The Activities And Expression Of CYP And UGT Enzymes In Human Primary Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a global health problem,representing the third cause of death for cancer and the fifth most common cancer worldwide.In all areas of the world the incidence is higher in males than in females(M/F ratio ranging from 1.4 to 3.3).The liver is a vital organ present in vertebrates and some other animals,plays a major role in metabolism and has a number of functions in the body,including glycogen storage,decomposition of red blood cells,plasma protein synthesis,hormone production,and detoxification.The liver is considered the major "metabolic clearing house" for both endogenous chemicals(e.g.,cholesterol,steroid hormones,fatty acids,and proteins)and xenobiotics,including intake of food and medicine.The biological transformation of these substances undergoing within the liver like oxidation,reduction,hydrolysis and combined reactions are almost mediated by Drug-Metabolizing Enzymes.People who have liver cancer cannot receive the normal dosage of drugs,mostly because of the metabolism of enzymes.Their sensitivity to such drugs as sedatives and narcotic becomes heightened by their liver impairment.Even the standard dose of these and other medications could result in encephalopathy,or the accumulation of fluid in the brain.Due to the risk of fatality,some drugs cannot be given at all.Beside surgical resection and liver transplantation,chemotherapy is of great importance included in the systemic treatments of HCC.But the clinical efficacy of anticancer therapy is severely limited by an inability to accurately predict outcomes for the patient in terms of both tumor response and toxicity.The lack of prediction is of even greater clinical significance with these agents given their narrow therapeutic index.Despite advances in molecular medicine resulting in new drugs with specific cellular targets,there are still major issues related to drug resistance and individual patient variability.We should evaluate the activities and expression of the major drug metabolism enzymes in tumor tissue in order to better understand the chemosensitivity of the HCC liver tumors.In this study,we selected CYP3A4-specific probe,testosterone;UGT1A-specific probe,genistein;UGT1A1-specific probe,SN-38 and UGTlA9-specific probe,propofol as model chemicals.There are endogenous hormone,natural product,anti-cancer drug and anesthetic,respectively.All of them are good in vitro substrate to determine their kinetic parameters in tumor and normal liver microsomes prepared from five paired HCC normal control liver tissue and tumoral liver tissue.Moreover,We analysed the expression of CYP3A4,UGT1A,UGT1A1 and UGT1A9 protein in five paired HCC normal control liver tissue and tumoral liver tissue by Western blotting.Methods1.Tissue Samples and Preparation of Human Liver MicrosomesHuman liver tissue from individuals(age from 41 to 58 years old,n=5)who had undergone surgery between May 2011 and July 2011 for resection of hepatocellular carcinoma were obtained from NanFang Hospital,Guangzhou,China.Approvals for tissue collection and in vitro xenobiotic metabolism studies were obtained from the NanFang Hospital Reasearch Ethics Committee.Tissue samples were keeped in ice-cold saline immediately following surgery and sent to laboratory within 30 min.Healthy tissue surrounding primary tumour was isolated and considered as normal human liver tissue.Normal and tumoral human liver tissues were used for preparation of microsomes(normal or tumoral human liver microsomes,N/THLM),respectively.Microsomes were prepared using a standard differential ultracentrifugation and microsomal pellets were resuspended in 250 mM sucrose then stored(10-50 mg of protein/mL)at-80℃ immediately,with total protein concentrations measured according to the Bradford method,using bovine serum albumin as standard.All of the liver tumors were primary hepatic carcinoma,moderately or lowly differentiated(grade Ⅲ～Ⅳ).2.UPLC AnalysisAnalytical methods for each present compound and its corresponding