Correlation Between CYPs Protein Expression And Metabolic Activities In Drug Disposition In Hepatocellular Carcinoma Patients

BackgroundHepatocellular carcinoma is a major global health problem with high rates of morbidity and mortality and the overall 5-year survival rate is less than 10%. Chronic infection with hepatitis B virus (HBV) is the major etiology factor for HCC. Over 100 million carriers were diagnosed in China and caused nearly 400 thousand new HCC cases per year, which account for 50% of the cases worldwide. Surgical resection remains the gold standard for HCC. However, more than 80% of patients are not surgical candidates and other options such as chemotherapy and chemoembolization are considered.The efficacy of anticancer therapy is usually limited by an inability to predict patient outcomes such as tumor response and toxicity. Drug-metabolising enzymes such as CYPs and UGTs in tumors or their pericarcinomatous tissues play a key role in the activation or inactivation of numbers of cytotoxics, and influence the susceptibility of host and neoplastic tissues to their effects. Furthermore, it has been suggested that the local expression of CYPs in tumors is very important for the management of cancer since these functionally associated enzymes might be also involved in the development of HCC by activation of potential (pro-) carcinogens and mutagens. Therefore, a detailed understanding of the differential expression and activity of CYPs and UGTs within livers of HCC patients may provide opportunities for improved therapeutic outcome. However, CYP enzymes are also subject to significant inter- and intra-individual variability, displaying genetic polymorphism and inducibility. So even if the expressions and activities of CYP enzymes in tumor cell being well described, it is still difficult to predict the tumor response and toxicity in each patient. Therefore, an approach to monitoring the expressions and activities of CYP enzymes in HCC tumors is necessary for individualized treatment of HCC.MethodsIn this study,26 moderately and/or poorly differentiated HCC tumors and matched pericarcinomatous tissues were collected, and microsomes of these liver tissues were processed using standard differential centrifugation procedures. Protein expression levels of 7 CYPs and 5 UGTs isoforms in microsmes were determined simultaneously by using an isotope label-free LC-MS/MS method. Activities of the seven most important CYPs enzymes in both tumors and pericarcinomatous tissues were measured using specific probe substrates and the enzyme kinetic profile of sorafenib and tegafur, two anticancer drugs used for HCC therapies were also determined. Additionally, to clarify the potential mechanism for alterations of CYPs activities, mRNA expression level of these CYPs enzymes were determined by using real-time PCR.Results and Conclusions1. Protein expression levels of CYPs and UGTs enzymes were significantly down-regulated in HCC tumors.In this study, we firstly characterized the absolute protein expressions of CYPs and UGTs enzymes in HCC tumors and matched pericarcinomatous tissues by using a robust isotope label-free LC-MS/MS method. The results showed that protein expression levels of all the seven measured CYPs isoforms was decreased in over 80% tumor tissues. The average expressions of CYP1A2 in 25 tumor samples were 20-fold lower than that in pericarcinomatous tissues, while CYP2A6 were decreased by only 2 fold. The differences were 4-to 10-fold for the other measured CYPs. Drastic decrease (2- to 7-fold) were also observed for UGT1A1,1A4,1A9 and 2B7. We were somewhat surprised by the increase in UGT1A6 expressions in large percentage of subjects (50%). In fact, UGT1A6 in tumors was not down-regulated in 80% of subjects, and the average was actually 1.5 fold higher than pericarcinomatous tissues.We also found that, in healthy human liver, CYP2E1 showed the highest expression levels accounting for 30% of total CYPs proteins, while CYP3A4 and CYP2D6, two of the most important isoforms for drug metabolism, accounted for only 14% and 5%, respectively. UGT1A4 (29%) were found the most abundant isoform of UGTs family in healthy human livers, and isoforms essential for detoxication of drugs such as UGT1A9 and UGT1A1 accounted for over 30% of the total UGTs proteins. Similar expression ratios of these enzymes were observed in healthy human livers and pericarcinomatous tissues, While UGT1A6 went from a minor contributor to overall CYPs expression (9%) in pericarcinomatous tissue to significant contributor (38%) in HCC tumors. On the contrary, the proportion of UGT2B7was remarkably declined from 22% to 8%.Among the measured CYPs and UGTs isoforms in pericarcinomatous, the greatest inter-individual variability in protein expression levels were observed for CYP1A2 and 2B7 (52 and 39-fold, respectively). Expression levels of CYP3A4,2D6, 2C9 and UGT1A1,1A9, which are essential for drug metabolism, varied from 4 to 17 fold among the 25 individuals. In HCC tumors, greater inter-individual variability were observed, which may caused by the different regulation pathway in HCC tumors.2. Activities of CYPs enzymes were decreased significantly in HCC tumorsIn this study, we systematically characterized the enzyme kinetics of seven major CYP enzymes in HCC tumors using probe substrates. The results showed that activities of all these CYPs enzymes were remarkably decreased in HCC tumors compared with those in matched pericarcinomatous tissues. The intrinsic clearance of testosterone and phenacetin metabolized respectively by CYP3A4 and CYP1A2 were over 20-fold lower in HCC tumors than in pericarcinomatous tissues. Decreased metabolic rate of probe substrates metabolized by other CYPs isofroms (2-to 8-fold) were also observed in HCC tumors. When comparing the activities of CYPs enzyme with their protein expression levels, high correlations were observed in both tumors and matched pericarcinomatous tissues. This result demonstrates that the drastic