Study On The Combination Of Anti Folic Acid Drugs And Specific Cell Cycle Inhibitors In The Treatment Of Bladder Cancer

PrefaceFolic acid is widely used in the treatment of malignant tumors,and the most commonly used anti-folic acid drugs are methotrexate(Methotraxate,MTX).It is mainly through the inhibition of dihydrofolate reductase(DHFR),thereby reducing the synthesis of tetrahydrofolate,and is important in vivo synthesis of purine nucleotides and pyrimidine deoxyribonucleotide.Thus,the transfer of one carbon group in the biosynthesis of purine nucleotides and pyrimidine nucleotides was blocked,resulting in the inhibition of DNA biosynthesis.Pralatraxate(Pralatraxate,PTX)is a new type of anti-folic acid treatment of cancer drugs,PTX is currently considered to be an upgrade of MTX.Is AllosTherapeutics developed methotrexate derivatives.As folic acid targeted agents,the drug can completely inhibit dihydrofolate reductase can competitively inhibit leaf acyl poly glutamine synthetase,blocking thymine and other biological molecules on single carbon transfer synthesis by interfering with DNA synthesis,promote tumor cell apoptosis.Palbociclib Isethionate(PAL)is a cell cycle inhibitor,specific inhibition of CDK4/6.In February 3,2015,Pfizer Inc R & D(PD-0332991,Ibrance)received FDA accelerated approval.Is also the first FDA approved CDKs(Cyclin-dependent kinases,CDKs)inhibitorBladder cancer is one of the common malignant tumors,of which 30% patients with a diagnosis of muscle invasive bladder cancer(Muscle-Invasive,Bladder,Cancer,MIBC)is a clinical stage T2 ~ T4 of bladder cancer.It has the characteristics of rapid progress,easy recurrence,distant metastasis,high malignant degree and high mortality.Bladder cancer is a malignant tumor,especially muscle invasive bladder cancer in a way can be regarded as a systemic disease,which means they have the ability of tumor metastasis,so the treatment of this cancer,in addition to the traditional radical cystectomy(Radical,Cystectomy,RC)at the same time for pelvic lymph node dissection(Pelvic Lymph Node Dissection,PLND),should also be systemic therapy,such as chemotherapy.At present,more and more attention has been paid to neo-adjuvant chemotherapy for bladder cancer.At the present stage,the first line of chemotherapy for bladder cancer is GC(gemcitabine gemcitabine and cisplatin cis-platinum)and MVAC(methotrexate,Changchun Vincristine,doxorubicin,cisplatin).However,due to the easy recurrence of bladder cancer and the emergence of drug resistance,there is often a case of ineffective chemotherapy.Therefore,at the present stage,we need to study a new simple and effective chemotherapy regimen for the resistance to chemotherapy.DHFR is one of those proteins expressed in the CDK4/6 downstream pathway.This experiment is to use CDK4/6 inhibitors to inhibit DHFR expression,reduce the target of antifolate drugs,so as to improve the effect of anti-folate drug.In this study,we verified the feasibility of the combination therapy of anti-folic acid and specific cell cycle inhibitor in the treatment of bladder cancer.It provides a new idea for future bladder cancer chemotherapyMaterials and methods1.Experimental materialsMTX from the MDAnderson cancer center drug sector.PTX comes from AllosTherapeutics company,we help the company to complete the efficacy comparison of different tumor cells.PAL purchased by www.selleckchem.com,manufactured from Pfizer Inc.Wild type bladder cancer cell line 5637 and T24These two kinds of cell lines from Houston University Professor Li hui2.The changes and differences of cell protein levels were detected by Western blotting method.3.Determination of IC50 in various cells by wst1 method4.Determination of drug concentration in cells per unit time by FITC microscopy5.Cell cycle,apoptosis and absolute number of cells were detected by flow cytometrResults First,to identify IC50 of various drugs in two cell lines and the correctness of experimental design ideas.1.IC50 and PTX cell lines of MTX and PAL were determined by WST1 method in5637 cell lines and T24 cell lines.The results show that MTX and PTX have different IC50 for the same drug,and T24 is more sensitive than the other,PAL’s IC50 is nearly stable.2.Western blot method was used to study the difference of the two cell lines IC50 at the protein level.The results showed that 5637 cell lines could express less RFC and more DHFR than T24 cells.Therefore,to some extent,explain the possible reasons for the different IC50.3,using FITC-MIX fluorescence imaging method.Treatment of 5637 cell line and T24 cell line by FITC-MTX.Determination of the amount of drug in the inhaled cells at 90mins and 24 hours.The results showed that at the same time,T24 cells could inhale more FITC-MIX into the cells.So we can explain the difference between the two cell lines IC50.4,using Western blot method to further verify the accuracy of the experimental ideas. Cells treated with different concentrations of PAL could induce a significant decrease of DHFR.The results showed that the two drugs acted on the same cell pathway.The correctness of the experimental design is verified.Two.To explore the characteristics of single agent in the treatment of tumor cells1.Using Western blot to detect the expression of DHFR in the cells of the two cell lines treated with PAL.The results showed that the expression of DHFR was decreased with the increase of PAL concentration.The results showed that if MTX and PTX were used to treat this two kinds of cells separately,that DHFR was significantly increased,and was positively correlated with concentration.2.Cell cycle and apoptosis were detected by flow cytometry.The results suggested that MTX and PTX could induce more apoptosis.However,PAL can cause more cells to stay in the G1 phase,but after the removal of PAL,the cells can re-enter the S phase,that is,PAL can only inhibit cell division,but can not cause more cell apoptosis.Three,combination of MTX or PTX woth PAL,and the best use of this combination1.Western blot method was used to verify the expression of DHFR in MXT,PTX,PAL and combination therapy.The results showed that the expression of DHFR was decreased after combined administration.And the longer the PAL administration time, the greater the drug concentration,the less the expression of DHFR.2.Flow cytometry was used to detect the number of tumor cells after Beads treatment.Results showed that in the single drug administration of two kinds of drugs and Drug administration and other two kinds of drugs in order to compare different injection methods,the first adding PAL then add MTX or PTX to more killing tumor cells,the curative effect is more effective.Conclusion1,DHFR and CDK4/6 in a cell pathway in the 5637 and T24,relative to other protein expressions in this cell pathway,CDK4/6 has greater impact on DHFR2,The combination of anti folic acid drugs and specific cell cycle inhibitors can play a synergistic role,increasing the role of anti folic acid drugs.3,Although the two drugs have a synergistic effect,but need to pay attention to adding order can get better curative effect.The level of cell experiment showed that adding PAL1 st then add MTX or PTX could get better drug effect...