Effect Of Exopolysaccharide From Trichoderma Pseudokoningii On The Apoptosis In Gastric Cancer

Objective: To observe Effect of exopolysaccharide from Trichoderma pseudokoningiion the proliferation and the possible apoptosis mechanism in human gastric cancer.Methods:1. The IR spectrum of EPS was determined using a Fourier transforminfrared spectrophotometer (FTIR). The purity and average molecular weight of EPSwas measured by high performance gel permeation chromatography (HPGPC). Themonosaccharide components of EPS was analysised by HPLC.2.The effect of EPS (exopolysaccharide) has been evaluated in various tumor cell linesderived from gastric, intestinal, lung cancers. The BGC-823, HT29, A549cells weretreated with increasing concentration of EPS for24,48,72h and cell proliferation invitro was measured by SRB (Sulforhodamine B) assay.3. To determine whether the anti-proliferative activity induced on BGC-823cell line byEPS was caused by induction of apoptosis, morphological changes after treatment ofEPS were observed by fluorescence microscope following Hoechst33258staining.4. The DNA was extracted from BGC-823cells after treated with increasingconcentration of EPS and agarose gel electrophoresis was performed. To furthercharacterize the apoptosis process, quantify different period apoptotic cells, we detectedapoptotic characterizations of BGC-823cells treated with EPS by Annexin V-FITC/PI double staining assay. Induction of apoptosis was also confirmed by flow cytometricanalysis of cell cycle.5. To examine the mechanisms of EPS induced apoptosis in BGC-823. Themitochondrial membrane potential of BGC-823cells were examined by usingrhodamine123staining, a dye accumulated in mitochondria is directly proportional toΔΨ m. The changes of2’,7’-dichlorofluorescein diacetate (DCFH-DA) fluorescence inBGC-823cells were observed to detected intracellular Reactive oxygen species (ROS).Caspase activity was also measured using Caspase Activity Kits according to themanufacturer’s instructions.6. The apoptotic-related gene expression in BGC-823cells was analysed by usingreverse transcription (RT)-PCR. Total RNA was extracted by TRIzol reagents accordingto the manufacturer’s instructions. The RT-PCR products were separated byelectrophoresis using1%agarose gel and stained with ethidium bromide.Results:1. Infrared spectrum shows that EPS had characteristic absorption peaks ofpolysaccharides and mainly β-glycosidic bond pyranose. EPS was eluted as asymmetrical peak on HPGPC, and its average molecular weight was estimated to be30.7kDa. HPLC analysis showed EPS was composed of Mannose, Rhamnose, Glucose,Galactose and Xylose with a molar ratio of2.5:1.2:2.1:3.5:1.0.2. EPS showed in vitro inhibition of tumor cells proliferations in a time-andconcentration-dependent compared with control cells. It was obviously that the growthof BGC-823cells was significantly inhibited, with the growth inhibition ratio of11.57-42.57%after treated with increasing concentration of EPS for72h. Treatmentwith1mg/mL EPS for24h、48h、72h, the inhibition rates were20.65%,36.88%and42.57%respectively.3. EPS-treated cells showed significant morphological changes including cellshrinkages, small vesicles, chromatin compaction and nuclear fragmented. The nuclei appeared to be slightly smaller and had a brighter fluorescence than the control.4. DNA electrophoresis revealed that DNA fragmentation ladders were clearly seen inthe group of BGC-823cells exposed to EPS for48h. The apoptotic rate of BGC-823cells treated with EPS at the concentration of0.2,0.6and1.0mg/mL for48h of wassignificantly increased to23.50%、26.08%and29.20%respectively, while the apoptoticrate of control untreated cells was0.71%. The result of cell cycle analysis showed thatBGC-823cells treated with EPS concentration-dependently induced a significantincrease in the population in S phase.5. Compared with control cells, the rhodamine-stained cells treated with EPS showeddecreased fluorescence of mitochondria remarkably in a concentration-dependentmanner. In the detection of ROS, the treatment with EPS increased the proportion ofcells with higher green fluorescence intensity in a dose-dependent manner, indicatingthat EPS induced the accumulation of intracellular ROS. The activities of caspase-3,-8,and-9were also significantly increased at a dose-dependent manner in BGC-823cellstreated with EPS.6. RT-PCR results demonstrated that the expression of Bax, Fas, FasL and p53wereup-regulated in BGC-823cells treated with EPS, while the level of Bcl-2mRNA wasdown-regulated compared with control group.Conclusion:1. EPS possessed significant tumor inhibition effect on BGC-823cells in atime-and concentration-dependent manner.2. EPS could inhibit the growth of BGC-823cells by induction of apoptosis.3. The most probably mechanisms of EPS induced apoptosis in BGC-823cells areinvolved in the mitochondria pathway and the death receptor pathway.