metabolite were validated for intra-day and inter-day by using 6 samples at three concentrations.Stability studies were couducted to ensure all of the modeling substrates in stable conditions during the procsesses of experiments.3.Enzymatic Activities of CYP3A4Kinetic parameters were determined in commercial pooled HLM(PHLM),pooled-NHLM(a pooled of five normal HLM samples)and pooled-THLM(a pool of five tumor HLM samples),respectively.For testosterone,a good specific probe for CYP3A4,60 min incubations at 37℃ were performed at 5 to 300 μM testosterone in three different kinds of HLM.To profile activities of CYP3A4 from tumor and normal microsomes for each case,three substrate concentrations,i.e.10,50 and 150μM of testosterone were used in both NHLM and THLM from five donors.The incubation procedures were the same as mentioned above,and all incubations were performed three times.4.Enzyme Assay for genistein,SN-38 and propofolGenistein,SN-3 8 and propofol were used as the substrates to determine the activities of UGT1A,UGT1A1 and UGT1A9 in N/THLM,respectively,while commercial pooled human liver microsome was used as a reference.The formations of genistein O-glucuronide,SN-38G and propofol O-glucuronide were determined according to the method of those published previously with slight modifications.5.Kinetic AnalysisRates of substrates metabolism by N/THLM or PHLM were expressed as amounts of metabolites formed per min per mg protein(pmol or nmol/min/mg).Kinetic parameters were then obtained according to the profile of Eadie-Hofstee plots.If the Eadie-Hofstee plot was linear or showed characteristic profiles of atypical kinetics,formation rates(V)of metabolin at respective substrate concentrations(C)were fit to the standard Michaelis-Menten equation or other atypical kinetics equations,using the ADAPT Ⅱ program.To determine the best-fit model,the model candidates were discriminated using the Akaike’s information criterion(AIC),and the rule of parsimony was applied.6.Western BlottingThe CYP3A4,UGT1A,UGT1A1 and UGT1A9 protein levels of N/THLM were determined by Western blotting.Microsomes(40-60 μg)were boiled for 5 min in 1X loading buffer and separated on a 10%sodium dodecyl sulfate polyacrylamide gel by electrophoresis and then transferred onto PVDF(polyvinylidene difluoride)membrane.Blots were blocked according to the antibody manufacturer’s directions with slight modifications.The intensities of stained bands were measured by densitometry using the Bio-Rad software program Quantity One software(v 4.2).7.Statistical AnalysesResults were expressed as mean ± SD.SPSS 13.0 for Windows was used as statistical software.An independent-samples t test was used to determine whether the reaction rates of oxidation or glucuronidation in tumor have significant differences from the rates in normal liver tissue.Differences were considered significant when p values were<0.05.Results1.Kinetic studies of testosterone metabolized by CYP3A4 from PHLM,pooled-NHLM and pooled-THLM.Comparison of the metabolism velocity in THLM and matched NHLM of each patient.The kinetics of testosterone metabolized to 6β-hydroxytestosterone by PHLM,pooled-NHLM and pooled-THLM was determined using Eadie-Hofstee plots.The kinetic parameters were determined for Michaelis-Menten kinetic profiles using ADAPT II kinetic modeling software.There were no significant differences for Km and Vmax values between PHLM(50.74 μM,2.33 nmol/min/mg)and pooled-NHLM(65.72 μM,2.43 nmol/min/mg).As expected,the Km’ value(40.2 μM)in pooled-THLM was much lower than that in PHLM and pooled-NHLM.The Vmax value in pooled-THLM(0.30 nmol/min/mg)was only one tenth to that of the other HLM.The results above have demonstrated the impairment of CYP3A4-mediated metabolism in tumor microsomes.In attempted to further determine the impairment,we comparison of the metabolism velocity in tumor microsome and in the matched nontumor micro some of each patient.The experiments were conducted in the two matcheds microsomes using 10,50,and 150 μM substrate concentrations.As expected,the metabolism rate(6β-hydroxytestosterone