Komal Ramani

S-adenosylmethionine inhibits lipopolysaccharide-induced gene expression via modulation of histone methylation†

We previously showed that S‐adenosylmethionine (SAMe) and its metabolite methylthioadenosine (MTA) blocked lipopolysaccharide (LPS)‐induced tumor necrosis factor α (TNFα) expression in RAW (murine macrophage cell line) and Kupffer cells at the transcriptional level without affecting nuclear factor κ B nuclear binding. However, the exact molecular mechanism or mechanisms of the inhibitory effect were unclear. While SAMe is a methyl donor, MTA is an inhibitor of methylation. SAMe can convert to MTA spontaneously, so the effect of exogenous SAMe may be mediated by MTA. The aim of our current work is to examine whether the mechanism of SAMe and MTAs inhibitory effect on proinflammatory mediators might involve modulation of histone methylation. In RAW cells, we found that LPS induced TNFα expression by both transcriptional and posttranscriptional mechanisms. SAMe and MTA treatment inhibited the LPS‐induced increase in gene transcription. Using the chromatin immunoprecipitation assay, we found that LPS increased the binding of trimethylated histone 3 lysine 4 (H3K4) to the TNFα promoter, and this was completely blocked by either SAMe or MTA pretreatment. Similar effects were observed with LPS‐mediated induction of inducible nitric oxide synthase (iNOS). LPS increased the binding of histone methyltransferases Set1 and myeloid/lymphoid leukemia to these promoters, which was unaffected by SAMe or MTA. The effects of MTA in RAW cells were confirmed in vivo in LPS‐treated mice. Exogenous SAMe is unstable and converts spontaneously to MTA, which is stable and cell‐permeant. Treatment with SAMe doubled intracellular MTA and S‐adenosylhomocysteine (SAH) levels. SAH also inhibited H3K4 binding to TNFα and iNOS promoters. Conclusion: The mechanism of SAMes pharmacologic inhibitory effect on proinflammatory mediators is mainly mediated by MTA and SAH at the level of histone methylation. (HEPATOLOGY 2008.)

S-adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model†

Hepatocellular carcinoma (HCC) remains a common cancer worldwide that lacks effective chemoprevention or treatment. Chronic liver disease often leads to impaired hepatic S‐adenosylmethionine (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 SAMes 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 SAMes 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. SAMes chemopreventive effect may be related to its proapoptotic action and its ability to inhibit angiogenesis. (HEPATOLOGY 2009.)

Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice†

Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver‐specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up‐regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh‐7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh‐7 cells did not affect proliferation significantly or sensitize cells to sorafenib‐induced apoptosis. Conclusion: Hepatocyte‐specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. (HEPATOLOGY 2010.)

Leptin's mitogenic effect in human liver cancer cells requires induction of both methionine adenosyltransferase 2A and 2β

Leptin is an adiopokine that plays a pivotal role in the progression of liver fibrogenesis and carcinogenesis. Recently, leptin was shown to be mitogenic in human liver cancer cell lines HepG2 and Huh7. Whether leptin can act as a mitogen in normal hepatocytes is unclear. Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes the formation of S‐adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, whereas a third gene (MAT2β) encodes for a regulatory subunit that modulates the activity of MAT2A‐encoded isoenzyme. The aims of this study were to examine whether leptins mitogenic activity involves MAT2A and MAT2β and whether this can be modulated. We found that leptin is mitogenic in HepG2 cells but not in primary human or mouse hepatocytes. Leptin induced the expression of MAT2A and MAT2β in HepG2 cells and normal human and mouse hepatocytes, but although it increased SAMe level in HepG2 cells, it had no effect on SAMe level in normal hepatocytes. Leptin‐mediated induction of MAT genes and growth in HepG2 cells required activation of extracellular signal‐regulated kinase and phosphatidylinositol‐3‐kinase signaling pathways. Treatment with SAMe or its metabolite methylthioadenosine (MTA) lowered expression of MAT2A and MAT2β and blocked leptin‐induced signaling, including an increase in MAT gene expression and growth. Increased expression of MAT2A and MAT2β is required for leptin to be mitogenic, although by entirely different mechanisms. Conclusion: Leptin induces MAT2A and MAT2β expression in HepG2 cells and normal hepatocytes but is mitogenic only in HepG2 cells. Pharmacological doses of SAMe or MTA lower expression of both MAT2A and MAT2β and interfere with leptin signaling. (HEPATOLOGY 2007.)

