姜黄素调控PI3K/AKT-Skp2-Cip/Kips通路诱导MCF-7，MDA-MB-231不同细胞毒性敏感性的分子机理研究
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摘要
姜黄素是植物姜黄的一个主要成分。它的药理作用广泛,主要特征为抗氧化和抗炎症。近些年大量文献报道姜黄素有抗癌活性,它能够抑制多种肿瘤细胞的增殖,侵袭,血管生成以及细胞代谢。与此同时,不同肿瘤细胞对姜黄素又表现出不同的敏感性,然而对其背后的机理却研究得很少。本课题探讨姜黄素作用于MCF-7和MDA-MB-231二种不同乳腺癌细胞的分子机理,从而进一步了解姜黄素的作用机制以及姜黄素成为潜在抗肿瘤药物的可能性。
WST-1细胞毒性试验,克隆群落试验,流式周期检测和caspase-3活性检测试验结果显示姜黄素对MCF-7细胞和MDA-MB-231细胞均有抗增殖及致凋亡作用,而姜黄素对MDA-MB-231细胞毒性明显大于其对MCF-7细胞毒性。进一步研究发现,MCF-7和MDA-MB-231细胞对姜黄素呈现不同敏感性与细胞周期蛋白抑制蛋白Cip/Kips家族的p27和p21的激活相关,而与p57不相关。在MDA-MB-231细胞上,p27和p21的表达随姜黄素浓度及作用时问的递增而上调,而调控p27和p21降解的泛素化连接酶skp2则呈现随姜黄素浓度和时间梯度下调。在MCF-7细胞株中,仅当姜黄素浓度高且作用时间较长时,才出现显著的p27,p21上调以及skp2下调,40μM姜黄素作用MCF-7细胞3hr,6hr观察到skp2反而上调。结果提示,姜黄素在MCF-7和MDA-MB-231细胞上对skp2-Cip/Kips信号通路有不同的调控作用,而这种调控作用的差异与MCF-7和MDA-MB-231细胞对姜黄素毒性呈现敏感性差异有着密切关系。这在skp2RNA干扰(skp2-siRNA)的MCF-7以及MDA-MB-231细胞中得到了证实,即skp2RNA干扰后,这二种细胞的p27和p21明显上调,且skp2-siRNA和姜黄素联用显著提高了MCF-7细胞对姜黄素毒性的敏感性,细胞毒性与姜黄素诱导MDA-MB-231细胞毒性相当。Skp2-siRNA和姜黄素联用处理MDA-MB-231细胞有药物协同作用,诱导细胞毒性更大。
为了解姜黄素作用下skp2-Cip/Kips的变化与其上游信号分子的作用关系,我们考察了作为skp2代谢的关键调控因子之一的AKT活性对姜黄素作用的响应。结果显示,姜黄素介导的AKT (ser473位点)磷酸化及其底物foxol及foxo3a磷酸化在MCF-7和MDA-MB-231细胞中呈相反的变化,提示PI3K/AKT信号通路也可能与姜黄素诱导的毒性敏感性差异存在重要作用关系。为确证姜黄素调控skp2与PI3K/AKT的作用关系,我们将wortmannin (PI3K特异性抑制剂)和姜黄素共同作用于耐药株MCF-7细胞,发现姜黄素引起的AKT磷酸化及其skp2上调都得到了逆转,提示姜黄素调控skp2的变化与AKT的磷酸化程度密切相关。使用wortmannin和姜黄素联合作用MCF-7细胞提高了MCF-7对姜黄素诱导细胞毒性的敏感性至姜黄素单独作用MDA-MB-231细胞毒性的水平。Wortmannin和姜黄素联用处理MDA-MB-231细胞有药物协同作用,联合用药诱导的MDA-MB-23细胞毒性更大
综上所述,我们的结果揭示skp2是姜黄素作用乳腺癌MCF-7细胞和MDA-MB-231细胞重要的分子靶标。姜黄素诱导的细胞毒性与PI3K-Skp2-Cip/Kips信号通路的抑制程度相关,提示P-AKT及其skp2在预测姜黄素用于乳腺癌治疗效果中是非常重要的药理学分子标记物。PI3K/AKT通路抑制剂以及skp2依赖的泛素蛋白酶降解途径抑制剂和姜黄素联合用药可能为其治疗乳腺癌提供更好的策略。
第一章姜黄素诱导MCF-7和MDA-MB-231细胞毒性研究
为了考察姜黄素对乳腺癌细胞的作用机制,选择了两种典型的细胞株MCF-7和MDA-MB-231作为细胞模型。我们分别运用WST-1细胞毒性检测,克隆群落实验,细胞周期流式以及细胞形态学观察研究姜黄素对MCF-7和MDA-MB-231细胞的毒性作用。试验结果显示,姜黄素对两种细胞株均有毒性作用,但姜黄素对两个细胞株毒性的敏感性不一样,WST-1细胞毒性检测,克隆群落实验以及流式周期实验一致表明姜黄素对MDA-MB-231细胞表现出更大的毒性。
第二章姜黄素对细胞凋亡调控因子caspase-3活性的影响
因caspase-3是细胞凋亡的关键执行者,故我们采用Western Blotting方法检测姜黄素对其表达活性的影响。结果表明,姜黄素均可以上调两个乳腺癌细胞的caspase-3活性,其中MDA-MB-231细胞caspase-3活性上调更明显。Western Blotting的结果和第一章细胞毒性结果一致,姜黄素对MDA-MB-231细胞表现出更大的细胞毒性。同时提示姜黄素诱导乳腺癌细胞毒性的作用与caspase-3激活的细胞凋亡途径有关。
第三章姜黄素调控MCF-7和MDA-MB-231细胞skp2-Cip/Kips信号通路的研究
