| ObjectiveApigenin belongs to flavonoids that can inhibit carcinogen carcinogenic activity which mainly existed in Stellera Verbenaceae,Selaginella plants,such as large content of Daphne genkwa,selaginella.With other flavonoids(quercetin,kaempferol flavonoids)compared with the characteristics of low toxicity,no mutagenicity.Colon(cancer of)is one of the most common malignant tumors in Western Europe,North America and other developed countries.In China,due to the death of colon cancer,fifth men died of malignant tumors,women ranked in the top sixth.Up to now,the 5 year survival rate is still hovering at about 50%,and the effective combination therapy or the appropriate adjuvant radiotherapy and chemotherapy is the important aspect of clinical attention.In recent years,many scholars have tried to seek a combination of traditional Chinese and Western medicine to improve the five year survival rate of patients.In this study,we used colon cancer cell line SW620 as a model to study the inhibitory effect of apigenin on SW620 cell polyamine metabolism and glucose uptake.Methods1 Cell culture conditionsCells were routinely cultured in Dulbecco modified Eagle medium(DMEM)supplemented with 10%foetal bovine serum(FBS)and equilibrated with humidified 5%CO2 in air at 37℃.2 Proliferation assay of SW620 cellsUsing SW620 cells as the research object,the experiment with 10～80 μM of different concentration of apigenin on the cell,DFMO(2.5mM)as a positive control medicine,cultured for 24,48,72 hr after inhibition of cell proliferation was detected by CCK-8;at the same time,the morphological changes were observed under inverted microscope.3 The effects on the SGLT1 pathway and glucose uptake on SW620 cellsReal-Time PCR and Western blot were used to detect the mRNA and protein expression levels of SGLT1 and GLUT1 in SW620 cells after administration of different concentrations of apigenin,and the effect of SW620 cells on glucose uptake was measured by glucose assay kit.4 The effects on polyamine content of SW620 cellsThe effect of different concentrations of apigenin on intracellular polyamine content was determined by pre column derivatization and high performance liquid chromatography(HPLC).5 The effects on the ODC/SSAT pathway on SW620 cellsReal-Time PCR and Western blot were used to detect the levels of mRNA and protein expression of ODC and SSAT in SW620 cells after administration of different concentrations of apigenin.6 The effects of reactive oxygen species content and apoptosis on SW620 cellsFlow Cytometer(FCM)was used to detect the relative level of reactive oxygen species and cell apoptosis rate,and the morphological changes of apoptotic cells were detected by Hoechst33258.Results1 Proliferation assay of SW620 cellsDifferent concentration of apigenin after treatment of SW620 cells,the cell growth was inhibited,the inhibitory effect was time and concentration dependent,and better than DFMO(2.5mmol· L-1)group,compared with the control group,the difference was statistically significant(P<0.05).After 48hr observation under inverted microscope,normal control group SW620 cells were short spindle cells,transparent,well refraction,abundant cytoplasm translucent,cell tight junctions,cell density,cell death and cell debris suspension rare.To observe the effect of apigenin after 48 hr in visible cell density decreased,intracellular particle enhancement,the cell body became long,cell gap widened gradually,the cell boundaries blur,cytoplasm decreased,cell shedding is suspended,cell debris can be seen,the survival cells decreased.2 The effects on the SGLT1 pathway and glucose uptake on SW620 cellsCompared with the control group,apigenin in SW620 cells 24,48,72h,the glucose consumption of SW620 cells decreased significantly(P<0.05),when the same apigenin concentration and action time prolonged gradually or at the same time apigenin concentration gradually increased,the inhibition of SW620 cell uptake of glucose effect gradually enhanced in a concentration and time dependent manner.However,the DFMO administration group of 2.5mM had a certain effect on glucose consumption of SW620 cells,no better than apigenin.Compared with the control group,the expression of GLUT1 and SGLT1 protein and mRNA decreased with the increase of apigenin concentration.3 The effects on polyamine content of SW620 cellsCompared with the control group,apigenin in SW620 cells after 48h,spermine content of cells decreased significantly(P<0.01,P<0.05),putrescine content increased significantly(P<0.01,P<0.05),reduce the total polyamine content in a concentration dependent.The 2.5mM DFMO administration group on SW620 cell,putrescine,spermine,spermidine content decreased(P<0.01).When the two combinations were used,the total polyamine content showed a significant decrease(P<0.01).4 The effects on the ODC/SSAT pathway on SW620 cellsCompared with the control group,the