Stigmasterol Simultaneously Induces Apoptosis and Protective Autophagy by Inhibiting Akt/mTOR Pathway in Gastric Cancer Cells

Abstract

Background: Stigmasterol (SS) has been proven to possess potential anticancer activities in several cancer cell lines, but its molecular mechanism is still unknown. Thus, we investigated whether SS has the capabilities of inducing autophagy and its molecular mechanisms in gastric cancer cells.

Methods: We used CCK8 assay, clone formation assay, and EdU proliferation assay to assess the effects of SS on cell proliferation in SGC-7901 and MGC-803 cells in vitro, and its inhibition on the tumor growth of gastric cancer was observed in vivo. Apoptosis induced by SS was demonstrated using Hoechst and TUNEL staining, annexin V-FITC/PI assay. Immunofluorescence staining is used to detect the formation of autophagosomes triggered by SS. Apoptosis and autophagy related proteins were analyzed by western blot.

Results: The results indicated that SS treatment inhibited cell proliferation in SGC-7901 and MGC-803 cells. Furthermore, SS treatment induced apoptosis and autophagy by blocking Akt/mTOR signaling pathway. The pretreatment with the Akt inhibitor MK-2206 could promote apoptosis and autophagy induced by SS, predicting that Akt/mTOR pathway is involved in SS-induced apoptosis and autophagy. In addition, blockade of autophagy with 3-MA (an inhibitor of autophagy) enhanced SS-induced apoptosis in SGC-7901 and MGC-803 cells, implying that autophagy mediated by SS plays a cytoprotective role against apoptosis. Finally, an in vivo study demonstrated that tumor growth of gastric cancer was suppressed by SS in a xenograft model.

Conclusion: Our findings illustrate for the first time that SS simultaneously induces apoptosis and protective autophagy by inhibiting Akt/mTOR pathway in gastric cancer cells, and SS may become a potential anticancer drug in treating gastric cancer in the future.

Keywords: Akt/mTOR signaling pathway; apoptosis; autophagy; gastric cancer cells; stigmasterol.

Figure 1

Stigmasterol (SS) inhibits viability and proliferation of gastric cancer cells. (A, B) Gastric cancer cells and normal cells were treated with indicated concentrations (2.5, 5, 10, 15, 20, 25, 30 µM) or without addition of SS for 24, 48, and 72 h, and the cell viability was evaluated by CCK8 assay. (C) SGC-7901 and MGC-803 cells were incubated with 0, 10, 20 µM SS for 10 days, and the number of colonies were calculated. (D) Cells proliferation was tested using EdU Cell Proliferation Kit with Alexa Fluor 594, and EdU-positive cells were monitored in five random fields/sample. Data are expressed as mean ± SD of triplicate experiment. **P < 0.01 vs. control (0 µM SS).

Figure 2

Stigmasterol (SS) induces apoptosis in gastric cancer cells. SGC-7901 and MGC-803 cells were treated with indicated dose or without addition of SS for 48 h. (A, B) Cells were stained with Hoechst 33258 and TUNEL, and morphological changes of gastric cancer cells were monitored under a fluorescence microscope. (C) Early and late apoptotic cells changes induced by SS was determined by Annexin V-FITC/PI. (D) The expression of caspase-3, cleaved caspase-3, PARP, cleaved-PARP, Bcl-2, Bax, and β-actin were measured by western blot analysis. Data are expressed as mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 vs. control (0 µM SS).

Figure 3

Stigmasterol (SS) induces autophagy in gastric cancer cells. (A) Cells were exposed to selected dose of SS and autophagy related proteins such as LC3, Beclin-1, and p62 were detected by western blot analysis. (B) Cells were treated as in A and stained with ant-LC3 antibody for observe of LC3 puncta under a confocal microscopy. (C) SGC-7901 and MGC-803 cells were treated with or without SS (20µM) in combination with or without Baf A1 (100 nM) for 48h, the expression of LC3-I, LC3-II, and p62 were detected by western blot. (D) Cells were treated as in C and the LC3 green fluorescent puncta formation in cytoplasm was monitored. (E) SGC-7901 and MGC-803 cells were treated with SS (20µM) in the absence or presence of Baf A1 (100 nM) for 48h, and the expression of LC3-I, LC3-II were detected by western blot. (F) Cells were treated as in (E) and the LC3 green fluorescent puncta formation in cytoplasm was monitored. Data are expressed as mean ± SD of triplicate experiment. *P < 0.05, **P < 0.01 vs. control (0 µM SS). ^#P < 0.05, ^##P < 0.01 vs. SS treated group.

Figure 4

Stigmasterol (SS) induces apoptosis and autophagy by inhibiting the Akt/mTOR pathway in gastric cancer cells. (A) The expression levels of AKT, p-AKT, mTOR, p-mTOR, and β-actin were measured by western blot. (B) Cells were treated with or without SS (20 µM) in combination with or without MK-2206 (8 mM), the cell viability was detected using CCK-8 assays. (C) Cells were treated as in B, the phosphorylated level of AKT and apoptotic related protein cleaved caspase-3, Bcl-2, Bax, and β-actin were determined by western blot. (D) Cells were treated as in B, the autophagy related protein LC3 and β-actin were determined by western blot. (E) Cells were treated as in B, and the LC3 green fluorescent puncta formation in cytoplasm was monitored. Data are expressed as mean ± SD of triplicate experiment. **P < 0.01 vs. control (0 µM SS). ^#P < 0.05, ^##P < 0.01 vs. SS treated group.

Figure 5

Inhibition of autophagy enhanced stigmasterol (SS) induced apoptosis in gastric cancer cells. Cells pretreated with 3-MA (5 mM), and then incubated with or without SS (20 µM) for 48 h. (A) Cell viability was observed by CCK-8 assay. (B) Morphological changes of gastric cells were monitor under a fluorescence microscope. (C) The expression of cleaved caspase-3, Bcl-2, Bax, and β-actin were determined by western blot. Data are expressed as mean ± SD of triplicate experiment. **P < 0.01 vs. control (0 µM SS). ^##P < 0.01 vs. SS treated group.

Figure 6

Stigmasterol (SS) inhibits gastric tumor growth in vivo. (A) Images of gastric tumors dissected from mice after treatment with indicated therapy. (B) The tumor volumes were detected every 3 days for 28 days. (C) The tumor weight was determined after mice treatment with indicated therapy. **P < 0.01 vs. control (0 µM SS).

Figure 7

A hypothetical molecular mechanisms of stigmasterol (SS) induced anticancer properties in gastric cancer cells. SS simultaneously induces apoptosis and protective autophagy by inhibiting Akt/mTOR pathway in gastric cancer cells.