bafilomycin-a1 and Stomach-Neoplasms

Autophagy is an intracellular degradation pathway that delivers organelles or protein to the lysosome and has been recently implicated in the resistance of gastric cancer to chemotherapy. This study aimed to investigate whether blocking autophagy is a new approach for the treatment of chemoresistant gastric cancer. SGC-7901 gastric cancer cell line was treated with 5-fluorouracil (5-FU) or/and autophagy inhibitor bafilomycin A1. Cell viability and growth were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and clonogenic assay. Apoptosis was evaluated by flow cytometry. Cell migratory and invasive ability were evaluated by migration and invasion assays. Autophagy was evaluated by scanning electron microscopic, acridine orange staining, and Western blot analysis. We observed that 5-FU induced autophagy in SGC-7901 cells. Bafilomycin A1 decreased the viability and clone formation, inhibited the invasive and migratory ability, and increased the apoptosis of SGC-7901 cells. Taken together, our data suggest that chemotherapy-induced autophagy contributes to gastric cancer chemoresistance, and the inhibition of autophagy is a promising strategy for gastric cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Enzyme Inhibitors; Fluorouracil; Humans; Macrolides; Stomach Neoplasms

To investigate the effect of bafilomycin A1 (BAF) on the cell proliferation, invasiveness, apoptosis, and oxaliplatin sensitivity in gastric cancer MGC-803 cells.. MGC-803 cells were divided into control group, BAF group, oxaliplatin group, and BAFµ oxaliplatin group. MTT assay and plate clone formation assay were used to assess the viability and colony forming ability of the cells after the treatments. The expression of nucleosomes in the cells was examined with ELISA. The cell migration and invasion after the treatments were evaluated. Western blotting was performed to detect the expression of Bcl-2 and Bax in the treated cells, and scanning electron microscopy, immunohistochemistry and Western blotting were employed to to observe the cell autophagy.. Compared with the control cells, the cells treated with BAF showed a substantial decrease in autophagosome accumulation with attenuated cell proliferation, migration and invasion. Compared with cells treated with oxaliplatin alone, the cells treated with both BAF and oxaliplatin showed significantly lowered autophagosome accumulation, suppressed cell proliferation, migration and invasion, increased cell apoptosis, increased Bax expression and lowered Bcl-2 expression.. BAF can inhibit the proliferation and invasiveness of MGC-803 cells, promote cell apoptosis by inhibiting autophagy, and enhances the sensitivity of the cells to oxaliplatin.

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Macrolides; Organoplatinum Compounds; Oxaliplatin; Stomach Neoplasms

Matrine has potent antitumor activity against a broad variety of cancer cells and our previous study showed that both autophagy and apoptosis were activated during matrine-induced gastric cancer cell death. The aim of the present study was to determine the significance of autophagy in antineoplastic effects of matrine and the molecular mechanism by which matrine induces autophagy in gastric cancer cells. Western blot analysis showed that exposure of gastric cancer cells to matrine resulted in the extent of autophagy increasing in a dose- and time-dependent manner by detecting micro-tubule-associated protein 1 light chain 3 (LC3). This induction was due to activation of autophagic flux, as supported using the lysosome inhibitor, bafilomycin A1, which produced an accumulation of LC3-II. Propidium iodide staining demonstrated that matrine induced cell death in a dose-dependent manner and the autophagy inhibitor 3-methyladenine (3-MA) or bafilomycin A1 enhanced lethality of matrine against gastric cancer cells. Moreover, after pretreatment with 3-MA, some of the gastric cancer cells treated with matrine exhibited prototypical characteristics of apoptosis by transmission electron microscopy. The ability of 3-MA to increase matrine-induced apoptosis was further confirmed by Annexin V-FITC/PI staining. Also, the combination of matrine and 3-MA was more potent than matrine alone in inhibiting the proliferation of SGC-7901 cells assessed by sulphorhodamine B assay. Furthermore, administration of the pan-caspase inhibitor zVAD-fmk or autophagy inducer rapamycin decreased the matrine-induced cell death. In addition, matrine treatment did not inhibit the phosphorylation of Akt and its downstream effectors mammalian target of rapamycin (mTOR) as well as p70 ribosomal protein S6 kinase (p70S6K), although the levels of the total Akt and mTOR were decreased. These results suggest that autophagy was activated as a protective mechanism against matrine-induced apoptosis and inhibition of autophagy may be an attractive strategy for enhancing the antitumor potential of matrine in gastric cancer.

Topics: Adenine; Alkaloids; Apoptosis; Autophagy; Cell Line, Tumor; Cell Nucleus; Cell Shape; Cell Survival; Drug Synergism; Humans; Immunoglobulin G; Macrolides; Matrines; Melphalan; Microtubule-Associated Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Quinolizines; Stomach Neoplasms

We investigated the effects of vacuolating cytotoxin (VacA) prepared from Helicobacter pylori on the metabolism of gastric epithelial cells, AZ-521. VacA caused the ATP levels to decrease in a time-dependent manner; by approximately 20% in 6 h, 35% in 12 h and 50% in 24 h, at a concentration of 120 nM. This decrease was also dependent on the concentration of VacA. To evaluate the impairment of mitochondria by VacA, mitochondrial membrane potential was estimated by flow cytometric analysis using 3, 3'-dihexyloxacarbocyanine iodide as a substrate. VacA decreased membrane potential with the relative fluorescence intensity of AZ-521 cells in 6 h from 52+/-3 to 24+/-1. Treatment of the cells with bafilomycin A1, a specific inhibitor of vacuolar ATPase proton pump, showed no apparent effect on these changes in the levels of ATP and the mitochondrial membrane potential. Secondly, we estimated the effect of VacA on oxygen consumption. VacA inhibited oxygen consumption in AZ-521 cells: the levels of PO 2 in the medium of control cells decreased by 73% in 3 h and 37% in 6 h, whereas those in VacA-treated cells were 84% in 3 h and 59% in 6 h. Flow cytometric analysis showed the number of cells in the G0/G1 phase was increased by VacA. Taken together, VacA induced an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells.

Topics: Adenocarcinoma; Adenosine Triphosphate; Anti-Bacterial Agents; Bacterial Proteins; Bacterial Toxins; Cell Cycle; Cell Respiration; Cytotoxins; Enzyme Inhibitors; Flow Cytometry; Gastric Mucosa; Helicobacter pylori; Humans; Macrolides; Membrane Potentials; Mitochondria; Oxygen Consumption; Stomach Neoplasms; Tumor Cells, Cultured