S期激酶相关蛋白2(skp2),作为细胞周期中重要的调节因子,广泛的参与到肿瘤的发生,转移,血管生成,以及药物抗逆性作用中。Skp2介导的泛素化降解底物Cip/Kips家族蛋白p27,p21,p57被广泛的报道与化疗药物作用肿瘤细胞的毒性敏感性相关,我们检测了姜黄素作用这两个细胞skp2-Cip/Kips蛋白的变化。结果显示,在mRNA和蛋白水平上姜黄素明显抑制了skp2的表达,伴随着p27和p21浓度梯度和时问梯度的上调。在MCF-7细胞,我们仅在药物高浓度作用长时间才观察到skp2的抑制作用并伴随p27和p21的上调。姜黄素处理MCF-7细胞和MDA-MB-231细胞,p57没有显著变化。与此同时40μM姜黄素作用MCF-7细胞3小时,skp2上调,6小时达到最高点。Skp2-SiRNA干扰skp2的表达后,MCF-7和MDA-MB-231细胞的skp2-SiRNA干扰株p27和p21均明显上调,P57变化不明显。结果提示姜黄素诱导细胞毒性的敏感性与其调控Skp2-Cip/Kips通路的抑制能力相关。同时WST-1细胞毒性测试结果显示姜黄素表现出对skp2-SiRNA干扰MCF-7细胞株和skP2-SiRNA干扰MDA-MB-231细胞株更强的细胞毒性,结果进一步肯定了skp2水平是姜黄素作用MCF-7和MDA-MB-231细胞毒性重要的决定因素。
第四章姜黄素诱导不同细胞毒性差异性的分子机制研究(PI3K/AKT-Skp2通路)
AKT磷酸化激活程度在决定细胞毒性的敏感性方面起重要的作用,同时AKT的活性是调节skp2变化的重要影响因子之一。我们进一步研究了姜黄素作用细胞PI3K/AKT相关信号通路的变化。结果显示姜黄素显著抑制了MDA-MB-231细胞AKT ser473位点磷酸化水平。相反,40μM姜黄素处理MCF-7细胞上调了其AKT ser473位点的磷酸化。同时我们观察到姜黄素处理两个细胞株AKT磷酸化底物p-foxol (Thr24), p-foxo3a(Ser318/321)的表达也呈现出相反的变化。1μM wortmannin (PI3K/AKT抑制剂)和40μM姜黄素联合处理MCF-7细胞6小时,扭转了姜黄素引起的p-AKT(ser473)以及skp2的上调。同时,我们观察到1μM wortmannin单独作用MCF-7细胞6小时,skp2蛋白表达并没有显著下调,结合第3章结果40μM姜黄素单独处理MCF-7细胞24小时,skp2下调,而p-AKT依旧维持在高表达水平,提示姜黄素调控skp2代谢除了p-AKT途径可能还有其他途径。进一步确认PI3K/AKT信号通路在决定姜黄素诱导MCF-7和MDA-MB-231细胞毒性敏感性中的作用。我们使用PI3K/AKT通路抑制剂(wortmannin)和姜黄素联用作用MCF-7和MDA-MB-231细胞。WST-l细胞毒性检测结果显示,对于抵抗株MCF-7细胞,药物联用克服(overcome)了姜黄素的抵抗且诱导的细胞毒性与姜黄度诱导敏感株细胞MDA-MB-231的毒性相当。对于敏感株细胞MDA-MB-231, wortmannin与姜黄素联用有药物协同作用,联合用药诱导的细胞毒性更大。
Curcumin, a major constituent of the spice turmeric, has been reported to inhibit proliferation, invasion, angiogenesis and metastasis in multiple tumor cells. Cell inhibition of curcumin also shows differently in multiple cancer cells, however the mechanism for the different cytotoxic effect of curcumin has been little explored. A better understanding of the downstream cellular targets of curcumin will provide information on its mechanism of action and help to explore the potential of using curcumin for chemotherapy.
We presented in vitro evidence that curcumin exhibited much cytotoxic in MDA-MB-231cells than in MCF-7cells characterized by measurement of cell viability, clonogenic ability, and activation of caspase-3. This different sensitivity was associated with the induction of Cip/Kips (CDK interacting protein/kinase inhibitory protein) p27, p21expressions. In MDA-MB-231cells, we demonstrated dose-and time-dependent up-regulations of p27and p21that correlated with the reduced expression of skp2, an important