Lysine-Specific Demethylase 1 (LSD1) Inhibitor S2101 Induces Autophagy via the AKT/mTOR Pathway in SKOV3 Ovarian Cancer Cells

Abstract

BACKGROUND S2101 is one of the most potent LSD1 inhibitors, which can inhibit ovarian cancer cells viability. This study aimed to detect the mechanism behind the anticancer properties of S2101 in SKOV3 ovarian cells. MATERIAL AND METHODS Cell viability was tested by Cell Counting Kit-8 (CCK-8) assay. Cellular apoptosis and autophagy were evaluated by flow cytometric analysis using Annexin-V/PI staining methods and Green fluorescent protein (GFP)-fused-LC3 (GFP-LC3), respectively. Western blotting was performed for analyzing the Bax, Bcl-2, mTOR, p- mTOR, p62, LC3-I, LC3-II, AKT, and p-AKT protein expression. RESULTS Our results show that the proportion of early apoptotic and late apoptotic cells increased significantly for cells treated with S2101 at a concentration of 100 μM for 48 h. Treatment of S2101 in SKOV3 cells resulted in upregulation of Bax and downregulation of Bcl-2 in a time-dependent manner, indicating that S2101 can induce apoptosis in SKOV3. There was a downward trend in the expression of p62 when the SKOV3cells were treated with 100 µm S2101 for 12 h, 24 h and 48 h. The conversion of LC3-I to LC3-II was increased significantly at 24 h and 48 h. Autophagy was induced by S2101 in SKOV3 cells, evidenced by an increase in punctuate localization of GFP-LC3 and a change in expression of autophagy-related proteins. CONCLUSIONS S2101 treatment decreased the levels of phosphorylated AKT and mTOR. S2101 inhibits SKOV3 cells viability and induces apoptosis and autophagy. The AKT/mTOR signaling pathway was found to be affected by S2101.

Figure 1

S2101 inhibits growth of SKOV3 cells. (A, B) The growth curve of SKOV3 cells treated with 0, 50, 100, 150 and 200 μm S2101 for 24 h and 48 h, respectively. (C) The flow cytometric analysis of apoptosis using Annexin-V-FITC/PI staining of SKOV3 cells treated with 100 μm S2101; (D) The detection of apoptosis-related protein Bax and Bcl-2 of SKOV3 cells treated with 100 μm S2101 by Western blot analysis.

Figure 2

S2101 induces autophagy in SKOV3 cells. (A) The detection of autophagy-related protein LC3-I, LC3-II and P62 of SKOV3 cells treated with 100 μm S2101 by Western blot analysis. (B) The percentage of SKOV3 cells with GFP-LC3 puncta treated with 100 μm S2101.

Figure 3

SKOV3 cell viability can be inhibited by blocking autophagy. (A) Pre-treatment with 3-MA resulted in reduced conversion of LC3-I to LC3-II in SKOV3 cells as compared with S2101 alone group. (B) S2101 treatment alone significantly inhibited cell proliferation compared with combination of S2101 and 3-MA

Figure 4

S2101 inhibits the AKT/mTOR/p70S6K signaling pathway.