Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

Gastric adenocarcinoma is among the top causes of cancer-related death and is one of the most commonly diagnosed carcinomas worldwide. Benzyl isothiocyanate (BITC) has been reported to inhibit the gastric cancer metastasis. In our previous study, BITC induced apoptosis in AGS cells. The purpose of the present study was to investigate the effect of BITC on autophagy mechanism in AGS cells. First, the AGS cells were treated with 5, 10, or 15 μM BITC for 24 h, followed by an analysis of the autophagy mechanism. The expression level of autophagy proteins involved in different steps of autophagy, such as LC3B, p62/SQSTM1, Atg5-Atg12, Beclin1, p-mTOR/mTOR ratio, and class III PI3K was measured in the BITC-treated cells. Lysosomal function was investigated using cathepsin activity and Bafilomycin A1, an autophagy degradation stage inhibitor. Methods including qPCR, western blotting, and immunocytochemistry were employed to detect the protein expression levels. Acridine orange staining and omnicathepsin assay were conducted to analyze the lysosomal function. siRNA transfection was performed to knock down the LC3B gene. BITC reduced the level of autophagy protein such as Beclin 1, class III PI3K, and Atg5-Atg12. BITC also induced lysosomal dysfunction which was shown as reducing cathepsin activity, protein level of cathepsin, and enlargement of acidic vesicle. Overall, the results showed that the BITC-induced AGS cell death mechanism also comprises the inhibition of the cytoprotective autophagy at both initiation and degradation steps.

Fluoxetine Simultaneously Induces Both Apoptosis and Autophagy in Human Gastric Adenocarcinoma Cells

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 μM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V-propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.