Mechanism Of HIF-1α Mediated Lactate Utilization In Potassium Dichromate Induced Proliferation Of A549 Cells

Objective:Chromium metal（Cr）is hard and brittle,corrosion resistance and other excellent properties,so it is widely used in metal plating,wood products anticorrosion,leather tanning,paint,plastic processing and other industrial fields^[1].In 1990,the International Agency for Research on Cancer（IARC）identified Cr（VI）compounds as Class I carcinogens^[2],mainly causing lung cancer.Malignant tumors have become one of the most serious public health problems worldwide,with lung cancer affecting more than 2.2 million people and being the second largest tumor worldwide.Although significant progress has been made in the diagnosis and treatment of lung cancer in recent years,the prognostic effect is still not ideal,so the prevention and treatment of lung cancer has become the focus of reducing the incidence of lung cancer and improving the survival level of patients.In our previous study,we found that chromium induced lactic acid utilization in human lung adenocarcinoma cells（A549 cells）played an important role in promoting glycolysis and cell proliferation.Meanwhile,chromium treatment of A549 cells resulted in an increase in hypoxic-inducer factor-1α（HIF-1α）.It has been reported that HIF-1αcan regulate lactic acid transporter（MCT1）.Therefore,in this study,the mechanism of HIF-1αregulation of MCT1 in chrome-induced lactic acid reuse of A549 cells was investigated.Methods:In this study,male BALB/c mice were used as the in vivo experimental model,human embryonic lung fibroblasts（HELF cells）and human lung adenocarcinoma cells（A549 cells）were used as the in vitro experimental model.Male BALB/c mice were given different concentrations of potassium dichromate by intranasal instillation.Western Blot was used to analyze the protein expression of MCT1and MCT4 in the lung tissue of mice.MTT assay was used to determine the difference in cell growth between A549 and HELF cells treated with potassium dichromate（Cr（VI））at different concentrations of lactic acid.Western Blot was used to analyze the protein expression of MCT1 and MCT4 in A549 and HELF cells exposed to potassium dichromatic acid for 0,6,12 and 24 hours.Western Blot was used to analyze the protein expression of HIF-1αin A549 and HELF cells exposed to potassium dichromate.Western Blot analysis and MTT assay were used to determine the protein expression of MCT1 and cell survival rate in A549 cells treated with HIF-1αinhibitor YC-1 and potassium dichromate,and in HELF cells treated with HIF-1αactivator Co Cl₂and potassium dichromate.Western Blot was used to analyze the changes of Cyclin E1protein induced by potassium dichromate in A549 cells treated with YC-1.Cell scratch assay was used to analyze the effect of MCT1 inhibitor AZD3965 on the migration ability of A549 cells co-treated with YC-1 and HELF cells co-treated with AZD3965and Co Cl₂.The relationship between HIF-1αand MCT1 regulation was analyzed by Chromatin immunoprecipitation（Ch IP）assay and real-time fluorescence quantitative PCR.The protein expression levels of p53 in A549 and HELF cells were analyzed by Western Blot assay.Western Blot assay was used to analyze the effect of p53 on HIF-1αinduced by potassium dichromate in A549 cells treated with RITA,an activator of p53.Results:In vivo experiments showed that the protein levels of MCT1 and MCT4in lung tissue of BALB/c mice were significantly increased.In vitro experiments showed that high concentration of lactic acid promoted the growth of A549 cells,and the promotion effect was more obvious after potassium dichromate exposure.In HELF cells,lactic acid did not promote the growth of HELF cells,and there was no significant difference in the growth of HELF cells under different lactic acid concentrations after potassium dichromate exposure.The results of in vitro and in vivo experiments showed that the protein levels of MCT1 and MCT4 in lung tissue and A549 cells of BALB/c mice were significantly increased with time after potassium dichromate exposure,while the protein levels of MCT1 were decreased and the protein levels of MCT4 were increased in HELF cells.Western Blot analysis showed that the HIF-1αprotein level of A549 cells infected with potassium dichromate increased significantly with the increase of time,while the HIF-1αprotein level of HELF cells did not increase significantly at 6and 12 h,but decreased significantly at 24 h.When HIF-1αinhibitor YC-1 was treated with potassium dichromate in A549 cells,MCT1 levels were significantly reduced.HELF cells treated with potassium dichromate and HIF-1αaccelerator Co Cl₂increased MCT1 protein level.MTT assay also showed that the survival rate of A549cells was lower in the group treated with potassium dichromate and YC-1.In HELF cells,cell growth was not evident in the potassium dichromate and Co Cl₂treated group.After A549 cells were treated with potassium dichromate and YC-1 in glucose-free medium containing sodium lactate for 24h,the protein level of cyclin E was significantly reduced.Cell scratch assay results showed that potassium dichromate induced A549 cell migration,YC-1 and MCT1 inhibitor AZD3965 both weakened potassium dichromate induced cell migration.Only AZD3965 treatment had a limited effect on HELF cell migration.Co Cl₂and AZD3965 treated HELF cells together significantly reduced the scratch healing rate.Ch IP assay showed that HIF-1αprotein was bound to MCT1 gene promoter sequence.The expression of p53 in A549 cells was significantly lower than that in HELF cells.Western Blot test analysis of p53 activation agent RITA processing A549 cell,HIF-1 alpha protein expression levels.Conclusion:Potassium dichromate treatment induced the utilization of lactic acid by A549 cells.The mechanism was that the deletion of p53 in A549 cells promoted the elevation of HIF-1αinduced by potassium dichromate,and HIF-1αincreased the utilization of lactic acid by binding to MCT1 promoter region to promote the transcription of MCT1,and finally promoted the growth of A549 cells.