| Background and aim:Primary liver cancer causes more than 8 million deaths worldwide each year,and has become the sixth most common cancer and the third leading cause of cancer-related deaths,becoming a serious global health burden.Hepatocellular carcinoma(HCC)accounts for 75%-85% of cases of primary liver cancer.Because HCC has no early typical symptoms,most HCC patients are diagnosed in the middle and late stages,losing the opportunity for radical surgery.Therefore,there is an urgent need to find a safe and effective treatment strategy for HCC patients.Aerobic glycolysis also known as the "Warburg effect",has been identified as one of the metabolic characteristics of HCC,which means in the presence of sufficient oxygen,cancer cells tend to convert glucose to lactate and produce ATP by glycolysis.In unit time,aerobic glycolysis produces energy faster than mitochondrial oxidative phosphorylation(OXPHOS).Moreover,the metabolic substrate and lactic acid produced by the glycolytic pathway can promote the progress of HCC by regulating the proliferation,growth,immune escape,invasion,metastasis,angiogenesis,and sorafenib resistance of HCC.Targeting the key enzymes in glycolytic pathway can exert antiHCC effects.Hexokinase 2(HK2)is the first rate-limiting enzyme in the glycolytic pathway,which converts glucose into glucose-6-phosphate(G-6-P).HK2 promotes the occurrence and progression of HCC by regulating glycolysis and mitochondriaassociated apoptosis.Inhibiting the expression of HK2 can interfere with the energy metabolism of HCC cells,inhibit their proliferation,induce their apoptosis,and increase the sensitivity of HCC cells to the first-line HCC targeted therapy drug sorafenib.More importantly,no adverse physiological consequences were found in mice with systematic HK2 deletion.Therefore,based on the metabolic characteristics of HCC cells and the important role of HK2,screening drugs that target glycolysis,especially HK2,may provide a safe and effective strategy for the treatment of HCC.Sodium butyrate(NaBu)is the sodium salt of short-chain fatty acid butyrate,which is mainly produced by Firmicutes of gut microbiota.It has anti-oxidation,anticancer,and immune-regulatory properties.NaBu exerts anti-HCC effects by targeting multiple phenotypes of HCC.In addition,NaBu regulates the glycolysis of breast cancer cells,dendritic epidermal T cells,and lung cancer cells.However,the effect of NaBu on glycolysis of HCC cells and whether the anti-HCC effect of NaBu is related to its regulation of key enzymes in glycolytic pathway remains unknown.In the present study,HCC cells and xenograft mouse model were used to investigate the modulatory effects of NaBu on glycolysis of HCC cells and its underlying mechanism.The results may help us better understand the role that gut microbiota and its metabolite play in the development and treatment of HCC,and may provide a theoretical basis for the clinical application of NaBu.Methods:1.Investigate the modulatory effect of NaBu on proliferation,apoptosis,and glycolysis of HCC cells both in vivo and in vitro:(1)in vitro: three kinds of HCC cells(LM3,SMMC-7721,Bel-7402),hepatoblastoma cell line Hep G2,and the normal liver cell line LO2 were cultured in oxygen conditions.The effect of NaBu(0-5 m M)on cell proliferation was detected by CCK-8 assay at 24 h,48 h,and 72 h.The IC50 on the above cells at 48 h was calculated.NaBu-sensitive HCC cell lines LM3 and Bel-7402 were selected for the subsequent experiments.The dose of NaBu for subsequent in vitro experiments was selected.Colony formation assay was employed to determine the effect of NaBu on the proliferation of LM3 and Bel-7402 cells.Annexin V-FITC/PI staining was used to examine the effect of NaBu on the apoptotic rate of LM3 and Bel-7402 cells.The effect of NaBu on the expression of proliferation-related and apoptosis-related proteins was detected by western blot.Glucose enzyme oxidation method and L-lactate colorimetric method were employed to determine the effect of NaBu on the glucose uptake and lactate production level of LM3 and Bel-7402 cells.PCR was used to detect the effect of NaBu on the m RNA expression of glycolytic enzymes in LM3 and Bel-7402 cells.The protein expression of OXPHOS,HK2,PFK1,and LDH-A in LM3 and Bel-7402 cells was detected by western blot.