Inhibition Of Brusatol On Proliferation,Invasion And Metastasis Of Gastric Cancer Cell SGC-7901

Gastric cancer is one of the common tumors,and its lethality ranks at the forefront of the disease.The occurrence and development of human gastric cancer are closely related to factors such as food,smoking,environment,and genetics.And with the acceleration of the pace of the whole society,work pressure,and irregular diet.The age of onset of gastric cancer gradually decreases.Therefore,human gastric cancer seriously harms human and social development.At present,to improve the survival rate of patients with gastric cancer,non-early human gastric cancer can be treated with surgery,mostly with traditional chemotherapy after surgical resection,which has a poor prognosis for eliminating infections and other injuries.In addition,the higher mortality rate for gastric cancer is due to the fact that the diagnosis is usually at an advanced stage.Tumor microenvironment can be understood as the soil for tumor survival.Inflammation is one of the important factors affecting tumor microenvironment,and it is also a factor leading to tumor spread.The Chinese medicine Brucea javanica has the effects of clearing away heat and detoxification,cutting off malaria,stopping diarrhea,and corroding warts.Brusatol is one of the effective active ingredients of Chinese medicine Brucea javanica,and is a kind of bitter wood lactone compound.Modern pharmacological studies have found that it has inhibitory effects on leukemia,liver cancer,pancreatic cancer,and melanoma cells.Apoptosis is a programmed death regulated by a variety of cellular signaling pathways,and it plays an important role in tissue development and immune response,as well as other physiological processe.This project intends to select different concentrations of brucereol and lipopolysaccharide to act on human gastric cancer SGC-7901 cells together to investigate the mechanism of brusatol inhibiting the proliferation of gastric cancer cells and reversing the increase in metastasis ability in an inflammatory environment.To study the inhibitory effect and mechanism of brusatol on the invasion,proliferation,and metastasis of gastric cancer cell SGC-7901,to clarify the role of PI3K/Akt signaling pathway in brusatol induced gastric cancer cell death,and to further explain its mechanism.To study the inhibitory effect of brusatol on the proliferation,and metastasis of gastric cancer cells SGC-7901 and its mechanism,the role of PI3K/Akt signaling pathway in brusatol induced gastric cancer cell death was elucidated,and its mechanism was further explored,and to further interpret its mechanism of action.Method:The MTT method screened the different time and concentration of brusatol and lipopolysaccharide on SGC-7901 cells,and determined the best treatment time and concentration of brusatol and lipopolysaccharide;observed the changes of SGC-7901 cell morphology with an inverted microscope;scratch experiment The effects of lipopolysaccharide and brusatol on the migration and invasion of gastric cancer cells SGC-7901 were tested with Transwell experiment;Observe DAPI by fluorescence microscope,the changes of cell fluorescence color after JC-1 staining,the changes of mitochondrial membrane potential（△Ψm）,the apoptotic rate and the changes of intracellular reactive oxygen species（ROS）are detected by flow cytometry;transmission electron microscopy to observe the structural changes of organelles,Western blot to investigate Bax,Bcl-2,Caspase-3,PI3K,AKT,NF-κB,E-cadherin,N-cadherin,MPP-9expression and nuclear translocation of NF-κB;fluorescence microscope observation of Vimentin protein expression;flow cytometry and MMT method to detect the effect of apoptosis inhibitor Z-VAD-FMK on cell apoptosis rate.Changes of related protein indexes after treatment with PI3K inhibitor LY294002.Results:1.Brusatol inhibits proliferation invasion and metastasis of gastric cancer cells induced by lipopolysaccharide（1）The IC₅₀ of brusatol was screened by the MTT method,and the optimal concentration of LPS was investigated.It was found that after 400 n M brusatol was treated with gastric cancer SGC-7901 cells for 24 h,the cell inhibition rate was almost 50%.When the lipopolysaccharide concentration was higher than 10μg/m L,it has an inhibitory effect on cell proliferation,（2）Observation by an inverted microscope showed that the cells in the normal group were normally adherent and