],并能明显抑制MGC803细胞的增殖[(52.94±6.15)vs(100±3.22),P<0.05)]。与处理前相比,50和100μmol/L水飞蓟宾处理对Akt磷酸化无明显影响[(0.16±0.09)、(0.17±0.06)vs(0.11±0.04),P>0.05],但250μmol/L水飞蓟宾处理后,细胞内Akt磷酸化水平明显增强[(0.33±0.06)vs(0.11±0.04),P<0.05]。结论:水飞蓟宾可能通过影响mTOR和HIF-1α的表达水平而发挥抗肿瘤活性。

Objective： To investigate the effect of Silibinin on hypoxia inducible factor-1α （HIF-1α） expression as well as its possible molecular mechanism. Methods：Gastric carcinoma cell line MGC803 was cultured in vitro under hypoxic condition. After treated with different concentration of Silibinin, the expression of HIF-1α mRNA was detected by Real- time PCR ; HIF-1α protein level and phosphorylation of mTOR and Akt were detected by Western blotting. Effect of Silibi- nin on proliferation of MGC803 cell was analyzed by MTT assay. Results ： After 4 h of incubation under hypoxia condition, the expression of HIF-1α protein was significantly increased in MGC803 cells, as compared with the normoxia group （ [ 0. 94± 0.16 ] vs [ 0. 015 ± 0.03 ], P 〈 0.01 ）. After treatment with 250μmol/L Silibinin, the expression of HIF-1 ot protein was significantly inhibited as compared to pre-treatment （ [ 0.24 ± 0.09 ] vs [ 0.94 ± 0.16 ], P 〈 0.01 ）. As com- pare with normoxia group, hypoxia couldn＇ t increase expression of HIF-1α mRNA （ [0. 074±0. 011 ] vs [0.07 ±0.02], P 〉 0. 05 ）, even though treatment with 250 μmol/L aqueous Silibinin also couldn＇ t significantly affect expression of HIF- 1 a mRNA （ [ 0. 081 ± 0.011 ] vs [0.07 ± 0.02 ], P 〉 0.05 ） , but Silibinin could significantly increase the expression of ubiquitinated HIF-1α protein （ [0.94 ±0.16] vs [0.24 ±0.09], P 〈0.01 ）. After incubation with Silibinin, phospho-rylation of roTOR was significantly inhibited （ [ 0. 17 ＋ 0.06 ） vs [ 0.53 ＋ 0. 141, P 〈 0.05 ）, and proliferation of MGC803 cell was significantly inhibited （ [ 52.94 ± 6.15 ] vs [ 100 ± 3.2 ], P 〈 0.05 ）, as compared with pre-treatment. In contrast, 50 and 100 μmol/L Silibinin had no obvious effect on phosphorylation of Akt （ [ O. 16 ± 0. 09 ], [ 0.17 ± 0. 06 ] vs [0.11 ± 0.04 ], P 〈 0.05 ）, but 250 μmol/L Silibinin could significantly increase the phosphorylation of Akt in the cells （ [0.33 ±0.06] vs [0.11 ±0.04], P 〈0.05）. Conelusion：Silibinin could exert antitumor action through to effect on expression of mTOR and HIF-1α.