乳腺癌拉帕替尼耐药模型建立及二甲双胍逆转其耐药性的初步探讨

Establishment of lapatinib-resistant breast cancer and preliminary study on the resistance-reversing function of metformin

摘要:

目的：建立乳腺癌拉帕替尼耐药模型并初步探讨二甲双胍对其耐药性的逆转作用。方法：体外建立乳腺癌拉帕替尼耐药模型；实验分为亲本组、空白耐药组、拉帕替尼组二甲双胍组和联合用药组（n=3）；CCK-8法检测并计算空白耐药组细胞耐药倍数及二甲双胍组细胞逆转耐药倍数；流式细胞术（flow cytometry，FCM）检测各组细胞凋亡率和周期；Western blot检测各组细胞PI3K信号通路相关蛋白的表达水平。结果：空白耐药组细胞对拉帕替尼敏感性降低；亲本组、空白耐药组及二甲双胍组细胞IC50值分别为（2.64±0.12）、（11.21±0.03）、（5.62±0.13） μmol/L，耐药倍数约为4.25，逆转耐药倍数约为2.0；二甲双胍组（0.73±0.04）细胞增殖指数较空白耐药组（1.11±0.02）明显降低（P=0.004），联合用药组（0.63±0.06）细胞增殖指数较空白耐药组（1.11±0.02）同样明显降低（P=0.031）；二甲双胍组[（10.70±0.76）%]细胞凋亡率较空白耐药组[（5.05±0.59）%]明显增高（P=0.007），联合用药组[（24.68±1.52）%]细胞凋亡率较空白耐药组[（5.05±0.59）%]同样明显增高（P=0.006）；联合用药组中PI3K信号通路相关磷酸化蛋白和非磷酸化蛋白表达较空白耐药组均下降。结论：成功建立乳腺癌拉帕替尼耐药模型；二甲双胍通过抑制PI3K信号通路逆转乳腺癌拉帕替尼耐药性。

Abstract:

Objective：To establish a model of lapatinib-resistance in breast cancer and explore the reversal effect of metformin on the resistance. Methods：The model of lapatinib-resistance in breast cancer was established in vitro. The experiment cells were divided into the parent group，blank resistance group，lapatinib group，metformin group and lapatinib-metformin group（n=3）. CCK-8 method was used to detect and calculate the resistance times of blank resistance group and the resistance-reversing times of metformin group. The apoptosis rate and cycle of each group were detected by FCM，and the expressions of proteins related to PI3K signaling pathway in each group by Western blot. Results：The sensitivity of cells to lapatinib in blank resistance group was lowered. IC50 values in the parent group，blank resistance group，and metformin group were （2.64±0.12），（11.21±0.03），and （5.62±0.13） μmol/L respectively. The drug resistance was about 4.25 and the reversal of drug resistance was approximately 2.0. Compared with the blank resistance group，the cell proliferation index significantly decreased in both metformin group（0.73±0.04 vs. 1.11±0.02）（P=0.004） and the lapatinib-metformin group（0.63±0.06 vs. 1.11±0.02）（P=0.031），while the cell apoptosis rate significantly increased in both metformin group（[（10.70±0.76）%] vs. [（5.05±0.59）%]）（P=0.007）and the lapatinib-metformin group（[（24.68±1.52）%] vs. [（5.05±0.59）%]）（P=0.006）. The expressions of PI3K signaling pathway-associated phosphorylated and non-phosphorylated proteins in lapatinib-metformin group were lower than those in the blank resistance group. Conclusion：The lapatinib-resistant model of breast cancer has been successfully established. Metformin can reverse the drug resistance of lapatinib in breast cancer cell by inhibiting the PI3K signaling pathway.