| Research one Expression and significance of PI3K/Akt signal pathway protein in gastric cancer tissueGastric cancer is a common malignant tumor around the world.The pathogenesis of gastric cancer is a complex process that involves changes in intracellular signal transduction pathways.In this study,gastric cancer patients with pathological diagnosis and initial surgical resection were selected.Immunohistochemistry was used to study the expression of PI3K/Akt protein in the upstream of PI3K/Akt/m TOR signaling pathway in gastric cancer tissues.To explore the correlation between the clinicopathological parameters such as tumor size,invasion depth,lymph node metastasis and differentiation degree and the expression levels of PI3 K and Akt.To explore whether PI3K/Akt/m TOR signaling pathway can be a new target for the treatment of gastric cancer.Aim:To study the expression of PI3 K and Akt protein in tumor tissues of patients with gastric cancer.And to investigate whether tumor size,depth of invasion,lymph node metastasis and differentiation were related to the expression levels of PI3 K and Akt.Method:1.The expression of p-Akt and p-PI3 K protein was detected by immunohistochemistryThis study collected 43 cases of gastric cancer(from ?the edge of the tumor 5cm)that were surgically resected from the Department of Gastrointestinal Surgery at the Third Affiliated Hospital of Anhui Medical University from August 2013 to May 2016.Patients were included in the criteria: 1.preoperative and pathological diagnosis clear 2.no preoperative radiotherapy,chemotherapy 3.no distant metastasis.The specimens were immediately fixed with 10% formaldehyde,using paraffin section and using streptavidin-perosidase(SP)to detect the expression level of p-Akt and p-PI3 K in tumor tissues and adjacent tissues.2.Clinical dataThere were 43 patients with gastric cancer,including 27 males and 16 females,aged 40-78 years with an average of 61.93±9.69.According to the 2014 International Union Against Cancer(UICC)stage,there were 16 cases in T1 and T2,27 cases in T3 and T4,14 cases in N0 and N1,and 29 cases in N3 and N4.According to histological grade,11 cases were high and moderately differentiated,and 32 cases were poorly differentiated.3.Statistical analysisSPSS 18.0 software was used for statistical analysis.Measured data were expressed as x ±s.The t-test was used to compare the mean values between the two groups.The comparison of the count data was corrected by the ?2 test.P<0.05 was considered statistically significant.Results:1.Expression of p-Akt and p-PI3 K in gastric carcinomaThe expression levels of p-Akt and p-PI3 K in gastric cancer tissues were higher,while the expression levels of p-Akt and p-PI3 K in the adjacent tissues were weak or negative.2.The expression of p-PI3 K and p-Akt in gastric cancer and adjacent tissuesThe positive expression rates of p-PI3 K and p-Akt in gastric cancer tissues were 81.40% and 74.42%,respectively.The positive expression rates of p-PI3 K and p-Akt in adjacent tissues were 23.26% and 20.93%,respectively.Compared with adjacent tissues,the expression level of p-PI3 K and p-Akt in gastric cancer tissues was significantly higher than that in adjacent tissues,and the difference was statistically significant(P<0.001).3.The relationship between the expression of p-PI3 K and p-Akt and the size of tumor in gastric cancer tissueIn 14 patients with tumor diameter ?5cm,the positive rate of p-PI3 K protein expression was 78.57%.The positive rate of p-PI3 K protein expression was 82.76% in 29 patients with tumor diameter >5cm.The difference was not statistically significant(P=1.000).In 14 patients with tumor diameter ?5cm,the positive rate of p-Akt protein expression was 71.43%.In 29 patients with tumor diameter >5cm,the positive rate of p-Akt protein expression was 75.86%.The difference was not statistically significant(P=1.000).4.The relationship between the expression of p-PI3 K and p-Akt and the depth of tumor invasion in gastric cancer tissueThe positive expression rate of p-PI3 K protein in 16 patients with T1 and T2 gastric cancer was 62.5%.In 27 patients with T3 and T4 gastric cancer,the positive expression rate of p-PI3 K protein was 92.59%.The difference was statistically significant(P<0.05).The positive expression rate of p-Akt protein in patients with stage T1 and T2 was 50%.The positive rate of p-Akt protein in patients with T3 and T4 was 88.89%,and the difference was statistically significant(P<0.05).5.The relationship between the expression of p-PI3 K and p-Akt and lymph node metastasis in gastric cancer tissueThe positive expression rate of p-PI3 K protein in 14 patients with gastric cancer staged as N0-N1 was 57.14%,and in 29 patients with stage N2-N3,the positive rate of p-PI3 K protein was 93.10%,and the difference was statistically significant(P<0.05).The positive expression rate of p-Akt protein in patients with stage N0-N1 was 50%.And the positive rate of p-Akt protein was 86.21% in the patients with N2-N3,and the difference was statistically significant(P<0.05).6.The relationship between the expression of p-PI3 K and p-Akt and the degree of differentiation