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.

Ameliorative Effects of Humulus japonicus Extract and Polysaccharide-Rich Extract of Phragmites rhizoma in Rats with Gastrointestinal Dysfunctions Induced by Water Avoidance Stress.

Chronic stress can cause the gastrointestinal disorders characterized by an altered bowel movement and abdominal pain. Studies have shown that Humulus japonicus extract (HJE) has anti-inflammatory and antidiarrheal effects, and Phragmites rhizoma extract (PEP) has antioxidative and antistress effects. The present study aimed to investigate the possible effects of HJE and PEP in rat models with stress-induced gastrointestinal dysfunctions. The rats were exposed to water avoidance stress (WAS, 1 h/day) for 10 days to induce gastrointestinal disorders. We found that WAS significantly increased fecal pellet output during 1 h stress, gastric emptying, colonic contractility, and permeability compared to the normal rats. Pretreatment with HJE and PEP (0.25 and 0.5 mL/kg, both administered separately) improved the increased gastric emptying and colonic contractility induced by electrical field stimulation, acetylcholine, and serotonin and also alleviated the increased colonic permeability. HJE and PEP also increased the claudin-1 and occludin expressions, reduced by WAS. WAS increased the concentration of TNF-α and TBARS and reduced FRAP. HJE and PEP recovered these effects. HJE and PEP improved the gastrointestinal disorders induced by WAS by upregulating the tight junction protein, possibly acting on cholinergic and serotonergic receptors to abolish the colonic hypercontractility and hyperpermeability and degradation of inflammatory cytokines via an antioxidant effect.

The effect of paclitaxel on apoptosis, autophagy and mitotic catastrophe in AGS cells.

Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

Signaling pathways underlying changes in the contractility of the stomach fundus smooth muscle in diabetic rats.

Dysfunction of gastrointestinal (GI) motility is a common complication in patients with diabetes mellitus (DM). Studies related to changes in fundus contraction induced by inhibitors in DM are not well known. Therefore, this study aimed to investigate the signaling pathways involved in the changes in the contraction of fundus smooth muscle obtained from control and DM rats. DM was induced by injecting streptozotocin (65 mg/kg) into Sprague-Dawley rats. The rats were sacrificed after 14 days. Fundus smooth muscle contraction was stimulated using electrical field stimulation (amplitude, 50 V; duration, 1 min; frequency, 2-20 Hz) and acetylcholine (0.1 mM). The inhibitor-mediated cell membrane was pre-treated with atropine, verapamil, methysergide, ketanserin, ondansetron, and GR 113808. Inhibitors related to intracellular signaling, such as U73122, chelerythrine, L-NNA, were also used. ML-9 and Y-27632 were identified as inhibitors of factors of myosin light chain (MLC). The contractility was observed to be lower in the DM group than in the control group. Further, the activities of phospholipase C (PLC), protein kinase C (PKC), and myosin light chain kinase (MLCK) were decreased in the DM group. DM reduced the activity of PLC, PKC, and MLCK, which resulted in a decrease in the contractility of the fundus smooth muscle. Therefore, our results present the mechanism of this DM-mediated GI disorder.

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.

Fluoxetine Enhances Anti-tumor Activity of Paclitaxel in Gastric Adenocarcinoma Cells by Triggering Apoptosis and Necroptosis.

BACKGROUND/AIM: Fluoxetine, an antidepressant, has cytotoxic effects on several cancer cell lines, while paclitaxel is an antineoplastic agent for various cancers. The aim of this study was to evaluate whether fluoxetine enhances the cytotoxic effect of paclitaxel in gastric adenocarcinoma cells and determine the mechanism of cell death. MATERIALS AND METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability and perform cell cycle analysis. Annexin V propidium iodide (PI) staining, 4',6-diamidino-2-phenylindole (DAPI) staining, caspase-3/7 assay, and western blot analysis were performed for determining cell death. RESULTS: Fluoxetine enhanced the anti-proliferative effect of paclitaxel. Fluoxetine-paclitaxel combination caused G2/M arrest and increased events in the sub G0/G1 phase in a time and dose-dependent manner, indicating apoptotic cell death. Combination treatment caused an increase in early apoptotic and late apoptotic cell death compared to single treatment alone. CONCLUSION: Fluoxetine enhanced the antiproliferation effect of paclitaxel in gastric adenocarcinoma AGS cells and the combination caused cell death by triggering apoptosis and necroptosis.