Laboratorio de Oncología, Unidad de Medicina Molecular, Centro Regional de Investigaciones Biomédicas Universidad de Castilla-La Mancha, Unidad Asociada de Biomedicina UCLM, Unidad asociada al CSIC, Albacete, Spain.
Departamento de ciencias Médicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Albacete, Spain.
PLoS One. 2018 Jul 26;13(7):e0200878. doi: 10.1371/journal.pone.0200878. eCollection 2018.
To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines.
An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA.
Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches.
The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.
为了充分阐明丝裂原活化蛋白激酶（Mitogen Activated Protein Kinase，MAPK）在索拉非尼治疗肾细胞癌（Renal Cell Carcinoma，RCC）中的治疗反应中的作用以及与该激酶抑制剂相关的细胞死亡机制，我们评估了几种丝裂原活化蛋白激酶在肾细胞癌衍生细胞系中的作用。
通过 MTT 测定法评估肾细胞癌衍生细胞系（ACHN 和 786-O 细胞）的细胞活力，通过 caspase 3/7 活性诱导细胞凋亡，通过 LC3 脂质化和 p62 降解评估自噬诱导，并使用磷酸化靶向抗体评估激酶活性。使用编码特异性 shRNA 的慢病毒载体敲低 ATG5 和 ERK5。
我们的数据排除了细胞外调节激酶 1/2 和 5 以及 p38 MAPK 途径作为索拉非尼毒性作用的介质，而是表明抑制作用是通过 PI3K/Akt 信号通路发挥的。此外，我们证明抑制 Akt 介导与索拉非尼相关的细胞死亡，而不激活 caspase，这与自噬的诱导一致，如通过药理学和遗传方法所示。
本报告表明，索拉非尼通过 Akt 依赖性方式诱导自噬而发挥其毒性作用，而不涉及丝裂原活化蛋白激酶。因此，我们的数据排除了 RAF-MEK-ERK1/2 信号通路抑制剂在 RCC 中的应用，并支持使用促进自噬的化合物，为肾细胞癌提供了新的治疗机会。
