Significantly Decreased and More Variable Expression of Major CYPs and UGTs in Liver Microsomes Prepared from HBV-Positive Human Hepatocellular Carcinoma and Matched Pericarcinomatous Tissues Determined Using an Isotope Label-free UPLC-MS/MS Method

Yan, Tingting; Gao, Song; Peng, Xiaojuan; Shi, Jian; Xie, Cong; Li, Qiang; Lu, Linlin; Wang, Ying; Zhou, Fuyuan; Liu, Zhongqiu; Hu, Ming

doi:10.1007/s11095-014-1525-x

Cited by 41 publications

(51 citation statements)

References 33 publications

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“…The biologic impact of locally reduced glucuronidation capacity in diseased tissues remains to be examined but could be of clinical relevance to susceptibility to kidney cancer or treatment of kidney cancer. The important loss of UGT1A9-and UGT2B7-conjugating activity in tumor kidneys is in line with the reduced expression of several UGT1A and UGT2B7 observed at both mRNA and protein levels in hepatocellular carcinomas (Strassburg et al, 1997;Yan et al, 2014;Ye et al, 2014), UGT1A10 and UGT2B7 in breast cancer Fig. 6.…”

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confidence: 55%

Quantitative Profiling of Human Renal UDP-glucuronosyltransferases and Glucuronidation Activity: A Comparison of Normal and Tumoral Kidney Tissues

et al. 2015

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Renal metabolism by UDP-glucuronosyltransferase (UGT) enzymes is central to the clearance of many drugs. However, significant discrepancies about the relative abundance and activity of individual UGT enzymes in the normal kidney prevail among reports, whereas glucuronidation in tumoral kidney has not been examined. In this study, we performed an extensive profiling of glucuronidation metabolism in normal (n = 12) and tumor (n = 14) kidneys using targeted mass spectrometry quantification of human UGTs. We then correlated UGT protein concentrations with mRNA levels assessed by quantitative polymerase chain reaction and with conjugation activity for the major renal UGTs. Beyond the wide interindividual variability in expression levels observed among kidney samples, UGT1A9, UGT2B7, and UGT1A6 are the most abundant renal UGTs in both normal and tumoral tissues based on protein quantification. In normal kidney tissues, only UGT1A9 protein levels correlated with mRNA levels, whereas UGT1A6, UGT1A9, and UGT2B7 quantification correlated significantly with their mRNA levels in tumor kidneys. Data support that posttranscriptional regulation of UGT2B7 and UGT1A6 expression is modulating glucuronidation in the kidney. Importantly, our study reveals a significant decreased glucuronidation capacity of neoplastic kidneys versus normal kidneys that is paralleled by drastically reduced UGT1A9 and UGT2B7 mRNA and protein expression. UGT2B7 activity is the most repressed in tumors relative to normal tissues, with a 96-fold decrease in zidovudine metabolism, whereas propofol and sorafenib glucuronidation is decreased by 7.6-and 5.2-fold, respectively. Findings demonstrate that renal drug metabolism is predominantly mediated by UGT1A9 and UGT2B7 and is greatly reduced in kidney tumors.

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confidence: 55%

Quantitative Profiling of Human Renal UDP-glucuronosyltransferases and Glucuronidation Activity: A Comparison of Normal and Tumoral Kidney Tissues

et al. 2015

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“…Liver microsomes of tumors (tHLMs-individual) and matched pericarcinomatous tissues (nHLMs-individual) from 25 donors were processed using standard differential centrifugation procedures, which is essentially the same as those described previously in our publication [19]. A portion of 15 tHLMs-individual or matched nHLMs-individual were pooled together, named as nHLMs-pooled or tHLMs-pooled, based on equivalent protein amount to ensure the mixture represent the average of 15 individuals.…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression levels of the seven CYP isoforms were determined simultaneously by using an isotope label-free LC-MS/MS method as described previously, with some minor modifications [19]. Protein expression levels were determined by quantifying proteotypic peptides produced by (high purity) trypsin digestion, using synthetic standard peptides (APeptide Co., Shanghai, China).…”

Section: Methodsmentioning

confidence: 99%

“…And these MS-based protein quantification methods were highlighted as a promising approach for basic research of drug development such as drug-drug interaction study and PK/PD modeling. We recently also developed an isotope label-free LC-MS/MS method to determine the absolute amounts of CYPs and UGTs isoforms in human liver tissues and the results showed that this method was accurate and reproducible, and could lead to a better understanding of hepatic disposition and metabolism of drugs [19]. …”

