The Effects Of HCC On The Glucuronidation Of UGT1A4 And UGT2B7 And Its Potential Mechanism Analysis

Background and objectiveThe liver has the function of biological transformation for endogenous chenicals and xenobiotics(including food and drugs).The biotransformation,namely metabolism,includes oxidation,reduction,hydrolysis and combined reactions and so on,the polarity of substrate changes after the reactions.Almost all of the metabolism require drug metabolism enzymes involved in,the liver is rich of metabolism enzyme.Liver cancer is malignant tumors in liver,it can devided into primary carcinoma of the liver and metastatic hepatic carcinoma according to the pathogeny.More than 90%of liver cancer patients are with primary liver cancer,primary carcinoma of the liver could devided into hepatocellular carcinoma,cholangiocellular carcinoma and mixed type of hepatic tumors.The incidence hepatocellular carcinoma(HCC)is high in China,and it has higher incidence in males than in females.HCC has become the second cause of death for malignancy in China.Some function of drug metabolism enzyme would changed when liver diseases happens,these changes would affect the metabolism of drugs in vivo,which leads to efficacy of the drug different,or drug accumulation in vivo,the sensitivity of organism to drugs might be heightened or reduced.In conclusion,the alteration of the enzyme will affect safe medication and human healthy.Hepatocellular carcinoma is a result of various liver diseases.Understanding of the changes of the drug metabolism enzyme in HCC patients and how these changes happen is meaningflull for keeping healthy.At present,the research about the effects of liver diseases on enzyme focus on cytochrome P450,little data presents the effects on UGTs.It is necessary to take a research on the regularity of HCC and UGTs,we should evaluate the activities of the major UGT isoforms in tumor tissue in order to provide more information about the changes of drug metabolism enzyme and changes of drug metabolism pathway.We choose dozens of HCC cases to study the differences of UGTIA4 and UGT2B7 in liver cells between pericarcinomatous tissue and tumor tissue in the catalytic activity and the amount of protein and gene expression as well as how the situations of two kinds of flavonoid compounds with antitumor activity in Tumor tissues was metabolize by UGT changes,to reveal metabolic influences caused by liver cancer.UGT1A4 mainly catalytic the N-Glucuronidation of amine compounds while UGT2B7 has a specific selectivity towards catechol structure,makes an important contribution to the regulation of estrogen activity and has a relatively high catalytic activity towards various exogenous drugs,including narcotic analgesics(morphine),carboxyl non-steroidal anti-inflammatory drugs(ketopmfen),cancer drugs(all trans retinoic acid).Natural flavonoids has hepatoprotective effects and is mainly metabolized through conjugation reactions,the metabolism changes can reflect the activity changes of metabolic enzyme.MethodsWith the approvals for tissue collection from NanFang Hospital Reasearch Ethics Committee,we used human liver tissue which were from HCC patients who had undergone surgery for resection to prepare liver microsome by differential centrifugation,and we extracted total RNA of the tissue.All of the liver tumors were primary hepatic carcinoma,moderately or lowly differentiated.We selected UGT1A4-specific probe,tamoxifen,UGT2B 7-specific probe,zidovudine and two flavonoids compounds which have antitumor activity,chrysin and wogonin as in vitro substrate to determine the rate of metabolism and kinetic parameters in pericarcinomatous and matched tumor liver microsomes,kinetic parameters were determined by HLM(C-HLM),pooled-PHLM(a mixture of 18 pericarcinomatous HLM sanples)and pooled-THLM(a mixture of 18 tumor HLM samples),respectively.Accordingly,we can evaluate the changes or differences of drug metabolism enzyme in tumor cell.To further explain the mechanism of UGTs alteration in the tumor tissue preliminary,we used western blot analyze UGT1A4,UGT2B7 enzyme protein expression in tumor and pericarcinomatous tissue,and we extracted total RNA in tumor and pericarcinomatous tissue,which reverse transcriptase into cDNA for real-time fluorescence quantitative polymerase chain reaction(real time-PCR),to clarify the changes of the two enzymes at the genetic level.Rates of substrates metabolism 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,using the XL-Kinetics program to determine the best-fit model,the model candidates were discriminated using the Akaike’s information criterion(AIC),and correlation coefficient square.Western blot data was analyzed by Quantity one.Real time PCR was performed using an ABI 7500 real-time PCR system,The levels of gene transcripts were quantified using the 2-△△CT method.SPSS 13.0 for Windows was used as statistical software.An independent-samples t test was used to determine whether the parameters in tumor have significant differences from the parameters in Pericarcinomatous tissue.Differences were considered significant when p values were<0.05.Results1.Kinetic studies of tamoxifen metabolized by UGT1A4 from C-HLM,pooled-PHLM and pooled-THLM.Comparison of the metabolism velocity in THLM and matched PHLM of each patient.The kinetics of tamoxifen metabolized to tamoxifen N-glucuronide by C-HLM,pooled-PHLM and pooled-THLM was determined using Eadie-Hofstee plots.The kinetic paraneters were determined as Substrate inhibition kinetic profiles using XL-Kinetics modeling software.The correlation of observed value and plotted line from calculated according to apparent kinetie 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,CLin,).The CLint of C-HLM is 9.68μL/min/mg,the CLint of pooled PHLM(9.6 μL/min/mg)is higher than pooled THLM(3.78 μL/min/mg).We also compared the metabolism velocity in tumor microsome and in the matched pericarcinomatous microsome