regulator of Cip/Kips, while in MCF-7cells, we found the similar effect of curcumin only at a higher concentration and a longer treatment time. Further, we found that curcumin showed converse effects on AKT phosphorylation and its substrates p-foxo3a and p-foxol in MCF-7and MDA-MB-231cells, suggesting PI3K/AKT pathway may contribute greatly to the curcumin-induced differential expressions of skp2and Cip/Kips in MCF-7and MDA-MB-231cells. Wortmannin, a PI3K inhibitor could reverse curcumin-induced phosphorylation of AKT and up-regulation of skp2in MCF-7cells, and hereby enhanced the susceptibility of MCF-7cells to curcumin. Silencing the expression of skp2with a small interfering RNA resulted in an accumulation of p27, p21and enhanced cytotoxicity of curcumin in MCF-7and MDA-MB-231cells.
Taken together, these results suggest that curcumin showed differential cytotoxicities in different kinds of human breast cancer cells, which were demonstrated to be associated, at least in part, with PI3K/AKT-Skp2-Cip/Kips pathway. These results also indicated that p-AKT and skp2levels were key determinants of antitumor responses to curcumin in breast cancer, highlighting them to be potential pharmacogenomic markers for predicting the sensitivity of human breast cancer cells to curcumin as well as skp2silencing and p-AKT inhibiting strategies.
1Curcumin exhibits differential cytotoxicity on MCF-7and MDA-MB-231cells.
To characterize the mechanisms of curcumin's action in breast cancer cells, two typical breast cell lines MCF-7and MDA-MB-231were used. The effects of curcumin on the morphology and cell cycle of MCF-7and MDA-MB-231cells were examined, respectively. Curcumin treatment caused MCF-7cells and MDA-MB-231cell shrinkage, rounding and partial-detachment, demonstrating the cytotoxic effects of curcumin on both MCF-7and MDA-MB-231cells. Curcumin arrested MCF-7cells at G2/M phase of the cell cycle. In MDA-MB-231cells, an obvious apoptotic peak, but no significant cell cycle arrest, was observed after curcumin treatment. The data obtained using two other experimental approaches (WST-1assays and clonogenic analysis) confirmed that MCF-7and MDA-MB-231cells responded to curcumin differentially, and MDA-MB-231cells were much more sensitive to curcumin than MCF-7cells.
