Alterations Of Sulfotransferases In Hepatocellular Carcinoma And On Sulfotransferases-mediated Drug Disposition

Background and ObjectivesHepatocellular carcinoma(HCC)is a major global health problem with high rates of morbidity and mortality.Study on the alteration of mRNA and functions of drug-metabolizing enzymes in HBV-positive HCC patients can provide a certain amounts of experimental data to the rational use of drugs and the early diagnosis of HCC.Sulfotransferase(SULT)enzymes are major members of phase? drug-metabolizing enzymes and thus play critical roles in the biotransformation of many endogenous and xenobiotics compounds.It is difficult to predict the efficacy of drugs on the tumor and pericarcinomatous tissues toxicity.In this study,we investigated the changes and the relationship of SULTs activity,protein amounts and mRNA expression levels in HCC tissues.Resveratrol was taken as an example to investigate the effect of SULTs enzyme activity on drug metabolism in patients with HCC,so as to provide reference for clinical rational drug use.Methods1:Isotope label-free UHPLC-MS/MS and Western-blot were used to quantify the protein amounts of 5 SULTs from healthy human liver S9 fraction.2:S9 fractions from both tumors and pericarcinomatous tissues were prepared using standard differential centrifugation procedures.Protein amounts of 5 SULTs in S9 were assessed simultaneously by using an isotope label-free UHPLC-MS/MS method.Activities of the five most important SULT enzymes were measured using specific probe substrates.At the same time,the enzyme kinetic parameters of resveratrol were determined in S9 fractions from healthy human liver,pericarcinomatous and tumor tissues.Additionally,to clarify the potential mechanism for alterations of SULTs activities,mRNA expression level of these SULT enzymes were determined by using real-time PCR.Results1.Comparison between Western-blot,and the isotope label-free UHPLC-MS/MS method with regard to analysis specificity and speed of analysis.To validate the specificity of analysis method,we used the Western-blot analysis and LC-MS/MS methods in analyzing human recombinant SULT proteins,namely,SULT1A1,SULT1A3,SULT1B1,SULT1E1,and SULT2A1.The results showed that only 40%of antibodies displayed specificities.In contrast,100%of the LC-MS/MS displayed absolute specificity.The Western-blot analysis took 24 h,whereas the LC-MS/MS method finished the analysis in less than 10 min,although sample preparation took about 6 h.2.SULT enzymes protein quantification and correlation plots in 10 healthy human liver S9.We then used LC-MS/MS and Western-blot to quantify 5 SULT isoforms,respectively.The results showed that the Western-blot analysis revealed relatively small differences among the five SULT isoforms.We then plotted the absolute protein amounts(from LC-MS/MS)and relative protein expression levels(from Western-blot).As expected,the plots of SULT1B1 and SULT1E1 showed excellent correlation coefficients because the antibodies were specific.By contrast,SULT1A3 and SULT2A1 showed poor correlation because the antibodies displayed poor specificities(r2=0.492,r2=0.546).SULT1A1,which was expressed at significantly higher levels in our LC-MS/MS measurement,displayed good correlation because its expression levels were much higher than those of the interfering SULT1A3 in human liver S9,thereby suppressing the impact of SULT1A3 interference.The present LC-MS/MS method was validated in terms of intra-and inter-day precision and accuracy,recovery,and matrix effect.3.Impaired SULT enzymatic protein amounts in tumor tissues of HCC patients.For SULT1A1 isoform(as quantified by LC-MS/MS),70%of the samples had higher amount in pericarcinomatous tissues than tumor tissues,and 30%had lower amounts in pericarcinomatous tissues than tumor tissues;for SULT1B1,20%had higher amounts in pericarcinomatous tissues than tumor tissues,70%had lower amounts in pericarcinomatous tissues than tumor tissues,and 10%were left unchanged;for