Wang Lin, Zhu Zhi-Xia, Zhang Wen-Ying, Zhang Wei-Min
Department of Oncology, Guangzhou General Hospital of Guangzhou Military Command (Guangzhou Liuhuaqiao Hospital), Guangzhou, Guangdong 510010, P.R. China.
Exp Ther Med. 2011 Sep;2(5):969-975. doi: 10.3892/etm.2011.293. Epub 2011 Jun 22.
Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR-HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G(0)/G(1) arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib, but not by concomitant or sequential incubation with erlotinib followed by pemetrexed. In summary, our results demonstrated that the combined use of erlotinib and pemetrexed exhibited a strong synergism in BXPC-3 and PANC-1 cells. The inhibitory effects were strongest after sequential administration of pemetrexed followed by erlotinib. The synergistic effects may be related to activation of the EGFR/HER3/AKT pathway induced by pemetrexed.
先前的研究表明，细胞毒性药物培美曲塞和表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）厄洛替尼均能抑制胰腺癌细胞的生长。然而，它们对胰腺癌细胞是否具有协同抗肿瘤作用仍不清楚。本研究旨在评估厄洛替尼与培美曲塞联合使用不同序贯给药方案对人胰腺癌BXPC-3和PANC-1细胞增殖的协同作用，并探究其细胞机制。通过定量PCR高分辨率熔解曲线分析（qPCR-HRM）检测EGFR和K-ras基因突变状态。使用不同给药方案将培美曲塞和厄洛替尼与BXPC-3和PANC-1细胞共同孵育。采用MTT法测定细胞毒性，通过流式细胞术测定细胞周期分布。使用蛋白质印迹法检测EGFR、HER3、AKT和MET的表达及磷酸化水平。体外实验中，培美曲塞和厄洛替尼均以剂量和时间依赖性方式抑制BXPC-3和PANC-1细胞的增殖。培美曲塞与厄洛替尼联合使用时，对细胞增殖具有协同作用。协同作用的程度取决于给药顺序，培美曲塞给药后24小时间隔依次给予厄洛替尼时协同作用最为明显。细胞周期研究表明，培美曲塞诱导S期阻滞，厄洛替尼诱导G(0)/G(1)期阻滞。培美曲塞给药后依次给予厄洛替尼诱导S期阻滞。蛋白质印迹分析表明，培美曲塞分别增加EGFR、HER3和AKT的磷酸化水平，而厄洛替尼降低其磷酸化水平。然而，培美曲塞和厄洛替尼对c-MET的磷酸化均无显著影响。培美曲塞给药后依次给予厄洛替尼可显著抑制EGFR、HER3和AKT的磷酸化，但厄洛替尼给药后依次给予培美曲塞或两者同时给药则无此作用。总之，我们的结果表明，厄洛替尼与培美曲塞联合使用在BXPC-3和PANC-1细胞中表现出强烈的协同作用。培美曲塞给药后依次给予厄洛替尼时抑制作用最强。协同作用可能与培美曲塞诱导的EGFR/HER3/AKT信号通路激活有关。
