| Objective: To study the effects of naringenin（NAR）on the growth,proliferation,apoptosis and cell cycle of As PC1 and Bx PC3 cells of human pancreatic cancer,to detect the changes in the activity of key molecules in the PI3K/AKT/m TOR signaling pathway of cells,and to elucidate their possible molecular mechanisms,so as to provide experimental and theoretical basis for the treatment of pancreatic cancer with naringenin.Methods:1.MTT assay was used to detect the effect of NAR（0,100,200,400,800,1000mol/L）concentrations on the survival rate of normal pancreatic duct cell line H9C6 and pancreatic cancer As PC1 and Bx PC3 cells for 24,48 h and 72 h.2.After 15 days of NAR（0,100,200,300,400 μmol/L）treatment on As PC1 and Bx PC3 cells of pancreatic cancer,the formation of cell clones was detected by crystal violet staining and photographed.After 48 h treatment with As PC1 and Bx PC3 cells of pancreatic cancer,the growth and proliferation of the cells were detected by digital camera.3.After NAR concentration（0,200,400 μmol/L）was applied to As PC1 and Bx PC3 cells of pancreatic cancer for 48 h,the cell cycle distribution was detected by flow cytometry with PI staining.Annexin V FITC/PI double staining was used to analyze the apoptosis rate by flow cytometry.Apoptosis and necrosis were detected by Hoechst/PI live staining and fluorescence microscopy.4.After NAR concentration（0,100,200,300 and 400 μmol/L）was applied to As PC1 and Bx PC3 cells of pancreatic cancer for 48 h,the cell cycline-related proteins Cyclin A,Cyclin D1,CDK2,P21 and PCNA were detected by Western blot.Expression of apoptotic proteins Cleaved-Caspase3,Bax,Bcl-2 and PI3 K,p-AKT,p-m TOR and p-4EBP1.Results:1.Determined by MTT experiment results show that the element of pomelo peel on the growth of normal pancreatic duct cell lines H9C6 proliferation and no obvious inhibitory effect,but the element of pomelo peel can obviously inhibit As PC1 and Bx PC3 pancreatic cancer cell survival,and with the increase of drug delivery dosage and function of the extension of time,pomelo peel,the more obvious inhibitory effect on pancreatic cancer cell survival rate（p<0.05）.2.The results of As PC1 and Bx PC3 cell cloning experiments showed that naringenin could significantly inhibit the growth and proliferation of As PC1 and Bx PC3 cells of pancreatic cancer.With the increase of naringenin dosage,the number and volume of cell clones decreased significantly,which was statistically significant compared with the normal control group（p<0.05）.Under the microscope,the results showed that with the increase of naringenin dose,the number and volume of the cells decreased significantly,and the cells separated from the surrounding cells and the permeability changed.3.The testing results of FCM show that the element of pomelo peel effect on pancreatic cancer As PC1 and Bx PC3 cells after 48 h,compared with control group,two cell lines and S phase cell percentage of the medicine group were significantly increased（p<0.05）,and the S phase cells proportion with pomelo peel pigment concentration increases,the percentage of cells in G2 / M phase were significantly reduced（p<0.05）,and the proportion of G2 / M phase cells with pomelo peel pigment concentration increase and decrease,shows that element of pomelo peel As PC1 and Bx PC3 cells can be induced to S phase.After 48 h,compared with normal control group,As PC1 cells to medicine group of early apoptosis rate and late apoptosis rate were significantly higher（p<0.01）,In addition,the higher the dose of naringenin,the more significant the increase of late apoptosis was,and the results showed a significant difference（p<0.01）,indicating that naringenin could induce apoptosis of As PC1 and Bx PC3 cells（p<0.05）.Live by Hoechst/PI dyeing-fluorescence microscope observation result,pomelo peel effect after 48 h,compared with normal control group,low concentration of pomelo peel group（100 μmol/L）with a small amount of cells appear the phenomenon of low blu-ray/High red-ray and suggests that some cell necrosis happened,high concentration of pomelo peel group（200,300,400 μmol/L）there is an obvious phenomenon of low blue/high red,that have taken place in the cell apoptosis,and pomelo peel element to the dose,the greater the apoptosis phenomenon more significantly.4.Through the WB results shows that compared with normal control group,the results show that compared with the control group,with increasing pomelo peel pigment concentration,amount of Cyclin A protein expression than the control group significantly decreased（p<0.05）,CDK2 and PCNA protein expression rate than the control group were significantly lower,and the high concentration（300,400 μmol/L）when the difference was statistically significant（p<0.05）,while the amount of P21 protein expression than the control group were significantly higher,and the high concentration（300,400 μmol/L）when the difference was statistically significant（p<0.05）,There was no significant change in the expression of Cyclin D1 protein（P>0.05）,as shown in the figure,indicating that naringenin can regulate the expression of Cyclin A in pancreatic cancer cell lines.When treated with naringenin,the levels of Cleaved-caspase3 and Bax proteins in As PC1 cells were up-regulated compared with the normal control group,and the differences were statistically significant at high concentration（400 μmol/L）（p<0.01）,while the expression levels of Bcl-2 protein were significantly lower than those in the control group（p<0.01）.After the treatment of Bx PC3 cells with different concentrations of naringenin,there was no significant difference in the expression level of Cleavedcaspase-3 protein（p>0.05）,and the expression level of Bax protein was significantly up-regulated（p<0.01）,while the expression level of Bcl-2 protein was significantly lower than that of the control group,and the difference was statistically significant（p<0.05）,The results showed that naringenin could regulate the expression of apoptotic proteins in pancreatic cancer cell lines.The protein expression levels of PI3 K,p-AKT,p-m TOR and p-4EBP1 in each concentration treatment group were significantly lower than those in the control group.With the increase of naringenin concentration,the decreasing trend was more significant,and the difference was statistically significant（p<0.01）.These results suggest that naringenin may induce pancreatic cancer cell cycle arrest and apoptosis by inhibiting the PI3K/AKT/m TOR signaling pathway.Conclusion:1.Naringenin has a significant inhibitory effect on the survival rate of As PC1 and Bx PC3 cells of pancreatic cancer,and it is dose-dependent and time-dependent manner.2.The inhibitory effect of naringenin on As PC1 and Bx PC3 cells of pancreatic cancer may be related to the induction of cell cycle arrest and apoptosis.3.The cell cycle arrest and apoptosis induced by naringenin in pancreatic cancer may be related to the inhibition of PI3K/AKT/m TOR signaling pathway. |
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