(2)in vivo: the xenograft nude mouse model was constructed by subcutaneous injection of LM3 cells.The mice received 200 mg/kg NaBu treatment by gavage for 16 days.H&E,Ki67,and TUNEL staining were used to determine the effect of NaBu on the necrosis,proliferation,and apoptosis of HCC cells in vivo,respectively.To evaluate the safety of NaBu in vivo,the levels of mice serum ALT,AST,and creatinine were determined.H&E staining of liver,heart,kidney,and lung sections of mice was observed.The expression of proliferation-related and apoptosis-related proteins was detected.The lactate level in the tumor tissues was determined.The effect of NaBu(200 mg/kg)on the expression of HK2,PFK1,and LDH-A was detected by western blot and immunohistochemistry.2.Verify whether the effect of NaBu on the proliferation,apoptosis,and glycolysis of HCC cells is related to its modulation of HK2: lentivirus overexpressing HK2 was used to transfect LM3 and Bel-7402 cells.The effect of NaBu on the glycolysis level in HK2-overexpressing HCC cells was detected.CCK-8 assay,western blot,and Annexin V-PE staining were used to detect the effect of NaBu on the proliferation and apoptosis of HK2-overexpressing HCC cells.3.Determine the upstream pathway involving in the regulation of HK2 by NaBu:PCR was used to screen the upstream pathway of HK2 after NaBu treatment for 48 h.The protein expression of c-myc in LM3 and Bel-7402 cells,and in tumor tissues after NaBu treatment was detected.Lentivirus overexpressing c-myc was used to transfect LM3 and Bel-7402 cells.The effect of NaBu on the expression of HK2 and glycolysis level in c-myc-overexpressing HCC cells was detected.c-myc overexpressing HCC cells were transfected with sh HK2 lentivirus,the effects of NaBu on the aerobic glycolysis of these HCC cells were detected.HCC cells were transfected with c-myc knockdown si RNAs,the effect of NaBu on the HK2 expression in c-myc knockdown HCC cells was detected.4.Investigate the effect of combined treatment of NaBu and sorafenib on HCC in vitro and in vivo:(1)in vitro: CCK-8 assay was used to detect the inhibitory effect of NaBu or/and sorafenib on the proliferation of LM3 cells.Sorafenib and NaBu were combined at a ratio of 1:500,the combination index(CI)and dose reduction index(DRI)were calculated to evaluate the combination effect.Colony formation assay,western blot,and Annexin V-FITC/PI staining were used to detect the effect of sorafenib or/and NaBu on the proliferation and apoptosis of LM3 cells.Glucose enzyme oxidation method,Llactate colorimetric method,and western blot were employed to determine the effect of sorafenib or/and NaBu on the glycolysis level and protein expression of rate-limiting enzymes in glycolytic pathway.(2)in vivo: the xenograft nude mouse model was constructed by subcutaneous injection of LM3 cells.The mice received 200 mg/kg NaBu or/and 10 mg/kg sorafenib treatment by gavage for 16 days.The effect of NaBu and sorafenib cotreatment on the necrosis,proliferation,and apoptosis of HCC cells in vivo was detected by H&E,Ki67,and TUNEL staining,respectively.The expression of apoptosis-related and proliferation-related proteins in the tumor tissues was detected by western blot.Results:1.in vitro study: CCK8 assay results showed that NaBu inhibited the proliferation of three HCC cell lines and Hep G2 cells in a time-dependent and dose-dependent manner,but