海参糖胺聚糖联合顺铂对肺腺癌A549细胞的增殖抑制及化疗增敏的作用机制

The roles of holothurian glycosaminoglycan combined with cisplatin on proliferation and chemotherapeutic response in A549 human lung adenocarcinoma cell

目的探讨海参糖胺聚糖（hGAG）与顺铂（DDP）单独或联合应用对人肺腺癌A549细胞株的增殖抑制作用及hGAG对DDP化疗的增敏机制。方法将对数生长期的A549细胞分为hGAG组、DDP组、hGAG和DDP联合组（联合组）以及对照组。倒置相差显微镜观察细胞的生长状态和形态变化，CCK-8法检测各组细胞的增殖抑制率，Hoeehst33258染色、annexinV-FITC和PI双染法检测细胞的凋亡情况，Westernblot检测各组凋亡蛋白Bel-2、Bax、survivin和easpase-3的表达。结果CCK-8检测结果显示，处理24h后，hGAG组、DDP组、联合组和对照组A549细胞的增殖抑制率分别为（19．74±5．39）％、（42．01±2．57）％、（53．89±4．58）％和0；处理48h后，分别为（23．17±4．78）％、（54．00±7．64）％、（77．58±4．26）％和0；处理72h后，分别为（29．17±4．21）％、（59．35±7．31）％、（79．94±4．58）％和0;同一时间hGAG组、DDP组和联合组A549细胞的增殖抑制率均高于对照组（均P〈0．05）．联合组A549细胞的增殖抑制率均高于DDP组（均P〈0．05）。Hoehest33258染色结果显示，对照组未见凋亡细胞，hGAG组、DDP组和联合组均可见凋亡细胞，其中联合组凋亡细胞数最多，且细胞密度最低。流式细胞术检测结果显示，hGAG组、DDP组、联合组和对照组细胞中早、中期细胞凋亡率分别为（12．59±4．22）％、（16．36±3．63）％、（44．60±5．45）％和（2．38±0．59）％，hGAG组、DDP组和联合组的细胞凋亡率均高于对照组（均P〈0．05），联合组高于DDP组（P〈0．05）。Westernblot检测显示．与对照组比较，hGAG组、DDP组和联合组细胞中Bax和easpase．3蛋白的表达均增高（均P〈0．05），联合组细胞中Bax和easpase-3蛋白的表达均高于DDP组（均P〈0．05）；而Bel-2和survivin蛋白的表达均降低（均P〈0．05），且联合组中Bel-2和survivin蛋白的表达均低于DDP组（均P〈0．05）。结论hGAG对人肺腺癌A549细胞的增殖具有抑制作用，且可增强A549细胞对DDP化疗的敏感性，其作用机制可能与上调Bax和caspase．3蛋白的表达以及下调Bcl-2和survivin蛋白的表达有关。

Objective To investigate the effects and mechanism of Holothurian Glycosaminoglycan （hGAG） alone in combination with cisplatin （DDP） on apoptosis of pulmonary adenoearcinoma cell A549. Methods A549 cells were separately treated with blank, hGAG, DDP and hGAG combined with DDP （hGAG ＋ DDP）. The cell morphology in 4 groups was observed using light microscope. CCK8 assay was used to determine the cell viability. Flow cytometry by Hoeehst 33258 and AnnexinV-FITC/PI staining was applied to detect cell apoptosis. Western blot was then used to detect the protein expression of Bax, Bcl-2, survivin and caspase-3. Results After treatment for 24 h, the inhibitory rates of A549 ceils in control, hGAG, DDP and hGAG ＋ DDP groups were 0, （ 19.74±5.39）%, （42.01±2.57）% and （53.89±4.58）%, respectively. Moreover, after treatment for 48 h and 72 h, the inhibitory rates in each group were 0, （23.17±4.78）% and （29.17±4.21）%, （54.00±7.64）% and （59.35±7.31）%, as well as （77.58±4.26）% and （79.94±4.58） %, respectively. The cell viability was significantly lower in drug treatment groups compared with those in control group at the same time point （P〈0.05）. Hochest 33258 staining showed that no obvious apoptotic cells were detected in the control group, while apoptotic cells were visible in hGAG, cisplatin and combination groups. Flow cytometry showed that cell apoptotic rates were （ 2. 38 ± 0.59 ） %, （ 12. 59 ±4.22）%, （16.36±3.63）% and （44.60±5.45）% in the control, hGAG, DDP and hGAG ＋ DDP groups, respectively. The cell apoptosis was significantly lower in drug treatment groups compared with those in control group at the same time point （P〈O.05）. Furthermore, western blot results showed that the expression of Bax and caspase-3 protein was increased （P〈0.05） , whereas Bcl-2 and survivin was decreased （P〈 0.05） in the hGAG＋DDP group compared with cisplatin alone （P〈0.05）. Conclusions HGAG can inhibit the proliferation and promote the apoptosis of human lung adenocarcinoma A549 cells. Meanwhile, it can strengthen the chemosensitivity of A549 cells to DDP via up-regulation of Bax, caspase-3 and down- regulation of Bcl-2 and survivin.