Metformin Upregulates SLCO1B3 Expression And Sensitizes A549 Cells To Paclitaxel Treatment

Background and Objective: The incidence and mortality of lung cancer are the top of malignant tumors and seriously threaten human health in Chinese men.Paclitaxel is currently a common drug for the treatment of non-small cell lung cancer(NSCLC),but the long-term use of drug resistance severely limits its clinical use.SLCO1B3(Solute Carrier Organic Anion Transporter Family,Member 1B3)is an organic anion transport peptide protein that is located on the hepatocyte membrane and is encoded by the SLCO1B3 gene,a transport protein(Gene bank: NM-019844).SLCO1B3 is present in various cancer cells and is a negative ion chemical anticancer drug transporter such as paclitaxel.LKB1 is a common tumor suppressor gene and its substrate includes AMPK.Studies have confirmed that the mutation rate of LKB1 is as high as 15-35% in non-small cell lung cancer.Inactivation of LKB1 is inactivated,which results in a decrease in AMPK activity and an increase in m TOR activity,thereby promoting tumorigenesis and metastasis and accelerating tumor progression.Metformin is the first-line anti-diabetic drug in clinical practice.It has attracted great attention in the treatment of cancer in recent years.It is an AMPK activator,and under many conditions metformin is mediated through AMPK.In this study,the relationship between metformin and paclitaxel transporter SLCO1B3 was studied using A549 and A549-LKB1 cells as models.Methods: 1.A549 and A549-LKB1 cells were treated with different concentrations of paclitaxel,and cell survival was observed.A549 cells were treated with metformin and paclitaxel to calculate the number of viable cells to reflect the inhibition rate of the drug on cells,and to explore whether AMPK increased the anti-tumor effect of paclitaxel.2.q-PCR and Western blot were used to detect the effect of paclitaxel on the expression of SLCO1B3 from m RNA and protein levels.3.Metformin was used to treat A549 cells and A549-LKB1 cells and total RNA was extracted.The effect of metformin on m RNA level of SLCO1B3 was detected by q-PCR.4.A549 paclitaxel-resistance cell line was established to observe cell morphological changes;cell resistance was tested by concentration test and drug resistance index was calculated.5.Total protein and total RNA were extracted,and the expression of SLCO1B3 in A549 paclitaxel-resistance cell line was detected by Western blot and q-PCR.Metformin acts on A549 paclitaxel-resistance cell line and explores whether metformin reverses paclitaxel resistance by affecting SLCO1B3 expression.Results: 1.Cell viability experiments showed that A549 cells highly expressing LKB1 were more sensitive to paclitaxel than wild-type cells,and paclitaxel inhibited tumor cell growth more efficiently(P<0.05 vs control).Using metformin and paclitaxel in combination A549 cells,the cell growth inhibition rate was higher than that of paclitaxel alone group,further suggesting that metformin can increase the sensitivity of paclitaxel and enhance its anti-tumor effect.2.A549 cells were treated with different concentrations of paclitaxel(2.5,5,10,20,40 n M)for 48 hours.Western blot and q-PCR showed that with the increase of paclitaxel concentration,expression of SLCO1B3 protein and m RNA were inhibited,and phosphorylated The expression of ACC was also inhibited;A549 cells were treated with 10 n M paclitaxel at different times(0,8,12,24,48,72 h).It was observed that with the prolongation of paclitaxel exposure,SLCO1B3 protein and m RNA were expressed.All of them were inhibited(p<0.01 vs control group)and the expression of p-AMPK was decreased in a time-dependent manner.3.A549 and A549-LKB1 were treated with 10 m M metformin to extract total RNA.q-PCR showed that the expression of SLCO1B3 m RNA in the metformin-treated group increased(P<0.01 vs control).Under the effect of metformin,the m RNA expression of SLCO1B3 in A549-LKB1 cells was higher than that in A549(P<0.01 vs control),suggesting that this may be related to AMPK.4.The A549 paclitaxel-resistance cell line was successfully constructed.Photomicrographs were taken to observe the morphology of the cells.The epithelial cells were transformed into similar fibroblasts.The cell resistance index was 2.3.5.Western blot and q-PCR results showed that the SLCO1B3 protein expression and m RNA expression in A549 paclitaxel-resistance cell line decreased compared with wild-type.Metformin was used to treat A549 paclitaxel-resistance cell line.Western blot results showed that the expression of SLCO1B3 protein in metformin group increased.Conclusion: 1.Metformin/AMPK can increase the sensitivity of paclitaxel to A549 cells and enhance the drug sensitivity of paclitaxel.2.With the increase of the time and concentration of paclitaxel,the expression of SLCO1B3 protein and m RNA were inhibited,SLCO1B3 could mediate the occurrence of paclitaxel resistance;and paclitaxel could inhibit the activation of AMPK.3.Metformin/AMPK up-regulated the expression of SLCO1B3 and reversed the resistance of A549 cells to paclitaxel.This study will provide experimental and theoretical basis for the use of metformin,an AMPK drug activator,as an anti-tumor agent,or in combination with other chemotherapeutic agents for anti-tumor therapy.