Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells
Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells
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DOI：
10.3892/etm.2011.293
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发表时间：
2011-09-01
影响因子：
2.7
通讯作者：
Zhang, Wei-Min
中科院分区：
文献类型：
--
作者：
Wang, Lin;Zhu, Zhi-Xia;Zhang, Wei-Min
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)厄洛替尼都能抑制胰腺癌细胞的生长。然而，它们是否对胰腺癌细胞起到协同抗肿瘤作用尚不清楚。本研究旨在评价厄洛替尼联合培美曲塞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，并探讨其细胞机制。采用定量聚合酶链式反应高分辨熔融法(qPCR-HRM)检测EGFR和K-ras基因突变情况。用培美曲塞和厄洛替尼按不同给药程序孵育BXPC-3和PANC-1细胞。用四甲基偶氮唑盐比色法测定细胞毒作用，用流式细胞仪测定细胞周期分布。Western blotting检测EGFR、HER3、AKT和MET的表达及磷酸化。培美曲塞和厄洛替尼在体外均能抑制BXPC-3和PANC-1细胞的增殖，且呈剂量和时间依赖关系。培美曲塞与厄洛替尼联合应用对细胞增殖有协同作用。协同作用的程度取决于给药顺序，以培美曲塞后24小时间隔顺序给药时最为明显。细胞周期分析显示培美曲塞诱导S细胞周期阻滞，厄洛替尼诱导G(0)/G(1)期细胞周期阻滞。培美曲塞后序贯应用厄洛替尼诱导S骤停。Western印迹分析显示培美曲塞使EGFR、HER3和AKT的磷酸化水平分别升高，而厄洛替尼则降低。但培美曲塞和厄洛替尼对c-MET的磷酸化均无明显影响。培美曲塞和厄洛替尼共同孵育可显著抑制EGFR、HER3和AKT的磷酸化，但与厄洛替尼和培美曲塞同时孵育或序贯孵育不能显著抑制EGFR、HER3和AKT的磷酸化。综上所述，我们的结果表明厄洛替尼和培美曲塞联合使用在BXPC-3和PANC-1细胞中显示出很强的协同作用。培美曲塞和厄洛替尼连续给药的抑制作用最强。培美曲塞的协同作用可能与其激活EGFR/HER3/AKT通路有关。
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.