Anti-tumor Activity Of Resveratrol Combined With 5-FU Based On Energy Metabolism In Vitro

Cancer is the second most deadly disease in the world and poses a serious threat to human health.At present,surgical treatment,radiotherapy and chemotherapy are the main methods for treating tumors.However,traditional treatment methods still have many drawbacks,such as poor specificity,large side effects and drug resistance.5-FU is one of the commonly used chemotherapeutic drugs in clinical practice,which has good curative effect on digestive system cancer and breast cancer.It also has strong toxic and side effects,which may cause hemorrhagic diarrhea,myelosuppression and liver and kidney toxicity.Therefore,the combination of 5-FU with other natural anti-tumor drugs is essential to enhance efficacy and reduce toxicity.Resveratrol is a non-flavonoid polyphenolic compound found in many natural foods and Chinese herbal medicines.Recent studies have confirmed that resveratrol has various pharmacological effects including anti-tumor,and has low toxic and side effects.It indicates that it is expected to become a promising anti-tumor drug in the future.Compared with normal tissue cells,tumor cells have special metabolic modes,such as abnormal glycolysis activity and dependence on glutamine.Therefore,targeting tumor energy metabolism may also be an effective anti-tumor method.In this study,resveratrol and 5-FU were used alone or in combination in tumor cells and vascular endothelial cells to observe their anti-tumor and anti-tumor angiogenic activities and explore the relationship with energy metabolism.At the same time,explore whether resveratrol combined with 5-FU has synergistic effect.In this experiment,human gastric cancer cells SGC7901,human breast cancer cells MCF-7 and human umbilical vein endothelial cells HUVEC were used to observe the proliferation inhibition activity,pro-apoptotic activity,morphological changes and effects on cellular energy metabolism after treated by resveratrol and 5-FU alone or in combination.The effects of resveratrol on cell viability of SGC7901,MCF-7 and HUVEC cells were detected by CCK-8 in vitro.The IC₅₀value was calculated according to the proliferation inhibition rate,and the optimal concentration was selected.The Q value method was used to calculate the combined drug effect according to the inhibition rate of three cells by resveratrol and 5-FU alone or in combination;The growth curve was drawn to observe the effect of resveratrol and 5-FU on cell proliferation at different time points;Flow cytometry PI single staining and Annexin V single staining were used to detect the effects on cell cycle and apoptosis,respectively;HE staining was used to observe the changes of cell morphology;The enzyme activity of phosphofructose kinase（PFK）,pyruvate kinase（PK）,succinic acid dehydrogenase（SDH）and glutamine enzyme（GLS）as well as the changes of metabolite lactate（LD）,ATP content and glucose consumption were detected by spectrophotometry;Western blotting was performed to detect the expression of energy metabolic-associated protein including hypoxia-inducible factor 1α（HIF-1α）,glucose transporter 1（GLUT-1）,and mono-carboxylic acid transporter 4（MCT-4）in cells.CCK-8 results showed that resveratrol inhibited the proliferation of SGC7901,MCF-7and HUVEC cells in a concentration-dependent manner,with IC₅₀values of 63.755μmol/L,65.592μmol/L and 78.679μmol/L,respectively.Finally,50μmol/L resveratrol was selected as the subsequent experimental concentration.The combined drug results showed that the proliferation inhibition rate of 50μmol/L resveratrol combined with 100μmol/L 5-FU was significantly increased.The Q values were 0.93,0.97 and 0.91,respectively,has an additive effect.The results of growth curve indicated that resveratrol and 5-FU alone or in combination had obvious inhibitory effect on SGC7901,MCF-7 and HUVEC cells in different time points.Cell cycle assay showed that compared with the control group,the percentage of G₀/G₁phase and S phase was increased（P<0.05）and the G2/M phase was decreased（P<0.01）in SGC7901cells;The percentage of G₀/G₁phase was increased（P<0.01）and the G2/M phase was decreased（P<0.01）in MCF-7 cells;The percentage of G₀/G₁phase of HUVEC cells was decreased（P<0.01）,and the percentage of S phase was increased significantly（P<0.01）;Simultaneously,the effect of cell cycle block in combined group is generally better than the single administration group.The results of apoptosis assay showed that the apoptosis rate of SGC7901,MCF-7 and HUVEC cells was significantly higher than that of the control group（P<0.01）,and the apoptosis rate of the combined group was higher than that of the single administration group（P<0.01）.The results of HE staining showed that resveratrol and 5-FU alone or in combination significantly reduced the number of cells,and observed that the cell volume was reduced,the edges were blurred,the nuclei were deeply stained,the cell morphology was changed,and the morphological changes of the cells in the combination group are more obvious.The results of energy metabolism showed that compared with the control group,the activities of PK,PFK and SDH in SGC7901 cells were significantly decreased（P<0.01,P<0.05）,the ATP content and LD excretion were significantly reduced（P<0.01,P<0.05）,and the activity of GLS and glucose consumption in the combined group were decreased（P<0.01）;The PK activity,ATP content,LD excretion and glucose consumption in MCF-7 cells were significantly decreased（P<0.01,P<0.05）,and the activities of PFK and SDH were decreased in the resveratrol group and the combined group（P<0.01）,but no significant change in the level of glutaminase activity was observed in each group（P>0.05）;The activities of PK,SDH and ATP in HUVEC cells were significantly decreased（P<0.01）,the activities of GLS,PFK and LD excretion in the resveratrol group and the combined group were significantly decreased（P<0.01,P<0.05）,the PFK activity increased significantly in 5-FU group（P<0.01）,but there was no significant change in glucose consumption（P>0.05）.Western blot result showed that compared with the control group,the expression of GLUT-1 in SGC7901 cells was significantly decreased after 48h treatment（P<0.01,P<0.05）,and the expression of MCT-4 in resveratrol group and combined group was decreased（P<0.01）,while HIF-1αwas only decreased in the combined group（P<0.05）;The expression levels of three proteins in MCF-7 cells were significantly decreased（P<0.01,P<0.05）;The expression of HIF-1αin HUVEC cells was decreased significantly（P<0.01,P<0.05）,and the expression of MCT-4 in resveratrol group and combined group was decreased（P<0.01,P<0.05）,however,the expression of GLUT-1 has not changed significantly（P>0.05）.Therefore,it is concluded that resveratrol combined with 5-FU has the effect of inhibiting the proliferation of tumor cells and vascular endothelial cells and promoting their apoptosis,and the combined effect is better than the single use,the mechanism is to reduce the energy metabolism-related enzyme activity and metabolite content by reducing the expression of HIF-1α,GLUT-1 and MCT-4.