2Curcumin activates caspase-3in MCF-7and MDA-MB-231cells
Substantive evidences have shown that curcumin could induce MCF-7and MDA-MB-231cells to apoptosis, while caspase-3activation has been shown to be one of the most important cell executioners for apoptosis. Therefore, we examined the effect of curcumin on the activation of caspase-3by western blotting analysis. Activation of caspase-3was observed in both MCF7and MDA-MB-231cells treated with curcumin. Notably, the activation of caspase-3seemed to be much more significant in MDA-MB-231than MCF-7cells, which was in agreement with the data showing differential cytotoxicity and clonogenic ability after curcumin treatment.
3PI3K/AKT-Skp2-Cip/Kips is involved in curcumin-induced cytotoxicity in MCF-7and MDA-MB-231cells
Since Cip/kips are often well positioned to function as both sensor and effectors of multiple cytotoxicity signals, we compared the changes in protein expression of p21, p27and p57following curcumin treatment by western blotting analysis to determine whether differences in the expression or activity of Cip/Kips could explain the heterogeneous response to curcumin in MCF-7and MDA-MB-231cells. Our results confirmed that the cellular response to curcumin may be deeply associated with the activation of p27and p21in MCF-7and MDA-MB-231cells, not p57. S-phase kinase-associated protein-2(skp2), a specific substrate-recognition subunit of the Skpl-Cullin-F-box protein (SCF) type ubiquitin ligase complex, has been reported to mediate ubiquitin-dependent degradation of some cell-cycle proteins, including p27Kip1,p21Cip1and p57Kip2. Results showed curcumin increased the protein level of both p27and p21, decreased the level of skp2simultaneously. It is indicated that eurcumin-elevated p27and p21level may result from the suppression of the negative regulatory protein skp2, which subsequently leads to the cytotoxicity in MCF-7and MDA-MB-231cells. In order to confirm the role of skp2in the regulation of p27and p21in MCF-7and MDA-MB-231cells, we next performed gene silencing experiments. Skp2siRNA knocked down the protein levels of skp2in cells compared with control siRNA transfectants and untreated ones (NEG). As expected, it indeed increased the protein levels of both p27and p21after transfection for48hr. The above results suggest curcumin-induced cytotoxicity is associated with the inhibition of skp2-Cip/Kips pathway. Silencing of skp2in MCF-7and MDA-MB-231cells increased cellular sensitivity to curcumin. In conclusion, our data indicates skp2level is a key determinant of cytotoxic responses to curcumin.
4Effects of curcumin on expression and activity of AKT (PKB) and its substrates in MCF-7and MDA-MB-231cells
It has been well verified that the activity of AKT is one of the key determinants in defining sensitivity to chemotherapeutic drugs and its role involved in skp2regulation by transcriptional level and post-translational modification has also been reported largely in various cell types. We found that curcumin showed converse effects on AKT phosphorylation and its substrates p-foxo3a and p-foxol, suggesting PI3K/AKT pathway may contribute greatly to the differential expression of skp2and Cip/Kips in MCF-7and MDA-MB-231cells treated with curcumin. We next investigated whether the combined treatment with curcumin and wortmannin, a selective PI3K inhibitor, would reverse curcumin-induced phosphorylation of AKT thus down-regulation of skp2in MCF-7cells. MCF-7cells were treated with both wortmannin and curcumin for6hr, wortmannin significantly down regulated curcumin-induced phosphorylation of AKT and reversed curcumin-caused up-regulation of skp2markedly. We did not find the use of combination of wortmannin and curcumin or single agent wortmannin could down regulate the expression of skp2significantly comparing to the control group. In order to confirm the relevance of PI3K/AKT pathway in the cellular response to curcumin in MCF-7and MDA-MB-231cells, we next performed cell viability assays to assess whether simultaneous treatment with curcumin and wortmannin could enhance curcumin-induced cytotoxicity. Results showed the combing using of curcumin with wortmannin enhanced the cytotoxicity of curcumin to MCF-7and MDA-MB-231cells.