had minor effect on normal liver cell LO2.The IC50 of them at 48 h were3.91 m M(LM3),2.61 m M(Bel-7402),4.18 m M(SMMC-7721),5.25 m M(Hep G2),19.68 m M(LO2),respectively.LM3 and Bel-7402 cells,which were the most sensitive to NaBu,were chosen for the subsequent experiments.2,3 and 4 m M of NaBu were chosen for the subsequent experiments.The results of colony formation assay showed that NaBu suppressed the proliferation of HCC cells.The results of flow cytometry showed that NaBu induced the apoptosis of HCC cells.NaBu inhibited the expression of PCNA and Bcl-2 while enhanced the expression of cleaved caspase 3/9.NaBu had no effects on the apoptosis-related proteins in LO2 cells.NaBu suppressed the glucose uptake and lactate production,while enhanced the protein expression of OXPHOS in HCC cells.m RNA expressions of HK2,phosphofructokinase-1(PFK1),and lactate dehydrogenase-A(LDH-A)were all significantly suppressed in both HCC-LM3 and Bel-7402 cells by treatment with NaBu for 48 h.Among these three genes,the m RNA level of HK2 decreased the most in both cell lines.NaBu suppressed the protein expression of HK2,PFK1 and LDH-A.2.in vivo study: NaBu(200 mg/kg)suppressed the growth of tumor,and induced necrosis and apoptosis,while inhibited proliferation of HCC cells in vivo.NaBu(200mg/kg)had no effect on mouse body weight,serum transaminase,and creatinine levels,and had no damage to heart,liver,lung,and kidney.NaBu(200 mg/kg)reduced the lactate level and the expression of HK2 in tumor tissues.3.HK2 overexpression promoted the proliferation and increased the glycolysis level of HCC cells,while inhibited their apoptosis.The modulatory effects of NaBu on the proliferation,apoptosis,and glycolysis in LM3 and Bel-7402 cells were reversed by HK2 overexpression,demonstrating that the anti-HCC effect of NaBu is related to its modulation of HK2,a key enzyme of glycolytic pathway.4.The results of PCR indicated that among the upstream pathway of HK2,the m RNA expression of c-myc was significantly inhibited in both HCC-LM3 and Bel-7402 cells by treatment with NaBu.NaBu suppressed the expression of total c-myc and nuclear c-myc in HCC cells.NaBu(200 mg/kg)suppressed the expression of c-myc in tumor tissues.C-myc overexpression dampened the inhibitory effect of NaBu on the HK2 expression,glucose uptake,and lactate production of HCC cells,which could be reversed by HK2 knockdown.HK2 protein expression couldn’t be affected by NaBu in c-myc si RNA knockdown HCC cells5.The results of CCK-8 assay indicated that the inhibitory effect of co-treatment on proliferation of LM3 cells was superior to that of NaBu or sorafenib alone,with CI< 1,and DRI > 1.Sorafenib inhibited the proliferation of LM3 cells and induced their apoptosis.The above effects could be further enhanced by NaBu.Sorafenib alone increased the glycolysis level in LM3 cells and enhanced the expression of key enzymes in glycolytic pathway,while this trend could be reversed by NaBu.Sorafenib(10 mg/kg)inhibited the growth of tumor in nude mice,promoted necrosis and apoptosis of HCC cells,and inhibited their proliferation in vivo.The anti-HCC effect of sorafenib in vivo was further enhanced by NaBu.Conclusion:1.NaBu inhibits proliferation and aerobic glycolysis of HCC cells,while induces their apoptosis both in vitro and in vivo.NaBu has no effects on normal liver cell LO2 and has no toxicity to normal organs of nude mice.2.The modulatory effects of NaBu on the proliferation,apoptosis,and glycolysis of HCC are related to its inhibitory effect of HK2 expression,3.By inhibiting c-myc/HK2 pathway,NaBu disturbs glycolysis of HCC cells.4.NaBu can reduce the enhanced glycolysis level in HCC cells induced by sorafenib in vitro.NaBu can enhance the anti-HCC effect of sorafenib both in vitro and in vivo. |
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