南蛇藤醇介导的PTEN过表达增强顺铂对食管癌细胞的化学敏感性并抑制裸鼠成瘤能力

PTEN Overexpression Mediated by Celastrus Orbiculatus Extracts Enhanced the Chemical Sensitivity of Cisplatin to Esophageal Cancer Cells

目的探究南蛇藤醇介导的PTEN过表达对食管癌细胞化学敏感性的影响。方法构建食管癌肿瘤模型小鼠,使用南蛇藤醇(1、5 mg/kg)和顺铂(5 mg/kg)单独或联合对其进行瘤内治疗,观察其肿瘤体积变化和检测30 d时肿瘤质量;免疫组化检测Ki67,VEGF和Caspase 3的表达情况。用不同剂量的南蛇藤醇(0.5、1、2.5、5、10、20、50、80、100μmol/L)处理人食管上皮细胞系(HET-1 A)24 h,CCK8分析检测细胞存活率。顺铂(4μg/m L)预处理EC109/DDP细胞后分别用不同剂量的南蛇藤醇(1、2.5、5μmol/L)处理EC109/DDP细胞,对照组用PBS代替顺铂,CCK8检测细胞存活率;Transwell检测细胞的侵袭情况,Western blot检测细胞中EMT相关蛋白以及凋亡相关蛋白表达情况,Hoechst染色检测细胞凋亡情况。结果南蛇藤醇1 mg/kg和5 mg/kg处理后的食管癌肿瘤模型小鼠体内肿瘤质量和体积均显著降低(P<0.05);PTEN蛋白表达水平显著升高(P<0.05);Ki67,VEGF和Caspase 3阳性细胞数均显著减少(P<0.05)。用南蛇藤醇(1、2.5、5μmol/L)处理后,与对照组比较,EC109细胞存活率和侵袭力均显著降低(P<0.05);Ki67、PCNA、N-cadherin和Vimentin蛋白表达水平显著降低(P<0.05);E-cadherin、cleaved PARP和cleaved caspase 3蛋白表达水平以及细胞凋亡率显著升高(P<0.05);干扰PTEN后细胞存活率以及侵袭力显著升高(P<0.05);凋亡率显著降低(P<0.05)。结论南蛇藤醇介导的PTEN过表达增强顺铂对食管癌细胞EC109生长、侵袭和EMT的抑制作用,促进顺铂诱导的食管癌细胞EC109凋亡,从而增强顺铂对食管癌细胞的化学敏感性。

Objective To explore the effect of PTEN over expression mediated by Celastrus orbiculatus extracts on the chemical sensitivity of esophageal cancer cells. Method The model mice of esophageal cancer were constructed,and they were treated intratumoral with Cel. orbiculatus extracts( 1,5 mg/kg) and cisplatin( 5 mg/kg) alone or in combination. The tumor volume was observed and the tumor weight was detected at 30 days. Immunohistochemistry detected the expression of Ki67,VEGF and Caspase 3. HET-1 A was treated with different doses of Cel. orbiculatus extracts( 0. 5,1,2. 5,5,10,20,50,80 and 100 μmol/L) for 24 h,and cell viability was determined by CCK8 assay. After pretreatment of EC109/DDP cells with cisplatin( 4 μg/m L),EC109/DDP cells were treated with different doses of Cel. orbiculatus extracts( 1,2. 5 and 5 μmol/L). The control group was replaced with PBS instead of cisplatin. CCK8 was used to detect cell survival. The transwell was used to detect the invasion of cells. The expression of EMT-related protein and apoptosis-related protein was detected by Western blot. The apoptosis of cells was detected by Hoechst staining. Result After treatment with Cel. orbiculatus extracts( 1 mg/kg and 5 mg/kg),the tumor weight and volume in the model mice were significantly reduced( P< 0. 05). The expression level of PTEN protein was significantly increased( P < 0. 05).Ki67,VEGF and Caspase 3 positive cells were significantly reduced( P<0. 05). Compared with the control group,the survival rate and invasiveness of EC109 cells were significantly decreased( P < 0. 05). Ki67,PCNA,ncadherin and Vimentin were significantly decreased( P < 0. 05). The expression levels of e-cadherin,cleaved PARP and cleaved caspase 3 protein and the apoptosis rate were significantly increased( P<0. 05). Cell survival rate and invasion force were significantly increased after PTEN interference( P <0. 05). Apoptosis rate decreased significantly( P < 0. 05). Conclusion PTEN overexpression mediated by Cel. orbiculatus extracts enhanced the inhibitory effect of cisplatin on EC109 growth,invasion and EMT of esophageal cancer cells,promoted cisplatin induced EC109 apoptosis of esophageal cancer cells,and thus enhanced the chemical sensitivity of cisplatin to esophageal cancer cells.