WST-1细胞毒性试验,克隆群落试验,流式周期检测和caspase-3活性检测试验结果显示姜黄素对MCF-7细胞和MDA-MB-231细胞均有抗增殖及致凋亡作用,而姜黄素对MDA-MB-231细胞毒性明显大于其对MCF-7细胞毒性。进一步研究发现,MCF-7和MDA-MB-231细胞对姜黄素呈现不同敏感性与细胞周期蛋白抑制蛋白Cip/Kips家族的p27和p21的激活相关,而与p57不相关。在MDA-MB-231细胞上,p27和p21的表达随姜黄素浓度及作用时问的递增而上调,而调控p27和p21降解的泛素化连接酶skp2则呈现随姜黄素浓度和时间梯度下调。在MCF-7细胞株中,仅当姜黄素浓度高且作用时间较长时,才出现显著的p27,p21上调以及skp2下调,40μM姜黄素作用MCF-7细胞3hr,6hr观察到skp2反而上调。结果提示,姜黄素在MCF-7和MDA-MB-231细胞上对skp2-Cip/Kips信号通路有不同的调控作用,而这种调控作用的差异与MCF-7和MDA-MB-231细胞对姜黄素毒性呈现敏感性差异有着密切关系。这在skp2RNA干扰(skp2-siRNA)的MCF-7以及MDA-MB-231细胞中得到了证实,即skp2RNA干扰后,这二种细胞的p27和p21明显上调,且skp2-siRNA和姜黄素联用显著提高了MCF-7细胞对姜黄素毒性的敏感性,细胞毒性与姜黄素诱导MDA-MB-231细胞毒性相当。Skp2-siRNA和姜黄素联用处理MDA-MB-231细胞有药物协同作用,诱导细胞毒性更大。
为了解姜黄素作用下skp2-Cip/Kips的变化与其上游信号分子的作用关系,我们考察了作为skp2代谢的关键调控因子之一的AKT活性对姜黄素作用的响应。结果显示,姜黄素介导的AKT (ser473位点)磷酸化及其底物foxol及foxo3a磷酸化在MCF-7和MDA-MB-231细胞中呈相反的变化,提示PI3K/AKT信号通路也可能与姜黄素诱导的毒性敏感性差异存在重要作用关系。为确证姜黄素调控skp2与PI3K/AKT的作用关系,我们将wortmannin (PI3K特异性抑制剂)和姜黄素共同作用于耐药株MCF-7细胞,发现姜黄素引起的AKT磷酸化及其skp2上调都得到了逆转,提示姜黄素调控skp2的变化与AKT的磷酸化程度密切相关。使用wortmannin和姜黄素联合作用MCF-7细胞提高了MCF-7对姜黄素诱导细胞毒性的敏感性至姜黄素单独作用MDA-MB-231细胞毒性的水平。Wortmannin和姜黄素联用处理MDA-MB-231细胞有药物协同作用,联合用药诱导的MDA-MB-23细胞毒性更大
综上所述,我们的结果揭示skp2是姜黄素作用乳腺癌MCF-7细胞和MDA-MB-231细胞重要的分子靶标。姜黄素诱导的细胞毒性与PI3K-Skp2-Cip/Kips信号通路的抑制程度相关,提示P-AKT及其skp2在预测姜黄素用于乳腺癌治疗效果中是非常重要的药理学分子标记物。PI3K/AKT通路抑制剂以及skp2依赖的泛素蛋白酶降解途径抑制剂和姜黄素联合用药可能为其治疗乳腺癌提供更好的策略。
第一章姜黄素诱导MCF-7和MDA-MB-231细胞毒性研究
为了考察姜黄素对乳腺癌细胞的作用机制,选择了两种典型的细胞株MCF-7和MDA-MB-231作为细胞模型。我们分别运用WST-1细胞毒性检测,克隆群落实验,细胞周期流式以及细胞形态学观察研究姜黄素对MCF-7和MDA-MB-231细胞的毒性作用。试验结果显示,姜黄素对两种细胞株均有毒性作用,但姜黄素对两个细胞株毒性的敏感性不一样,WST-1细胞毒性检测,克隆群落实验以及流式周期实验一致表明姜黄素对MDA-MB-231细胞表现出更大的毒性。
第二章姜黄素对细胞凋亡调控因子caspase-3活性的影响
因caspase-3是细胞凋亡的关键执行者,故我们采用Western Blotting方法检测姜黄素对其表达活性的影响。结果表明,姜黄素均可以上调两个乳腺癌细胞的caspase-3活性,其中MDA-MB-231细胞caspase-3活性上调更明显。Western Blotting的结果和第一章细胞毒性结果一致,姜黄素对MDA-MB-231细胞表现出更大的细胞毒性。同时提示姜黄素诱导乳腺癌细胞毒性的作用与caspase-3激活的细胞凋亡途径有关。
第三章姜黄素调控MCF-7和MDA-MB-231细胞skp2-Cip/Kips信号通路的研究
S期激酶相关蛋白2(skp2),作为细胞周期中重要的调节因子,广泛的参与到肿瘤的发生,转移,血管生成,以及药物抗逆性作用中。Skp2介导的泛素化降解底物Cip/Kips家族蛋白p27,p21,p57被广泛的报道与化疗药物作用肿瘤细胞的毒性敏感性相关,我们检测了姜黄素作用这两个细胞skp2-Cip/Kips蛋白的变化。结果显示,在mRNA和蛋白水平上姜黄素明显抑制了skp2的表达,伴随着p27和p21浓度梯度和时问梯度的上调。在MCF-7细胞,我们仅在药物高浓度作用长时间才观察到skp2的抑制作用并伴随p27和p21的上调。姜黄素处理MCF-7细胞和MDA-MB-231细胞,p57没有显著变化。与此同时40μM姜黄素作用MCF-7细胞3小时,skp2上调,6小时达到最高点。Skp2-SiRNA干扰skp2的表达后,MCF-7和MDA-MB-231细胞的skp2-SiRNA干扰株p27和p21均明显上调,P57变化不明显。结果提示姜黄素诱导细胞毒性的敏感性与其调控Skp2-Cip/Kips通路的抑制能力相关。同时WST-1细胞毒性测试结果显示姜黄素表现出对skp2-SiRNA干扰MCF-7细胞株和skP2-SiRNA干扰MDA-MB-231细胞株更强的细胞毒性,结果进一步肯定了skp2水平是姜黄素作用MCF-7和MDA-MB-231细胞毒性重要的决定因素。