expression of SSAT and mRNA increased with the increase of apigenin concentration,but there was no significant difference between ODC and mRNA.The results indicated that apigenin could promote the expression of polyamine by promoting the expression of SSAT,which was consistent with the result of HPLC detection.5 The effects of reactive oxygen species content and apoptosis on SW620 cellsFCM results showed that compared with control group,the ROS content and apoptosis rate of SW620 cells increased with the increase of apigenin concentration.Compared with the control group,the apoptosis and necrosis of the cells were significantly higher than those of the control group,and the number of apoptotic cells increased with the concentration of apigenin.After Hoechst 33258 staining,the nucleus of the control group was large,and the fluorescence in the nucleus was light,and the brightness was uniform.Different concentration of apigenin treated cells after apoptosis,the number of cells with apigenin concentration increased and decreased gradually,appear apoptosis morphological changes including apoptosis,nuclear pyknosis,nuclear fragmentation into massive,deep colored with light blue,formation of apoptotic bodies.Conclusion1 Proliferation assay of SW620 cellsApigenin can inhibit the proliferation of human colon cancer cell line SW620,and has a concentration and time dependence.Effect of apigenin treated 24,48,72hr,with the increase of concentration of apigenin inhibited cells increased significantly,10μM of apigenin 48h,the proliferation of SW620 cells was inhibited,showed that 10μM is the lowest concentration of apigenin may play a pharmacodynamics in vitro.It is helpful for the further study andclinical application of apigenin in colon cancer.2 The effects on the SGLT1 pathway and glucose uptake on SW620 cellsGlucose transporter 1(GLUT1)and sodium dependent glucose transporter(SGLT1)is currently the two most known distribution in vivo glucose transporter widely,normal physiological processes which are involved in glucose transport,cellular uptake of glucose increased mainly by increasing the transmembrane transport of GLUT1 and SGLT1,to achieve their high uptake for energy.This experiment through the detection of glucose consumption of SW620 cells,SW620 cells showed that the glucose effect of celery consumption were significantly reduced,and with apigenin concentration increased,the inhibition of SW620 cell glucose uptake effect more obvious,in a dose dependent manner.Accordingly,apigenin also reduced SW620 cell GLUT1 and the expression level of SGLT1 protein,so we speculate that the effect of apigenin on glucose uptake in SW620 cells,may be related to inhibition of cell proliferation,regulation of GLUT1 and SGLT1 protein expression,which may-be one of the anticancer mechanism of apigenin.3 The effects on polyamine content of SW620 cellsPolyamines are a kind of natural small molecular aliphatic substances widely distributed in tissues,including putrescine(putrescine,Put),spermidine(spermidine,Spd)and spermine(spermine,Spm),the activity and function of spermine strongest.Polyamines play an important role in regulating the growth,proliferation and differentiation of mammalian cells,and the content of polyamines in tumor tissues is much higher than that in normal tissues.Through this experiment,polyamine content detected in SW620 cells,SW620 cells of spermine and spermidine effects after celery decreased putrescine increased slightly,reducing the total polyamine content,that promotes polyamine catabolism,and with apigenin concentration increased,the SW620 to promote the role of polyamine metabolism is more obvious and in a dose dependent manner.4 The effects on the ODC/SSAT pathway on SW620 cellsOrnithine decarboxylase is a rate limiting enzyme in polyamine synthesis,to the important role of intracellular polyamine synthesis,while spermidine/spermine-N1-acetyltransferase is the rate limiting enzyme for the catabolism of polyamines.Through the experiments of mRNA and protein expression of ODC and SSAT in the detection of SW620 cells,suggesting that the upregulation of SSAT protein in SW620 cells of celery and mRNA in the role of the ODC and the control group had no significant difference,and this change also showed the same polyamine content.5 The effects of reactive oxygen species content and apoptosis on SW620 cellsPolyamine metabolism decomposition process will produce secondary metabolites of ROS cell apoptosis induced by the inner product party ROS fluorescent staining to evaluate the morphological changes effect of apigenin on the apoptosis of SW620 cells.The results showed that the effect of apigenin on SW620 cell 48h was significantly higher than that of the control group,while the apoptosis of ROS cells.This suggests that apigenin induces cell apoptosis by inducing polyamine catabolism in SW620 cells. |
