Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis
Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis
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DOI：
10.1007/s10495-013-0835-5
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发表时间：
2013-07-01
期刊：
影响因子：
7.2
通讯作者：
Chung, Kyung-Sook
中科院分区：
文献类型：
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作者：
Ahn, Jiwon;Won, Misun;Chung, Kyung-Sook
紫草素衍生物具有很强的细胞毒作用，可诱导细胞凋亡，并可逃避肿瘤的多药耐药。然而，其抗癌活性的各种机制尚未完全了解。在这里，我们证明了紫草素诱导的细胞凋亡是由活性氧（ROS）介导的Akt/ASK 1/p38丝裂原活化蛋白激酶（MAPK）的激活和p21（Cip 1）的下调引起的。在紫草素的存在下，Akt的失活导致凋亡信号调节激酶1（ASK 1）的Ser 83去磷酸化，这与ASK 1的激活有关。紫草素诱导的细胞凋亡通过抑制Akt而增强，而组成性激活Akt的过表达通过调节ASK 1磷酸化而阻止细胞凋亡。通过siRNA沉默ASK 1和MKK 3/6分别降低MAPK激酶（MKK）3/6和p38 MAPK的活化和细胞凋亡。抗氧化剂N-乙酰半胱氨酸（N-acetylcysteine）可抑制Akt/ASK 1/p38信号通路中ASK 1的去磷酸化和p38 MAPK的激活，表明紫草素诱导的ROS参与了Akt/ASK 1/p38信号通路的激活。p21（Cip 1）的表达在早期反应中显着诱导，但随着紫草素暴露时间的延长而逐渐降低。p21（Cip 1）的过表达使细胞保持在G1期的时间延长，并减弱了紫草素诱导的凋亡。p21（Cip 1）的缺失促进了紫草素诱导的细胞凋亡，这意味着p21（Cip 1）通过G1期阻滞延迟了紫草素诱导的细胞凋亡。免疫组织化学和体外结合试验表明，紫草素，这是由Akt抑制阻断的p21（Cip 1）的细胞质定位的瞬时改变。细胞质p21（Cip 1）实际上结合并抑制ASK 1的活性，调节G1期细胞周期进程。这些结果表明，紫草素诱导的活性氧激活ASK 1通过减少Ser 83磷酸化和解离的负调节p21（Cip 1），导致p38 MAPK激活，并最终，促进细胞凋亡。
Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.