formation rate)of testosterone in THLM of each patient was always lower than that in the matched NHLM at allsubstrate concentrations(p<0.01,compared t-test).The metabolism rate of testosterone in NHLM has obvious substrate dependence while the situation in THLM is not significant,and the rate difference between NHLM and THLM is more significant along with the increase of substrate concentration.The maximum difference is 100 times in 150μM(No.3).2.Kinetics of genistein glucuronidation by 5 paired N/T HLM from HCC tissue.The kinetics of genistein metabolized to genistein O-glucuronide by PHLM,NHLM and THLM was according to their Eadie-Hofstee plots and determined as Biphasic kinetics.There were no significant differences for Km and Vmax values between PHLM(0.20 μM,0.83 nmol/min/mg)and the mean of 5 independent NHLM(0.27 ± 0.21 μM,0.81± 0.19 nmol/min/mg).However,the average value of Vmax(0.35±0.18 nmol/min/mg)for 5 independent THLM was lower than that in PHLM and NHLM.The value of Km performance in THLM is large inter-individual differences while compared to the paired NHLM,the Km value is higher in patient No.1,2,3,4 but lower in 5.3.Kinetics of SN-38 glucuronidation by 5 paired N/T HLM from HCC tissue.The curves of SN-3 8 glucuronidation rate didn’t fit any known model and hence kinetic parameters were not obtained,we showed the observation Rmax(The maximum reaction rate,Rmax)of SN-38G The value of Rmax in PHLM is 79.27 pmol/min/mg,the average Rmax of 5 samples NHLM is 90.39 ± 36.3 pmol/min/mg and THLM is 32.87 ± 35.9 pmol/min/mg.The value of Rmax in individual NHLM is much higher(N/T:No.1 to 4,5.1,7.3,4.8,3.5 fold,respectively and No.5 has not significant difference)compared to matched THLM.4.Kinetics of propofol glucuronidation by 5 paired N/T HLM from HCC tissue.The Eadie-Hofstee plot for the rates versus rates over concentration,indicative of the reaction kinetic of propofol is Substration inhibition enzyme kinetic model.The correlation of observed value and plotted line from calculated according to apparent kinetic parameters is fited well but the simulation parameters like Km and Vmax are greater different from our observed value.Therefore,we emphasised on the rate of clearance(Vmax/Km,CLint)consideration.The CLint of PHLM is 10.92μL/min/mg and the CLint average of 5 independent NHLM(16.83±8.38 μL/min/mg)is higher than THLM(3.21±0.87 μL/min/mg).In addition,the value of CLint in individual NHLM is much higher(N/T:No.l to 5,3.2,10.1,2.75,4.7,6.1 fold,respectively)than matched THLM.5.Western blot analyses of UGT1A,UGT1A1 and UGT1A9 protein expression in five paired HCC normal control liver tissue and tumoral liver tissue.Samples were quantified by laser densitometry in duplicate and used Quantity One sofeware.Significant differences(p<0.05,compared T-test)were found in the levels of CYP3A4,UGT1A,UGT1A1,UGT1A9 protein expression between tumor tissue and normal tissue for each case and the expressions of these four enzyme proteins in tumors were significantly lower(p<0.05)than that in normal livers.ConclusionsIn conclusion,the study verified the expressions of CYP3A4,UGT1A,UGT1A1,UGT1A9 in tumors were significantly lower(p<0.05)than that in adjacent nonneoplastic liver tissue and their mediated metabolism were evidently reduced in human hepatocellular carcinoma tissue.These data are of great importance to further understand the mechanisms of drug resistance in human liver cancer.The enzyme activities in regions distant from the tumor were within the normal range.Hence,changes in primary HCC may have significant effects on the pharmacokinetics of other specific substrates(including endogenous substances such as testosterone,natural products such as soybeans and other food and commonly used drugs such as intravenous anesthetics,anti-cancer agents,etc.)in vivo and may contribute to alterations in the endocrine status of these patients.Therefore,this study suggests that we should considered on drug side effects and drug-drug interactions not only from the pharmacodynamics but also pharmacokinetic when we chose drug and dosage.