well attached;after being treated with lipopolysaccharide,the cells were mostly spindle-shaped and the cell density increased significantly.After administration of brusatol for 24 hours,the cells shrank and ruptured.And the number of cells is reduced;（3）Through the cell scratch test,the effect of brusatol and lipopolysaccharide on the metastasis of SGC-7901 cells.Compared with the control group,the cells showed increased migration ability after LPS.And this shift.The migration ability decreases with the increase of the concentration of brusatol;（4）Transwell experiment found that lipopolysaccharide can induce the invasion and migration ability of SGC-7901 cells.The above results indicate that brusatol can significantly inhibit the migration and invasion of SGC-7901 cells induced by LPS,and brusatol can significantly reverse this reality.2.The molecular mechanism of brusatol in inhibiting the proliferation of gastric cancer cells（1）Cell apoptosis was detected by DAPI staining.Fluorescence microscopy showed that the chromatin was uniform in the normal group,while after the treatment of brusatol,some cells showed bright blue,and the proportion of blue dense cells increased with the increase of dosage;（2）Transmission electron microscope observation results showed that the internal structure of the blank control group was complete,and the mitochondria and nuclei were relatively intact.Being treated with brusatol 24 hours later,the intracellular blebbing was evident,and the chromatin gathered by the nuclear membrane,and part of the intracellular Apoptotic bodies appear;（3）JC-1 fluorescence staining found that brusatol induced the decrease of mitochondrial membrane potential,fluorescence microscope observation revealed that the fluorescence color changed from red to green,and the green fluorescence intensity increased with the increase of the dose;（5）The H₂DCF-DA test found that the production of reactive oxygen species in the cells increased significantly,which was significantly different from that of the blank control group.The results showed that brusatol affect mitochondrial function,promote production of ROS（6）Flow cytometry detection revealed that brusatol could significantly induce apoptosis of gastric cancer cells compared with the normal group,and the apoptotic rate increased with the dose;（7）Western blot test results show that after brusatol acts on SGC-7901 cells,the expression of Caspase-3 and Bax increase with the increase of dose,the expression of Bcl-2 decreased,and the expression of Bcl-2/Bax ratio;（8）The results of the MTT method showed that the Caspase-3 inhibitor Z-VAD-FMK can also reverse the apoptosis of gastric cancer cells induced by brusatol,which was verified by flow cytometry.3.The molecular mechanism of brusatol in inhibiting the invasion and metastasis of gastric cancer cells（1）Elisa kit detects inflammatory factors.Lipopolysaccharide can inhibit the anti-inflammatory factor IL-10 and promote the secretion of the pro-inflammatory factor IL-17.Compared with the blank control group,there is a significant difference,brusatol can reverse this phenomenon.（2）Western blot experiment results showed that compared with the blank group,the expression of N-cadherin and MMP-9 protein in the cells was up-regulated after lipopolysaccharide stimulation,while the expression of E-cadherin decreased（P<0.05）,after treated by brusatol,the expression of N-cadherin and MMP-9 protein decreased,while the expression of E-cadherin increased,and there was a statistical difference（P<0.01）,（3）Observe the expression of Vimentin protein by fluorescence microscope;compared with the normal group,the fluorescence intensity increased after treatment with lipopolysaccharide,while the expression decreased after administration of brusatol,（4）The results of Western blot experiments showed that compared with the control group,the expression of PI3K/AKT pathway protein increased after lipopolysaccharide stimulation,and its expression decreased with the increase of concentration after administration of brusatol.This was verified by PI3K pathway inhibitor LY294002.（5）The results of Western blot showed that lipopolysaccharide up-regulated the expression of NF-κB protein in the nucleus;brusatol inhibited its nuclear translocation.Conclusion:The research results show that brusatol can induce apoptosis of SGC-7901cells and inhibit its invasion and metastasis through PI3K/Akt/NF-кB signaling pathway.