Liu F et al. (2022), Stichoposide C Exerts Anticancer Effects on Ova...

ECB-ART-50535

Onco Targets Ther 2022 Jan 01;15:87-101. doi: 10.2147/OTT.S340556.

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Stichoposide C Exerts Anticancer Effects on Ovarian Cancer by Inducing Autophagy via Inhibiting AKT/mTOR Pathway.

Liu F , Tang L , Tao M , Cui C , He D , Li L , Liao Y , Gao Y , He J , Sun F , Lin H , Li H .

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PURPOSE: Stichoposide C (STC) is a triterpene glycoside isolated from Thelenota ananas, which is previously demonstrated to wide spectrum of anticancer effects against various tumor cells. However, the antitumor effects and underlying molecular mechanisms in ovarian cancer (OC) cells are not fully understood. Here, we examined if and through which mechanisms STC exerts anticancer effects on OC. METHODS: CCK-8 and colony formation assays were used to detect cell viability and proliferation. Flow cytometry was used to detect apoptosis and cell cycle arrest. Protein expression and phosphorylation were measured by Western blotting analysis. Confocal fluorescence microscopy was used to observe the autophagy flux. Autophagosome formation was observed via transmission electron microscopy. Antitumor effect of STC was investigated in patient-derived organoids (PDOs) and A2780 subcutaneous xenograft tumors. RESULTS: STC was found that not only exerted antiproliferation activity and apoptosis but also induced autophagy. Mechanistically, STC induced autophagy via inhibited the AKT/mTOR signaling pathway in ovarian cancer cells. In addition, STC and an autophagy inhibitor 3-methyladenine (3-MA) combination treatment showed significant synergetic effects on inhibiting proliferation and promoting apoptosis in vitro. Consistent with cell experiments, STC also inhibited the growth of two OC PDOs. Finally, STC markedly reduced the growth of A2780 subcutaneous xenograft tumors without organ toxicity and activated autophagy in vivo. CONCLUSION: Stichoposide C exerts in vitro and in vivo anticancer effects on ovarian cancer by inducing autophagy via inhibiting AKT/mTOR pathway. The findings warrant further prove for STC as a potential therapeutic agent for ovarian cancer.

