Role of PINCH and Its Partner Tumor Suppressor Rsu-1 in Regulating Liver Size and Tumorigenesis

Particularly interesting new cysteine-histidine-rich protein (PINCH) protein is part of the ternary complex known as the IPP (integrin linked kinase (ILK)-PINCH-Parvin-α) complex. PINCH itself binds to ILK and to another protein known as Rsu-1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu-1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu-1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu-1 suppressed cell proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu-1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype. © 2013 Donthamsetty et al

Modulation of Cytochrome P450 Metabolism and Transport across Intestinal Epithelial Barrier by Ginger Biophenolics

Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE) confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural ‘‘milieu’’ confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G), 8-gingerol (8G), 10-gingerol (10G) and 6-shogaol (6S), through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP) enzyme activity in human liver microsomes by monitoring metabolites of CYPspecific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE’s inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp). Intriguingly, however, 10G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an indepth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens

Differential alterations in mitochondrial function induced by a choline-deficient diet: Understanding fatty liver disease progression

http://www.sciencedirect.com/science/article/B6W8G-4T72WXD-1/2/734055ec11eadc8100fee13e06c683d

Nonalcoholic steatohepatitic (NASH) mice are protected from higher hepatotoxicity of acetaminophen upon induction of PPARa with clofibrate

The objective was to investigate if the hepatotoxic sensitivity in nonalcoholic steatohepatitic mice to acetaminophen (APAP) is due to downregulation of nuclear receptor PPARa via lower cell division and tissue repair. Male Swiss Webster mice fed methionine and choline deficient diet for 31 days exhibited NASH. On the 32nd day, a marginally toxic dose of APAP (360 mg/kg, ip) yielded 70% mortality in steatohepatitic mice, while all non steatohepatitic mice receiving the same dose survived. 14C-APAP covalent binding, CYP2E1 protein, and enzyme activity did not differ from the controls, obviating increased APAP bioactivation as the cause of amplified APAP hepatotoxicity. Liver injury progressed only in steatohepatitic livers between 6 and 24 h. Cell division and tissue repair assessed by 3H-thymidine incorporation and PCNA were inhibited only in the steatohepatitic mice given APAP suggesting that higher sensitivity of NASH liver to APAP-induced hepatotoxicity was due to lower tissue repair. The hypothesis that impeded liver tissue repair in steatohepatitic mice was due to downregulation of PPARa was tested. PPARa was downregulated in NASH. To investigate whether downregulation of PPARa in NASH is the critical mechanism of compromised liver tissue repair, PPARa was induced in steatohepatitic mice with clofibrate (250 mg/kg for 3 days, ip) before injecting APAP. All clofibrate pretreated steatohepatitic mice receiving APAP exhibited lower liver injury, which did not progress and the mice survived. The protection was not due to lower bioactivation of APAP but due to higher liver tissue repair. These findings suggest that inadequate PPARa expression in steatohepatitic mice sensitizes them to APAP hepatotoxicity. © 2008

Nonalcoholic fatty liver sensitizes rats to carbon tetrachloride hepatotoxicity

This study tested whether hepatic steatosis sensitizes liver to toxicant-induced injury and investigated the potential mechanisms of hepatotoxic sensitivity. Male Sprague-Dawley rats were fed a methionine- and choline-deficient diet for 31 days to induce steatosis. On the 32nd day, administration of a nonlethal dose of CCl4 (2 mL/kg, intraperitoneally) yielded 70% mortality in steatotic rats 12-72 hours after CCl4 administration, whereas all nonsteatotic rats survived. Neither CYP2E1 levels nor covalent binding of [14C]CCl4-derived radiolabel differed between the groups, suggesting that increased bioactivation is not the mechanism for this amplified toxicity. Cell division and tissue repair, assessed by [3H]thymidine incorporation and proliferative cell nuclear antigen assay, were inhibited in the steatotic livers after CCl4 administration and led to progressive expansion of liver injury culminating in mortality. The hypothesis that fatty hepatocytes undergo cell cycle arrest due to (1) an inability to replenish ATP due to overexpressed uncoupling protein-2 (UCP-2) or (2) induction of growth inhibitor p21 leading to G1/S phase arrest was tested. Steatotic livers showed 10-fold lower ATP levels due to upregulated UCP-2 throughout the time course after CCl 4 administration, leading to sustained inhibition of cell division. Western blot analysis revealed an up-regulation of p21 due to overexpression of TGF ß1 and p53 and downregulation of transcription factor Foxm1b in steatotic livers leading to lower phosphorylated retinoblastoma protein. Thus, fatty hepatocytes fail to undergo compensatory cell division, rendering the liver susceptible to progression of liver injury. Conclusion: Impaired tissue repair sensitizes the steatotic livers to hepatotoxicity. Copyright © 2007 by the American Association for the Study of Liver Diseases