The Molecular Mechanism Of Polyphenol From Foxtail Millet Bran Reversing Drug Resistance In Human Colorectal Cancer By Regulating Glycosphingolipid Metabolism Through C-Myc/miR-149/AKT

Colorectal cancer(CRC)is one of the most common malignant tumors,with the third highest incidence and second mortality.With the rapid economic development in China,people’s living standard and the diet structure have been greatly improved,which results in significant increases in the number of CRC patients in the past 20 years.Although substantial progress has been made in surgical techniques and postoperative chemotherapy in recent years,the prognosis for CRC is still not optimistic due to the disadvantage of the first-line chemotherapy drugs.For example,molecular mechanisms of these drugs are limited,and long-term use not only leads to drug resistance of CRC,but also causes tremendous side effects to patients.According to statistics analysis,the development of drug resistance causes chemotherapy failure of over 90 % of patients with metastatic cancer.The high expression of some miRNAs boosts chemotherapeutic drugs efflux out of cancer cells,which brings about the decrease of intracellular accumulation of drugs,and the proliferation of tumor cells could not be inhibited,but the other miRNAs possess the completely opposite biological characteristics.P-glycoprotein(P-gp),a member of the ABC transporter family of proteins,is an ATP-dependent drug efflux pump that confers multidrug resistance in neoplastic cells by catalyzing chemotherapeutic agent efflux.Furthermore,the concentrations of chemotherapeutic agents intracellular were affected by the expression of miRNAs,which are relative with the abnormal expression of P-gp.Glycolipids are glycocon-jugates of lipids that generally exist in the extracellular surface of eukaryotic cellular membranes and take part in regulating considerable number of biological processes and events,including morphology of cell,proliferation and differentiation of cell,etc.The expression level of P-gp in the neoplastic cells is correlated with the glycosphingolipid metabolism.That is to say,the key enzymes and metabolites involved in the metabolism of glycosylsphingolipid are correlated with the over-expression of P-gp.These discoveries suggest that it is a new scheme to reverse drug resistance by intervening glycosylsphingolipid metabolism in cancer cells.Our previous evidences suggested that bound polyphenol from millet bran(BPIS)possesses the potential of inhibiting cancer cells proliferation.Interestingly,there is no side effect on the growth of normal cells and mice.Recently,traditional chemotherapy drugs combined with novel chemosensitizers has become a prevalent trend to overcome tumor drug-resistance.Based on the preceding studies,we further investigate how BPIS treatment reverses drug resistance of cancer and the possible mechanism.The main results are as follows.(1)The drug resistance reverses effect of BPIS on drug-resistance colon cancer to oxaliplatin.Data showed that BPIS possessed synergistic effects with OXA on the proliferation of CRC cells(CI<1).Further study found that,the proliferation was inhibited and the apoptosis was induced in drug-resistance HCT-116/L cells with BPIS treatment.Simultaneously,the fluorescence intensity of Rhodamine 123 had a significant elevation with BPIS incubation,which implied that BPIS promoted the intracellular accumulation of chemotherapeutic drugs.These phenomena were primarily attribute to the destruction of transport function of P-gp and reduction level of this protein in drug-resistance HCT-116/L cells by BPIS treatment.(2)BPIS increases the sensitivity of drug-resistant in colon cancer cells to oxaliplatin by regulating the expression of miR-149/AKT axis.Over-expressed miR-149 in drug-resistant cells using miR-149 mimics,and then we observed that the proliferation ability in drug-resistant cells was significantly inhibited,apoptosis phenomenon occurred,and the Rhodamine123 intracellular accumulation showed an obvious increased.However,the proliferation ability was enhanced in drug-resistant cells transfection with inhibitor of miR-149.Data showed that BPIS remarkably increases the expression level of miR-149 in drug-resistant cells.Subsequently,we found the expression levels of AKT and FOXM1,the target genes of miR-149,were reduced by BPIS treatment.After treating the drug-resistant cells with specific inhibitors of AKT(LY294002)and FOXM1(FDI-6),the expression levels of P-gp and BCRP showed a significantly down-regulated.The above results suggest that BPIS up-regulates the expression of miR-149 in drug-resistant cells and results in down-regulating the expression of AKT and FOXM1,thus suppresses expression levels of drug resistant-relative proteins and reverses the chemo-resistance of colon cancer.