Transporter Expression in Noncancerous and Cancerous Liver Tissue from Donors with Hepatocellular Carcinoma and Chronic Hepatitis C Infection Quantified by LC-MS/MS Proteomics

Abstract

Protein expression of major hepatobiliary drug transporters (NTCP, OATPs, OCT1, BSEP, BCRP, MATE1, MRPs, and P-gp) in cancerous (C, = 8) and adjacent noncancerous (NC, = 33) liver tissues obtained from patients with chronic hepatitis C with hepatocellular carcinoma (HCV-HCC) were quantified by LC-MS/MS proteomics. Herein, we compare our results with our previous data from noninfected, noncirrhotic (control, = 36) and HCV-cirrhotic ( = 30) livers. The amount of membrane protein yielded from NC and C HCV-HCC tissues decreased (31%, 67%) relative to control livers. In comparison with control livers, with the exception of NTCP, MRP2, and MATE1, transporter expression decreased in NC (38%-76%) and C (56%-96%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP expression increased (113%), MATE1 expression decreased (58%), and MRP2 expression was unchanged relative to control livers. In C HCV-HCC tissues, NTCP and MRP2 expression decreased (63%, 56%) and MATE1 expression was unchanged relative to control livers. Compared with HCV-cirrhotic livers, aside from NTCP, OCT1, BSEP, and MRP2, transporter expression decreased in NC (41%-71%) and C (54%-89%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP and MRP2 expression increased (362%, 142%), whereas OCT1 and BSEP expression was unchanged. In C HCV-HCC tissues, OCT1 and BSEP expression decreased (90%, 80%) relative to HCV-cirrhotic livers, whereas NTCP and MRP2 expression was unchanged. Expression of OATP2B1, BSEP, MRP2, and MRP3 decreased (56%-72%) in C HCV-HCC tissues in comparison with matched NC tissues ( = 8), but the expression of other transporters was unchanged. These data will be helpful in the future to predict transporter-mediated hepatocellular drug concentrations in patients with HCV-HCC.

Protein Abundance of Hepatic Drug Transporters in Patients With Different Forms of Liver Damage

“…In addition to characterizing the state of hepatic drug transporters according to the degree of hepatic insufficiency, the impact of specific types of liver disease on the transporters was analyzed. A clear conclusion is that protein values do not always correlate with mRNA expression, which is in agreement with other reports . Therefore, findings based only on transcriptome analysis do not reflect real changes in transporter abundance.…”

“…In this study, and in other published reports, dissected samples from cirrhotic livers were analyzed, which could have an impact on the reported results. Progression of liver dysfunction is associated with reduction in a number of hepatocytes, while other components of liver structure may expand (depending on liver pathology type and disease stage), i.e., nonparenchymal cells (sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells), cellular components of the extracellular space (fibroblasts and transdifferentiated myofibroblasts) and fibrous connective tissue .…”

“…There is, however, a lack of quantitative data on protein abundance of hepatic transporters in patients with compromised liver function, and most of the studies have been performed using nonprecise methods, such as immunoblotting . In fact, precise quantitative data are limited to hepatitis C–induced cirrhosis and alcoholic cirrhosis, but those studies did not analyze the functional state of the liver. It is an important issue, as function of hepatocytes may be determined by membrane transporters, and liver dysfunction may impact abundance of the transporters.…”

Interindividual and Regional Variability in Drug Transporter Abundance at the Human Blood–Brain Barrier Measured by Quantitative Targeted Proteomics

“…The protein concentration of cell and tissue lysates was measured by the Pierce BCA Protein Assay. The 100–220 μg of protein was reduced (dithiothreitol), denatured (sodium deoxycholate and heat), alkylated (iodoacetamide), precipitated (methanol‐chloroform), and trypsin digested over 16 hours at 37° (enzyme: substrate ratio, 3.2 μg: 220–100 μg) according to our previously published protocol . The tryptic digests were combined with labeled internal standard peptides and formic acid, centrifuged at 4,000 g for 5 minutes, and then the supernatant was analyzed by LC‐MS/MS.…”

Extrahepatic Drug Transporters in Liver Failure: Focus on Kidney and Gastrointestinal Tract

“…However, more precise proteomic quantitative information (mass spectrometry based methods) is also available. Those reports indicate no correlation between expression and protein abundance in the case of many transporters [13,[42][43][44][45][46][47]. Therefore, findings based only on transcriptome analysis may not reflect the real status of transporter abundance.…”