Mechanism Of Alisol B Acetate On The Apoptosis, Invasiveness And Migration Of SGC-7901 Cells

IntroductionGastric cancer is one of the common malignancies in gastrointestinal tract.Though extensive clinical research has been carried out with numerous combinations of cytotoxic agents,the overall prognosis of advanced gastric cancer still remains poor. Natural products are potential sources of novel anticancer drugs over the decades and contribute a lot to the cancer chemotherapy.Herbal medicines have always held an attraction..Alisol B acetate is a major ingredient isolated from Alismatis rhizoma and has been used for urological disease in traditional Chinese medicine.In recent years,the pharmacological characterization of alisol B acetate has been identified and several biological activities have been defined,such as the inhibitory effects on lipopolysaccharide-induced mRNA expression of inducible nitric oxide synthase and nitric oxide production,the inhibition of complementary activity and antibody-mediated allergic reaction.Furthermore,it has been demonstrated that alisol B acetate induces cell death in hepatoma and leukemia cells.Furthermore,alisol B acetate induces apoptosis in PC-3 cells via a mitochondria-mediated mechanism with activation of caspase-8,-9 and -3.Alisol B acetate induced Bax up-regulation and nuclear translocation;Up to date,there is no report concerning the role of this natural triterpene on the Gastric Cancer Cell Lines.In order to know the chemopreventive potentials of pseudolaric acid B on gastric carcinoma.,we examinated the effects of alisol B acetate on the growth and apoptosis of the gastric cancer lines SGC7901,and the change of mitchondrial potential and the protein expression of caspase3,caspase9,Bax,Bcl-2 and PI3 K/Akt signal pathway.We analyze MMP-2,MMP-9 activities and protein expression while investigated the effects of Alisol B acetate on the invasive activity and motility of tumor.The data offer a potential mechanism for Alisol B acetate-induced apoptosis in adenocarcinoma SGC7901 cells,suggesting that Alisol B acetate may severve as an effective reagent for the treatment of gastric cancer.Materials and MethodsMaterial1.Human gastric carcinoma cell line SGC-79012.Experimental medicine Alisol B acetate3.Staining reagents for flowcytometry4.Reagents for Western blot5.Reagents for invasion and migrationMethods1,Inhibition of cell proliferation was determined by MTT assay.2,Cells were stained with PI and cell cycle analysis was performed by Flow cytometry3,AnnexinV-FITC/PI fluorescent staining was used to tested early cell apoptosis rate.4,The morphology of SGC7901cells exposed to alisol B acetate for different time was observed first under inverted microscope.Then electronic microscope was used to observe nuclear morphological changes and the apoptotic morphology.5,DNA fragmentation analysis was assessed by agarose gel electrophoresis.6,Changes of mitochondrial membrane potential(ΔΨm) were monitored by Rhodamine-123 staining.7,The effects of Alisol B acetate on invasion and migration of SGC7901 cells were determined in a transwell Boyden chamber8,Genistein zymography assay were to determine MMP-2 and MMP-9 activities.9,Western blot was performed to test the influence of Alisol B acetate on Caspase-3,Caspase-9,Bax,Bcl-2,PI3 K/Akt,MMP-2 and MMP-9 protein levels. Results1,MTT methods indicated alisol B acetate(over 30μmol/L) had significant growth inhibition effect on SGC7901cells in a dose-and time-dependent manner.2,Size and number of SGC7901 cells were significantly reduced by incubation with alisol B acetate under inverted microscope.Marked morphological changes of cell apoptosis such as condensation of chromatin and apoptosis body were found clearly using electronic microscope.3,The characteristic "ladder" of DNA fragments representing integer multiples of the internucleosomal DNA length(about 180～200 bp) was observed by agarose gel electrophoresis in a time-dependent manner.4,Early Apoptoti cells(AnnexinV/FITC(+),PI(-)) gradually increased in time-dependent manner in SGC7901 cells staining by AnnexinV-FITC/PI fluorescent and analyzing by FCM5,SGC7901 cells were stained with PI and analyzed by FCM.The percentage of sub-G1 cells in SGC7901 cells was increased in a time-dependent manner.Along with the enhancement of cell growth inhibition,apoptotic cells gradually increased6,The changes in the membrane potential of the mitochondria were examined by Rhodamine-123 staining.After Alisol B acetate treatment for 12 hours,the cells exhibited a significant alterations inΔΨm,and the percentage of disruption ofΔΨm gradually increased in a time-dependent manner.7,Western blot analysis of Bcl-2 and Bax expression.Bcl-2 was upregulated while Bax was down-regulated in a time-dependent manner.8,The proteolytic cleavage of procaspase-3 and-9 also were detected by Western blotting.caspase-3 and caspase-9 were activated,as measured by the appearance of its caspase-3 17 kDa and caspase-9 37 kDa subunit.Caspase-3 and caspase-9 activation increased concomitantly with increased time of alisol B acetate treatment.9,Protein levels with Western blot analysis the impact of alisol B acetate on PI3K and Akt expressions.Rapid phosphorylations of PI3K and Akt kinase were detected while the total proteins of PI3K and Akt kinase showed no change in alisol B acetate treatment. 10,Effect of alisol B acetate on the Invasion and Migration of SGC7901Cells was observed by Transwell Boyden chamber.Invasion and migration alibility of SGC7901Cells were decreased significantly.Alisol B acetate was able to reduce the number of SGC7901 cells that could penetrated Matrigel membranes in a dose-dependent manner.11,Western blotting was performed to determine MMP-2 and MMP-9 proteins expression.The expressions of MMP-2 and MMP-9 proteins and activities were significantly decreased in a time-dependent mannerConclusion1,Alisol B acetate had significant growth inhibition effect on SGC7901cells in a dose-and time-dependent manner.2,SGC7901 cells apoptosis inducing by Alisol B acetate were confirmed by inverted microscope,electronic microscope,DNA fragments,AnnexinV-FITC/PI fluorescent staining.3 Activations of caspase-3 and caspase-9,PI3K/Akt kinase,disruption of the membrane potential of the mitochondria,the changes of Bcl-2,Bax protein level might be involved in alisol B acetate-induced SGC7901 cells apoptosis4 Alisol B acetate changes the cell cycle phase distribution and arrested SGC7901 cells at G0/G1 phase.The change of the cell cycle phase distribution involved in cells apoptosis5,Alisol B acetate inhibited invasion and migration of SGC7901 cells.The decreasion of protein level and active enzyme of MMP-2,MMP-9 play a important role in invasion and migration of SGC7901Cells...