Study On The Effect Of Platycodin D On GAGs,MUC1 And STs In Colon Cancer Cells Based On The Phlegm Pathogenic Theory

BackgroundTumor is a global malignant disease with high morbidity and mortality,which seriously affects people's health and quality of life.Colon cancer is a common malignant tumor of the digestive tract.It has not obvious clinical symptoms in the early stage and has not attracted people's attention.It is often found in the advanced stage of cancer and cannot be completely eradicated by surgery.Therefore,chemotherapy is the main treatment for colon cancer.However,chemotherapy has a large toxic side effect on the body,is relatively expensive,and is prone to drug resistance.It is a realistic and urgent need to find drugs with obvious efficacy and less side effects from Traditional Chinese Medicine(TCM)to treat colon cancer.With the continuous development of research on tumor mechanism,many studies have shown that changes in glycosaminoglycans(GAGs),mucins(MUCs)and sialic acid of colon cancer cell seriously affect the function of colon cancer cells,such as proliferation,migration and signal transduction.TCM believes that tumors are closely related to the 'phlegm'.Since phlegm masses are the main pathogenesis of tumors,reducing phlegm and resolving masses is the important treatment for tumors.The phlegm has the characteristics of sticky and turbidity and it has similarity with the increase of the content of carbohydrate macromolecules represented by GAGs,MUCs and sialic acid in colon cancer,which can cause the accumulation of pathological substances and promote the development of tumors.In this experiment,the main active ingredient of Chinese medicinal peony platycodon grandiflorum D(PD)is applied to colon cancer cell HCT116 to study the effects of PD on colon cancer cells GAGs,MUC1 and sialyltransferase(STs).This experiment links the TCM 'phlegm' theory to modern medical pathology,provides a new idea for studying colon cancer,and provides a certain experimental basis for the development of the TCM 'phlegm' theory.ObjectiveTo observe the effect of PD on the activity of human colon cancer cells HCT116 and human normal colonic epithelial cells HCoEpiC.To observe the changes of GAGs content after PD intervene to HCT116 cells.To observe the changes in cellular GAGs content after PD intervene to HCT116 cells.To detact the expression of chondroitin sulfate synthase(CHSYs)protein and mRNA after PD intervene to HCT116 cells.To observe the changes of MUC1 protein and mRNA levels in HCT116 cells treated with PD.To observe the different expression of STs between human normal colonic epithelial cells HCoEpiC and human colon cancer cells HCT116.To detect the effect of PD on the highly expressed STs gene in HCT116 cells.MethodsThe effects of different concentrations of PD on the activity of HCT116 and HCoEpiC were determined by CCK-8 method.After HCT116 cells were treated with PD,GAGs were extracted,the content of uronic acid in GAGs samples was detected by hydroxybenzidine method,and the content of N-acetylgalactosamine(Ga1NAC)was detected by High Performance Liquid Chromatography(HPLC).Western Blot and Real-Time PCR were used to detect the changes in chondroitin synthase 1(CHSY1)and chondroitin synthetase 2(CHSY2)protein levels and mRNA levels in colon cancer cells after PD intervention.Western blot and Real-Time PCR were used to detect the levels of MUC1 protein and mRNA in HCT116 cells after PD intervention.The differential genes of 20 STs in human normal colonic epithelial cells HCoEpiC and human colon cancer cells HCT116 were screened by Real-Time PCR.Real-Time PCR was used to detect the effect of different concentrations of PD on the highly expressed STs gene in HCT116 cells.Results1.The results of CCK-8 assay showed that different concentrations of PD could inhibit the growth of HCT116 cells,and the inhibitory effect depend on the concentration of PD.The different concentrations of PD had different effects in HCoEpiC cells,and had no effects at low concentrations.At higher concentrations,HCoEpiC cells had a certain inhibitory effect.According to a=0.05 level,4 ?M PD group and the blank control group(P=0.963),8 ?M PD group and the blank control group(P=0.022),16 ?M PD group and the blank control group(P<0.001),32 ?M PD group and the blank control group(P<0.001),64 ?M PD group and the blank control group(P<0.001)were statistically significant,indicating that the inhibition of HCT116 cells was different through compared with the PD groups and the blank control group.In HCoEpiC cells,4 ?M PD group and the blank control group(P<=0.291),8 ?M PD group and the blank control group(P=1.000),16 ?M PD group and the blank control group(P<0.671)were no statistically significant,32 ?M PD group and the blank control group(P<0.001),64 ?M PD group and the blank control group(P<0.001)were statistically significant,indicating that PD had no effect in HCoEpiC cells at lower concentrations and had an inhibitory effect at high concentrations.According to the absorbance,cell inhibition rate was calculated,and the IC50 value of HCT116 cells after PD treatment for 48 h was 13 ?M,and the IC50 value of HCoEpic cells was 37 ?M.2.The content of uronic acid was detected by hydroxybiphenyl method.The results showed that the uronic acid content decreased after PD intervention.According to a=0.05 level,there was significant difference between PD group and the blank control group(P<0.001).3.The content of GalNAC was detected by HPLC.The results showed that the content of GalNAC decreased after PD intervention in HCT116 cells.According to ?