Protective Macroautophagy Is Involved in Vitamin E Succinate Effects on Human Gastric Carcinoma Cell Line SGC-7901 by Inhibiting mTOR Axis Phosphorylation

Abstract

Vitamin E succinate (VES), a potential cancer therapeutic agent, potently induces apoptosis and inhibits the growth of various cancer cells. Autophagy has been supposed to promote cancer cell survival or trigger cell death, depending on particular cancer types and tumor microenvironments. The role of autophagy in the growth suppressive effect of VES on gastric cancer cell is basically unknown. We aimed to determine whether and how autophagy affected the VES-induced inhibition of SGC-7901 human gastric carcinoma cell growth. SGC-7901 cells were treated with VES or pre-treated with autophagy inhibitor, chloroquine (CQ) and 3-methyladenine (3-MA). Electron microscopy, fluorescence microscopy and Western blot were used to study whether VES induced autophagy reaction in SGC-7901 cells. Western blot evaluated the activities of the mammalian target of rapamycin (mTOR) axis. Then we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry to detect the level of cell viability and apoptosis. Collectively, our data indeed strongly support our hypothesis that VES treatment produced cytological variations that depict autophagy, increased the amount of intracellular green fluorescent protein-microtubule associated protein 1 light chain 3 (GFP-LC3) punctate fluorescence and the number of autophagic vacuoles. It altered the expression of endogenous autophagy marker LC3. VES activated the suppression of mTOR through inhibiting upstream regulators p38 MAPK and Akt. mTOR suppression consequently inhibited the activation of mTOR downstream targets p70S6K and 4E-BP-1. The activation of the upstream mTOR inhibitor AMPK had been up-regulated by VES. The results showed that pre-treatment SGC-7901 with autophagy inhibitors before VES treatment could increase the capacity of VES to reduce cell viability and to provoke apoptosis. In conclusion, VES-induced autophagy participates in SGC-7901 cell protection by inhibiting mTOR axis phosphorylation. Our findings not only strengthen our understanding of the roles of autophagy in cancer biology, but may also be useful for developing new treatments for gastric cancer patients.

Fig 1. Inhibition of gastric cancer cell SGC-7901 viability by VES.

(A) Incubation of VES for 24 h decreased cell viability in a concentration-dependent manner as determined by MTT assay in SGC-7901. (B) Cells were treated with or without different concentrations of VES for 24 h. DNA contents were determined by flow cytometry analysis. (C) DNA histogram shows the accumulation of S and G2/M phases cells induced by VES. *p < 0.05 means significantly different from the control group.

Fig 2. VES induces an autophagic response in SGC-7901 cells.

(A) Ultrastructural observations in SGC-7901 cells with transmission electron microscope. (a) Untreated SGC-7901 cells exhibited normal of cytoplasm, cell organelles, and nuclei morphologies. VES-treated SGC-7901 cells showed characteristic ultrastructural morphology of autophagy, (b to c) a significant number of autophagic vacuoles, (d) a giant autophagic vacuole filled with abundant degraded organelles, (e) autophagic vacuoles fused with multivesicular endosome. (a, c, & d, 20 000×; b, 30 000×; e, 12 000×). Typical changes were indicated by arrows. (B) Treatment of SGC-7901 cells with VES for 24 h prominently enhanced formation of autophagic vacuoles and fluorescent intensity, as determined by transfection of GFP-LC3 (C) Protein levels of LC3-I and LC3-II were measured using Western blot analysis. Data was representative of three individual experiments with similar results. (a) SGC-7901 cells were treated with 0, 5, 10, 15, and 20 μg/mL VES for 24 h. (c) SGC-7901 cells were treated with 20 μg/mL VES for 0, 6, 12, 18, and 24 h. (b) and (d) Relative densitometry of protein expression was determined by LC3-II protein densitometry with LC3-I. Actin was used as a loading control.* means p < 0.05 compared with control.

Fig 3. Effects of VES on mTOR axis.

SGC-7901 cells were treated with 0, 5, 10, 15, and 20 μg/mL VES for 24 h, and activation of p38 MAPK (p-p38 MAPK), Akt (p-Akt), AMPK (p-AMPK), mTOR (p-mTOR), p70S6K (p- p70S6K), and 4E-BP-1 (p-4E-BP-1) were examined using Western blot. The data in (A) and (C) were representative of three individual experiments with similar results. The data in (B) and (D) were expressed as mean ± S.D from three individual experiments. Actin was used as a loading control. * means p < 0.05 compared with control.

Fig 4. Autophagy inhibition enhances VES-induced viability suppression in SGC-7901.

SGC-7901 cells were exposed to VES, CQ, or 3-MA alone or in presence of 10 mM 3-MA or 20 μM CQ for 24 h. (A) MTT assay show that combined action of both VES and CQ and VES and 3-MA significantly suppress cell viability compared with VES treatment alone (*, compared with control group, p < 0.05; #, compared with VES group, p < 0.05). (B) and (C) DNA histogram shows that accumulation of S phase cells induced by VES was enhanced by CQ and 3-MA (*, compared with control group, p < 0.05; #, compared with VES group, p < 0.05).

Fig 5. Autophagy inhibition enhances VES-induced apoptosis in SGC-7901.

(A) SGC-7901 cells were exposed to VES, CQ, or 3-MA alone or in presence of 10 mM 3-MA or 20 μM CQ for 24 h, respectively, stained with Hoechst 33343, and visualized under a confocal microscopy (1000×) (B) SGC-7901 human gastric cancer cells were treated as (A), stained with Annexin V-FITC/PI, and detected through flow cytometry analysis. The significance of the obtained four quadrants: FITC Annexin V-negative/PI-negative cells as viable cells, FITC Annexin V-positive/PI-negative cells as early apoptotic cells, FITC Annexin V-positive/PI-positive cells as late apoptotic cells, and FITC Annexin V-negative/PI-positive cells as necrosis cells. (C) Columns: mean of triplicate treatments; bars: ± SD. (*, compared with control group, p < 0.05; #, compared with VES group, p < 0.05).

Fig 6. Hypothetical model of VES autophagy mechanism in human gastric carcinoma SGC-7901 cells.

VES induced the suppression of both Akt and p38 MAPK, and up-regulated AMPK, leading to the inhibition of mTOR activity. And downstream regulators p70S6K and 4E-BP-1 were suppressed consequently, resulting in autophagy in SGC-7901 cells. VES cooperated with autophagy inhibitors to induce increased anticancer effect compared with VES alone.