依维莫司对顺铂产生抗人皮肤鳞状细胞癌COLO-16细胞效应的增效作用研究

Synergistic effect of everolimus on cisplatin-mediated effect against human cutaneous squamous ceil carcinoma COLO-16 ceils

目的探讨依维莫司是否增加顺铂对人皮肤鳞状细胞癌COLO-16细胞的杀伤效应。方法分别用50、100、200nmol／L的依维莫司和25μmol／L顺铂处理人皮肤鳞状细胞癌系COLO-16细胞12、24h，用AO标记自噬体囊泡并结合溶酶体酶抑制剂分析自噬和自噬流水平。Western印迹分析自噬标记性分子LC3．Ⅰ→LC3-Ⅱ转化、Caspase3和PARP剪切；LDH释放实验分析细胞死亡；AnnexinV．EGFP染色分析细胞凋亡。结果50、100、200nmol／L的依维莫司处理COLO．16细胞后，12hLC3．Ⅰ→Lc3-Ⅱ转换值分别为3．52±0．21、4．03±0．39、5．05±0．22，两两之间比较，差异无统计学意义（P〉0．05），与未用药物处理的对照组（2．07±0．05）比较，差异有统计学意义（P〈0．05）。24hLC3-Ⅰ→LC3-Ⅱ转换值分别为3．38±0．26、3．29±0．06、6．57±0．16，两两之间比较，差异无统计学意义（P〉0．05），与对照组（2．61％±0．16％）比较，差异有统计学意义（P〈0．05）。50、100、200nmol／L的依维莫司联合E64d、pepstatin处理COLO-16细胞12h后，LC3-Ⅱ与B肌动蛋白的比值分别为1．26±0．40、1．16±0．34、1．21±0．39，与E64d、pepstatin单独处理细胞组（1．19±0．27）比较，差异无统计学意义（P〉0．05）。100nmol／L依维莫司联合E64d、pepstatin自噬体囊泡表达阳性率2．06-4-0．61，与E64d、pepstatin单独处理细胞组（1．68±0．62）比较，差异无统计学意义（P〉0．05）。依维莫司与顺铂联合处理24h细胞死亡率42．58％±0．93％，高于顺铂单独处理的细胞（死亡率18．20％±1．46％），差异有统计学意义。依维莫司联合顺铂处理与顺铂单独处理细胞相比，Caspase3和PARP剪切、AnnexinV标记细胞数以及LC3-Ⅱ形成均无统计学差异（P〉0．05）。结论依维莫司增强顺铂诱导COLO-1细胞死亡，这种效应不依赖于凋亡和自噬调控。

Objective To evaluate the synergistic effect of everolimus on cisplatin- mediated cytotoxicity against human cutaneous squamous cell carcinoma COLO-16 ceils. Methods Cultured COLO- 16 cells were divided into several groups to be treated with everolimus at different concentrations of 50, 100 and 200 nmol/L or 25 μmol/L cisplatin for 12 and 24 hours. Acridine orange （AO）-labeled autophagic vesicles combined with lysomal enzyme inhibitors （E64d and pepstatin） were used to detect the levels of autophagy and autophagic flow. Western blot analysis was performed to track the conversion of the autophagosome marker microtubule-associated protein 1 light chain-3 （LC3） - I to LC3- 11, as well as to detect cleavage levels of Caspase 3 and poly-ADP-ribose polymerase （PARP）. Lactate dehydrogenase （LDH） assay was conducted to detect cell death, and Annexin V-EGFP staining to evaluate cell apoptosis. Results The LC3- H / LC3- I ratios （LC3- I conversion to LC3- lI ） after 12- and 24-hour treatment did not differ among the 50-, 100- and 200-nmol/L everolimus groups （12 hours： 3.52 ± 0.21 vs. 4.03 ± 0.39 vs. 5.05± 0.22, P 〉 0.05; 24 hours： 3.38± 0.26 vs. 3.29 ± 0.06 vs. 6.57 ± 0.16, P 〉 0.05）, but were significantly higher in the three everolimus groups than in the control group receiving no treatment （12 hours： 2.07 ± 0.05, P 〈 0.05; 24 hours： 2.61 ＋ 0.16, P 〈 0.05）. After 12-hour treatment, no significant differences were observed in the ratio of LC3- H to 13-actin between the 50-nmol/L everolimus ＋ E64d ＋ pepstatin group （1.26 ± 0.40）, 100-nmol/L everolimus ＋ E64d ＋ pepstatin group （1.16 ~ 0.34）, 200-nmol/L everolimus ＋ E64d ＋ pepstatin group （1.21 ± 0.39） and E64d ＋ pepstatin group （1.19 ± 0.27, P 〉 0.05）. Moreover, there was no significant difference in the percentages of autophagic vesicle-positive cells between the 100-nmol/L everolimus ＋ E64d ＋ pepstatin group and E64d ＋ pepstatin group （2.06% ± 0.61% vs. 1.68% ± 0.62%, P 〉 0.05）. After 24-hour treatment, the everolimus ＋ cisplatin group showed significantly increased rate of cell death compared with the cisplatin alone group （42.58%± 0.93% vs. 18.20% ±1.46%）. However, no significant differences were observed in the cleavage levels of Caspase 3 and PARP, the number of annexin V-labelled cells and ratio of LC3-ⅡI to 15-actin between the everolimus ＋ cisplatin group and the cisplatin-alone group （P 〉 0.05）. Conclusion Everolimus has a synergistic effect on the cisplatin-mediated COLO-16 cell death, and this effect does not depend on cell apoptosis or autophagy.