Mcl-1 determines the imiquimod-induced apoptosis but not imiquimod-induced autophagy in skin cancer cells
Mcl-1 determines the imiquimod-induced apoptosis but not imiquimod-induced autophagy in skin cancer cells
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DOI：
10.1016/j.jdermsci.2011.11.001
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发表时间：
2012-03-01
影响因子：
4.6
通讯作者：
Shieh, Jeng-Jer
中科院分区：
文献类型：
--
作者：
Huang, Shi-Wei;Chang, Chia-Che;Shieh, Jeng-Jer
背景：咪喹莫特可诱导多种皮肤癌细胞的凋亡和自噬，尤其是基底细胞癌细胞。目的：探讨咪喹莫特诱导皮肤癌细胞凋亡和自噬的分子机制。方法：免疫印迹法检测Mcl-1、Bcl2和Bclxl蛋白的表达。用逆转录聚合酶链式反应和实时荧光定量聚合酶链式反应检测Mcl-1mRNA水平。通过加入放线菌酮，探讨咪喹莫特降低Mcl-1蛋白表达的机制。MG132蛋白酶体抑制剂或泛caspase抑制剂。免疫印迹法检测咪喹莫特对eIF4E、4E-BP1和eEF2的磷酸化作用。通过XTT试验、线粒体膜电位测定、DNA含量测定、LC3免疫印迹、EGFP-LC3斑点形成和吖啶橙染色定量等方法评价咪喹莫特对Mcl-1高表达细胞的诱导凋亡和自噬作用。结果：咪喹莫特作用后Mcl-1蛋白水平下降的速度和强度均快于Bcl2和Bclxl。咪喹莫特诱导的Mcl-1蛋白表达下降不是由于转录受阻或促进降解所致，而是与BCC细胞中翻译因子失活有关。咪喹莫特作用下高表达Mcl-1的BCC细胞对内源性细胞凋亡的抵抗力明显高于对照BCC细胞。结论：咪喹莫特可能通过抑制皮肤癌细胞的翻译而迅速下调Mcl-1蛋白水平。在咪喹莫特治疗皮肤癌细胞的过程中，MCL-1可能起到保护细胞不发生自噬和自噬细胞死亡的作用。(C)2011年日本皮肤病研究学会。爱思唯尔爱尔兰有限公司出版。版权所有。
Background: Imiquimod had been shown to induce apoptosis and autophagy in several skin cancer cells, especially basal cell carcinoma (BCC) cells.Objective: We evaluate the molecular mechanisms of imiquimod-induced apoptosis and autophagy in skin cancer cell lines.Methods: The Mcl-1, Bcl-2 and Bcl-xL proteins were determined by immunoblotting. The Mcl-1 mRNA level was examined by RT-PCR and real-time PCR. The mechanisms of imiquimod-induced decrease in Mcl-1 protein were evaluated by addition of cycloheximide. MG132 proteasome inhibitor or pan-caspase inhibitor. The phosphorylation of eIF4E, 4E-BP1 and eEF2 in imiquimod treated cells were examined by immunoblotting. The imiquimod-induced apoptosis and autophagy were evaluated in Mcl-1-overexpressing cells by XTT test, mitochondrial membrane potential measurement, DNA content assay, LC3 immunoblotting, EGFP-LC3 puncta formation and quantification of acidic vesicular organelle with acridine orange staining.Results: The decrease in the Mcl-1 protein level was faster and stronger than the decrease in Bcl-2 and Bcl-xL in imiquimod-treated skin cancer cells. The imiquimod-induced decrease in Mcl-1 protein was not caused by blocked transcription or the promotion of degradation but was associated with inactivation of translation factors in BCC cells. The Mcl-1-overexpressing BCC cells were more resistant to intrinsic cellular apoptosis than control BCC cells during imiquimod treatment. Mcl-1 overexpression in BCC cells resulted in the basal activation of autophagy but did not modulate imiquimod-induced autophagy or rescue imiquimod-induced autophagic cell death in BCC cells.Conclusions: Imiquimod may rapidly downregulate Mcl-1 protein levels by inhibiting translation in skin cancer cells. Mcl-1 may act to protect against apoptosis but not autophagy and autophagic cell death during imiquimod treatment in skin cancer cells. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.