Dysregulation of glutathione synthesis during cholestasis in mice: Molecular mechanisms and therapeutic implications

Glutathione (GSH) provides important antioxidant defense and regulates multiple critical processes including fibrogenesis. There are conflicting literature studies regarding changes in GSH during cholestasis. Here we examined changes in the GSH synthetic enzymes during bile duct ligation (BDL) in mice and how treatment with ursodeoxycholic acid (UDCA) and/or S‐adenosylmethionine (SAMe) affects the expression of these enzymes and liver injury. The hepatic expression of glutamate‐cysteine ligase (GCL) subunits and GSH synthase (GS) increased transiently after BDL but fell to 50% of baseline by 2 weeks. Nuclear factor‐erythroid 2‐related factor 2 (Nrf2) trans‐activates gene expression by way of the antioxidant response element (ARE), which controls the expression of all three genes. Despite increased Nrf2 nuclear levels, Nrf2 nuclear binding to ARE fell 2 weeks after BDL. Nuclear levels of c‐Maf and MafG, which can negatively regulate ARE, were persistently induced during BDL and the dominant proteins bound to ARE on day 14. UDCA and SAMe induced the expression of GCL subunits and raised GSH levels. They increased nuclear Nrf2 levels, prevented c‐Maf and MafG induction, and prevented the fall in Nrf2 nuclear binding to ARE. Combined treatment had additive effects, reduced liver cell death, and prevented fibrosis. Conclusion: GSH synthesis falls during later stages of BDL due to lower expression of GSH synthetic enzymes. UDCA and SAMe treatment prevented this fall and combined therapy was more effective on preserving GSH levels and preventing liver injury. (HEPATOLOGY 2009.)

S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers.

Ubiquitin‐conjugating enzyme 9 (Ubc9) is required for sumoylation and is overexpressed in several malignancies, but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S‐adenosyl methionine (SAMe) levels decrease in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol‐fed mice. We examined the regulation of Ubc9 by SAMe in murine liver and human HCC, breast, and colon carcinoma cell lines and specimens. Real‐time polymerase chain reaction and western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by western blotting examined protein‐protein interactions. Ubc9 expression increased in HCC and when hepatic SAMe levels decreased. SAMe treatment in Mat1a KO mice reduced Ubc9 protein, but not messenger RNA (mRNA) levels, and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh7, colon cancer cell line RKO, and breast cancer cell line MCF‐7 with SAMe or its metabolite 5′‐methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 posttranslational regulation is unknown. Ubc9 sequence predicted a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice had higher phosphorylated (phospho)‐Ubc9 levels, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, as well as phospho‐Ubc9 and protein sumoylation in liver, colon, and breast cancer cells. Serine 71 of Ubc9 was required for phosphorylation, interaction with Cdc2, and protein stability. Cdc2, Ubc9, and phospho‐Ubc9 levels increased in human liver, breast, and colon cancers. Conclusion: Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers, and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA. (HEPATOLOGY 2012;56:982–993)

Changes in the expression of methionine adenosyltransferase genes and S-adenosylmethionine homeostasis during hepatic stellate cell activation†

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Methionine adenosyltransferase (MAT) catalyzes biosynthesis of S‐adenosylmethionine (SAMe), the principle methyl donor. SAMe metabolism generates two methylation inhibitors, methylthioadenosine (MTA) and S‐adenosylhomocysteine (SAH). Liver cell proliferation is associated with induction of two nonliver‐specific MATs: MAT2A, which encodes the catalytic subunit α2, and MAT2β, which encodes a regulatory subunit β that modulates the activity of the MAT2A‐encoded isoenzyme MATII. We reported that MAT2A and MAT2β genes are required for liver cancer cell growth that is induced by the profibrogenic factor leptin. Also, MAT2β regulates leptin signaling. The strong association of MAT genes with proliferation and leptin signaling in liver cells led us to examine the role of these genes during HSC activation. MAT2A and MAT2β are induced in culture‐activated primary rat HSCs and HSCs from 10‐day bile duct ligated (BDL) rat livers. HSC activation led to a decline in intracellular SAMe and MTA levels, a drop in the SAMe/SAH ratio, and global DNA hypomethylation. The decrease in SAMe levels was associated with lower MATII activity during activation. MAT2A silencing in primary HSCs and MAT2A or MAT2β silencing in the human stellate cell line LX‐2 resulted in decreased collagen and alpha‐smooth muscle actin (α‐SMA) expression and cell growth and increased apoptosis. MAT2A knockdown decreased intracellular SAMe levels in LX‐2 cells. Activation of extracellular signal‐regulated kinase and phosphatidylinositol‐3‐kinase signaling in LX‐2 cells required the expression of MAT2β but not that of MAT2A. Conclusion: MAT2A and MAT2β genes are induced during HSC activation and are essential for this process. The SAMe level falls, resulting in global DNA hypomethylation. (HEPATOLOGY 2010.)