in gastric cancer tissueIn 11 cases of highly differentiated gastric cancer patients,the expression rate of p-PI3 K protein was 55.55%.In 32 cases of poorly differentiated gastric cancer patients,the expression of p-PI3 K protein was 90.63%,and the difference was statistically significant(P<0.05).The positive expression rate of p-Akt protein in 11 cases of highly differentiated gastric cancer was 45.45%.In 32 cases of poorly differentiated gastric cancer,the positive rate of p-Akt protein was 84.38%.The difference was statistically significant(P<0.05).Conclusion:1.The expression level of PI3 K and Akt was higher in gastric cancer tissues than that in adjacent tissues.2.The expression levels of PI3 K and Akt in gastric cancer were correlated with tumor differentiation,lymph node metastasis and tumor invasion,but not with tumor size.Research two Mangiferin Inhibits Gastric Cancer Cell Proliferation and Its Mechanism of Regulating PI3K/Akt Signaling PathwayMangiferin is a bipyrone compound,which is also known as C-glycoside xanthone.The mangiferin can be extracted from the araceae,mango,almond and gentian plants.Recent studies have shown that mangiferin has many pharmacological effects such as antioxidation and immune regulation.At present,the research on the role of mangiferin in tumors is less,and the effect on gastric cancer has not yet been clearly reported.In this study,MTT,flow cytometry,TUNEL staining,Western Blot and other methods were used to observe the effects of mangiferin on proliferation inhibition and apoptosis induction of human gastric cancer cell lines in vitro,and to study the effect of mangiferin on apoptosis-related protein expression levels.The regulation of PI3K/Akt signaling pathway provides a theoretical basis for studying the mechanism and target of mangiferin in gastric cancer.Aim:To study the effect of mangiferin on gastric cancer cells and its effect on PI3K/Akt signaling pathway.Method:1.Detection of the inhibitory effect of mangiferin on proliferation of human gastric cancer cell line SGC-7901 and BGC-823 by MTT assayAfter 2.5-200 ?mol/L mangiferin was added to human gastric cancer cell line SGC-7901 and BGC-823 for 24-72 hours respectively,the absorbance of each well was measured by ELISA at OD 490 nm.Cell growth curves were plotted and IC50 was calculated using Graphpad prisms5 software.2.Comparison of the inhibitory effect of mangiferin on the proliferation of human gastric cancer cell line SGC-7901?BGC-823 and human gastric mucosa cell line GES-1 by MTT assayMangiferin at a concentration of 2.5-200?mol/L was applied to human gastric cancer cell line SGC-7901?BGC-823 and normal human gastric mucosal cell line GES-1,respectively.After 48 hours,culture was terminated.OD 490 nm was selected to measure the absorbance of each well in an enzyme-linked immunosorbent assay.Cell growth curves were plotted and IC50 was calculated using Graphpad prisms5 software.3.Annexin V-FITC/PI Double Staining Method for Detecting the Apoptosis of Manganin in Human Gastric Cancer Cell Line SGC-7901Different concentrations(5 and 10 ?mol/L)of mangiferin were applied to human gastric cancer cell line SGC-7901 for 48 h.After the cells were digested with trypsin,Annexin V-FITC/PI double staining was used to detect apoptosis.4.Detection of Apoptosis of Human Gastric Cancer Cell Line SGC-7901 by TUNEL MethodDifferent concentrations of mangiferin(5,10 ?mol/L)were added to gastric cancer cells SGC-7901 for 48 hours,and TUNEL assay kit was used to detect apoptotic cells.The confocal microscopy was used to observe and photograph.5.Detection of apoptosis-related proteins in human gastric cancer cell line SGC-7901 by Western BlotDifferent concentrations(5,10 ?mol/L)of mangiferin were applied to human gastric cancer cell line SGC-7901 respectively,and the action time was 48 h.Western Blot detected apoptosis-related protein expression.6.Detection of PI3K/Akt/m TOR Signaling Pathway in Human Gastric Cancer Cell Line SGC-7901 by Western BlotDifferent concentrations(5,10 ?mol/L)of mangiferin were applied to human gastric cancer cell line SGC-7901 respectively,and the action time was 48 h.Western Blot detected PI3K/Akt/m TOR signaling pathway protein expression.7.Western Blot detects the effect of mangiferin on PI3K/Akt/m TOR signaling pathway in human gastric cancer cell line SGC-7901 after adding EGFDifferent concentrations(5,10?mol/L)of mangiferin were added to EGF at 100 ng/ml and incubated in human gastric cancer cell line SGC-7901 for 48 hours.Western Blot detected PI3K/Akt/m TOR signaling pathway protein expression.8.Effect of Mangiferin Combined with PI3K/Akt Regulator on Human Gastric Cancer Cell Line SGC-7901The concentration of 10 ?mol/L PI3 K inhibitor Ly294002 and 20 ?mol/L Akt agonist SC79 were incubated with 5 ?mol/L mangiferin in human gastric cancer cell line SGC-7901 for 48 h.Western Blot was used to determine the effect of mangiferin on PI3K/Akt/m TOR signaling pathway related proteins in human gastric cancer cell line SGC-7901.MTT assay was used to detect the inhibitory effect of PI3K/Akt modulator combined with mangiferin on the proliferation of human cancer cell line SGC-7901.TUNEL assay was used to detect the apoptosis of human gastric cancer cell line SGC-7901 induced by PI3K/Akt modulator combined with mangiferin.9.Statistical analysisSPSS 18.0 software was used for statistical analysis.Measured data were expressed as (?)