Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tertButylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidantresponsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-B family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-B binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-B family members.Glutathione (GSH) is the main nonprotein thiol in mammalian cells that participates in many critical cellular functions, including antioxidant defense and cell growth (14,24,28). The synthesis of GSH from its constituent amino acids involves two ATP-requiring enzymatic steps: the formation of ␥-glutamylcysteine from glutamate and cysteine and the formation of GSH from ␥-glutamylcysteine and glycine. The first step of GSH biosynthesis is rate limiting and catalyzed by glutamatecysteine ligase (GCL, also known as ␥-glutamylcysteine synthetase), while the second step is catalyzed by GSH synthetase (14). The GCL enzyme is composed of a catalytic (GCLC, M r of ϳ73,000) and a modifier (GCLM, M r of ϳ30,000) subunit which are encoded by different genes and dissociate under reducing conditions (7,27,35). The catalytic subunit exhibits all of the catalytic activity of the isolated enzyme as well as feedback inhibition by GSH (27). The modifier subunit is enzymatically inactive but plays an important regulatory function by lowering the K m of GCL for glutamate and raising the K i for GSH (7,8). GCL is a major determinant of the overall GSH synthesis capacity, and changes in GCL activity can result from regulation at multiple levels affecting only the catalytic or modifier subunit or both (14). Both human GCLC and GCLM promoters have been cloned (4,5,16,18,34). Antioxidantresponse element (ARE, also known as electrophile response element, EpRE) and activator protein 1 (AP-1) are two cisacting elements present in the promoter of both human GCL subunits that have been implicated in their transcriptional regulation by oxidants and -naphthoflavone (5, 14, 16, 18).Nrf1 and Nrf2, members of the cap 'n' collar-basic leu...
Hepatocellular carcinoma (HCC) remains a common cancer worldwide that lacks effective chemoprevention or treatment. Chronic liver disease often leads to impaired hepatic Sadenosylmethionine (SAMe) biosynthesis, and mice with SAMe deficiency develop HCC spontaneously. SAMe is antiapoptotic in normal hepatocytes but proapoptotic in cancerous hepatocytes. The present study investigated SAMe's effectiveness in prevention and treatment of HCC. Two weeks after injecting 2.5 million H4IIE cells into the liver parenchyma of ACI rats, they typically form a 1-cm tumor. When SAMe (150 mg/kg/day) was delivered through continuous intravenous infusion, hepatic SAMe levels reached 0.7 mM (over 10-fold) 24 hours later. This regimen, started 1 day after injecting H4IIE cells and continued for 10 days, was able to reduce tumor establishment and growth. However, if intravenous SAMe was started after HCC had already developed, it was ineffective in reducing tumor growth for 24 days. Although plasma SAMe levels remained elevated, hepatic SAMe levels were minimally increased (30% higher). Chronic SAMe administration led to induction of hepatic methyltransferases, which prevented SAMe accumulation. To see if SAMe's preventive effect on tumor establishment involves angiogenesis, the effect of SAMe on angiogenesis genes was studied. SAMe treatment of H4IIE cells altered the expression of several genes with the net effect of inhibiting angiogenesis. These changes were confirmed at the protein level and functionally in human umbilical vein endothelial cells. Conclusion: SAMe is effective in preventing HCC establishment but ineffective in treating established HCC because of induction of hepatic methyltransferases, which prevents SAMe level to reach high enough to kill liver cancer cells. SAMe's chemopreventive effect may be related to its proapoptotic action and its ability to inhibit angiogenesis. (HEPATOLOGY 2009;50:462-471.) H epatocellular carcinoma (HCC) is the fifth most common cancer and the third most frequent cause of cancer death worldwide. 1 The incidence of HCC is expected to rise due to the high prevalence of chronic hepatitis C in many parts of the world. 2 Only 30% of HCC patients are considered candidates for surgical resection, and the overall 5-year survival for HCC patients is less than 10%. 3 Treatments for advanced HCC
GSH synthesis occurs via two enzymatic steps catalysed by GCL [glutamate-cysteine ligase, made up of GCLC (GCL catalytic subunit), and GCLM (GCL modifier subunit)] and GSS (GSH synthetase). Co-ordinated up-regulation of GCL and GSS further enhances GSH synthetic capacity. The present study examined whether TNFalpha (tumour necrosis factor alpha) influences the expression of rat GSH synthetic enzymes. To facilitate transcriptional studies of the rat GCLM, we cloned its 1.8 kb 5'-flanking region. TNFalpha induces the expression and recombinant promoter activities of GCLC, GCLM and GSS in H4IIE cells. TNFalpha induces NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein 1) nuclear-binding activities. Blocking AP-1 with dominant negative c-Jun or NF-kappaB with IkappaBSR (IkappaB super-repressor, where IkappaB stands for inhibitory kappaB) lowered basal expression and inhibited the TNFalpha-mediated increase in mRNA levels of all three genes. While all three genes have multiple AP-1-binding sites, only GCLC has a NF-kappaB-binding site. Overexpression with p50 or p65 increased c-Jun mRNA levels, c-Jun-dependent promoter activity and the promoter activity of GCLM and GSS. Blocking NF-kappaB also lowered basal c-Jun expression and blunted the TNFalpha-mediated increase in c-Jun mRNA levels. TNFalpha treatment resulted in increased c-Jun and Nrf2 (nuclear factor erythroid 2-related factor 2) nuclear binding to the antioxidant response element of the rat GCLM and if this was prevented, TNFalpha no longer induced the GCLM promoter activity. In conclusion, both c-Jun and NF-kappaB are required for basal and TNFalpha-mediated induction of GSH synthetic enzymes in H4IIE cells. While NF-kappaB may exert a direct effect on the GCLC promoter, it induces the GCLM and GSS promoters indirectly via c-Jun.
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