decline in activity of CYPs in tumor tissues is mainly due to a decrease in the amount of the enzymes present in HCC.Metabolic rate can be also influenced by the catalytic efficiency of an enzyme, which is commonly modulated by post translational modifications and/or noncovalent binding of allosteric effectors. In this study, the catalytic efficiency of each CYP isoforms was assessed by calculating the turnover numbers (TON) of its probe substrate. Similar TONs (with maximal difference of～2 fold) were observed in microsomes prepared form HCC tumors, percicarcinomatous tissues and healthy liver tissues, suggesting that the catalytic efficiencies of CYP enzymes were not seriously impaired in HCC tumors. Moreover, kinetic analysis also showed same kinetic mechanisms with similar km values in all three groups, indicating that the functional properties of CYPs enzymes were not impaired seriously by HCC.Metabolism of sorafenib and tegafur, two anticancer drugs commonly used for HCC therapies, were also tested in HCC tumors. The results showed that the inactivation rate of sorafenib was significantly decreased in tumor tissues compared with pericarcinomatous tissues, which may lead to its accumulation in tumor cells and causing more toxicity. This effect may explain why sorafenib is better tolerated in patients and is not associated with liver toxicities. On the other hand, the activation rate of tegafur to cytotoxic metabolite 5-fluorouracil were 3-fold lower in HCC tumors, which may obviously reduce the curative effect.3. mRNA expression levels of CYPs enzymes were decreased significantly in HCC tumorsTo clarify the potential mechanism for alterations of CYPs activities, mRNA expression level of 7 CYPs enzymes were also determined by using real-time PCR. The results showed that all these CYPs isoforms was unequivocally expressed in both HCC tumors and pericarcinomatous tissues. We found in pairwise comparisons in most of cases a loss of CYPs expressions in tumor tissues. The expression of CYP3A4 were drastically decreased by 23-fold in tumors of all subjects. On the other hand, mRNA expression levels of CYP2D6 were decreased less than 3 fold in 42% of subjects by only a more modest extent of 2.5-fold. The average mRNA expression levels of other CYPs isoforms were also found decreased significantly by more than 6 fold. The decrease in CYP mRNA levels in HCC tumors was comparable to that in protein levels determined by LC-MS/MS, suggesting that the downregulation of CYPs levels might result from either decreased transcription of CYP genes or decreased mRNA stability. However, in some tumors, alteration in mRNA of CYPs was not accompanied by a corresponding level of change in its protein amount. This might be due to translation regulation or alteration of protein stability, suggesting that CYP protein synthesis in HCC tumors could be regulated at both the transcriptional and translational levels.We also found that mRNA expressions of seven measured CYPs isoforms were generally correlated to each other in pericarcinomatous tissues. High correlationship was observed between mRNA expressions of CYP2C8 and CYP2C9, which are both transcribed from a cluster of cytochrome P450 genes located on chromosome 10q24, with a correlation coefficient of 0.906. mRNA expression of CYP2A6, which is located on chromosome 19q13, was also found to be highly correlated to that of CYP2C9 and CYP2C8 (r2=0.866 and 0.794, respectively). Medium correlations were observed between CYP3A4 and the other isoforms except for CYP2E1. It is universally acknowledged that CYPs gene expressions are predominantly regulated by nuclear receptors and the target genes of nuclear receptors are usually overlapped. For example, CYP 2C9,2C8 and 2A6 could be regulated by the same nuclear receptors including PXR, CAR, PPARa, and GR, which might explain the high correlation between their mRNA expression levels. However, the correlationships between CYPs mRNA expressions were generally weakened or even lost in HCC tumors, which may caused by the alteration of nuclear receptors involved in the regulations of each CYP expression.4. Correlation of protein expressions level with activities of CYPs enzymes involved in drug metabolism.In this study we have measured the activities and protein expression levels of CYPs enzymes in tumor tissues and matched pericarcinomatous tissues form the same patients with HCC. For all the measured CYP enzymes in pericarcinomatous tissues, high correlations between activities and protein expressions were observed with correlation coefficient values of more than 0.69 (in particular, correlation for CYP2D6 and 2A6 were 0.928 and 0.917, respectively). In HCC tumor tissues, CYPs enzyme showed a similar correlation with correlation coefficient of better than 0.59. This results suggesting that the decline in activity of CYPs enzyme in tumor tissues is due to a decrease in the amount of the enzyme, and the enzyme activities could be well predicted by measuring their protein amounts. Moreover, slopes of correlation between protein and activity of CYP enzymes in tumor tissues was nearly identical to that in pericarcinomatous, suggesting the same catalytic efficiency of CYP enzymes in tumors and pericarcinomatous tissues.High correlationships were also observed between CYP3A4 acitivity and metabolic rate of sorafenib, as well as between CYP2A6 and metabolic rate of tegafur. These results indicated that alterations of CYPs enzymes would strongly impact the metabolic rate of anticancer drugs and their efficacy. Therefore, measuring the activities and protein amounts of these enzymes could well predict the toxicity and efficiency of drug. In this study, a robust, reproducible and reliable LC-MS/MS method capable of simultaneously quantifying 9 or more CYPs and 5 or more UGTs in human liver microsomes. We proposed that protein expression profile of drug metabolism enzymes could be determined by this novel approach in each patient for individualized treatment of HCC.