SULT1A3,50%had higher amounts in pericarcinomatous tissues than tumor tissues,30%had lower amounts in pericarcinomatous tissues than tumor tissues,and 20%were left unchanged;for SULT1E1,40%had higher amounts in pericarcinomatous tissues than tumor tissues,40%had lower amounts in pericarcinomatous tissues than tumor tissues,and 20%were left unchanged;for SULT2A1,70%had higher amounts in pericarcinomatous tissues than tumor tissues,10%had lower amounts in pericarcinomatous tissues than tumor tissues,and 20%were left unchanged.4.Decrease in SULT enzymatic activities in tumor tissues of HCC patients and the regulation on resveratrol metabolism.Using probe substrates we systematically measured the metabolic functions of SULTs in tumor S9(tHLS9-pooled),pericarcinomatous S9(nHLS9-pooled),and reference pooled normal human S9(rHLS9-pooled).The results showed that the protein amounts of SULTs in S9 from tumor tissues were significantly decreased except for SULT1A3 and SULT1B1.On the other hand,the Vmax values derived using different pooled S9 preparations were highly variable when using different probe substrates.We used the probe substrates at predetermined low concentrations to measure SULT enzymatic activities in the hHLS9-individual,nHLS9-individual and tHLS9-individual.The results showed that,for SULT1A1,the activities in peri carcinomatous S9 were all higher than those in tumor S9;for SULT1B1,20%were higher in pericarcinomatous S9 than in tumor S9,70%were lower in pericarcinomatous S9 than in tumor S9,and 10%were left unchanged in HCC patients;for SULT1A3,30%were higher in pericarcinomatous S9 than in tumor S9,and 70%were lower in pericarcinomatous S9 than in tumor S9;for SULT1E1,50%were higher in pericarcinomatous S9 than in tumor S9,40%were lower in pericarcinomatous S9 than in tumor S9,and 10%were left unchanged;for SULT2A1,80%were higher in pericarcinomatous S9 than in tumor S9,10%were lower in pericarcinomatous S9 than in tumor S9,and 10%were left unchanged.The turnover numbers(or TONs)of SULTs other than SULT1A1 in the tumor tissues were nearly identical to the values observed in pericarcinomatous tissues and healthy samples(P>0.05),thereby suggesting that the catalytic efficiency of SULTs was not seriously impaired in tumor S9.Inactivation rate of resveratrol was significantly decreased in tumor tissues compared with the healthy and pericarcinomatous tissues,which may lead to its accumulation in tumor cells and causing more toxicity.5.Downregulated SULT enzymatic mRNA expression levels in tumor tissues of HCC patients.The results showed that the mRNA expression levels of SULT1A1,SULT1A3,SULT1E1,and SULT2A1 were decreased or drastically decreased in 90%of the tumor samples in comparison with the matched pericarcinomatous samples(except for SULT1B1).6.Correlation of protein amounts and mRNA expression levels with SULT enzymatic activities.The results showed that there was a strong correlation between SULT enzymatic activities and the protein amounts was better than that between the activities and the mRNA expression levels for a majority of the SULT enzymes in HCC tumor and pericarcinomatous tissues(except for SULT1A3).SULTs are involved in the metabolism of resveratrol in vivo.Compared with the pericarcinomatous tissues,the metabolic rate of resveratrol in S9 from HCC tissue was significantly correlated with the enzyme activity and protein content of SULT1A1(r2>0.6).ConclusionsTo sum up,the drastic decline in activity of SULTs in tumor tissues is mainly due to a decrease in the protein amounts of the enzymes present in HCC,while the catalytic efficiencies of SULTs were not seriously impaired in HCC tumors.This study is the first to demonstrate the absolute quantification of SULT enzymes through isotope label-free UHPLC-MS/MS can be achieved using human liver S9 samples.Resveratrol was taken as an example for the evaluation of the effect of SULTs activity on drug metabolism in HCC,so as to provide reference for clinical rational drug use.