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	Figure 1. STC repressed ovarian cancer cell proliferation. (A) Two human ovarian cancer cell lines A2780 and SKOV3 cells were untreated or treated with different concentrations (0â10 Î¼M) of STC for 24 and 48 hours, and the cell viability was measured by CCK-8 assay. (B) A2780 and SKOV3 cells were treated with STC at 0, 0.5 and 1Î¼M for 48 hours, and the proliferation-related protein PCNA detected by Western blotting. (C) STC inhibited the colony formation ability of A2780 and SKOV3 cells. Data are presented as the mean Â± SEM of three independent experiments, P <0 0.05, P <0.01, **P<0 0.001, n=3.
	Figure 2. STC induced cell cycle arrest and promoted apoptosis in ovarian cancer cells. (A) Cell cycle distributions of A2780 and SKOV3 treated with STC for 48 hours were detected by flow cytometric assay. (B) A2780 and SKOV3 cells were treated with various concentrations (0, 0.5 and 1Î¼M) for 48 hours, and the expression of CDK2 and CDK4 were compared by Western blot analysis. (C) A2780 and SKOV3 cells were treated with STC at 0, 0.5 and 1Î¼M for 48 hours, and the expression of Î³H2AX were compared by Western blot analysis. (D) A2780 and SKOV3 cells were treated with STC at 0, 0.5 and 1Î¼M for 48 hours, and then apoptotic cells were detected with the Annexin V-PI kit and analyzed by flow cytometry. Data are presented as the mean Â± SEM of three independent experiments, P <0.05, P <0.01, **P <0.001, n=3.
	Figure 3. STC induced autophagy in ovarian cancer cells. (A) The expression of autophagy-associated protein (Beclin1 and ATG7) and autophagic substrates (LC3B and p62) were analyzed in A2780 and SKOV3 cells after STC treatment at 0, 0.5, and 1Î¼M for 48 hours by Western blotting. (B) A2780 and SKOV3 cells overexpressing GFP-mRFP-LC3 were treated with STC (0 and 1Î¼M) for 24 hours and then subjected to confocal microscopy. Scale bar: 10Î¼m. (C) Ultrastructural features of A2780 and SKOV3 cells treated with STC (0 and 1Î¼M) for 48 hours were analyzed by electron microscopy. Autophagosomes (Red arrows) are shown at high magnification. Scale bar: 5Î¼m. The number of autophagosomes in A2780 and SKOV3 cells is presented. Data are presented as the mean Â± SEM of three independent experiments, P <0.01, *P <0.001, n=3.
	Figure 4. STC-induced autophagy could be inhibited by autophagy inhibitors. (A) The expression of autophagy-associated proteins (Beclin1 and ATG7) and autophagic substrate (LC3B and p62) were analyzed by Western blotting in A2780 and SKOV3 cells after treatment with STC (1 Î¼M), 3-MA (5 mM) or combination for 48 hours. (B) A2780 and SKOV3 cells overexpressing GFP-mRFP-LC3 were treated with STC (1Î¼M), 3-MA (5 mM) or combination for 24 hours and then subjected to confocal microscopy. Scale bar: 10Î¼m. Data are presented as the mean Â± SEM of three independent experiments, #P<0.05, ***P <0.001, n=3.
	Figure 5. Autophagy inhibitors enhanced STC-induced apoptosis and growth inhibition. (A) CCK-8 assays of A2780 and SKOV3 cells after STC treatment with STC (1 Î¼M), 3-MA (5 mM) or 3-MA plus STC (5 mM+1 Î¼M) for 48 hours. (B) Apoptosis assays of A2780 and SKOV3 cells after STC treatment with STC (1 Î¼M), 3-MA (5 mM) or 3-MA plus STC (5 mM+1 Î¼M) for 48 hours by flow cytometry. Data are presented as the mean Â± SEM of three independent experiments, compare to STC, ***P <0.001; compared to 3-MA plus STC, #P <0.05, n=3.
	Figure 6. STC induced autophagy by inhibiting the AKT/mTOR signaling pathway. (A) The expression of AKT, p-AKT (Thr 308), mTOR and p-mTOR (Ser 2448) were analyzed by Western blotting in A2780 and SKOV3 cells after exposed to various concentrations of STC (0, 0.5, and 1 Î¼M) for 48 hours. (B) A2780 and SKOV3 cells were treated with or without STC (1 Î¼M) in combination with MK2206 (8 nM) for 48 hours. AKT, p-AKT (Thr 308), mTOR, p-mTOR (Ser 2448) and LC3B-II/I were detected by Western blotting. (C) A2780 and SKOV3 cells were treated with or without STC (1 Î¼M) in combination with Rapa (5 Î¼M) for 48 hours. AKT, p-AKT (Thr 308), mTOR, p-mTOR (Ser 2448) and LC3B-II/I were detected by Western blotting. Data are presented as the mean Â± SEM of three independent experiments, P <0.05, P <0 0.01, **P <0 0.001, n=3.
	Figure 7. Effects of STC on patient-derived ovarian cancer organoids. (A) Two OC PDOs (HGSOC-105 and ENOC-107) were treated with various concentrations of STC and cisplatin (0, 0.001, 0.01, 0.1, 1, 10, 100 Î¼M) for 5 days. The cell viability was measured by CellTiter-Lumiâ¢. (B) Representative photomicrographs of two OC PDOs treated with or without STC and cisplatin. Scale bar = 100 Î¼m. Data were expressed as mean Â± SEM of three independent experiments.
	Figure 8. STC exhibited antitumor efficacy in OC subcutaneous xenograft models. (A) Representative images of subcutaneous tumors after treatment with STC (0.5mg/kg) or cisplatin (2mg/kg) (n = 7). (B) The volume and (C) weight of tumors was calculated in control, STC and Cisplatin group. Tumor volumes was calculated at different time points. (D) Body weightâtime curve in control, STC and Cisplatin group. (E) The expression of PCNA, Î³H2AX, LC3B I/II and p62 in control and STC group were detected by Western blotting. (F) PCNA, Î³H2AX, LC3B I/II in STC and cisplatin group tumor tissues compared with the control group were detected by IHC staining. Original magnification: Ã40. (G) Histopathology of the important organs. Original magnification: Ã40. Data are presented as the mean Â± SEM (n=7), ***P <0.001, n=3.

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