±s.The t-test was used to compare the mean values between the two groups.The comparison of the count data was corrected by the ?2 test.P<0.05 was considered statistically significant.Result:1.Inhibitory Effect of Mangiferin on the Proliferation of Gastric Cancer Cell Line SGC-7901 and BGC-823Mangiferin has an inhibitory effect on the proliferation of human gastric cancer cell line SGC-7901 and BGC-823.With the increase of mangiferin treatment concentration and the prolongation of the action time,the survival rate of tumor cells gradually decreases.The half inhibitory concentration of mangiferin on gastric cancer cell line SGC-7901 was significantly lower than that of gastric cancer cell line BGC-823.2.Comparison of the inhibitory effect of mangiferin on proliferation of human gastric cancer cell line SGC-7901?BGC-823 and human gastric mucosal cell line GES-1Mangiferin has a significant inhibitory effect on human gastric cancer cell line SGC-7901 and BGC-823,while it has almost no inhibitory effect on normal human gastric mucosa cell line GES-1.3.Annexin V-FITC/PI Double Staining Method for Detecting the Apoptosis of Manganin in Gastric Cancer Cell Line SGC-7901Mangiferin has apoptotic effect on human gastric cancer cell line SGC-7901.With the increase of the concentration of mangiferin,the apoptosis rate of cells increases.4.Detection of apoptosis of human gastric cancer cell line SGC-7901 by TUNEL methodA large number of apoptotic cells appeared in the visual field after the action of mangiferin.The chromatin condensed in the apoptotic cells.The cells shrunk and deformed,and the volume became smaller,showing apoptotic bodies.The cells in the control group were tightly packed and apoptotic cells could not be observed.With the increase of the concentration of mangiferin,the rate of apoptosis increased.5.Detection of apoptosis-related proteins in human gastric cancer cell line SGC-7901 by Western BlotMangiferin can down-regulate the expression levels of Bcl-x L,Bcl-2,Mcl-1 in human gastric cancer cell line SGC-7901.The effect of Mangiferin increases with the concentration of Mangiferin.Mangiferin increases Bad,Bax and human gastric cancer cell lines.The expression levels of pigment C,Caspase-9,and Capase-3 were increased with increasing concentrations of mangiferin.6.Detection of PI3K/Akt/m TOR Signaling Pathway in Human Gastric Cancer Cell Line SGC-7901 by Western BlotMangiferin can down-regulate the expression of phosphorylated PI3 K,phosphorylated Akt,and phosphorylated m TOR in the human gastric cancer cell line SGC-7901 PI3K/Akt/m TOR signaling pathway.The effect of mangiferin is enhanced with the increasing concentration of mangiferin.7.Western Blot detects the effect of mangiferin on PI3K/Akt/m TOR signaling pathway in human gastric cancer cell line SGC-7901 after adding EGFEGF can increase the expression of phosphorylated PI3 K and phosphorylated Akt in the PI3K/Akt/m TOR signaling pathway of human gastric cancer cell lines.Mangiferin can still down-regulate the phosphorylation of PI3K/Akt/m TOR signaling pathway in human gastric cancer cell lines after EGF stimulation.The expression levels of PI3 K and phosphorylated Akt were increased with the increase of the concentration of mangiferin.8.Effect of Mangiferin Combined with PI3K/Akt Regulator on Human Gastric Cancer Cell Line SGC-7901The PI3 K inhibitor Ly294002 inhibits the expression of phosphorylated PI3 K in the PI3K/Akt/m TOR signaling pathway in human gastric cancer cells.Akt agonist SC79 can up-regulate the expression of phosphorylated Akt in the PI3K/Akt/m TOR signaling pathway.Ly294002 combined with mangiferin can inhibit the expression of phosphorylated PI3 K in the PI3K/Akt/m TOR signaling pathway in human gastric cancer cell lines.When SC79 and mangiferin act on human gastric cancer cell line SGC-7901,down-regulation of phosphorylated Akt in PI3K/Akt/m TOR signaling pathway by mangiferin can be antagonized by SC79.Ly294002 combined with mangiferin can significantly reduce the half inhibitory concentration of mangiferin.When SC79 and mangiferin act on human gastric cancer cell line SGC-7901,the half inhibitory concentration of mangiferin is increased.Ly294002 combined with mangiferin can enhance the inducing apoptosis of mangiferin.SC79 combined with mangiferin can reduce the induction of apoptosis of mangiferin.Conclusion:1.Mangiferin has a strong inhibitory effect on human gastric cancer cell line SGC-7901 in vitro.2.Mangiferin has an apoptosis-inducing effect on human gastric cancer cell line SGC-7901 in vitro,and mangiferin can mediate apoptosis-related protein regulation.3.Mangiferin has apoptosis-inducing effect on human gastric cancer cell line SGC-7901 and inhibits tumor cell proliferation in vitro,which may be related to its regulation of PI3K/Akt signaling pathway. |
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