| Objective:Chemotherapy is one of the main treatment methods for non-small cell lung cancer.However,the emergence of drug resistance has become the bottleneck of clinically effective chemotherapy.Cancer stem cells are considered to be one of the most important mechanisms of chemotherapeutic resistance and a key factor in promoting tumor development and recurrence.Metformin,as an anticancer adjuvant,has been proved to have synergistic anti-tumor effects with a variety of chemotherapy drugs,but the molecular basis of its regulation mechanism on paclitaxel sensitization is still unclear.This study aimed to explore the mechanism of metformin reversing the stemness to inhibit the development of drug resistance by constructing a paclitaxel-resistant non-small cell lung cancer A549 cell line.Methods:1.A549 paclitaxel-resistant strains were constructed by intermittent induction with high concentrations of paclitaxel.Once the resistant cells were established,they were maintained at a low concentration of paclitaxel;2.Cell Counting Kit-8(CCK-8)and plate cloning were used to observe the effects of metformin and paclitaxel on the cell viability and colony formation of A549 cells and A549/PTX drug-resistant cells;3.The stemness of A549/PTX drug-resistant cells was verified by sphere-formation assay and Western Blot;4.Flow cytometry,qPCR and Western Blot were used to detect the effects of metformin on the stemness of A549/PTX drug-resistant cells;5.Western Blot was used to detect changes in markers of cancer stem cells.Results:1.The cell inhibition rates of A549 cells and A549/PTX drug-resistant cells were measured by CCK-8 method;and the IC50 values of A549 cells and A549/PTX drug-resistant cells were calculated as 580 n M and 9317 n M,respectively,and the resistance index RI=16,indicating the successful construction of drug resistant strains.The morphology of A549/PTX drug-resistant cells changed from epithelial cells to conglobate cells.2.A549 cells and A549/PTX drug-resistant cells were treated with the single-agent,paclitaxel or metformin,and the combination of the two.The results with CCK-8 and plate cloning experiments showed that the combined group exhibited significantly lower cell viability and colony-forming ability than the monotherapy group(***p<0.001),suggesting that metformin could enhance the sensitivity to paclitaxel;3.Sphere-forming experiment showed more sphere-forming quantity and larger sphere-forming diameter with that A549/PTX drug-resistant cells than those with A549 parent cells(***p<0.001,*p<0.05),indicating that drug-resistant cells had significant self-renewal ability.In conjunction,Western blot revealed that the expression of cancer stem cell marker genes was significantly increased in A549/PTX drug-resistant cells(***p<0.001),further confirming that A549/PTX drug-resistant cells are enriched with stemness of cancer stem cells;4.Data from both qPCR and flow cytometry showed that upon treatment with metformin the expression of cancer stem cell marker genes was significantly decreased in the drug-resistant cells compared with the control group(**p<0.01);the Western Blot results revealed that the expression of cancer stem cell markers was reduced by metformin in a dose-dependent fashion(1,5,10 m M).5.Western blot results showed that metformin reduced the expression level of p-FOXO1/3a in A549/PTX drug-resistant cells,suggesting that metformin might reverse the stemness by regulating FOXO1/3a activity.The Akt inhibitor MK2206 was used to further verify that the inhibition of metformin on the expression of cancer stem cell marker genes in drug-resistant cells may be regulated by the PI3K/Akt/FOXO signaling pathway.Conclusion:Metformin enhances sensitivity to paclitaxel in A549 cells and A549/PTX drug-resistant cells.A549/PTX drug-resistant cells are enriched with stemness of cancer stem cells,and metformin selectively targets cancer stem cells,possibly by regulating FOXO1/3a activity to reduce the expression of cancer stem cell marker genes,thereby reversing the development of drug resistance. |
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