| BackgroundGastric cancer remains the second leading cause of cancer death in the worldwide. As one of the most frequently occurring cancer, gastric caner is mainly treated with surgery and combination chemotherapies which improved the overall survival. However, poor prognosis caused by multiple drug resistance still the major problem in the cancer treatment. Improved or novel combination chemotherapies are needed to be developed for overcoming drug resistance and improving the prognosis of gastric cancer patients.Flavonoid, mainly extracted from plants or fruits, have multiple biological activities and have been widely studied in cancer therapy. As a flavonoid, naringenin extracted from citrus fruits also have anti-mutagenic and anti-carcinogenic activities. Recently studies have shown that naringenin could induce cancer growth inhibition, migration or cell cycle arrest in various cancer cells including epidermoid carcinoma cells, human hepatocellular carcinoma cells, bladder cancer cells, breast tumor cells. However, the anti-cancer activity of naringenin in gastric cancer cells has not been studied.In various cancer cells, the overexpression of anti-apoptotic protein Bcl-2 has been commonly observed and is associated with drug resistance and poor prognosis. ABT-737, a small mimic of BH-3 only protein, play as an inhibitor of Bcl-2 and its anticancer activity has been well studies in lung cancer, acute myeloid leukemia, multiple myeloma, and lymphoma. However, the effect of ABT-737 on gastric cancer cells has not been well studied.In the present study, we demonstrated the combinative effect of naringenin and ABT-737 on gastric cancer cells. The results showed that naringenin or ABT-737 alone can induce the inhibition of cancer cell growth and colony formation and that the combinative use of them further increases the inhibition in gastric cancer cell line SGC7901. Meanwhile, the inhibition induced by naringenin and ABT-737 is related to the Akt and p53 pathways.Materials and MethodsCell culture and regentsHuman gastric cancer cell line SGC7901 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, glutamine, and antibiotics at 37℃ in 5% CO2. Naringenin and ABT-737 were obtained from Sigma (USA) and Selleck Chemicals (USA), respectively.Antibodies and regentsAntibodies to poly ADP-ribose polymerase (PARP), p-Akt, and Akt were bought from Cell Signaling Technology (USA). Antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Caspase-3, p53, as well as horseradish peroxidase (HRP)-linked anti-rabbit IgG, were purchased from Beyotime Institute of Biotechnology (Shanghai, PR China).Cell viability assayThe effect of naringenin or ABT-737 on SGC7901 cell growth was measured by Cell Counting Kit-8 (Beyotime, PR China). In brief,2000 cells cultured in a 96-well plate were treated with naringenin or ABT-737 for 48hours. After the treatment,10μl WST-8 solution was added and the cells were further incubated for another 2 hours. The number of cells was measured with a microplate reader at a test wavelength of 450 nm. Cell viability was normalized with the control and presented by histograms. All cell viability assays were performed in triplicate and repeated in 3 independent experiments.Colony formation assay500 SGC7901 cells were seeded in the well of 6-well plate and treated with naringenin or ABT-737 or both for 6 days. The colonies were fixed with methanol for 15 min at room temperature and stained with crystal violet for 30min. The number of colonies containing more than 50 cells was quantified and presented by histogram. This experiment were performed in triplicate and repeated in 3 independent experiments.Western blotsWhole cell lysates were extracted from cells suspended in cell lysis buffer supplemented with protease inhibitor cocktail. The lysates were resolved by electrophoresis on polyacrylamide gels containing SDS and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk. The blots were incubated with the appropriate primary antibody after washing and then exposed to secondary antibody conjugated with HRP. The bands on the membrane were visualized and captured using the ECL reagent (Beyotime, China) and X-ray films (Kodak, USA).Data analysisStatistical significance was determined using the Student’s t-test or One-way ANOVA. The standard deviations were demonstrated as a bar in the histogram.ResultsNaringenin and ABT-737 inhibit SGC7901 cell viabilitySince both naringenin and ABT-737 have anti-cancer activity, we evaluated the effect of them on SGC7901 cell growth using Cell Counting Kit-8 assay. Narigenin inhibited the SGC7901 cell growth in a dose-dependent manner and ABT-737 also inhibited the cell viability dose-dependently. Next, combination effect of naringenin and ABT-737 on the cell growth also was determined and further inhibition on cell growth was observed when SGC7901 cells were treated with both drugs. These results indicated that the naringenin could inhibit gastric cancer cell growth and that the inhibition induced by naringenin can be enhanced by ABT-737.Naringenin and ABT-737 inhibit SGC7901 cell colony formationNaringenin and ABT-737 could inhibit SGC7901 cell growth and we next investigated this inhibition using a colony formation assay. Lowe density of SGC7901 cell were seeded and treated with naringenin or/and ABT-737 for 6 days. Naringenin and ABT-737 alone decreased the clone numbers by about 40% and the combination of them induced a decrease of 90% colonies compared with control. Together, colony formation of SGC7901 could be inhibited by naringenin, ABT-737 or both.Naringenin and ABT-737 induced more cleaved caspase-3 and PARPNaringenin and ABT-737 could induce apoptosis in many other cancer cells and the apoptosis in SC7901 cells treated with these two drugs was examined. Apoptosis related protein caspase-3 and PARP was detected in SGC7901 cells using Western blot. Naringenin or ABT-737 alone increased the activated form of caspase-3, cleaved caspase-3 and the increased PARP was also detected. Combination of naringenin and ABT-737 further increased the cleavage of caspase-3 and PARP, suggesting an enhancement of apoptosis in SGC7901 cells.Naringenin and ABT-737 increase p53 expression and reduce Akt activationNaringenin could induce cell apoptosis through multiple pathways. The pathways involved in naringenin and ABT-737-induced cell apoptosis were also investigated. p53 and Akt are important pathways in cell apoptosis and survival. Our results showed that naringenin inhibit the phosphorylation of Akt but ABT-737 didn’t. Expression and phosphorylation of Akt in SGC7901 cells were significantly reduced by the use of naringenin and ABT-737. Meanwhile, both naringenin and ABT-737 increased the expression of p53 protein. These results suggested that p53 and Akt pathways are involved in cell apoptosis induced by combination of narigenin and ABT-737 in SGC7901 cells.Conclusions1. Naringenin have anticancer activity in gastric cancer cells.2. ABT-737 have anticancer activity in gastric cancer cells.3. ABT-737 in combination with naringenin have anticancer activity in gastric cancer cells.4. Narigenin alone or in combination with ABT-737 could induce the inhibition of gastric cancer cell growth and colony formation.5. Naringenin and ABT-737 induce activation of caspase-3 and cleavage of PARP, leading to cell apoptosis.6. Akt and p53 pathways are involved in the combination effect of naringenin and ABT-737 in gastric cancer cells. |
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