基于CDK16/MYC探讨葛根芩连汤含药血清抑制糖酵解促进结直肠癌细胞5-氟尿嘧啶敏感性的作用机制OA

Objective:To explore the molecular mechanisms by which serum containing Gegen Qinlian Tang(GQT)inhibits glycolysis and enhances chemotherapy sensitivity in 5-fluorouracil(5-FU)-resistant colorectal cancer(CRC)cells based on the cyclin-dependent kinase 16(CDK16)/MYC proto-oncogene(MYC)pathway.Methods:HCT-116/5-FU cells were treated with different concentrations(5％,10％,20％,30％)of GQT-containing serum.Cell viability and 5-FU sensitivity were assessed using the cell counting kit-8(CCK-8)assay,and the experimental concentrations of 5-FU and GQT for subsequent experiments were determined.Cell proliferation and apoptosis under individual 5-FU,GQT,and combined 5-FU+GQT treatments were evaluated using 5-ethynyl-2′-deoxyuridine(EDU)staining and annexin V-FITC/PI double staining,respectively.Glucose consumption,adenosine triphosphate(ATP)production,and lactate levels were measured by colorimetric assays.Expression levels of glycolysis-related proteins,CDK16,MYC,and phosphorylated MYC were detected by Western blot.Co-immunoprecipitation(CoIP)was used to examine the protein interaction between CDK16 and MYC,and cycloheximide(CHX)treatment was applied to assess the effect of CDK16 overexpression on MYC protein stability.Results:CCK-8 assays showed that 2.5 mg·L-1 5-FU significantly inhibited HCT-116 cell viability in a dose-dependent manner.In HCT-116/5-FU cells,significant inhibition was observed only at 5 mg·L-1 5-FU(P＜0.05),which was used for model establishment.Compared with 5-FU alone,addition of 5％GQT-containing serum significantly suppressed HCT-116/5-FU cell viability(P＜0.05),with stronger inhibition at higher serum concentrations.Thus,5％GQT-containing serum was used in subsequent experiments.Compared with the control group,5-FU,GQT,and 5-FU+GQT treatments all significantly reduced cell proliferation(P＜0.05)and increased apoptosis(P＜0.01).The 5-FU+GQT combination showed superior inhibition of proliferation compared with 5-FU or GQT alone(P＜0.01),accompanied by more pronounced reductions in glucose consumption,ATP production,and lactate generation(P＜0.01).Additionally,compared with control,5-FU,and GQT groups,the 5-FU+GQT group exhibited stronger suppression of MYC and its phosphorylated forms(P＜0.01)and greater inhibition of glycolytic enzymes,including hexokinase 2(HK2),3-phosphoinositide-dependent protein kinase 1(PDK1),lactate dehydrogenase A(LDHA),and pyruvate kinase M2(PKM2)(P＜0.01).CDK16,MYC,and MYC phosphorylation expression levels were significantly downregulated in the 5-FU+GQT group compared with the 5-FU group(all P＜0.01).MYC protein stability decreased in a time-dependent manner in the 5-FU+GQT group(P＜0.05),which was rescued by CDK16 overexpression(P＜0.05).Conclusion:GQT significantly enhances the sensitivity of HCT-116/5-FU cells to 5-FU,potentially by inhibiting CDK16 and thereby reducing MYC-mediated glycolysis.