=0.05 level,the difference between PD group and blank control group(P<0.001)was statistically significant.4.The effect of PD on the expression level of CHSYs proteins were determined by Western Blot.The results showed that the expression of CHSY1 and CHSY2 protein were decreased in each concentration group compared with the blank control group.According to ??0.05 level,in CHSY1,6.5 ?M PD group and blank control group(P=0.019),13 p M PD group and the blank control group(P=0.001),and 26 ?M PD group and the blank control group(P<0.001)were statistically significant.In CHSY2,6.5 ?M PD group and blank control group(P=0.021),13 ?M PD group and the blank control group(P=0.007),and 26 ?M PD group and the blank control group(P=0.003)were statistically significant.5.The results of Real-Time PCR showed that the levels of CHSY1 and CHSY2 mRNA was down-regulated in the 26 ?M PD group compared with the blank control group,and there were no different in the 6.5 and 13 ?M PD groups.At the level of ?=0.05,6.5 ?M PD group and the blank control group(P=0.548)and 13 ?M PD group and the blank control group(P=0.103)in CHSY1 were not statistically significant,26 ?M PD group and the blank control group(P=0.002)was statistically significant.In CHSY2,the 6.5 ?M PD group and the blank control group(P=0.977),13 ?M PD group and the blank control group(P=0.052)were not statistically significant,26?M PD group and the blank control group(P<0.001)was statistically significant.6.Western Blot was used to determine the MUC1 expression in HCT116 cells.The results showed that the expression levels of MUC1 protein were down-regulated in each concentration groups compared with the blank control group.According to ?=0.05 level,6.5 ?M PD group and the blank control group(P=0.007),13 ?M PD group and the blank control group(P=0.001),26 ?M PD group and the blank control group(P<0.001)were statistically significant.7.Real-Time PCR was used to detect the expression of MUC1 mRNA.The expression of MUC1 mRNA were down-regulated in each concentration groups compared with the blank control group.According to ?=0.05 level,6.5 ?M PD group and the blank control group(P=0.001),13 ?M PD group and the blank control group(P<0.001),26 ?M PD group and the blank control group(P<0.001)were statistically significant.8.Real-Time PCR was used to detect the expression of STs differential genes in HCoEpiC and HCT116 cells.The results showed that ST3GAL1(P<0.001),ST3GAL6(P<0.001),ST6GALNAC2(P<0.001),ST8SIA3(P=0.014),and ST8SIA5(P<0.001)were highly expressed in HCT116 cells,and low expression of other genes(P<0.05).9.Real-Time PCR was used to detect the effect of different concentrations of PD on high expression of STs in HCT116 cells.The results showed that compared with the blank control group,the mRNA levels of ST3GAL1,ST6GALNAC2,ST8SIA3,and ST8SIA5 were down-regulated in the 6.5,13 and 26 ?M PD groups.The expression of ST3GAL6 was not different in the 6.5 ?M PD group,and the mRNA expression were down-regulated in the 13 and 26 ?M PD groups.According to?=0.05 level,in ST3GAL1,6.5 ?M PD group and the blank control group(P=0.004),13 ?M PD group and the blank control group(P<0.001),and 26 ?M PD group and the blank control group(P<0.001)were statistically significant.In ST3GAL6,there was no significant difference between the 6.5 ?M PD group and the blank control group(P=0.855),13 ?M PD group and the blank control group(P=0.003)and 26 ?M PD group and the blank control group(P<0.001)were statistically significant.In ST6GALNAC2,6.5 ?M PD group and the blank control group(P=0.008),13 ?M PD group and the blank control group(P=0.004),26 ?M PD group and the blank control group(P=0.001)were statistically significant.In ST8SIA3,6.5 ?M PD group and the blank control group(P<0.001),the 13 ?M PD group and the blank control group(P<0.001),and 26 ?M PD group and the blank control group(P<0.001)were statistically significant.In ST8SIA5,6.5 ?M PD group and the blank control group(P=0.022),13 ?M PD group and the blank control group(P<0.001),and 26 ?M PD group and the blank control group(P<0.001)were statistically significant.ConclusionPD significantly inhibits the proliferation of human colon cancer cells and has less effect on human normal colonic epithelial cells.PD reduced the content of GAGs,decreased the expression of CHSY1 and CHSY2,decreased the expression of MUC1,and down-regulated the mRNA levels of ST3GAL1,ST3GAL6,ST6GALNAC2,ST8SIA3,and ST8SIA5 in colon cancer cells.Since the GAGs,MUC1 and STs can affect the proliferation,invasion and metastasis of tumor cells.PD is an effective component of the Chinese medicinal platycodon grandiflorum,it exerts anti-colon cancer effects by acting on carbohydrate macromolecules such as GAGs,MUC1 and sialylation.It is indicated that there may be a certain correlation between the TCM 'phlegm ' theory and the carbohydrate macromolecules in colon cancer cells.The treatment of reducing phlegm and resolving masses may affect the expression of carbohydrate macromolecules in tumor cells.