拉帕替尼耐药乳腺癌细胞株的建立及耐药机制的研究

Establishment of lapatinib-resistant breast cancer cells and primary exploration for mechanism of resistance

目的 :建立对拉帕替尼(lapatinib)耐药的乳腺癌SKBR3细胞株并筛选与拉帕替尼耐药相关的基因。方法:采用间隙大剂量冲击和逐步增加剂量相结合的方法诱导建立拉帕替尼耐药的乳腺癌细胞模型;应用CCK-8(cell counting kit-8)和FCM法检测亲本、耐药细胞增殖及凋亡的情况,蛋白质印迹法检测人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)通路中相关蛋白的表达情况,应用人类全基因组表达谱芯片检测亲本和耐药细胞中基因表达的差异。结果 :CCK-8和FCM法检测结果显示,耐药SKBR3细胞对拉帕替尼的敏感性明显降低,拉帕替尼对亲本和耐药细胞的半数抑制浓度(half inhibition concentration,IC50)分别为(0.35±0.07)μmol/L和(3.89±0.09)μmol/L,经计算耐药倍数约为11.11。蛋白质印迹法检测结果显示,耐药细胞中磷酸化HER2、HER3、蛋白激酶B(protein kinase B,PKB,又称Akt)和细胞外信号调节激酶1/2(extracellular signal-regulated protein kinase1/2,ERK1/2)的表达水均明显低于亲本细胞。全基因组芯片检测结果显示,差异表达基因(上调或下调)共1 598个,其中上调的基因为989个,下调的基因为609个。对差异表达基因进行功能分类,涉及氨基酸代谢、糖代谢、脂类代谢、细胞凋亡和细胞间黏附等相关细胞转导通路共391条。结论:成功建立了拉帕替尼获得性耐药的乳腺癌细胞株,细胞代谢等通路中基因的表达异常可能与乳腺癌细胞对拉帕替尼的耐药有关。

ObJective： To establish lapatinib-resistant breast cancer cells and to screen the genes related to acquired resistance to lapatinib in breast cancer SKBR3celIs. Methods： The lapatinib-resistant SKBR3 cells were established by continuously exposing parent SKBR3 cells to high-dose of lapatinib over a period of 18 months. The effect of lapatinib on growth of parent and resistant SKBR3 cells was assayed by cell counting kit-8 （CCK-8）. The effect of lapatinib on apoptosis in parent and resistant SKBR3 cells was assessed by flow cytometry （FCM）. The expressions of human epidermal growth factor receptor 2 （HER2） pathway-related proteins were detected by Western blotting. The differentially expressed genes between parent and resistant cells were screened by human whole genome expression microarray. Results： The CCK-8 assay showed that the viability of parent SKBR3 cells was lower than that of the resistant SKBR3 cells after exposing to different doses of lapatinib. The flow cytometry showed that the percentage of apoptotic cells in parent SKBR3 cells was higher than that in resistant SKBR3 cells （P 〈 0.05）. The half inhibition concentration （IC50） values of lapatinib for parent SKBR3 cells and resistant SKBR3 cells were （3.35±0.07）μmol/L and （3.89±0.09）μmol/L, respectively. The resistant SKBR3 cells were less sensitive to lapatinib than SKBR3 cells with 11.11-fold increase in resistance. The expression levels of phospho-HER2 （p-HER2）, p-HER3, p-protein kinase B （PKB, Akt） and p-extracellular signal-regulated protein kinase 1/2 （ERK1/2） proteins in resistant SKBR3 cells were lower than those in the parent SKBR3 cells. Gene expression data from microarray showed that there were 1 598 genes differently expressed between the parent and the resistant SKBR3 cells. Among the 1 598 genes, the expression levels of 989 genes were up-regulated, and 609 genes were down- regulated. The differently expressed genes between the parent and the resistant SKBR3 cells were classified according to their functions. These genes were involved in amino acid metabolism, lycometabolism, lipid metabolism, apoptosis, intercellular adhesion signal transduction, and so on. Conclusion： Lapatinib-resistant SKBR3 cells were established. Abnormal gene expressions in cell metabolism and other pathways might be responsible for lapatinib resistance in SKBR3 cells.