of each patient.The experiments were conducted in the two matched microsomes using 10,37.5,and 100 substrate concentrations.As expected,the metabolism rate(tamoxifen N-glucuronide formation rate)of tamoxifen in THLM of each patient was always lower than that in the matched PHLM at all substrate concentrations(p<0.05,compared t-test).2.Kinetic studies of zidovudine metabolized by UGT2B7 from C-HLM,pooled-PHLM and pooled-THLM.Comparison of the metabolism velocity in THLM and matched PHLM of each patient.The kinetics of zidovudine metabolized to zidovudine O-glucuronide by C-HLM,pooled-PHLM and pooled-THLM was determined using Eadie-Hofstee plots.The kinetic parameters were determined as Michaelis-Menten kinetic profiles using XL-Kinetics modeling software.There were no significant differences for Km and Vmax values between C-HLM(1844.6 μM,1830.8 pmol/min/mg)and pooled-PHLM(1759.4 μM,2050.8 pmol/min/mg).The Km value in pooled-THLM was 2885.3 μM,the Vmax value in pooled-THLM(405.4 pmol/min/mg)was only one fifth to that of the other HLM.We compared of the metabolism velocity in tumor microsome and in the matched nontumor microsome of each patient.The experiments were conducted in the two matched microsomes using 250,1500,and 3500 μM substrate concentrations.The metabolism rate of zidovudine in THLM of each patient was always lower than that in the matched PHLM at all substrate concentrations(p<0.05,compared t-test).3.Kinetic studies of chrysin metabolized by C-HLM,pooled-PHLM and pooled-THLM.Comparison of the metabolism velocity in THLM and matched PHLM of each patient.The kinetics of chrysin metabolized to chrysin O-glucuronide by C-HLM,pooled-PHLM and pooled-THLM was determined using Eadie-Hofstee plots.The kinetic parameters were determined as Michaelis-Menten kinetic profiles using XL-Kinetics modeling software.There were no significant differences for Km and Vmax values between C-HLM(3.7 5.2 nmol/min/mg)and pooled-PHLM(4.8μM,5.6 nmol/min/mg).The Km value in pooled-THLM was 4.7 μM,the Vmax value in pooled-THLM(2.7 pmol/min/mg)was only half to that of the other HLM.We compared the metabolism velocity in tumor microsome and in the matched pericarcinomatous microsome of each patient.The experiments were conducted in the two matched microsomes using 1,25 μM substrate concentrations.The metabolism rate of chrysin in THLM of 11 HCC patients was lower than that in the matched PHLM at all substrate concentrations,one had contrary condition.(p<0.05,compared t-test).4.Kinetic studies of wogonin metabolized by C-HLM,pooled-PHLM and pooled-THLM.Comparison of the metabolism velocity in THLM and matched PHLM of each patient.The kinetics of wogonin metabolized to Wogonoside by C-HLM,pooled-PHLM and pooled-THLM was determined using Eadie-Hofstee plots.The kinetic parameters were determined as Michaelis-Menten kinetic profiles using XL-Kinetics modeling software.There were no significant difirerences for Km and Vmax values between C-HLM(2.4 μM,4.4 nmol/min/mg)and pooled-PHLM(2.0 μM,4.2 nmol/min/mg).The Km value in pooled-THLM was 2.8 μM,the Vmax value in pooled-THLM(2.0 pmol/min/mg)was only half to that of the other HLM.We compared the metabolism velocity in tumor microsome and in pericarcinomatous microsome of each patient.The experiments were conducted in the two matched microsomes using 0.5,15 μM substrate concentrations.The metabolism rate of wogonin in THLM of 10 HCC patients was lower than that in the matched PHLM at all substrate concentrations,two had contrary condition.(p<0.05,compared t-test).5.Western blot analyses of UGT1A4,UGT2B7 protein expression in 12 paired HCC pericarcinomatous liver tissue and tumoral liver tissue.Samples were quantified by laser densitometry in duplieate and used Quantity One sofeware.UGT2B7 protein expression was significantly lower in tumor tissue(p<0.05,compared t-test)than that in pericarcinomatous livers for each case.For UGTIA4,the protein expression was significantly lower in tumor tissue than that in pericarcinomatous livers for 9 cases,the other 3 had contrary condition.The expression of UGT1A4 and UGT2B7 were significantly lower in pooled-THLM than that in pooled-PHLM.6.Real time PCR analyses of UGT1A4 and UGT2B7 mRNA expression in 12 paired HCC pericarcinomatous 1 and tumoral liver tissue.UGT1A4 mRNA expression was significantly lower in tumor tissue(p<0.05,compared t-test)than that in pericarcmomatous livers for each case.For UGT2B7,the mRNA expression was significantly lower in tumor tissue than that in pericarcinomatous livers for 11 cases,excepted 1 case had contrary condition.ConclusionsAt different concentrations of substrates,the catalytic activities of UGT1A4 in the tumor tissue of patients with HCC decrease to 1/2-1/20 of those in the tissue adjacent to the tumors,the quantity of mRNA or enzyme protein expression as well,suggesting that the mRNA of UGT1A4 is affected during the occurrence and development of HCC,which lead to the reduced protein expression and metabolism activity.At difterent concentrations of substrates,the catalytic activities of UGT2B7 in the tumor tissue of patients with HCC decrease to 1/3-1/70 of those in the tissue adjacent to the tumors,the quantity of mRNA or enzyme protein expression as well,suggesting that the mRNA of UGT1A4 is affected during the occurrence and development of HCC,which leads to the reduced protein expression and metabolism activity.In liver cancer tissues,metabolic changes in chrysin and wogonin,which are metabolized by various isoforms of UGT,can indirectly reflect the changes in the overall activity of UGTs.According to the results,the overall velocity of change of UGT activity is lower than either UGT1A4 or UGT2B7,and the specific substrates of other subtypes are required to individually research the activity changes of corresponding subtypes and provide more specific information for clinical medicine in the future.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.