NaB Induces Autophagy In Colorectal Cancer Cells Via The CaMKK?-AMPK Signaling Pathway

BackgroundColorectal cancer is one of the most common tumors which is characterized by a high incidence,low cure rate,high recurrence rate and poor prognosis.It has become a burden and hindering economic development.In recent years,with the combined application of surgery,radiotherapy and chemotherapy,the five-year survival rate of patients with colorectal cancer has increased,but the serious side effects caused by radiotherapy and chemotherapy are unavoidable.Therefore,we urgently need to find a relatively safe and effective drug to prevent and treat colorectal cancer.As a product of soluble dietary fiber fermentation in the intestine,sodium butyrate(NaB)acts primarily as a histone deacetylase inhibitor(HDACi)and inhibits histone deacetylase activity leading to histone which is highly acetylated,thus playing an preventing and treating role in colorectal cancer.It has been found that NaB induced autophagy in colorectal cancer cells at a lower concentration(2 mmol/L),while inhibition of NaB-induced autophagy increased colorectal cancer cell apoptosis.Higher concentrations(10 mmol/L)of NaB promoted calcium imbalance in the endoplasmic reticulum and cytoplasm through the store-operated calcium entry(SOCE)pathway,by down-regulating the endoplasmic reticulum,increasing the concentration of calcium in the cytoplasm,and eventually inducing colorectal cancer cell apoptosis.So,is there a correlation between NaB-induced calcium homeostasis and cell autophagy?The CaMKK?-AMPK pathway is the classical signaling pathway of autophagy,and the activity of CaMKK? is closely related to the intracellular Ca2+concentration and is also the upstream signal molecule of AMPK.So,can NaB activate CaMKK?-AMPK signaling pathway by triggering Ca2+ signal and exert its anti-colorectal cancer effect?PurposeTo investigate the possible molecular mechanism of NaB-induced autophagy in colorectal cancer cells and to provide a theoretical basis for the prevention and treatment of colorectal cancer.MethodHCT-116 and HT-29 cells were treated with different concentrations of NaB and the expressions of LC3,CaMKK?,AMPK? and ACC were detected by Western Blot and real-time fluorescence quantitative PCR.The formation of autophagosomes was observed by transmission electron microscope(TEM).The effects of BAPTA-AM(intracellular Ca2+ chelator),STO-609(CaMKK? inhibitor),Compound C(AMPK inhibitor,abbreviated as Com.C)and RNA interference with CaMKK? and AMPK?on NaB-induced autophagy in colorectal cancer were observed.The expression of LC3,CaMKK?,AMPK? and ACC were detected by Western Blot and RT-PCR.Results1 NaB induced autophagy in colorectal cancer cells1 mmol/L NaB could promote LC3 ? protein and LC3 mRNA expression in HCT-116 cells(P<0.05;P<0.01);2 mmol/L NaB could promote LC3? protein and LC3 mRNA expression in HT-29 cells(P<0.05;P<0.05).In addition,colorectal cancer cells were treated with 2 mmol/L NaB for 24 hours,and obvious formation of autophagosomes was observed.2 AMPKa signal was involved in NaB-induced colorectal cancer cell autophagy2.1 NaB increased phosphorylation of AMPKa and ACC proteins1 mmol/L NaB could significantly promote the expression of p-AMPK? protein in HCT-116 cells(P<0.01);0.5 mmol/L NaB could significantly increase the expression of p-AMPKa protein in HT-29 cells(P<0.05).5 mmol/L NaB promoted the expression of p-ACC protein in HCT-116 cells(P<0.05).Both 0.5 mmol/L,1 mmol/L and 2 mmol/L NaB could increase the expression of p-ACC protein in HT-29 cells for 2h(P<0.01;P<0.01;P<0.05).2.2 Com.C pretreatment could inhibit NaB-induced colorectal cancer cell autophagy and related signal moleculesThe LC3? protein and LC3 mRNA levels of the Com.C-NaB combined group were significantly lower than that of NaB group(P<0.05;P<0.01);the expression levels of p-AMPKa and p-ACC protein of the Com.C-NaB combined group were lower than that of NaB group(P<0.01;P<0.01).2.3 RNA interference targeting AMPKa could inhibit NaB-induced autophagy in colorectal cancer cells and their related signal moleculesThe levels of LC3? protein and LC3 mRNA in the AMPKa interference-NaB combined group were significantly lower than that in NaB group(P<0.01;P<0.05).The expression of p-AMPKa and p-ACC protein in the AMPKa interference-NaB combined group was lower than that in NaB group.Group(P<0.01;P<0.01).3 CaMKK?-AMPK signaling pathway was involved in NaB-induced autophagy in colorectal cancer cell3.1 NaB could increase the phosphorylation of CaMKK? protein0.5 mmol/L NaB treatment up-regulated the expression of p-CaMKK? protein in HCT-116 cells(P<0.05);2 mmol/L NaB stimulated the expression of p-CaMKK?protein in HT-29 cells(P<0.05).3.2 STO-609 pretreatment could inhibit NaB-induced autophagy in colorectal cancer cells and its related signal moleculesThe expression of LC3? protein and LC3 mRNA in STO-609-NaB combined group was significantly lower than that in the NaB group(P<0.05;P<0.05);STO-609 pretreatment reduced the expression of p-CaMKK?,p-AMPK? and p-ACC protein(P<0.01;P<0.001;P<0.05).3.3 RNA interference targeting CaMKK? could inhibit NaB-induced autophagy in colorectal cancer cells and its related signal moleculesThe levels of LC3? protein and LC3 mRNA in the CaMKK? interference-NaB combined treatment group were significantly lower than NaB treatment group(P<0.05;P<0.05).Interfering with CaMKK? could significantly down-regulate protein expression of NaB-induced p-CaMKK?,p-AMPK? and p-ACC.(P<0.01;P<0.05;P<0.01).3.4 Chelated intracellular Ca2+ could inhibit NaB-induced colorectal cancer cell autophagy and its related signal moleculesPretreatment with BAPTA-AM significantly reduced the LC3? protein and LC3 mRNA levels in NaB-induced colorectal cancer cells(P<0.05;P<0.01);Proteins expression of p-CaMKK?,p-AMPK? and p-ACC in the BAPTA-AM+NaB group was lower than in the NaB group(P<0.05;P<0.01;P<0.05).conclusion1.NaB can induce autophagy in colorectal cancer cells.2.CaMKK?-AMPK signaling pathway was involved in NaB induced-autophagy in colorectal cancer cells.