Folic Acid Ameliorates Renal Injury in Experimental Obstructive Nephropathy: Role of Glycine N-Methyltransferase.

Folic acid exerts both anti-inflammatory and antifibrotic effects. Glycine N-methyltransferase (GNMT), the major folic acid-binding protein in the liver, is a crucial enzyme that regulates the cellular methylation process by maintaining S-adenosylmethionine levels. However, as yet neither the therapeutic effects of folic acid in renal fibrosis nor whether GNMT is involved in these folic acid-associated mechanisms has been investigated. First, the expression of GNMT was examined in human kidneys with or without obstructive nephropathy. Later, wild-type and GNMT knockout (GNMT-/-) mice were subjected to unilateral ureteral obstruction (UUO) and then treated with either folic acid or vehicle for 14 days. Renal tubular injury, inflammation, fibrosis, and autophagy were evaluated by histological analysis and Western blotting. We observed increased expression of GNMT in humans with obstructive nephropathy. Furthermore, UUO significantly increased the expression of GNMT in mice; in addition, it caused renal injury as well as the development of both hydronephrosis and tubular injury. These were all alleviated by folic acid treatment. In contrast, GNMT-/- mice exhibited exacerbated UUO-induced renal injury, but the protective effect of folic acid was not observed in GNMT-/- mice. We propose a novel role for folic acid in the treatment of renal fibrosis, which indicates that GNMT may be a therapeutic target.

Reversal of High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Metformin Combined with PGG, an Inducer of Glycine N-Methyltransferase.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidities and mortality, and no effective drug treatment currently exists. We aimed to develop a novel treatment strategy to induce the expression of glycine N-methyltransferase (GNMT), which is an important enzyme regulating S-adenosylmethionine metabolism whose expression is downregulated in patients with NAFLD. Because 1,2,3,4,6-pentagalloyl glucose (PGG) is a GNMT inducer, and metformin was shown to upregulate liver mitochondrial GNMT protein expression, the effect of PGG and metformin was evaluated. Biochemical analysis, histopathological examination, immunohistochemical staining, reverse transcription-quantitative PCR (RT-qPCR), Western blotting (WB), proteomic analysis and Seahorse XF Cell Mito Stress Test were performed. The high-fat diet (HFD)-induced NAFLD mice were treated with PGG and metformin. Combination of PGG and metformin nearly completely reversed weight gain, elevation of serum aminotransferases, and hepatic steatosis and steatohepatitis. In addition, the downregulated GNMT expression in liver tissues of HFD-induced NAFLD mice was restored. The GNMT expression was further confirmed by RT-qPCR and WB analysis using both in vitro and in vivo systems. In addition, PGG treatment was shown to increase oxygen consumption rate (OCR) maximum capacity in a dose-dependent manner, and was capable of rescuing the suppression of mitochondrial OCR induced by metformin. Proteomic analysis identified increased expression of glutathione S-transferase mu 4 (GSTM4), heat shock protein 72 (HSP72), pyruvate carboxylase (PYC) and 40S ribosomal protein S28 (RS28) in the metformin plus PGG treatment group. Our findings show that GNMT expression plays an important role in the pathogenesis of NAFLD, and combination of an inducer of GNMT and metformin can be of therapeutic potential for patients with NAFLD.

Discovery of an Orally Efficacious MYC Inhibitor for Liver Cancer Using a GNMT-Based High-Throughput Screening System and Structure-Activity Relationship Analysis.

Glycine-N-methyl transferase (GNMT) downregulation results in spontaneous hepatocellular carcinoma (HCC). Overexpression of GNMT inhibits the proliferation of liver cancer cell lines and prevents carcinogen-induced HCC, suggesting that GNMT induction is a potential approach for anti-HCC therapy. Herein, we used Huh7 GNMT promoter-driven screening to identify a GNMT inducer. Compound K78 was identified and validated for its induction of GNMT and inhibition of Huh7 cell growth. Subsequently, we employed structure-activity relationship analysis and found a potent GNMT inducer, K117. K117 inhibited Huh7 cell growth in vitro and xenograft in vivo. Oral administration of a dosage of K117 at 10 mpk (milligrams per kilogram) can inhibit Huh7 xenograft in a manner equivalent to the effect of sorafenib at a dosage of 25 mpk. A mechanistic study revealed that K117 is an MYC inhibitor. Ectopic expression of MYC using CMV promoter blocked K117-mediated MYC inhibition and GNMT induction. Overall, K117 is a potential lead compound for HCC- and MYC-dependent cancers.

Utilizing an Animal Model to Identify Brain Neurodegeneration-Related Biomarkers in Aging.

Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAMe), a methyl donor in methylation. Over-expressed SAMe may cause neurogenic capacity reduction and memory impairment. GNMT knockout mice (GNMT-KO) was applied as an experimental model to evaluate its effect on neurons. In this study, proteins from brain tissues were studied using proteomic approaches, Haemotoxylin and Eosin staining, immunohistochemistry, Western blotting, and ingenuity pathway analysis. The expression of Receptor-interacting protein 1(RIPK1) and Caspase 3 were up-regulated and activity-dependent neuroprotective protein (ADNP) was down-regulated in GNMT-KO mice regardless of the age. Besides, proteins related to neuropathology, such as excitatory amino acid transporter 2, calcium/calmodulin-dependent protein kinase type II subunit alpha, and Cu-Zn superoxide dismutase were found only in the group of aged wild-type mice; 4-aminobutyrate amino transferase, limbic system-associated membrane protein, sodium- and chloride-dependent GABA transporter 3 and ProSAAS were found only in the group of young GNMT-KO mice and are related to function of neurons; serum albumin and Rho GDP dissociation inhibitor 1 were found only in the group of aged GNMT-KO mice and are connected to neurodegenerative disorders. With proteomic analyses, a pathway involving Gonadotropin-releasing hormone (GnRH) signal was found to be associated with aging. The GnRH pathway could provide additional information on the mechanism of aging and non-aging related neurodegeneration, and these protein markers may be served in developing future therapeutic treatments to ameliorate aging and prevent diseases.

Chemical Constituents with GNMT-Promoter-Enhancing and NRF2-Reduction Activities from Taiwan Agarwood Excoecaria formosana.

Hepatocellular carcinoma (HCC) is considered to be a silent killer, and was the fourth leading global cause of cancer deaths in 2018. For now, sorafenib is the only approved drug for advanced HCC treatment. The introduction of additional chemopreventive agents and/or adjuvant therapies may be helpful for the treatment of HCC. After screening 3000 methanolic extracts from the Formosan plant extract bank, Excoecaria formosana showed glycine N-methyltransferase (GNMT)-promoter-enhancing and nuclear factor erythroid 2-related factor 2 (NRF2)-suppressing activities. Further, the investigation of the whole plant of E. formosana led to the isolation of a new steroid, 7α-hydroperoxysitosterol-3-O-ß-d-(6-O-palmitoyl)glucopyranoside (1); two new coumarinolignans, excoecoumarin A (2) and excoecoumarin B (3); a new diterpene, excoeterpenol A (4); and 40 known compounds (5-44). Among them, Compounds 38 and 40-44 at a 100 µM concentration showed a 2.97 ± 0.27-, 3.17 ± 1.03-, 2.73 ± 0.23-, 2.63 ± 0.14-, 6.57 ± 0.13-, and 2.62 ± 0.05-fold increase in GNMT promoter activity, respectively. In addition, Compounds 40 and 43 could reduce NRF2 activity, a transcription factor associated with drug resistance, in Huh7 cells with relative activity of 33.1 ± 0.2% and 45.2 ± 2.5%. These results provided the basis for the utilization of Taiwan agarwood for the development of anti-HCC agents.

Utilizing Experimental Mouse Model to Identify Effectors of Hepatocellular Carcinoma Induced by HBx Antigen.

Hepatocellular carcinoma (HCC) is among the ten most commonly diagnosed cancers and the fourth leading cause of cancer-related death. Patients with hepatitis B virus (HBV) infection are prone to developing chronic liver diseases (i.e., fibrosis and cirrhosis), and the HBV X antigen plays an important role in the development of HCC. The difficulty in detecting HCC at the early stages is one of the main reasons that the death rate approximates the incidence rate. The regulators controlling the downstream liver protein expression from HBV infection are unclear. Mass spectrometric techniques and customized programs were used to identify differentially expressed proteins which may be involved in the development of liver fibrosis and HCC progression in hepatitis B virus X protein transgenic mice (HBx mice). FSTL1, CTSB, and TGF-ß enhanced the signaling pathway proteins during the pathogenesis of HBx. Missing proteins can be essential in cell growth, differentiation, apoptosis, migration, metastasis or angiogenesis. We found that LHX2, BMP-5 and GDF11 had complex interactions with other missing proteins and BMP-5 had both tumor suppressing and tumorigenic roles. BMP-5 may be involved in fibrosis and tumorigenic processes in the liver. These results provide us an understanding of the mechanism of HBx-induced disorders, and may serve as molecular targets for liver treatment.

