[The role of endoplasmic reticulum IP(3)R calcium channel in vitamin E succinate induced autophagy of human gastric cancer cell].

Abstract

Objective: To investigate the role of vitamin E succinate (VES) in inducing autophagy of human gastric cancer cells by activating calcium redistribution through inositol 1, 4, 5-trisphosphate receptors (IP(3)R) pathway. Methods: Human gastric cancer lines MKN28 (moderately differentiated) and MKN45 (poorly differentiated) cells were cultured in vitro in March 2022. Gastric cancer cells were treated with VES at different doses for 24 h, and cell viability was measured by CCK-8 method to determine VES dose for subsequent study. The experiment was set up with solvent control group (0.1% ethanol), VES dose groups, 100 nmol/L rapamycin (RAPA) as autophagy positive control group (RAPA group), 15 μg/ml tunicamycin (TM) was used as the endoplasmic reticulum stress (ERS) positive control group (TM group), 10 μmol/ml 2-aminoethyl diphenylborinate (2-APB group) was used to inhibit IP(3)R (2-APB group) and VES+2-APB group. The occurrence of autophagosomes in gastric cancer cells was observed by transmission electron microscopy, and microtubule associated protein 1 light chain 3 (LC3), Beclin1, IP(3)R, glucose-regulated protein 75 (Grp75), voltage-dependent anion channel 1 (VDAC1) protein expression was detected by western blotting. Fluo-4 AM was used to label intracellular calcium ions, Rhod-2 AM was used to label mitochondrial calcium ions, and the fluorescence intensity of calcium ions was observed by fluorescence microscope. One-way analysis of variance was used to compare the means among multiple groups, and LSD-t method was used for pairwise comparison. Results: CCK-8 results showed that compared with solvent control group, the proliferation rates of MKN28 cells in 10-100 μg/ml VES group and MKN45 cells in 20-100 μg/ml VES group were significantly decreased (P<0.05). Subsequent VES dosages were determined according to the growth curve, MKN28 was 5, 10, 20, 40 μg/ml, and MKN45 was 10, 20, 40, 80 μg/ml. The results of transmission electron microscopy and fluorescence showed that autophagosomes were formed in MKN28 cells in 5 and 20 μg/ml VES groups and MKN45 cells in 10 and 40 μg/ml VES groups, and the fluorescence intensity of calcium ions in cytoplasm and mitochondria was significantly higher than that in solvent control group (P<0.05). Compared with solvent control group, LC3, Beclin1, IP(3)R, Grp75 and VDAC1 protein expressions of MKN28 cells in 20 and 40 μg/ml VES groups and MKN45 cells in 40 and 80 μg/ml VES groups were significantly increased (P<0.05). After inhibiting IP(3)R with 2-APB, the expression levels of IP(3)R, Grp75 and VDAC1 in two kinds of gastric cancer cells in VES+2-APB group were significantly decreased compared with VES group (P<0.05). The fluorescence results showed that the fluorescence intensity of cytoplasmic and mitochondrial calcium ions in VES+2-APB groups was significantly lower than that in VES group (P<0.05). Compared with VES group, LC3 and Beclin1 protein expressions in two kinds of gastric cancer cells in VES+2-APB groups were significantly decreased (P<0.05) . Conclusion: VES may activate intracellular calcium redistribution through IP(3)R-Grp75-VDAC1 calcium channel and induce autophagy in gastric cancer cells.