Inactivation of Ras and changes of mitochondrial membrane potential contribute to oridonin-induced autophagy in A431 cells
Inactivation of Ras and changes of mitochondrial membrane potential contribute to oridonin-induced autophagy in A431 cells
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DOI：
10.1254/jphs.fpj06022x
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发表时间：
2007-09-01
影响因子：
3.5
通讯作者：
Ikejima, Takashi
中科院分区：
文献类型：
--
作者：
Li, Dan;Cui, Qiao;Ikejima, Takashi
我们以前已经表明，冬凌草甲素增强细胞凋亡，同时抑制自噬24小时内在HeLa细胞。然而，冬凌草甲素诱导A431细胞凋亡和自噬的机制尚不清楚。在此，发现当用manumycin A（Ras特异性抑制剂）预处理A431细胞时，与单独的冬凌草甲素处理相比，自噬水平显著上调，而用GW 5074（Raf抑制剂）或PD 98059（ERK抑制剂）预培养的细胞没有表现出这种效果。Ras而不是Raf或ERK参与了冬凌草甲素诱导的自噬的控制。同时，manumycin A有助于冬凌草甲素诱导的Ras蛋白表达下调。用冬凌草甲素和manumycin A的组合治疗下调了磷脂酰肌醇3-OH激酶（PI 3-K）下游Akt的磷酸化。与PI 3-K抑制剂wortmannin和Akt抑制剂KP 372 -1预孵育增强了冬凌草甲素诱导的细胞凋亡，而抑制了冬凌草甲素诱导的自噬。然而，在冬凌草甲素处理下，具有自噬诱导活性的Beclin-1的表达降低，表明Beclin-1不参与冬凌草甲素诱导的自噬。形态学观察、DNA片段化分析和LDH活性测定结果表明，自噬抑制剂3-甲基腺嘌呤（3-MA）可增加冬凌草甲素诱导A431细胞凋亡的敏感性。此外，manumycin A有助于冬凌草甲素诱导的线粒体膜电位（Δ psi m）的降低，与Bax/Bcl-2比值的上调一致。总之，Ras负性调节了冬凌草甲素处理的A431细胞的自噬，这可能与I类PI 3-K的激活有关。自噬过程的启动可能与Δ psi m的下调和Bax/Bcl-2比值的升高有关。
We have previously shown that oridonin isolated from Rabdosia rubescens augmented apoptosis while inhibiting autophagy within 24 h in HeLa cells. However, the mechanisms between apoptosis and autophagy induced by oridonin in A431 cells are largely unknown. Here, it was found that autophagic level is significantly upregulated when A431 cells are pretreated with manumycin A (Ras specific inhibitor) compared with oridonin alone treatment, whereas cells precultured with GW5074 (Raf inhibitor) or PD98059 (ERK inhibitor) did not exhibit such an effect. Ras, but not Raf or ERK, was engaged in the control of oridonin-induced autophagy. At the same time, manumycin A contributes to oridonin-induced down-regulation of Ras protein expression. Treatment with the combination of oridonin and manumycin A downregulated phosphorylation of Akt, downstream of phosphatidylinositol 3-OH kinase (PI3-K). Preincubation with the PI3-K inhibitor wortmannin and Akt inhibitor KP372-1 enhanced oridonin-induced apoptosis, whereas it inhibited oridonin-induced autophagy. However, under oridonin treatment, the expression of Beclin-1, which has autophagy-inducing activity, was reduced, suggesting that Beclin-1 did not participate in the oridonin-induced autophagy. Morphologic observations, DNA fragmentation analysis, and LDH activity-based assay showed that 3-methyladenine (3-MA), an inhibitor of autophagy, increased the apoptotic sensitivity of A431 cells to oridonin. In addition, manumycin A contributed to oridonin-induced decrease of mitochondrial membrane potential (Delta psi m), consistent with the upregulation of Bax/Bcl-2 ratio. In conclusion, Ras negatively regulated autophagy in oridonin-treated A431 cells, which might be associated with activation of class I PI3-K. Downregulation of Delta psi m and increasing of the ratio of Bax/Bcl-2 might also be partially responsible for the initiation of the autophagic process.