FoxM1 Inhibitor Sensitizes Resistant Osteosarcoma Cells To Cisplatin By Down-regulation Of Rad51

Objective: Osteosarcoma is the most common primary malignant bone tumor,mainlyoccurring in adolescents.The survival rate of osteosarcoma was significantly improved after neoadjuvant chemotherapy.However,the treatment of osteosarcoma has not been significantly improved since the 80 s of last century,whichmay be associated with drug resistance in osteosarcoma.There has been found a variety of mechanisms of chemotherapy resistance.In recent years,it has been found that DNA damage repair is one of the factors that lead to drug resistance.DNA repair gene Rad51 is the key protein of homologous recombination repair,which is an important repair form of DNA damage repair.Several studies have shown that Rad51 is highly expressed in tumor,and is closely related to drug resistance.Fox M1 is a transcription factor that intimately correlates with proliferation and plays an important role in the regulation of cell cycle.In recent years,it has been found that Fox M1 is closely related to the occurrence and development of tumors,andparticipate in drug resistance through regulating DNA damage repair genes.This experiment aimsto investigate whether Fox M1 participates in the inhibitory effect of cisplatin(CDP)in resistant osteosarcoma cell lines by down-regulation of Rad51.Methods: With myositis ossificans tissues used as the control group and osteosarcoma tissues as the experimental group,the Fox M1 expression level was detected by immunohistochemical method.Osteosarcoma cell lines MG-63 and HOS-MNNG were used as parental control,and the resistant cell lines were established by a combinatory approach of high dose shock and gradually increasing dose.Cells were cultured in 0.1μg/m L CDP in vitro for 24 h,and then culture cells in culture medium without CDP until cell generation remains stable.Repeat the above steps 3 times,increase the drug dose to 0.2μg/m L CDP,and then substitute medium for CDP-free medium until cell generation remains stable.According to the above methods,increase the dose of CDP gradually and the resistant cells were established after nearly 4 months.MTT method was used to detect the drug resistance index.The m RNA expression of Fox M1 and Rad51 was detected by q RT-PCR method,and the protein expression of Fox M1 and Rad51 was detected by Western blot in resistant cells and parental cells.The m RNA expression andthe protein expression of Fox M1 and Rad51 were detected in cells treated with Fox M1-sh RNAs or NTC.The m RNA expression andthe protein expression of Fox M1 and Rad51 were detected in cells treated with 4μmol/L Thiostrepton or not.Cell counting method was used to determine the proliferation rate of CDP-resistant osteosarcoma cell lines treated with 4μmol/L Thiostrepton and 2μg/m L CDP only or both.Results:Immunohistochemistry showed that the positive rate of Fox M1 in osteosarcoma was significantly higher than that inossificans.The resistant cell lines that grow stable in culture medium with 2μg/m L CDP were established by a combinatory approach of high dose shock and gradually increasing the drug dose,named MG-63/R and HOS-MNNG/R respectively.The resistance index of MG-63/R and HOS-MNNG/Rwere detected by MTT assay,30.5 and 37.87,respectively.Two resistant cell lines showed significant resistance to CDP.The m RNA level of Fox M1 and Rad51 was significantly higher in resistant cells than in parental cells by q RT-PCR.The protein level of Fox M1 and Rad51 was significantly higher in resistant cells than in parental cells by Western blot.The m RNA and protein level of Fox M1 and Rad51 were significantly decreased after Fox M1-sh RNAsor 4μmol/L Thiostrepton treatment in CDP-resistant cells.The proliferation rate of resistant cells treated with 4μmol/L Thiostrepton and 2μg/m L CDP was significantly lower than the two drugs single-treated group.Conclusion:The study found that Fox M1 and Rad51 washighexpressedin CDP-resistant cells.Rad51 expression was down-regulated by Fox M1-sh RNAsand Fox M1 inhibitor Thiostrepton in the resistant cells.The proliferation rate of resistant cells treated with Thiostrepton and CDP was significantly lower than the two drugs single-treated group.It suggests that inhibition of Fox M1 may increase the sensitivity of resistant cells to CDP chemotherapy.In short,the curative effect of the combination of Fox M1 inhibitors and chemotherapy drugs can improve the treatment of drug-resistant osteosarcoma,providing a new insights for the mechanism and treatment of osteosarcoma.