Autophagy Induction And The Underlying Molecular Mechanism In HeLa And MCF7 Cells Upon Cucurbitacin E Treatment

Aim: Cucurbitacins belong to a large family of triterpenoids present in Cucurbitaceae plants, and possess many biological activities, including anti-inflammatory, hepatoprotective, anti-diabetic, and anticancer activities. Recent studies show that cucurbitacins B and I of cucurbitacins family can induce autophagy in tumor cells. However, whether cucurbitacin E(Cu E) induces autophagy is unknown. In this study, we aimed to explore the molecular mechanism of autophagy induced by Cu E in human breast cancer MCF7 and cervical cancer He La cells, in order to provide a basis for further research on the action mechanism(s) by which cucurbitacins(including Cu E) act as anti-inflammatory, anti-diabetic, and anticancer agents.Method: 1. The effect of Cu E treatment on He La, MCF7 and DU145 cells’ viability was evaluated by WST-1 assay. 2. Western blotting was used to analyze the expression of LC3-II and p62/SQSTM1, which were the markers of autophagy, and that of other autophagy-related proteins(ATG) including Beclin1. The phosphorylation of m TORC1 substrates p70S6 K, 4E-BP1 and ULK1S758, as well as that of p70S6 K substrate S6 were also determined by Western blotting. This method was also recruited in the evaluating the effect of Cu E treatment on the activities of AMPK and Akt kinases, as indicated by the phosphorylation of ULK1S555、RaptorS792、AktT308 and AktS473. 3. Immunofluorescence microscopy was used to evaluate the fusion of autophagosomes with lysosomes by observing the colocalization of LC3 and LAMP2, indicating the on-going autophagy. 4. Specific inhibitors of autophagy pathway and small interfering RNAs targetingATG proteins were used in order to block the autophagic flow, which further verified the induction of autophagy under the treatment of Cu E.Result: 1. Cu E treatment resulted in a dose-dependent inhibition of the proliferation of He La, MCF7 and DU145 cells. The IC50(50% inhibiting concentration) value of Cu E for 24 h and 48 h treatment was 4.01 μmol/L and 0.06 μmol/L, respectively. 2. Western blotting showed that Cu E increased the level of LC3-II, an autophagy marker, in He La and MCF7 cells. When the cells were co-treated with chloroquine(CQ), an autopahgic flow blocker, the levels LC3-II was further upregulated. Immunofluorescence staining showed that LC3-II was co-localized with the lysosome marker LAMP2, indicating the fusion of autophagosome and lysosome(resulting in autolysosome). p62/SQSTM1 was significantly decreased during Cu E treatment, indicating the degradation of this protein and the existence of autophagic flow. All these indicated that autophagy had been induced by Cu E treatment. 3. Next, the effect of Cu E on autophagic pathway was determined in DU145 cells, which have been reported being lacking a key autophagic protein ATG5. As expected, the expression of LC3-II and the colocalization of LC3 and LAMP2 could not be detected in Cu E-treated DU145 cells. In addition, the expression of LC3-II was inhibited by si RNA to knockdown ATG5 in He La and MCF7 cells; however only a slight change of Beclin 1 could be detected. These results indicated that Cu E-induced autophagy was dependent on ATG5. 4. Cu E inhibited the phosphorylation of p70S6 K and S6 in a time- and dose-dependent manner, but upregulated the expression of 4E-BP1T37/46 and p-4E-BP1 T37/46. In addition, Cu E treatment decreased the phosphorylation of m TORC1 substrate ULK1S758. Immunofluorescence microscopy showed that the co-localization of m TOR and LAMP2 were decreased by Cu E, suggesting that Cu E could suppress the activity of m TORC1, thereby activating ULK1 to induced autophagy. 5. Western blotting showed that the phosphorylation of AMPKα and AMPKβ were significantly increased at the early time points, but were then declined to basal levelsat later time points in Cu E-treated He La, MCF7 and DU145 cells. Moreover, the phosphorylation of AktT308 was not changed, but the phosphorylation of AktS473 was slightly upregulated in Cu E treated cells. 6. In the presence of AMPK specific inhibitor compound C, Cu E-induced phosphorylation of AMPK substrate ULK1S555 and RaptorS792 was significantly inhibited, whereas the ratio of p-p70S6K/p70S6 K was upregulated in He La cells. But the phosphorylation of ULK1S758 was not restored. These results further indicated that Cu E induced autophagy by through activating AMPK and its downstream ULK1, therefore leading to the inhibition of m TORC1. 7. After knockdown of AMPKα by si RNAs, Cu E-induced suppression of m TORC1/p70S6 K was attenuated and the formation of LC3-II was decreased. However, Cu E has no effect on m TORC1/ULK1S758 signaling.Conclusion: Cu E induced autophagy via downregulation of m TORC1 signaling and upregulation of AMPK activity in human He La and MCF7 cells...