Study On The Pharmacodynamic Mechanism Of Puerarin Synergistically With 5-FU Against Colon Cancer

Colon cancer is a common malignant tumor of the digestive tract occurring in the colon,and its incidence has also shown a marked upward trend in recent years at home and abroad.Until now,5-FU has always been used as a first-line treatment both in singly and combination forms.While the natural drug resistance makes their overall effectiveness reduced,which remains a significant limitation to the clinical use of 5-FU.Puerarin is known as the main active ingredient of pueraria lobata.Modern pharmacological studies have shown that puerarin can play the role of specific anti-cancer by inhibiting the synthesis of DNA and the expression of COX-2 gene.It can inhibit the growth and promote apoptosis of multiple cell lines,such as human cholangiocarcinoma QBC939 cells,pancreatic cancer PANC-1 cells and lung cancer H446 cells.In addition,combined with other chemotherapeutic drugs,puerarin shows a synergistic effect and can increase the apoptotic rate of cancer cells.The resistance of chemotherapeutic drugs is mostly related to the expression of efflux protein,so,elucidating the mechanism from the perspective of efflux protein becomes very important.Drug transporters are a class of transmembrane proteins that seriously affect drug uptake and distribution.They are widely distributed in intestinal cells.P-glycoprotein can actively pump out chemotherapeutic drugs from tumor cells and reduce its accumulation.Overexpression of P-glycoprotein is one of the important mechanisms of multidrug resistance in tumors.Most of the chemical drugs found to reverse multidrug resistance are highly toxic and costly,which limits their clinical application.While the monomer components of traditional Chinese medicine have certain potential as chemosensitizer.By comparing the inhibitory effects of pentafluorouracil singly and together with puerarin treatment on human colon adenocarcinoma cell line Caco-2 and solid tumors,we tried to explore whether puerarin can increase the killing rate of chemotherapeutic drugs on cancer1.Pharmacodynamical studyHuman colon cancer Caco-2 cells were cultured stably,with a good growth and passage performance.MTT assay was used to investigate the inhibition rate of puerarin and 5-FU only and together on Caco-2 cells.Compared with single treatment,combination of puerarin and 5-FU could significantly reduce the survival rate of Caco-2 cells.The IC50 of puerarin and5-FU singly used for Caco-2 cells was 3404.53 ?M and 190.64 ?M,respectively.Yet,when used in combination,the values changed to 1475.06 ?M and 92.19 ?M.Thus suggesting that puerarin combined with 5-FU had synergistic effect on the inhibition of Caco-2 cells.Hoechst 33258 and flow cytometry were used to investigate the effects of combination and single administration on the apoptosis and cycle of Caco-2 cells.After Hoechst 33258 staining,compared with the control group,puerarin had no significant apoptotic effect on Caco-2 cells.While,5-FU had significant apoptotic effect on Caco-2 cells.There was no significant difference in apoptotic rate between the combined group and 5-FU itself,which showed that puerarin combined with 5-FU had no synergistic apoptotic effect on Caco-2 cells.The same results were obtained in flow experiments.As compared with the blank control group,puerarin group could significantly increase the proportion of GO/G1 phase cells(P<0.001),and reduce the proportion of S phase cells(P<0.05).The proportion of GO/G1 phase cells in single drug administration was significantly different from that in combination(P<0.001).Indicating that puerarin could block the cell cycle at GO/G1phase and cooperate with 5-FU to block the cell cycle.The combination of 5-FU alone with puerarin and 5-FU significantly inhibited the growth of subcutaneous tumors in nude mice.Although there was no statistically significant difference in puerarin,the average tumor volume in the puerarin group was 0.59 times that of the control group.The average tumor weight was 0.57 times that of the control group.The mean volume of tumor and tumor weight were smaller than the control group.The analysis may be due to the small number of samples,and the difference in tumor growth in the body is large,which affects the statistical difference.2.Research on mechanismsBy studying the effect of puerarin on the expression of Caco-2 MDR1 gene and P-glycoprotein,tried to elucidate the effect of puerarin on P-gp from the point of gene of protein.Compared with the blank group,the results of Q-PCR showed that the expression of MDR1 gene was significantly decreased when puerarin was administered alone or combined with 5-FU.By studying the effect of puerarin on the activity of P-gp ATPase and the content of ATP in cells,tried to elucidate its effect on the efflux function of P-gp from the perspective of energy dependence.According to the principle that the intensity of light emitted is proportional to the concentration of ATP,the ATP content was determined by measuring light intensity with chemiluminescent fluorescence spectrophotometer.The standard curve of ATP was constructed by diluting the luciferase buffer to different concentration,and thus the ATP content was calculated.The results showed that puerarin combined with 5-FU could significantly reduce the intracellular ATP content.By comparing the signal differences between the treated and untreated samples with Na3VO4,a selective inhibitor of P-gp ATPase,the effects of the compounds on the activity of P-glycoprotein ATPase were tested.The results showed that puerarin combined with 5-FU could significantly reduce the activity of ATPase and inhibit protein expression.Docking analysis was used to compare the binding ability of ATP and puerarin to protein.The Docking Score value of puerarin to P-glycoprotein was 6.435,while that of ATP as control group of positive drug was 5.700.Indicating that the binding activity of puerarin to P-glycoprotein was higher than ATP and thus it was easier to bind to protein.