| Aim: Cucurbitacins are a group of diverse triterpenoid compound, originally isolated from Cucurbitaceae plants and having diverse pharmacological and biological activities, including anticancer, antiviral and anti-inflammatory effects. In recent years, it has been found that cucurbitacins B and I could induce autophagy through different mechanisms. As an important member of cucurbitacin family and a close analog of cucurbitacin B, it is still unknown that whether cucurbitacin E can induce autophagy and what is the potential molecular mechanism. In this study, we aimed to explore the mechanism of cucurbitacin E-induced autophagy in He La cells. Methods: 1. He La cells were maintained in DMEM supplemented with 10% FBS, 100 U/m L penicillin and 100 μg/m L streptomycin at 37°C in a humidified incubator with 5% CO2. 2. Improved MTT(WST-1) assay was adopted to measure the effect of cucurbitacin E on cell proliferation. 3. Western blot was used to determine the phosphorylation levels of downstream signaling proteins of m TORC1 and the expression of autophagy associated proteins. 4. Immunofluorescence microscopy was adopted to measure the co-localization of autophagosome marker LC3 and the lysosome. Results: 1. Cucurbitacin E inhibited the proliferation of He La cells in a dose-dependent manner and the 24-h IC50 of Cu E was 4.01 μmol/L. 2. Cucurbitacin E could induce an elevation in the autophagosome marker LC3-II, and the levels of LC3-II was further increased in the presence of chloroquine. Furthermore, Cu E reduced the levels of p62/SQSTM1. These results indicated that Cu E induced autophagy in He La cells. 3. Cu E significantly inhibited the phosphorylation of p70S6 K in a time- and dose-dependent manner as evidenced by decreased phosphorylation levels of the m TORC1 substrate. 4. The decreased levels of phosphorylated ULK1S757 were positively correlated with autophagy induction in He La cells. Conclusion: Cu E is likely to induce autophagy through inhibiting m TORC1 activity. |
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