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.

Autophagy and Digestive Disorders: Advances in Understanding and Therapeutic Approaches.

The gastrointestinal (GI) tract is a series of hollow organs that is responsible for the digestion and absorption of ingested foods and the excretion of waste. Any changes in the GI tract can lead to GI disorders. GI disorders are highly prevalent in the population and account for substantial morbidity, mortality, and healthcare utilization. GI disorders can be functional, or organic with structural changes. Functional GI disorders include functional dyspepsia and irritable bowel syndrome. Organic GI disorders include inflammation of the GI tract due to chronic infection, drugs, trauma, and other causes. Recent studies have highlighted a new explanatory mechanism for GI disorders. It has been suggested that autophagy, an intracellular homeostatic mechanism, also plays an important role in the pathogenesis of GI disorders. Autophagy has three primary forms: macroautophagy, microautophagy, and chaperone-mediated autophagy. It may affect intestinal homeostasis, host defense against intestinal pathogens, regulation of the gut microbiota, and innate and adaptive immunity. Drugs targeting autophagy could, therefore, have therapeutic potential for treating GI disorders. In this review, we provide an overview of current understanding regarding the evidence for autophagy in GI diseases and updates on potential treatments, including drugs and complementary and alternative medicines.

The Altered Signaling on EFS-Induced Colon Contractility in Diabetic Rats.

Diabetes mellitus affects the colonic motility developing gastrointestinal symptoms, such as constipation. The aim of the study was to examine the role of intracellular signaling pathways contributing to colonic dysmotility in diabetes mellitus. To generate diabetes mellitus, the rats were injected by a single high dose of streptozotocin (65 mg/kg) intraperitoneally. The proximal colons from both normal and diabetic rats were contracted by applying an electrical field stimulation with pulse voltage of 40 V in amplitude and pulse duration of 1 ms at frequencies of 1, 2, 4, and 6 Hz. The muscle strips from both normal rats and rats with diabetes mellitus were pretreated with different antagonists and inhibitors. Rats with diabetes mellitus had lower motility than the control group. There were significant differences in the percentage of inhibition of contraction between normal rats and rats with diabetes mellitus after the incubation of tetrodotoxin (neuronal blocker), atropine (muscarinic receptor antagonist), prazosin (α1 adrenergic receptor antagonist), DPCPX (adenosine A1 receptor antagonist), verapamil (L-type Ca2+ channel blocker), U73122 (PLC inhibitor), ML-9 (MLCK inhibitor), udenafil (PDE5 inhibitor), and methylene blue (guanylate cyclase inhibitor). The protein expression of p-MLC and PDE5 were decreased in the diabetic group compared to the normal group. These results showed that the reduced colonic contractility resulted from the impaired neuronal conduction and decreased muscarinic receptor sensitivity, which resulted in decreased phosphorylation of MLC via MLCK, and cGMP activity through PDE5.

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.

Apoptotic effect of fluoxetine through the endoplasmic reticulum stress pathway in the human gastric cancer cell line AGS.

Gastric cancer is the fourth most common cancer in the world. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, can inhibit the growth of cancer cells by inducing apoptotic cell death through various signaling pathways. This study was aimed to determine the mechanism of apoptotic cell death induced by FLX in AGS cells. MTT assay for cell viability test and colony forming assay was performed for detection of cell proliferation. Western blot analysis was conducted for protein expression. Increased fluorescence intensity and chromatin condensation were observed using DAPI staining. Production of reactive oxygen species (ROS) was measured by DCFDA assay. AGS cell proliferation was remarkedly inhibited by FLX in a dose-dependent manner starting at a concentration of 20 µM. The expression of death receptors was increased, which resulted in elevated expression of activated caspases and cleaved PARP, leading to FLX-induced apoptosis. Moreover, FLX significantly increased production of ROS, and N-acetyl cysteine, which scavenges ROS, attenuated the cytotoxic effects of FLX. In addition, treatment with FLX increased the expression of the endoplasmic reticulum (ER) stress marker, CHOP. P53 protein expression in AGS cells also decreased significantly with FLX treatment. Inhibition of ER stress significantly decreased the expressions of death receptor 5 (DR5), cleaved caspase 3, and cleaved PARP, but not to control levels. FLX-induced apoptosis in AGS involved upregulation of death receptors, ROS generation, and activation of ER stress.

Benzyl Isothiocyanate Induces Apoptosis via Reactive Oxygen Species-Initiated Mitochondrial Dysfunction and DR4 and DR5 Death Receptor Activation in Gastric Adenocarcinoma Cells.

Benzyl isothiocyanate (BITC) is known to inhibit the metastasis of gastric cancer cells but further studies are needed to confirm its chemotherapeutic potential against gastric cancer. In this study, we observed cell shrinkage and morphological changes in one of the gastric adenocarcinoma cell lines, the AGS cells, after BITC treatment. We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, a cell viability assay, and found that BITC decreased AGS cell viability. Reactive oxygen species (ROS) analyses using 2',7'-dichlorofluorescin diacetate (DCFDA) revealed that BITC-induced cell death involved intracellular ROS production, which resulted in mitochondrial dysfunction. Additionally, cell viability was partially restored when BITC-treated AGS cells were preincubated with glutathione (GSH). Western blotting indicated that BITC regulated the expressions of the mitochondria-mediated apoptosis signaling molecules, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cytochrome c (Cyt c). In addition, BITC increased death receptor DR5 expression, and activated the cysteine-aspartic proteases (caspases) cascade. Overall, our results showed that BITC triggers apoptosis in AGS cells via the apoptotic pathways involved in ROS-promoted mitochondrial dysfunction and death receptor activation.

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.