Portulaca oleracea L. polysaccharide inhibits ovarian cancer via inducing ACSL4-dependent ferroptosis

Abstract

The antitumor effect of Portulaca oleracea L. polysaccharide (POL) has been demonstrated, but whether it curbs the development of ovarian cancer has not been reported. Here, we treated ovarian cancer cells with different concentrations of POL, detected cell activity by CCK-8 assay, and apoptosis rate by flow cytometry. The results showed that SKOV3 and Hey cell survival decreased with increasing POL concentration in a dose-dependent manner. POL significantly inhibited ovarian cancer cell migration and increased cell death compared with the control group. Ferroptosis inhibitors, but not apoptosis, necrosis, and autophagy inhibitors, reversed POL-induced cell death. Further studies revealed that POL promoted the accumulation of lipid reactive oxygen species (ROS), Fe2+, malondialdehyde (MDA), and decreased glutathione (GSH) production. Moreover, POL significantly increased the mortality of ovarian cancer cells. In vivo studies confirmed that POL reduced the volume and weight of tumors and increased the levels of Fe2+ and MDA in mice in vivo. Western blot assay revealed that POL increased the expression of ACSL4 in ovarian cancer cells as well as in tumors in mice in vivo. More importantly, the POL-mediated increase in lipid ROS, Fe2+, MDA, and decrease in GSH were significantly reversed after knocking down ACSL4 in ovarian cancer cells. Thus, POL can effectively inhibit ovarian cancer development, which may be achieved by increasing ACSL4-mediated ferroptosis. These results suggest that POL has the potential to be a potential drug for targeted treatment of ovarian cancer.

Keywords: ACSL4; Portulaca oleracea L. polysaccharide; ferroptosis; ovarian cancer.

Figure 1

POL reduced the proliferation of ovarian cancer cells in a dose-dependent manner. CCK-8 was assayed whether 25, 50, 100, 200, 400, 800, 1600 μg/ml of POL reduced the cell viability of SKOV3 (A), Hey (B) as well as HOSE (C). At 24 h, 48 h, and 72 h, 50 and 100 μg/ml of POL attenuated the viability of SKOV3 (D) and Hey (E) cells. ^**P < 0.01; ^***P < 0.001 vs. Con.

Figure 2

POL inhibits migration and induces cell death in ovarian cancer cells. Scratch assay revealed that 50 and 100 μg/ml of POL significantly inhibited the migration of SKOV3 (A) and Hey (B) cells at 48 h (4×). Transwell assay revealed that POL effectively inhibited the migration of SKOV3 (C) and Hey (D) cells (20×). Flow cytometry results assay revealed that 50 and 100 μg/ml of POL significantly increased the mortality of SKOV3 (E) and Hey (F) cells. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Con.

Figure 3

POL induced ferroptosis in SKOV3 and hey cells. CCK-8 assay showed that ferroptosis inhibitor, Fer-1 and DFO, were able to reverse the POL-induced decrease in cell viability of SKOV3 (A) and Hey (B) cells. RT-PCR analysis showed that POL was able to significantly increase the mRNA levels of ptgs2 and Chac1 in SKOV3 (C, D) and Hey (E, F) cells. ^*P < 0.05, ^***p < 0.001 vs. Con; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. POL.

Figure 4

POL increased ACSL4 expression in a concentration gradient-dependent manner. JC-1 analysis showed that POL decreased MMP in SKOV3 (A) and Hey (B) cells (20×). FerroOrange staining showed that POL increased Fe²⁺ levels in SKOV3 (C) and Hey (D) cells (20×). Western blot assay revealed that POL increased the protein expression level of ACSL4 in SKOV3 (E) and Hey (F) in a concentration gradient-dependent manner. ^**P < 0.01, ^***p < 0.001 vs. Con.

Figure 5

Knockdown of ACSL4 reverses POL-induced ferroptosis in ovarian cancer cells. Western blot assay revealed that POL-induced high expression of ACSL4 was reversed to some extent by si-ACSL4 in SKOV3 (A) and Hey (B) cells. EdU staining showed that POL decreased the proliferation of SKOV3 (C) and Hey (D) cells, and si-ACSL4 could reverse this effect (20×). Silencing ACSL4 also reduced POL-induced lipid peroxidation in SKOV3 (E) and Hey (F) cells (40×). ^*P < 0.05, ^**p < 0.01 vs. Con; ^#P < 0.05, ^##P < 0.01 vs. POL.

Figure 6

POL inhibited tumor growth in mice in vivo. POL significantly reduced the weight (A) and volume (B) of mouse tumors. POL increased the levels of ROS (C) and MDA (D) in mouse tumor tissues. (E) POL decreased GSH levels in mouse tumor tissues. (F) TUNEL staining revealed that POL increased the cell death rate in tumor tissues (20×). (G) IHC staining revealed that POL increased the level of ACSL4 in tumor tissues (20×). (H) RT-PCR results revealed that POL increased the mRNA levels of ptgs2 as well as chac1 in mouse tumor tissues. (I) Western blot similarly confirmed that POL increased the protein expression level of ACSL4 in mouse tumor tissues. ^**P < 0.01, ^***p < 0.001 vs. Con.