Department of Life Sciences, National Chung-Hsing University, No. 250 Kuo-Kuang Rd, Taichung 402, Taiwan, ROC.
J Neurooncol. 2011 Dec;105(3):507-22. doi: 10.1007/s11060-011-0632-3. Epub 2011 Jul 9.
Gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor, is under clinical testing and use in cancer patients, including glioma. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma remain largely uncharacterized. Gefitinib inhibits cell growth and induces apoptosis in human glioma cells. Gefitinib also induces death of H4 cells with characteristics of the intrinsic apoptotic pathway, including Bax mitochondrial translocation, mitochondrial outer membrane permeabilization, cytochrome c cytosolic release, and caspase-9/caspase-3 activation. The importance of Bax in mediating gefitinib-induced apoptosis was confirmed by the attenuation of apoptosis by Bax siRNA and Bax channel blocker. Gefitinib caused Bad dephosphorylation, particularly in serine-112, and increased its binding preference to Bcl-2 and Bcl-xL. The dephosphorylation of Bad in gefitinib-treated cells was accompanied by reduced intracellular cyclic AMP content and protein kinase A (PKA) activity. Adenylyl cyclase activator forskolin attenuated, but PKA inhibitor H89 augmented, gefitinib-induced Bad dephosphorylation, Bax mitochondrial translocation, caspase-9/caspase-3 activation, and viability loss. Intriguingly, a nonselective protein phosphatase inhibitor okadaic acid alleviated gefitinib-induced alterations, except Bad dephosphorylation. In parallel with the higher basal PKA activity, response of U87 cells to gefitinib treatment was delayed and relatively resistant compared with that of H4 and T98G cells. Inactivation of PKA sensitized H4, T98G, and U87 cells to gefitinib cytotoxicity, Bad dephosphorylation in serine-112, and caspase-9/caspase-3 activation. Our findings suggest the involvement of the Bad/Bax signaling pathway in gefitinib-induced glioma apoptosis. Furthermore, the inactivation of PKA was shown to play a role in triggering the proapoptotic function of Bad.
吉非替尼是一种选择性表皮生长因子受体酪氨酸激酶抑制剂，正在进行癌症患者（包括脑胶质瘤患者）的临床测试和使用。然而，吉非替尼介导的抗脑胶质瘤作用的分子机制在很大程度上仍未被阐明。吉非替尼抑制人脑胶质瘤细胞的生长并诱导其凋亡。吉非替尼还诱导具有内在凋亡途径特征的 H4 细胞死亡，包括 Bax 线粒体易位、线粒体外膜通透性、细胞色素 c 胞浆释放和 caspase-9/caspase-3 激活。通过 Bax siRNA 和 Bax 通道阻滞剂减弱凋亡证实了 Bax 在介导吉非替尼诱导的凋亡中的重要性。吉非替尼引起 Bad 的去磷酸化，特别是丝氨酸 112 上的去磷酸化，并增加其与 Bcl-2 和 Bcl-xL 的结合偏好。在吉非替尼处理的细胞中，Bad 的去磷酸化伴随着细胞内环磷酸腺苷含量和蛋白激酶 A（PKA）活性的降低。腺苷酸环化酶激活剂 forskolin 减弱，但 PKA 抑制剂 H89 增强，吉非替尼诱导的 Bad 去磷酸化、Bax 线粒体易位、caspase-9/caspase-3 激活和细胞活力丧失。有趣的是，非选择性蛋白磷酸酶抑制剂 okadaic 酸减轻了吉非替尼诱导的改变，除了 Bad 的去磷酸化。与较高的基础 PKA 活性平行，与 H4 和 T98G 细胞相比，U87 细胞对吉非替尼处理的反应延迟且相对耐药。PKA 的失活使 H4、T98G 和 U87 细胞对吉非替尼的细胞毒性、Bad 在丝氨酸 112 上的去磷酸化和 caspase-9/caspase-3 激活敏感。我们的研究结果表明，Bad/Bax 信号通路参与了吉非替尼诱导的脑胶质瘤细胞凋亡。此外，PKA 的失活被证明在触发 Bad 的促凋亡功能中起作用。
