Total Astragalus saponins promote ferroptosis in gastric cancer cells by upregulating SIRT3

Abstract

Background: Gastric cancer (GC) is a malignant tumor of the digestive tract originating from the epithelial cells of the gastric mucosa, which is highly invasive and heterogeneous, posing a serious threat to human health. In recent years, ferroptosis, as a novel mode of programmed cell death, has shown potential anticancer effects in tumor therapy. Total Astragalus saponins (TAS), a natural product derived from Astragalus membranaceus, have been shown to possess various pharmacological activities, including anticancer effects. This study aimed to investigate the effects of TAS on GC cells, focusing on the mechanism of action of its regulation of the silent information regulator 3 (SIRT3) in inducing ferroptosis in GC cells.

Methods: We treated SGC-7901 cells with TAS at concentrations of 50, 100, and 200 µg/mL. After TAS treatment, the SGC-7901 cells were transfected with a vector designed to knock down SIRT3 expression. We assessed cell proliferation, viability, and apoptosis using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay), colony formation assay, and flow cytometry. SIRT3 expression was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fe²⁺, malondialdehyde (MDA), lactate dehydrogenase (LDH), and superoxide dismutase assay kits were used to detect the level of reactive oxygen species (ROS) by fluorescent probe assay. Western blot was used to detect apoptosis-related proteins and SIRT3 protein expression.

Results: TAS dose-dependently inhibited SGC-7901 cell proliferation and viability (P<0.05) and induced apoptosis (P<0.05). TAS promoted the expression of SIRT3 and ACSL4 proteins (P<0.05), inhibited the expression of SLC7A11 and GPX4 proteins (P<0.05), and induced ferroptosis of SGC-7901 cells (P<0.05). Knockdown of the SIRT3 gene attenuated the effect of TAS treatment on ferroptosis (P<0.05).

Conclusions: TAS has therapeutic potential for GC and can effectively inhibit the proliferation and viability of SGC-7901 cells, and the mechanism may be that TAS upregulates SIRT3 to promote the ferroptosis of SGC-7901 cells.

Keywords: Gastric cancer (GC); ferroptosis; silent information regulator 3 (SIRT3); total Astragalus saponins (TAS).

Figure 1

TAS was found to suppress the proliferation of SGC-7901 cells while enhancing their apoptosis. (A) The impact of different TAS concentrations on SGC-7901 cell proliferation was evaluated using the MTT assay. (B) Effects of different concentrations of TAS on the viability of SGC-7901 cells observed by crystal violet assay, magnification 1×. (C) The effect of varying TAS concentrations on the apoptosis levels of SGC-7901 cells was analyzed using flow cytometry. **, P<0.01, vs. TAS 0 µg/mL; ^##, P<0.01, vs. TAS 50 µg/mL; ^&, P<0.05, vs. TAS 100 µg/mL (n=3). TAS, total astragalus saponin; SGC, stomach gastric carcinoma.

Figure 2

TAS induces ferroptosis in SGC-7901 cells. (A) The effects of different concentrations of TAS on ROS levels in SGC-7901 cells were detected and analysed by fluorescent probe method, magnification 200×. (B-D) The effect of varying TAS concentrations on (B) Fe²⁺, (C) MDA, and (D) LDH levels in SGC-7901 cells. (E-I) Western blot analysis was conducted to examine the impact of different TAS concentrations on the expression of ferroptosis-related proteins (SIRT3, SLC7A11, ACSL4, and GPX4) in SGC-7901 cells. **, P<0.01, vs. TAS 0 µg/mL; ^##, P<0.01, vs. TAS 50 µg/mL; ^&, P<0.05, ^&&, P<0.01, vs. TAS 100 µg/mL (n=3). TAS, total astragalus saponin; SGC, stomach gastric carcinoma; ROS, reactive oxygen species; MDA, malondialdehyde; LDH, lactate dehydrogenase; β-actin, internal reference.

Figure 3

TAS increased the ferroptosis of SGC-7901 cells by upregulating SIRT3. (A) The impact of varying TAS concentrations on SIRT3 expression in SGC-7901 cells was assessed through qRT-PCR analysis, **, P<0.01, vs. TAS 0 µg/mL; ^##, P<0.01, vs. TAS 50 µg/mL; ^&, P<0.05, vs. TAS 100 µg/mL (n=3); (B) the expression levels of SIRT3 in SGC-7901 cells following SIRT3 knockdown were quantified using qRT-PCR; (C) ROS levels were detected and SGC-7901 cells by fluorescent probe method, 200×; (D) detection of (D) Fe²⁺, (E) MDA, and (F) LDH levels in cells of different treatment groups; (G-K) Western blot analysis was employed to evaluate the expression levels of ferroptosis-related proteins (SIRT3, SLC7A11, ACSL4, and GPX4) across different treatment groups. **, P<0.01, vs. Control; ^##, P<0.01, vs. TAS + vector (n=3). TAS, total astragalus saponin; SGC, stomach gastric carcinoma; SIRT3, silent information regulator 3; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; MDA, malondialdehyde; LDH, lactate dehydrogenase; β-actin, internal reference.