紫杉醇联合SB203580对胃印戒细胞癌细胞增殖和凋亡的影响

Effect of paclitaxel combined with SB203580 on cell growth and apoptosis of gastric signet ring cell carcinoma

目的探讨紫杉醇联合P38MAPK通路抑制剂SB203580对胃印戒细胞癌细胞KATO Ⅲ增殖和凋亡的影响。方法通过CCK8方法检测不同浓度紫杉醇(0.1、1、10μg/mL)和不同浓度P38MAPK通路抑制剂SB203580(5、20、40μM)对KATO Ⅲ细胞生长曲线的影响,同时挑选合适的作用浓度用于后续的研究;将细胞分为4组:对照组、紫杉醇组(紫杉醇1μg/mL)、SB203580组(SB20358040μM)、紫杉醇+SB203580组(紫杉醇1μg/mL、SB20358040μM),采用CCK8方法检测紫杉醇和SB203580分别单独和联合使用对KATO Ⅲ细胞增殖的影响;细胞克隆形成观察KATO Ⅲ细胞在紫杉醇、SB203580、紫杉醇+SB203580作用下单细胞的克隆情况;采用Annexin V/PI试剂盒检测各组细胞在不同的处理下细胞的凋亡情况;通过蛋白免疫印迹法(Western blot)检测在不同的处理下,各组细胞中相关凋亡蛋白的表达水平。结果细胞生长曲线显示,紫杉醇和SB203580对KATO Ⅲ细胞具有生长抑制的效果,具有浓度依赖性。细胞增殖实验显示,紫杉醇[(0.73±0.00)vs(1.12±0.01),P<0.05]和SB203580[(0.75±0.00)vs(1.12±0.01),P<0.05]明显的抑制KATO Ⅲ细胞增殖,紫杉醇联合SB203580使用效果优于单独使用紫杉醇[(0.57±0.02)vs(0.73±0.00),P<0.05]。克隆形成结果显示,紫杉醇和SB203580对KATO Ⅲ细胞单细胞克隆具有明显的抑制效果,同时紫杉醇联合SB203580使用效果更明显[(0±0.00)vs(13.67±1.15),P<0.05]。Annexin V/PI和western blot检测细胞凋亡情况,紫杉醇和SB203580能明显的诱导相关凋亡蛋白的表达和促进细胞的凋亡。与单独紫杉醇相比,紫杉醇+SB203580组促进细胞凋亡的效果更强早期凋亡[(28.01±0.02)%vs(12.39±0.02)%],晚期凋亡[(29.77±0.01)%vs(15.17±0.01)%,P<0.05],Bax[(1.30±0.13)vs(1.01±0.05),P<0.05]、cleave-caspase3[(1.26±0.11)vs(1.07±0.06),P<0.05]相关凋亡蛋白的表达水平更高。结论紫杉醇联合SB203580能增强紫杉醇对胃印戒细胞癌细胞增殖的抑制作用,同时可促进胃印戒细胞癌KATO Ⅲ细胞的凋亡。

Objective To investigate the effects of paclitaxel combined with P38MAPK pathway inhibitor SB203580 on the proliferation and apoptosis of gastric signet ring cell carcinoma cellsKATO Ⅲ.Methods The effects of different concentrations of paclitaxel(0.1,1,10μg/mL)and different concentrations of P38MAPK pathway inhibitor SB203580(5,20,40μM)on the growth curve of KATO Ⅲ cells were detected by CCK8 method,and appropriate effective concentrations were selected for subsequent studies.The cells were divided into 4 groups:control group,paclitaxel group(paclitaxel 1μg/mL),SB203580 group(SB20358040μM),and paclitaxel+SB203580 group(paclitaxel 1μg/mL,SB20358040μM).The effects of paclitaxel and SB203580 on KATO Ⅲ cell proliferation were detectedby CCK8 method.The single cell cloning of KATO Ⅲ cells under the effects of paclitaxel,SB203580 and paclitaxel+SB203580 was observed.Annexin V/PI kit was used to detect apoptosis of cells in each group under different treatments.Western blotting was used to detect the expression levels of apoptotic proteins in each group under different treatments.Results The cell growth curve showed that taxol and SB203580 had growth inhibition effect on KATO Ⅲ cellswith concentration-dependence.The cell proliferation assay showed that taxol[(0.73±0.00)vs(1.12±0.01),P<0.05]and SB203580[(0.75±0.00)vs(1.12±0.01),P<0.05]significantly inhibited the proliferation of KATO Ⅲ cells.Taxol combined with SB203580 was more effective than taxol alone[(0.57±0.02)vs(0.73±0.00),P<0.05].The results of clone formation showed that taxol and SB203580 had significant inhibitory effect on KATO Ⅲ cell clone,while taxol combined with SB203580 had more obvious effect[(0±0.00)vs(13.67±1.15),P<0.05].Annexin V/PI and Western blotting showed that paclitaxel and SB203580 significantly induced the expression of apoptotic proteins and promoted cell apoptosis.Compared with paclitaxel alone,paclitaxel+SB203580 group showed stronger apoptotic effects:early apoptosis[(28.01±0.02)%vs(12.39±0.02)%],late apoptosis[(29.77±0.01)%vs(15.17±0.01)%,P<0.05],Bax[(1.30±0.13)vs(1.01±0.05),P<0.05],and Cleave-Caspase-3[(1.26±0.11)vs(1.07±0.06),P<0.05].And the expression level of apoptotic proteins was higher.Conclusion The combination of paclitaxel and SB203580 can enhance the inhibition effect of paclitaxel on the proliferation of signet ring cell cancer cells and promote the apoptosis of KATO Ⅲ cell of signet ring cell carcinoma.