(3)BPIS reverses chemo-resistance in drug-resistance cells to oxaliplatin through remodeling acidic glycosphingolipid(ganglioside)GM3 metabolism.Data showed that the expression level of neuraminidase 3(NEU3)was abnormally increased,which led to the disorder of GM3 metabolic in drug-resistance HCT-116/L cells.Meanwhile,the levels of Cer and GM3 in drug-resistance HCT-116/L cells were reduced,and the level of Gb3 was increased compared to HCT-116 cells.Further studies showed that the expression level of NEU3,a key enzyme of GM3 catabolism,had a significantly rise in the intestinal tissues of AOM/DSS-induced colitis associated colon cancer mice and CRC patients.These results suggest that disorder of GM3 metabolism is associated with the deterioration of CRC and its chemotherapy resistance.Further,we observed that the levels of NEU3 and the ganglioside GM3 related metabolites(Cer,GM3 and Gb3)were restored in drug-resistance HCT-116/L cells with different concentration of BPIS treatment.Meantime,knocking-down of NEU3 using si RNA,the reversal effect of BPIS on chemo-resistance was intensified in HCT-116/L cells.These results indicate that NEU3-mediated GM3 catabolism make a valuable contribution in the process of BPIS enhancing the chemo-sensitivity of CRC.(4)AKT regulates the metabolism of acidic glycosphingolipid(ganglioside)GM3.The expression level of NEU3 was markedly suppressed in drug-resistance HCT-116/L cells treatment with specific inhibitor of AKT,and followed by the accumulation of GM3 was augmented and the level of Gb3 was decreased.At the same time,a consistent phenomenon was observed after knocking-down the expression of AKT using si RNA in drug-resistant cells.In contrast,the levels of NEU3 and Gb3 were up-regulated and the accumulation of GM3 was down-regulated in the cells incubation with activator(SC79)of AKT,which led to the effect was abolished of BPIS on remodeling the ganglioside GM3 metabolism.These results suggest that AKT,the target gene of miR-149,takes part in regulating the downstream metabolism of ganglioside GM3.(5)c-Myc is a transcription factor of miR-149.Database analysis predicted that miR-149 promoter region contains a putative c-Myc-binding site.Data showed that the suppression of c-Myc using si RNA greatly increased the expression of miR-149.Subsequently,a luciferase reporter assay further confirmed that c-Myc was a direct transcription factor of miR-149.Knocking-down the expression of c-Myc significantly cut down the expression levels of P-gp,AKT and FOXM1.The results indicated that the expression level of c-Myc was dramatically suppressed in cells with BPIS treatment.These data indicate that BPIS inhibits the expression of c-Myc,miR-149 and its target genes,then down-regulates the expression levels of drug resistance proteins.(6)BPIS reverses drug resistance to oxaliplatin in nude mice.Drugresistance HCT-116/L cells were injected into the subcutaneous to establish drug-resistant nude mice model.We observed that there was no significant change in the volume and weight of nude mice in oxaliplatin alone group compared to the control group,but the growth of tumor was remarkably inhibited with co-treatment of BPIS combination oxaliplatin.Furthermore,results from immunohistochemistry and western blot displayed that the expression levels of c-Myc,miR-149,AKT,P-gp and NEU3 were changed,and it is consistent with the data from cell levels.In conclusion,BPIS inhibits the expression of c-Myc,which cripples the ability of c-Myc binding to the promoter region of miR-149 and then promotes the expression of miR-149.The target genes of miR-149 are inhibited,which results in the suppression of the downstream metabolism of acidic glycosphingolipid(ganglioside)GM3.Subsequently,the expression level of P-gp is repressed,and the chemotherapy resistance to oxaliplatin is reversed in colon cancer cells.