阿司匹林联合吉西他滨对胆囊癌GBC-SD细胞增殖及凋亡的影响

Influence of aspirin plus gemcitabine on proliferation and apoptosis of GBC-SD cells

目的 观察应用不同剂量阿司匹林、吉西他滨处理后胆囊癌GBC-SD细胞增殖与凋亡情况,探讨阿司匹林对GBC-SD细胞吉西他滨化疗敏感性的影响及可能机制。方法 取对数生长期GBC-SD细胞,分别应用浓度为0、0.2、1.0、5.0、10.0μmol/L吉西他滨处理24、48、72 h,应用0、0.5、1.0、4.0 mmol/L阿司匹林处理24、48 h,采用CCK-8法检测细胞生长抑制率。结合前期预实验结果,采用1.0μmol/L吉西他滨、0.5 mmol/L阿司匹林进行后续实验。取对数生长期GBC-SD细胞分为阴性对照组、吉西他滨组、阿司匹林组、联合组。待细胞贴壁后,阴性对照组加入DMSO溶液1μL;阿司匹林组加入0.5 mmol/L阿司匹林1μL;吉西他滨组加入1.0μmol/L吉西他滨0.4μL;联合组先加入0.5 mmol/L阿司匹林1μL,1 h后加入1.0μmol/L吉西他滨0.4μL。药物处理48 h, 4组采用CCK-8法检测细胞生长抑制率,采用流式细胞术检测细胞凋亡率,采用Western blot法检测p-PI3K、PI3K蛋白相对表达量。结果 吉西他滨处理24、48、72 h, 0.2、1.0、5.0、10.0μmol/L浓度的细胞生长抑制率依次增高(P<0.05);阿司匹林处理24、48 h, 0.5、1.0、4.0 mmol/L浓度的细胞生长抑制率依次增高(P<0.05)。药物处理48 h,吉西他滨组、阿司匹林组、联合组细胞生长抑制率[(29.7±2.9)%、(26.9±2.5)%、(59.3±5.4)%]、细胞凋亡率[(15.3±2.1)%、(12.9±3.1)%、(36.1±8.6)%]均高于阴性对照组[0、(4.5±1.1)%](P<0.05),联合组均高于吉西他滨组和阿司匹林组(P<0.05)。药物处理48 h,吉西他滨组p-PI3K蛋白相对表达量(3.49±0.14)高于阴性对照组(2.83±0.17)和联合组(2.69±0.11)(P<0.05),阿司匹林组p-PI3K蛋白相对表达量(1.97±0.13)低于阴性对照组和联合组(P<0.05),联合组p-PI3K蛋白相对表达量与阴性对照组比较差异无统计学意义(P>0.05);4组PI3K蛋白相对表达量比较差异无统计学意义(P>0.05)。结论 吉西他滨、阿司匹林均可抑制胆囊癌GBC-SD细胞生长、促进GBC-SD细胞凋亡,二者联合具有协同效应,其机制可能与阿司匹林抑制PI3K通路相关。

Objective To observe the proliferation and apoptosis of GBC-SD cells after treatment with aspirin plus gemcitabine in different doses, and to investigate the influence of aspirin on the chemotherapy sensitivity of GBC-SD to gemcitabine and its potential mechanism. Methods GBC-SD cells in logarithmic growth phase were treated with 0, 0.2, 1.0, 5.0 and 10.0 μmol/L gemcitabine for 24, 48 and 72 h, and 0, 0.5, 1.0 and 4.0 mmol/L aspirin for 24 and 48 h. The inhibitory rate was detected by CCK-8 assay. The subsequent experiment was done by using 1.0 μmol/L gemcitabine plus 0.5 mmol/L aspirin on the basis of pilot trials. GBC-SD cells in logarithmic growth phase were divided into negative control group, gemcitabine group, aspirin group, and gemcitabine + aspirin group, which were added with 1 μL of DMSO, 0.4 μL of 1.0 μmol/L gemcitabine, 1 μL of 0.5 mmol/L aspirin, and 1 μL of 0.5 mmol/L aspirin followed by 0.4 μL of 1.0 μmol/L gemcitabine 1 h later, respectively, after cell attachment. CCK-8 assay was used to defect the proliferation rate and flow cytometry was used to detect the apoptosis rate 48 h later. Western blot was used to detect the relative expressions of p-PI3 K and PI3 K proteins. Results The growth inhibitory rate increased gradually in turn in cells in concentration of 0, 0.2, 1.0, 5.0 and 10.0 μmol/L after 24-, 48-and 72-h treatment with gemcitabine(P<0.05), and also increased gradually in turn in cells in concentration of 0.5, 1.0 and 4.0 mmol/L after 24-and 48-h treatment with aspirin(P<0.05). After 48-h treatment, the growth inhibitory rate and apoptosis rate were higher in gemcitabine group [(29.7±2.9)%,(15.3±2.1)%], aspirin group [(26.9±2.5)%,(12.9±3.1)%] and gemcitabine + aspirin group [(59.3±5.4)%,(36.1±8.6)%] than those in negative control group [0,(4.5±1.1)%](P<0.05),and higher in gemcitabine+ aspirin group than those in gemcitabine group and aspirin group(P<0.05).The relative expression of p-PI3K protein was higher in gemcitabine group(3.49±0.14)than that in negative control group(2.83±0.17)and gemcitabine+ aspirin group(2.69±0.11)(P<0.05),was lower in aspirin group(1.97±0.13)than that in gemcitabine + aspirin group and negative control group(P<0.05),and showed no significant difference between gemcitabine+ aspirin group and negative control group(P>0.05).There was no significant difference in the relative expression of PI3K protein among four groups(P>0.05).Conclusions Both gemcitabine and aspirin can inhibit the growth of GBC-SD cells and promote the apoptosis of GBC-SD.The combination of them two has a synergistic effect,and the mechanism may be correlated with the inhibition of PI3K pathway by aspirin.