MCF-7 和 MDA-MB-231 细胞对姜黄素细胞毒性作用的差异敏感性与 PI3K/Akt-SKP2-Cip/Kips 通路有关。
The differential susceptibilities of MCF-7 and MDA-MB-231 cells to the cytotoxic effects of curcumin are associated with the PI3K/Akt-SKP2-Cip/Kips pathway.
机构信息
Key Laboratory of Brain Functional Genomics, Ministry of Education Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, School of Life Sciences, Shanghai, 200062 China ; Current address: INSERM U823, Grenoble, F-38042 France.
Key Laboratory of Brain Functional Genomics, Ministry of Education Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, School of Life Sciences, Shanghai, 200062 China.
出版信息
Cancer Cell Int. 2014 Nov 30;14(1):126. doi: 10.1186/s12935-014-0126-4. eCollection 2014.
BACKGROUND
The mechanism underlying the differential cytotoxicity of curcumin in various cancer types, however, remains largely unclear. The aims of this study is to examine the concentration- and time-related effects of curcumin on two different breast cancer cells, MCF-7 and MDA-MB-231, and investigated the functional changes induced by curcumin treatment, as well as their relationship to the PI3K/Akt-SKP2-Cip/Kips pathway.
METHODS
First, WST-1 and clonogenic assay were performed to determine the cytotoxicity of curcumin in MCF-7 and MDA-MB-231 cells. Then, the expression of CDK interacting protein/Kinase inhibitory protein (Cip/Kips) members (p27, p21 and p57) and S-phase kinase-associated protein-2 (SKP2) was investigated by QRT PCR and Western Blotting. Curcumin's effect on PI3K (phosphatidylinositol 3-kinase) /Akt and its substrates Foxo1 and Foxo3a were then studied by Western Blotting. Small interfering RNAs (siRNAs) targeting SKP2 was used to explore the relationship between SKP2 and Cip/Kips members. Finally, WST-1 assay was tested to explore the concomitant treatment with curcumin and the inhibition of PKB or SKP2 signaling on curcumin sensitivity in MCF-7 and MDA-MB-231 cells.
RESULTS
We demonstrated MCF-7 and MDA-MB-231 cells exhibited differential responses to curcumin by WST-1 and clonogenic assay (MDA-MB-231 cells was sensitive, and MCF-7 cells was resistant), which were found to be related to the differential curcumin-mediated regulation of SKP2-Cip/Kips (p21 and p27 but not p57) signaling. The differential cellular responses were further linked to the converse effects of curcumin on PI3K/Akt and its substrates Foxo1 and Foxo3a. Importantly, PI3K inhibitor wortmannin could counteract both curcumin-induced phosphorylation of Akt and up-regulation of SKP2 in MCF-7 cells. Subsequent WST-1 assay demonstrated concomitant treatment with curcumin and wortmannin or SKP2 siRNA not only further augmented curcumin sensitivity in MDA-MB-231 cells but also overcame curcumin resistance in MCF-7 cells.
CONCLUSIONS
Our study established PI3K/Akt-SKP2-Cip/Kips signaling pathway is involved in the mechanism of action of curcumin and revealed that the discrepant modulation of this pathway by curcumin is responsible for the differential susceptibilities of these two cell types to curcumin.
背景
然而，姜黄素在不同癌症类型中产生差异细胞毒性的机制在很大程度上仍不清楚。本研究旨在研究姜黄素对两种不同乳腺癌细胞 MCF-7 和 MDA-MB-231 的浓度和时间相关作用，并研究姜黄素处理诱导的功能变化，以及它们与 PI3K/Akt-SKP2-Cip/Kips 途径的关系。
方法
首先，通过 WST-1 和集落形成实验确定姜黄素对 MCF-7 和 MDA-MB-231 细胞的细胞毒性。然后，通过 QRT-PCR 和 Western Blotting 研究 CDK 相互作用蛋白/激酶抑制蛋白（Cip/Kips）成员（p27、p21 和 p57）和 S 期激酶相关蛋白-2（SKP2）的表达。然后通过 Western Blotting 研究姜黄素对 PI3K（磷脂酰肌醇 3-激酶）/Akt 及其底物 Foxo1 和 Foxo3a 的影响。用靶向 SKP2 的小干扰 RNA（siRNA）探索 SKP2 与 Cip/Kips 成员之间的关系。最后，通过 WST-1 测定法研究姜黄素与抑制 PKB 或 SKP2 信号转导同时处理对 MCF-7 和 MDA-MB-231 细胞中姜黄素敏感性的影响。
结果
我们通过 WST-1 和集落形成实验证明 MCF-7 和 MDA-MB-231 细胞对姜黄素的反应不同（MDA-MB-231 细胞敏感，而 MCF-7 细胞耐药），这与姜黄素对 SKP2-Cip/Kips（p21 和 p27，但不是 p57）信号的差异调节有关。细胞反应的差异进一步与姜黄素对 PI3K/Akt 及其底物 Foxo1 和 Foxo3a 的相反作用有关。重要的是，PI3K 抑制剂wortmannin 可以逆转姜黄素诱导的 MCF-7 细胞中 Akt 磷酸化和 SKP2 上调。随后的 WST-1 测定表明，同时用姜黄素和 wortmannin 或 SKP2 siRNA 处理不仅进一步增强了 MDA-MB-231 细胞中姜黄素的敏感性，而且克服了 MCF-7 细胞中姜黄素的耐药性。
结论
本研究确立了 PI3K/Akt-SKP2-Cip/Kips 信号通路参与了姜黄素的作用机制，并揭示了姜黄素对该通路的不同调节是这两种细胞类型对姜黄素敏感性差异的原因。
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