| Objective:To investigate the exposure change of cytarabine due to its resistance and explore the possible mechanism.Method:(1)Pharmacokinetics change of cytarabine induced by its resistance in whole cells:Establishing UPLC-MS/MS method for analyzing Ara-C and its metabolites;Culturing sensitive HL-60 cells and resistant HL-60/Ara-C cells.After administration 10μM Ara-C,determining Ara-C,Ara-U,Ara-CMP,Ara-CDP and Ara-CTP in HL-60 and HL-60/Ara-C cells at 1,2,3,5,6,8 and 12h,respectively.The exposure of Ara-C and its metabolites in HL-60 and HL-60/Ara-C cells were compared.(2)Pharmacokinetics change of cytarabine induced by its resistance in subcellular level:Establishing the way to separate subcellular organelles and UPLC-MS/MS method to determine Ara-C and its metabolites in cytosol or nuclear.After administration 10μM AraC,determining cytosol and nuclear Ara-C,Ara-U,Ara-CMP,Ara-CDP and Ara-CTP level in HL-60 and HL-60/Ara-C cells at 1,2,3,5,6,8 and 12h,respectively.Calculating AUC to compare the exposure difference of intracellular and nuclear Ara-C and its metabolites,especially Ara-CTP.(3)Exploring the mechanism of cytarabine cellular pharmacokinetics change induced by resistance based on RNA-Seq technique:RNA-Seq technique was used to compare the transcriptome of HL-60 and HL-60/Ara-C cells.Candidate genes that participated in Ara-C cellular pharmacokinetics change induced by resistance were found by GO and KEGG analysis.After finding candidate genes,qPCR and western blotting were used for further validation.Results:(1)Pharmacokinetics change of cytarabine induced by its resistance in whole cell:The exposure of Ara-C,Ara-CMP,Ara-CDP and Ara-CTP in HL-60 cells are 851.6,4847,675.1 and 23.23 nM/mg protein*min,respectively.Them in HL-60/Ara-C cells are 249.2,1533,242.4 and 10.08 nM/mg protein*min,respectively.It is indicated that Ara-C and its metabolites have a higher exposure in HL-60 cells than in HL-60/Ara-C cells.The exposure of total Ara-C(Sum of Ara-C and its metabolites)in HL-60 cells is higher than it in HL-60/Ara-C cells(Ratio C/R=3.14),and HL-60 and HL-60/Ara-C cells have a similar ration of active metabolite Ara-CTP exposure to total Ara-C exposure(Ratio C/R=0.72).(2)Pharmacokinetics change of cytarabine induced by its resistance in subcellular level:In HL-60 cells,the cytosol exposure of Ara-C,Ara-CMP,Ara-CDP and Ara-CTP is 278.5,1205,4042 and 85.74 nM/mg protein*min,respectively.In HL-60/Ara-C cells,the cytosol exposure of Ara-C,Ara-CMP,Ara-CDP and Ara-CTP is 113.1,788.1,1308 and 41.67 nM/mg protein*min,respectively.It is indicated that Ara-C and its metabolites also have a higher cytosol exposure in HL-60 cells than in HL-60/Ara-C cells.Nuclear active metabolite Ara-CTP has a higher exposure in HL-60 cells(24.1 nM/mg protein*min)than in HL60/Ara-C cells(9.42 nM/mg protein*min).Correlation analysis showed that intracellular Ara-CTP level is not correlated with nuclear Ara-CTP level(p=0.43,r2=0.14).Results of AUC comparison showed that the nuclear Ara-CTP level of HL-60 and HL-60/Ara-C cells has a bigger gap than intracellular Ara-CTP level of HL-60 and HL-60/Ara-C cells(AUCNU Ratio C/R=2.56,AUCCY Ratio C/R=2.05).(3)Exploring the mechanism of cytarabine cellular pharmacokinetics change induced by resistance based on RNA-Seq technique:There were 2403 different expressed genes that were found according to the standard of p<0.05,and fold change>2 or fold change<0.5.Nuclear hydrolase SAMHD1 and efflux transporter ABCC1 and ABCC10 were found as the candidate genes that participated in Ara-C cellular pharmacokinetics change induced by resistance based on GO,KEGG and PPI analysis.Further validation supported a higher expression of SAMHD1 in both mRNA(p<0.01)and protein level in resistant HL-60/Ara-C cells.However,ABCC1 showed a decrease mRNA expression in HL60/Ara-C cells while its encoding protein MRP1 have no change in both two cells expression.ABCC10 also showed no change in both two cells mRNA expression.Results of RNA-Seq showed that none of known Ara-C metabolism related genes as DCK,NT5C2,RRM1 and RRM2,and transport related gene SLC29A1 have different mRNA expression in both HL60 and HL-60/Ara-C cells(0.5<fold change<2),while transport related gene SLC22A4 have a higher expression in resistant cells(fold change>2).However,qPCR results showed none of Ara-C metabolism related gene and transport related gene as DCK,NT5C2,RRM1,RRM2,SLC29A1 and SLC22A4 have a different expression in both cells(p>0.05).Conclusion:Ara-C resistance is related with the decrease of Ara-C and its metabolites exposure,especially related with the decrease of Nuclear Ara-CTP level.High expression of SAMHD1 in HL-60/Ara-C cells might be the one reason for the decrease of nuclear AraCTP exposure. |
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