第四章姜黄素诱导不同细胞毒性差异性的分子机制研究(PI3K/AKT-Skp2通路)
AKT磷酸化激活程度在决定细胞毒性的敏感性方面起重要的作用,同时AKT的活性是调节skp2变化的重要影响因子之一。我们进一步研究了姜黄素作用细胞PI3K/AKT相关信号通路的变化。结果显示姜黄素显著抑制了MDA-MB-231细胞AKT ser473位点磷酸化水平。相反,40μM姜黄素处理MCF-7细胞上调了其AKT ser473位点的磷酸化。同时我们观察到姜黄素处理两个细胞株AKT磷酸化底物p-foxol (Thr24), p-foxo3a(Ser318/321)的表达也呈现出相反的变化。1μM wortmannin (PI3K/AKT抑制剂)和40μM姜黄素联合处理MCF-7细胞6小时,扭转了姜黄素引起的p-AKT(ser473)以及skp2的上调。同时,我们观察到1μM wortmannin单独作用MCF-7细胞6小时,skp2蛋白表达并没有显著下调,结合第3章结果40μM姜黄素单独处理MCF-7细胞24小时,skp2下调,而p-AKT依旧维持在高表达水平,提示姜黄素调控skp2代谢除了p-AKT途径可能还有其他途径。进一步确认PI3K/AKT信号通路在决定姜黄素诱导MCF-7和MDA-MB-231细胞毒性敏感性中的作用。我们使用PI3K/AKT通路抑制剂(wortmannin)和姜黄素联用作用MCF-7和MDA-MB-231细胞。WST-l细胞毒性检测结果显示,对于抵抗株MCF-7细胞,药物联用克服(overcome)了姜黄素的抵抗且诱导的细胞毒性与姜黄度诱导敏感株细胞MDA-MB-231的毒性相当。对于敏感株细胞MDA-MB-231, wortmannin与姜黄素联用有药物协同作用,联合用药诱导的细胞毒性更大。
Curcumin, a major constituent of the spice turmeric, has been reported to inhibit proliferation, invasion, angiogenesis and metastasis in multiple tumor cells. Cell inhibition of curcumin also shows differently in multiple cancer cells, however the mechanism for the different cytotoxic effect of curcumin has been little explored. A better understanding of the downstream cellular targets of curcumin will provide information on its mechanism of action and help to explore the potential of using curcumin for chemotherapy.
We presented in vitro evidence that curcumin exhibited much cytotoxic in MDA-MB-231cells than in MCF-7cells characterized by measurement of cell viability, clonogenic ability, and activation of caspase-3. This different sensitivity was associated with the induction of Cip/Kips (CDK interacting protein/kinase inhibitory protein) p27, p21expressions. In MDA-MB-231cells, we demonstrated dose-and time-dependent up-regulations of p27and p21that correlated with the reduced expression of skp2, an important regulator of Cip/Kips, while in MCF-7cells, we found the similar effect of curcumin only at a higher