Identification of signal transduction that induces anticancer drug resistance for clinical application

吉非替尼是一种表皮生长因子受体（EGFR）酪氨酸激酶的选择性抑制剂，虽然已被临床证明对某些类型的癌细胞有效，但其抗肿瘤活性的分子机制尚未完全阐明。在本研究中，我们研究了吉非替尼诱导HAG-1人胆囊腺癌细胞生长抑制和凋亡的机制。1 mM剂量的吉非替尼对细胞生长有明显的抑制作用，培养72 h后细胞数量不可逆地下降，120 h后凋亡细胞群逐渐扩大。治疗2小时后，吉非替尼显著抑制EGFR自磷酸化及随后通过Erk和Akt的下游信号通路，并在24小时诱导细胞在细胞周期的G0/G1期积累，同时p21转录增加，p27表达增加。吉非替尼对总Bax和p53丝氨酸15的磷酸化量没有影响，但上调了总Bax的表达，随后增加了Bax的活性形式p18 Bax的表达。Bcl-2和Bad的表达没有变化。吉非替尼诱导的总Bax表达的增加可能是由于Bax的降解减少，因为吉非替尼治疗没有改变Bax mRNA的水平。吉非替尼促进线粒体富集部分全长p21 Bax裂解为p18 Bax，这是Bax向凋亡激活的一个特征。此外，使用抗Bax小干扰双链RNA （siRNA）阻断Bax可显著减少吉非替尼诱导的细胞凋亡。综上所述，这些数据表明p18 Bax在吉非替尼诱导的细胞凋亡中起关键作用。接下来，我们研究了Src和Ras在吉非替尼敏感性中的机制作用，这两种主要的致癌基因产物与许多人类癌症的发病机制有关。以亲代和v-src或c- h - Ras转染的人胆囊腺癌HAG-1细胞系为研究对象，通过WST-1法、流式细胞术和Western blots检测吉非替尼对细胞毒性、细胞周期阻滞和凋亡以及EGFR、Akt和Erk酪氨酸磷酸化的影响，激活的Ras和Src对吉非替尼分别产生了近30倍和200倍的强抗性。吉非替尼在24小时诱导细胞周期的G0/G1期细胞积累，亲代HAG-1细胞中凋亡细胞群逐渐扩增，但这些影响在转染v-src或c- h -ras的细胞系中完全消除。经吉非替尼处理后，HAG-1细胞中EGFR的激活以及随后通过Erk和Akt的下游激活均被显著抑制。相比之下，吉非尼布在v-src转染的细胞和Erk中未能抑制Akt和Erk的激活，但在c- h -ras转染的细胞中没有抑制Akt，尽管在这些细胞系中阻断了EGFR的激活。用herbimycin A（一种Src酪氨酸激酶抑制剂）处理v-sre转染的细胞，部分揭示了吉非尼替的耐药性，同时抑制了Akt和Erk。我们的研究结果表明，激活的Ras和Src可以通过激活Akt和Erk信号通路中的一种或两种来诱导吉非尼布耐药，从而为评估EGFR下游的这些特异性信号分子以定制治疗提供了战略依据。少

Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been clinically demonstrated to be effective for certain cancer cell types, the molecular mechanisms of the anti-tumor activity have not been fully elucidated. In this study, we investigated the mechanism of gefitinib-induced growth inhibition and apoptosis in HAG-1 human gallbladder adenocarcinoma cells. Treatment of gefitinib at a dose of 1 mM resulted in a significant growth inhibition, and the cell number irreversibly declined after 72-h incubation, with a progressive expansion of apoptotic cell population over 120-h. Following 2-h treatment, gefitinib significantly inhibited EGFR autophosphorylation and subsequent downstream signaling pathway through Erk and Akt, and induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, accompanied by a concomitant increase in p21 transcript and increased expression of p27. Gefitinib did not affect the amount of total and … More phosphorylated p53 at serine 15, but upregulated the expression of total Bax, with subsequent increase in p18 Bax, an active form of Bax. The expression of Bcl-2 and Bad was unchanged. An increase in gefitinib-induced expression of total Bax might be due to the decreased degradation of Bax, because the level of Bax mRNA has not been altered by gefitinib treatment. Gefitinib promoted the cleavage of full-length p21 Bax into p18 Bax in mitochondrial-enriched fraction, a characteristic feature of Bax activation toward apoptosis. Moreover, blockade of Bax by using anti-Bax small interfering double stranded RNA (siRNA) significantly reduced gefitinib-induced apoptosis. Taken together, these data suggest a critical role of p18 Bax in gefitinib-induced apoptosis.Next, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Geftinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-sre-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially revearsed the gefinitib resustance, with concomitant inhibition of Akt and Erk. Our results suggest that activated Ras and Src could induce gefinitib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationate for assessment of these specific signaling molecules downstream of EGFR to customize treatment. Less