乏氧诱导CDK6与SUMO1解离对鼻咽癌化疗抵抗的作用

Hypoxia increases chemotherapy resistance in nasopharyngeal carcinoma via inducing CDK6 deSUMOylation

目的通过蛋白质类泛素化修饰角度解读乏氧条件下鼻咽癌化疗抵抗的新机制。方法氯化钴(CoCl2)化学诱导法建立人鼻咽癌CNE1细胞乏氧模型,流式细胞术检测细胞周期变化,Western blot法检测小泛素相关修饰蛋白(SUMO)1和细胞周期蛋白依赖激酶6(CDK6)蛋白表达水平;四甲基偶氮唑盐比色法(MTT)检测顺铂对CNE1细胞的半数致死浓度(LC50),酶联免疫吸附实验(ELISA)测定乳酸脱氢酶(LDH)含量,流式细胞术检测细胞凋亡情况。两组间比较采用t检验。结果乏氧致CNE1细胞周期阻滞于G0/G1期。Western blot结果显示,乏氧组鼻咽癌细胞中CDK6的蛋白表达水平低于对照组(0.43±0.21比0.83±0.25,t=14.67,P=0.003),共价结合状态的SUMO1的蛋白水平明显低于对照组(1.38±0.31比2.69±0.48,t=17.22,P=0.001),而游离状态的SUMO1蛋白水平明显高于对照组(2.60±0.59比2.01±0.43,t=15.45,P=0.002)。顺铂对对照组CNE1细胞的LC50明显低于乏氧组(29.44 μg/ml比97.72 μg/ml,t=12.79,P=0.001)。CNE1细胞接受50 μg/ml的顺铂作用48 h后,对照组上清液中LDH含量明显高于乏氧组[(541.49±64.59)ng/ml比(234.67±41.03)ng/ml,t=11.94,P=0.007]。对照组CNE1细胞的细胞凋亡率明显高于乏氧组(76.64%±5.37%比32.84%±4.77%,t=8.49,P=0.003)。结论乏氧能够解离CDK6与SUMO1的共价修饰,抑制细胞周期,增加鼻咽癌化疗抵抗水平。

Objective To understand the mechanism of chemotherapy resistance in nasopharyngeal carcinoma under hypoxic conditions through the perspective of protein SUMOylation modification. Methods Cobalt chloride (CoCl2) was used to establish the hypoxic model of human nasopharyngeal carcinoma CNE1 cells. Then, the cell cycle was detected by flow cytometry, and the expression level of small ubiquitin-related modifier(SUMO) and cyclin-dependent kinase 6 (CDK6) proteins were detected by western blotting. MTT assay was used to determine the median lethal dose (IC50) of cancer cells against cisplatin, and enzyme-linked immunosorbent assay (ELISA) was used to determine lactate dehydrogenase (LDH) level. Results The cell cycle of CNE1 induced by hypoxia was arrested in G0/G1 phase.The results of Western blot showed that the protein expression level of CDK6 in CNE1 cells was lower than that in the control group (0.83±0.25 vs. 0.43±0.21, t=14.67, P=0.003). The protein level of conjugated SUMO1 was significantly lower than that in the control group (2.69±0.48 vs. 1.38±0.31, t=17.22, P=0.001), while the level of free SUMO1 protein was significantly higher than that in the control group (2.01±0.43 vs. 2.60±0.59, t=15.45, P=0.002).The LC50 of CNE1 cells in the control group was significantly lower than that in the hypoxic group (29.44 μg/ml vs. 97.72 μg/ml, t=12.79, P=0.001). After CNE1 cells received 50 μg/ml cisplatin for 48 h, the LDH content in the supernatant of the control group was significantly higher than that in the hypoxic group ((541.49±64.59) ng/ml vs.(234.67±41.03) ng/ml, t=11.94, P=0.007)). The apoptosis rate of CNE1 cells in the control group was significantly higher than that in the hypoxic group ((76.64±5.37)% vs.(32.84±4.77) ng/ml, t=8.49, P=0.003)). Conclusion Hypoxia can dissociate the covalent modification of CDK6 and SUMO1, inhibit cell cycle and increase the chemotherapy resistance of nasopharyngeal carcinoma.