concentration and a longer treatment time. Further, we found that curcumin showed converse effects on AKT phosphorylation and its substrates p-foxo3a and p-foxol in MCF-7and MDA-MB-231cells, suggesting PI3K/AKT pathway may contribute greatly to the curcumin-induced differential expressions of skp2and Cip/Kips in MCF-7and MDA-MB-231cells. Wortmannin, a PI3K inhibitor could reverse curcumin-induced phosphorylation of AKT and up-regulation of skp2in MCF-7cells, and hereby enhanced the susceptibility of MCF-7cells to curcumin. Silencing the expression of skp2with a small interfering RNA resulted in an accumulation of p27, p21and enhanced cytotoxicity of curcumin in MCF-7and MDA-MB-231cells.
Taken together, these results suggest that curcumin showed differential cytotoxicities in different kinds of human breast cancer cells, which were demonstrated to be associated, at least in part, with PI3K/AKT-Skp2-Cip/Kips pathway. These results also indicated that p-AKT and skp2levels were key determinants of antitumor responses to curcumin in breast cancer, highlighting them to be potential pharmacogenomic markers for predicting the sensitivity of human breast cancer cells to curcumin as well as skp2silencing and p-AKT inhibiting strategies.
1Curcumin exhibits differential cytotoxicity on MCF-7and MDA-MB-231cells.
To characterize the mechanisms of curcumin's action in breast cancer cells, two typical breast cell lines MCF-7and MDA-MB-231were used. The effects of curcumin on the morphology and cell cycle of MCF-7and MDA-MB-231cells were examined, respectively. Curcumin treatment caused MCF-7cells and MDA-MB-231cell shrinkage, rounding and partial-detachment, demonstrating the cytotoxic effects of curcumin on both MCF-7and MDA-MB-231cells. Curcumin arrested MCF-7cells at G2/M phase of the cell cycle. In MDA-MB-231cells, an obvious apoptotic peak, but no significant cell cycle arrest, was observed after curcumin treatment. The data obtained using two other experimental approaches (WST-1assays and clonogenic analysis) confirmed that MCF-7and MDA-MB-231cells responded to curcumin differentially, and MDA-MB-231cells were much more sensitive to curcumin than MCF-7cells.