Section: Introductionmentioning

confidence: 99%

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Severely Impaired and Dysregulated Cytochrome P450 Expression and Activities in Hepatocellular Carcinoma: Implications for Personalized Treatment in Patients

Yan

Xie

et al. 2015

Molecular Cancer Therapeutics

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This study aims to systematically determine the activities and expressions of cytochrome P450s (CYP) in hepatocellular carcinoma (HCC) patients to support their optimal use in personalized treatment of HCC. Activities of seven major drug-metabolizing CYP enzymes (CYP1A2, 2A6, 2C8, 2C9, 2D6, 2E1, and 3A4) were determined in tumors and pericarcinomatous tissues harvested from 26 patients with hepatitis B virus-positive HCC using probe substrates. Protein and mRNA levels of these CYPs were also measured using isotope label-free LC/MS-MS method and realtime PCR, respectively. Maximal metabolic velocity (V max ) of CYP probe substrates was decreased by 2.5-to 30-fold in tumor microsomes, accompanied by a corresponding decrease in their protein and mRNA expression levels. However, K m values and turnover numbers of substrates in tumor microsomes were not changed. High correlations between activities and CYP protein levels were also observed, but the correlation between activities and mRNA levels was often poor. There was a major decrease in the degree of correlation in CYP expression in tumor tissues, suggesting that CYP expression levels are greatly disrupted by the tumorigenic process. Our unprecedented systemic study of the effects of HCC on CYPs demonstrated that activities of CYPs were seriously impaired and their expression patterns were severely altered by HCC. We proposed that determination of the CYP protein expression profile by LC/MS-MS in each patient is a promising approach that can be clinically used for individualized treatment of HCC.

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“…However, significant quantitative inaccuracy arises from the lack of correlation between mRNA and protein expression levels and the high sequence similarity among UGTs (Margaillan et al, 2015). A number of research groups including ours have developed mass spectrometry-based methods to quantify UGTs in human tissues (Table 1) (Harbourt et al, 2012;Ohtsuki et al, 2012;Fallon et al, 2013b;Achour et al, 2014;Groer et al, 2014;Sato et al, 2014;Yan et al, 2015). Despite the higher precision and specificity in UGT quantification, a strong interindividual variability in the expression profile of all UGTs has been highlighted by all studies; consequently, substantial differences in UGT quantification have arisen from the different studies.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential

et al. 2015

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Phase II metabolism is prominently governed by UDPglucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30-34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%-184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential. IntroductionThe liver is a key organ for the metabolism of drugs and endogenous compounds such as hormones and bile acids. Through the expression of a complex cocktail of enzymes such as uridine 59-diphosphoglucuronosyltransferases (UGTs), the biochemical properties and bioactivity of these compounds are highly regulated by the liver. Glucuronidation contributes 35% of the phase II drug metabolic pathways and is involved in the clearance of 55% of the 200 most prescribed drugs (Guillemette et al., 2014). The liver expresses a diversified array of UGTs, with 12 of the 16 UGT1A and UGT2B proteins (Guillemette et al., 2014).The expression profile of UGTs was first described using various techniques based on mRNA and immunochemical quantification (Court et al., 2012). However, significant quantitative inaccuracy arises from the lack of correlation between mRNA and protein expression levels and the high sequence similarity among UGTs (Margaillan et al., 2015). A number of research groups including ours have developed mass spectrometry-based methods to quantify UGTs in human tissues (Table 1) (Harbourt et al., 2012;Ohtsuki et al., 2012;Fallon et al., 2013b;Achour et al., 2014; Gr...

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Significantly Decreased and More Variable Expression of Major CYPs and UGTs in Liver Microsomes Prepared from HBV-Positive Human Hepatocellular Carcinoma and Matched Pericarcinomatous Tissues Determined Using an Isotope Label-free UPLC-MS/MS Method

Cited by 41 publications

References 33 publications

Quantitative Profiling of Human Renal UDP-glucuronosyltransferases and Glucuronidation Activity: A Comparison of Normal and Tumoral Kidney Tissues

Quantitative Profiling of Human Renal UDP-glucuronosyltransferases and Glucuronidation Activity: A Comparison of Normal and Tumoral Kidney Tissues

Severely Impaired and Dysregulated Cytochrome P450 Expression and Activities in Hepatocellular Carcinoma: Implications for Personalized Treatment in Patients

Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential

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