| Objective: Gastric cancer is one of the most common cancer, and it is the second rank for the death caused by malignant tumor. The incidence and mortality of gastric cancer are very high in China. Although the surgery, radiotherapy, chemotherapy, molecular targeted therapy and biotherapy are considered as the most common methods to treat gastric cancer, the 5-year disease-specific survival was less than 20%.It is well known that chemotherapy and radiotherapy exert severe side effects in clinic cancer therapy. Therefore, it is necessary to explore more effective chemotherapeutic agents for the prevention and treatment of cancer. Metabolites form natutal plant and fungal is the choice of the drugs. A study reported that among 65 new drugs registered for cancer treatment during 1981-2002, 48 drugs were obtained from natural products. Recently, new drug development approaches have been made in extraction of active component from mushroom metabolites to treat various clinical diseases including cancers.Iso-suillin, a petroleum ether extracted from of Suillus flavus, molecular weight as 440.29 Kd, is belong to the prenylphenol class. To date, iso-suillin has been shown the activity of antioxidant and antitumor. In the present study, we aimed to investigate the effects of iso-suillin on cell proliferation of gastric cancer BGC-823 cell, and to determine the underlying mechanism iso-suillin induced apoptosis.Methods:1 Cell culture: BGC-823 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 μg/ml at 37 ℃ and 5% CO2 atmosphere.2 Measured the inhibition ratios of BGC-823 cell by MTT assay: BGC-823 cells in exponential growth phase were planted into 96-well multiplates. The cells were cultured for different time periods(24, 48 and 72 h). After the supernatant were discarded, 150 μl DMSO was added into each well, allowed to stand up for 10 min in the dark. The absorbance at 490 nm were measured using a microplate reader. The 50% inhibitory concentration(IC50) was calculated dependent on the inhibitory rates.3 Detection of morphologic changes: BGC-823 cells were cultured on 6-well plates and exposed to iso-suillin for 48 h.(1) After durg treatment, the cells were observed using a optical microscope.(2) Through stained with 4’, 6-diamidino-2-phenylindole(DAPI, Amresco, USA), morphological changes of the nuclear were observed using a fluorescence microscope(Olympus, Japan).(3) The ultrastructure changes in the cells were investigated under transmission electron microscope.4 Detected apoptosis by flow cytometry(FCM): the cells were collected, fixed with 70% ethanol for 24 h at 4 ℃, then stained with PI kit according to the manufacturer’s instructions. The apoptotic rates of the cells were measured by flow cytometry.5 Examined the related protein expressions by western blot: after the cells collection, proteins were abstracted using lysed buffer. Protein concentration for each sample was determined using Coomassie brilliant blue G250 protein assay kit. Samples(50 μg) were separated by SDS-PAGE and transferred to a nitrocellulose filter(NC filter). The membranes were then blocked for 1 h at room temperature with blocking solution containing 5% nonfat milk. After incubated with primary antibody, the membrane were washed and treated with secondary antibody(goat antimouse Ig G-horseradish peroxidase) at 37 ℃ for 1 h. The samples were reacted using the ECL kit followed the supplier’s instructions. Finally, the membrane were photographed and analysed using a gel image analysis system. The proteins expression ratio were analyzed by the integrated optical density(IOD) of aimed protein bands compared to that of β-actin.6 Statistical analyses: the data was measured as mean ± standard deviation evaluated with SPSS 13.0 solftware. The variances were analyzed by One-Way ANOVA, P<0.05 was considered statistically significant.Results:1 MTT results showed that iso-suillin inhibited BGC-823 cell growth in a concentration and time-dependent manner.2 Observation of morphologic changes:(1) Optical microscope:BGC-823 cells treated by iso-suillin became shrunk and round, and floated from the attachment.(2) Fluorescence microscope:Followed the increase of iso-suillin concentration, apoptosis became apparent. Apoptotic features such as chromatin condensation, nuclear shrinkage were increased in the cells treated with iso-suillin.(3) Transmission electron microscopy: apoptotic cells characterized by nuclear shrinkage, condensed chromatin and high electron density, were increased in the sample treated by iso-suillin.3 The apoptosis rate by FCM: the apoptosis rates are(0.97±0.3)%,(14.18±1.2)%,(38.34±2.3)%,(50.72±1.7)% each for the control, and 8, 16, 32 μmol/L of drug groups, respectively. The significant difference were present between experimental and control groups(P<0.01).4 The resuls of western blot: the results indicated that Caspase-3, Caspase-8, Caspase-9 expression were up-regulated, suggesting apoptosis occured in the cells after iso-suillin treatment.5 Observation of autophgosome: compared with the control group, more autophgosomes were exist in the treated cells using electron microscopy.Conclusions:1 Iso-suillin inhibites the proliferation of BGC-823 cells in a concentration and time-dependent manner.2 Iso-suillin induces apoptosis of BGC-823 cells.3 Iso-suillin induces apoptosis via activation of Caspase-3, Caspase-8, Caspase-9.4 Iso-suillin may lead to autophagy. |
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