Differential Susceptibilities Of MCF-7and MDA-MB-231Cells To Curcumin-induced Cytotoxicity Are Associated With PI3K/AKT-Skp2-Cip/Kips Pathway

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-231cellsSubstantive 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-231cellsSince 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-231cellsIt 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.