Forced Expression of Methionine Adenosyltransferase 1A in Human Hepatoma Cells Suppresses in Vivo Tumorigenicity in Mice

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the principal methyl donor, and is encoded by MAT1A and MAT2A in mammals. Normal liver expresses MAT1A, which is silenced in hepatocellular carcinoma. We have shown that hepatoma cells overexpressing MAT1A grew slower, but whether this is also true in vivo remains unknown. To investigate the effect of overexpressing MAT1A on in vivo tumorigenesis, we generated stable transfectants of Huh7 cells overexpressing either MAT1A or empty vector. Real-time PCR and Western blotting were used to measure expression, and BALB/c nude mice were injected subcutaneously with untransfected or Huh7 cells transfected with empty or MAT1A expression vector to establish tumors. Tumor properties such as proliferation, angiogenesis, and apoptosis were compared, and microarray analysis was performed. Huh7 cells overexpressing MAT1A had higher S-adenosylmethionine levels but lower bromodeoxyuridine incorporation than control cells. Tumor growth rates and weights were lower in MAT1A transfected tumors. In addition, microvessel density and CD31 and Ki-67 staining were lower in MAT1A transfected tumors than control tumors, whereas the apoptosis index was higher in MAT1A-transfected tumors. Forced expression of MAT1A induced genes related to apoptosis and tumor suppression and lowered expression of cell growth and angiogenesis proteins. Our data demonstrate in vivo overexpression of MAT1A in liver cancer cells can suppress tumor growth. They also suggest inducing MAT1A expression might be a strategy to treat hepatocellular carcinoma.

S‐adenosylmethionine regulates dual‐specificity mitogen‐activated protein kinase phosphatase expression in mouse and human hepatocytes

Increased mitogen‐activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal‐regulated kinase [ERK]1/2) and the dual‐specificity MAPK phosphatase MKP‐1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S‐adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin‐like and caspase‐like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin‐like and caspase‐like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion: DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. HEPATOLOGY 2010

S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.

BACKGROUND & AIMS Genomic instability participates in the pathogenesis of hepatocellular carcinoma (HCC). Apurinic/apyrimidinic endonuclease 1 (APEX1) participates in the base excision repair of premutagenic apurinic/apyrimidinic (AP) sites. Mice deficient in methionine adenosyltransferase 1a (Mat1a KO) have chronic hepatic deficiency of S-adenosylmethionine (SAMe) and increased oxidative stress, and develop HCC. We examined livers of Mat1a KO mice for genomic instability and dysregulation of APEX1. METHODS Studies were conducted using Mat1a KO mice livers and cultured mouse and human hepatocytes. RESULTS Genomic instability increased in the livers of 1-month-old Mat1a KO mice, compared with wild-type mice, whereas Apex1 mRNA and protein levels were reduced by 20% and 50%, respectively, in Mat1a KO mice of all ages. These changes correlated with increased numbers of AP sites and reduced expression of Bax, Fas, and p21 (all APEX targets). When human and mouse hepatocytes were placed in culture, transcription of MAT1A mRNA decreased whereas that of APEX1 and c-MYC increased. However, the protein levels of APEX1 decreased to 60% of baseline. Addition of 2 mmol/L SAMe prevented increases in APEX1 and c-MYC mRNA levels, as well as decreases in MAT1A expression and cytosolic and nuclear APEX1 protein levels. CONCLUSIONS By 1 month of age, genomic instability increases in livers of Mat1a KO mice, possibly due to reduced APEX1 levels. Although SAMe inhibits APEX1 transcription, it stabilizes the APEX1 protein. This novel aspect of SAMe on APEX1 regulation might explain the chemopreventive action of SAMe and the reason that chronic SAMe deficiency predisposes to HCC.