2Curcumin activates caspase-3in MCF-7and MDA-MB-231cells
Substantive evidences have shown that curcumin could induce MCF-7and MDA-MB-231cells to apoptosis, while caspase-3activation has been shown to be one of the most important cell executioners for apoptosis. Therefore, we examined the effect of curcumin on the activation of caspase-3by western blotting analysis. Activation of caspase-3was observed in both MCF7and MDA-MB-231cells treated with curcumin. Notably, the activation of caspase-3seemed to be much more significant in MDA-MB-231than MCF-7cells, which was in agreement with the data showing differential cytotoxicity and clonogenic ability after curcumin treatment.
3PI3K/AKT-Skp2-Cip/Kips is involved in curcumin-induced cytotoxicity in MCF-7and MDA-MB-231cells
Since Cip/kips are often well positioned to function as both sensor and effectors of multiple cytotoxicity signals, we compared the changes in protein expression of p21, p27and p57following curcumin treatment by western blotting analysis to determine whether differences in the expression or activity of Cip/Kips could explain the heterogeneous response to curcumin in MCF-7and MDA-MB-231cells. Our results confirmed that the cellular response to curcumin may be deeply associated with the activation of p27and p21in MCF-7and MDA-MB-231cells, not p57. S-phase kinase-associated protein-2(skp2), a specific substrate-recognition subunit of the Skpl-Cullin-F-box protein (SCF) type ubiquitin ligase complex, has been reported to mediate ubiquitin-dependent degradation of some cell-cycle proteins, including p27Kip1,p21Cip1and p57Kip2. Results showed curcumin increased the protein level of both p27and p21, decreased the level of skp2simultaneously. It is indicated that eurcumin-elevated p27and p21level may result from the suppression of the negative regulatory protein skp2, which subsequently leads to the cytotoxicity in MCF-7and MDA-MB-231cells. In order to confirm the role of skp2in the regulation of p27and p21in MCF-7and MDA-MB-231cells, we next performed gene silencing experiments. Skp2siRNA knocked down the protein levels of skp2in cells compared with control siRNA transfectants and untreated ones (NEG). As expected, it indeed increased the protein levels of both p27and p21after transfection for48hr. The above results suggest curcumin-induced cytotoxicity is associated with the inhibition of skp2-Cip/Kips pathway. Silencing of skp2in MCF-7and MDA-MB-231cells increased cellular sensitivity to curcumin. In conclusion, our data indicates skp2level is a key determinant of cytotoxic responses to curcumin.
4Effects of curcumin on expression and activity of AKT (PKB) and its substrates in MCF-7and MDA-MB-231cells
It has been well verified that the activity of AKT is one of the key determinants in defining sensitivity to chemotherapeutic drugs and its role involved in skp2regulation by transcriptional level and post-translational modification has also been reported largely in various cell types. We found that curcumin showed converse effects on AKT phosphorylation and its substrates p-foxo3a and p-foxol, suggesting PI3K/AKT pathway may contribute greatly to the differential expression of skp2and Cip/Kips in MCF-7and MDA-MB-231cells treated with curcumin. We next investigated whether the combined treatment with curcumin and wortmannin, a selective PI3K inhibitor, would reverse curcumin-induced phosphorylation of AKT thus down-regulation of skp2in MCF-7cells. MCF-7cells were treated with both wortmannin and curcumin for6hr, wortmannin significantly down regulated curcumin-induced phosphorylation of AKT and reversed curcumin-caused up-regulation of skp2markedly. We did not find the use of combination of wortmannin and curcumin or single agent wortmannin could down regulate the expression of skp2significantly comparing to the control group. In order to confirm the relevance of PI3K/AKT pathway in the cellular response to curcumin in MCF-7and MDA-MB-231cells, we next performed cell viability assays to assess whether simultaneous treatment with curcumin and wortmannin could enhance curcumin-induced cytotoxicity. Results showed the combing using of curcumin with wortmannin enhanced the cytotoxicity of curcumin to MCF-7and MDA-MB-231cells.
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