HIV-1 Tat Interacts with a Kaposi's Sarcoma-Associated Herpesvirus Reactivation-Upregulated Antiangiogenic Long Noncoding RNA, LINC00313, and Antagonizes Its Function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining cancer with abnormal angiogenesis. The high incidence of KS in human immunodeficiency virus (HIV)-infected AIDS patients has been ascribed to an interaction between HIV type 1 (HIV-1) and KSHV, focusing on secretory proteins. The HIV-1 secreted protein HIV Tat has been found to synergize with KSHV lytic proteins to induce angiogenesis. However, the impact and underlying mechanisms of HIV Tat in KSHV-infected endothelial cells undergoing viral lytic reactivation remain unclear. Here, we identified LINC00313 as a novel KSHV reactivation-activated long noncoding RNA (lncRNA) that interacts with HIV Tat. We found that LINC00313 overexpression inhibits cell migration, invasion, and tube formation, and this suppressive effect was relieved by HIV Tat. In addition, LINC00313 bound to polycomb repressive complex 2 (PRC2) complex components, and this interaction was disrupted by HIV Tat, suggesting that LINC00313 may mediate transcription repression through recruitment of PRC2 and that HIV Tat alleviates repression through disruption of this association. This notion was further supported by bioinformatics analysis of transcriptome profiles in LINC00313 overexpression combined with HIV Tat treatment. Ingenuity Pathway Analysis (IPA) showed that LINC00313 overexpression negatively regulates cell movement and migration pathways, and enrichment of these pathways was absent in the presence of HIV Tat. Collectively, our results illustrate that an angiogenic repressive lncRNA, LINC00313, which is upregulated during KSHV reactivation, interacts with HIV Tat to promote endothelial cell motility. These results demonstrate that an lncRNA serves as a novel connector in HIV-KSHV interactions.IMPORTANCE KS is a prevalent tumor associated with infections with two distinct viruses, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the virus-virus interaction associated with KS formation has focused on secretory factors. HIV Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migration-regulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection.

Hepatoma-derived growth factor participates in Helicobacter Pylori-induced neutrophils recruitment, gastritis and gastric carcinogenesis.

Helicobacter pylori (Hp) infection and overexpression of hepatoma-derived growth factor (HDGF) are involved in gastric carcinogenesis. However, the relationship between Hp-induced gastric diseases and HDGF upregulation is not yet completely clear. This study aimed to elucidate the role of HDGF in Hp-induced gastric inflammation and carcinogenesis. HDGF expression in gastric biopsy and serum from patients was analyzed by immunohistochemical and ELISA analysis, respectively. Hp and gastric cells coculture system was employed to delineate the mechanism underlying HDGF overexpression during Hp infection. The gastric pathologies of wild type and HDGF knockout mice after Hp infection were investigated by immunohistochemical, immunoblot, and immunofluorescence analyses. HDGF level was significantly elevated in patients with Hp infection or intestinal metaplasia (IM, a precancerous lesion), and HDGF overexpression was positively correlated with Hp load, IM, and neutrophil infiltration in gastric biopsy. Consistently, patients with Hp infection or IM had significantly higher serum HDGF level. By using coculture assay, Hp infection led to HDGF upregulation and secretion in gastric cells. In mice model, HDGF ablation significantly suppressed the Hp-induced neutrophil infiltration and inflammatory TNF-α/COX-2 signaling, thereby relieving the tissue damage in stomach. This was further supported by that recombinant HDGF (rHDGF) stimulated the differentiation/chemotaxis of cultured neutrophils and oncogenic behaviors of gastric cells. Time series studies showed that Hp infection elicited an inflammatory TNF-α/HDGF/COX-2 cascade in stomach. HDGF secretion by Hp infection promotes the neutrophils infiltration and relays Hp-induced inflammatory signaling. Thus, HDGF may constitute a novel diagnostic marker and therapeutic target for Hp-induced gastritis and carcinogenesis.

Tumor suppressor gene glycine N-methyltransferase and its potential in liver disorders and hepatocellular carcinoma.

Glycine N-methyltransferase is a protein with many functions. In addition to catalyzing the production of sarcosine in the one carbon metabolism pathway, it plays a role in the detoxification of environmental carcinogens such as benzo[a]pyrene, aflatoxin B1, and aristocholic acid. There is also increasing evidence suggesting a role of GNMT deficiency in liver carcinogenesis. In this review, we discuss the role of GNMT in the detoxification of xenobiotics and the mechanism of GNMT suppression during liver tumorigenesis. The protective role of GNMT in the liver allows GNMT to not only serve as a marker of liver disease, but also potentially be applied in the treatment of liver disorders and hepatocellular carcinoma. We describe the potential use of GNMT in gene therapy and we introduce the development of a GNMT promoter reporter assay that can be used to screen medicinal drugs and herbal libraries for natural compounds with anti-cancer properties.

Utilizing proteomic approach to identify nuclear translocation related serine kinase phosphorylation site of GNMT as downstream effector for benzo[a]pyrene.

Glycine N-methyltransferase (GNMT) protein is highly expressed in certain tissues, such as liver, pancreas, and prostate. GNMT serves multiple roles which include a methyl group transfer enzyme and a liver tumor suppressor. Benzo(a)pyrene (BaP), a family member of polycyclic aromatic hydrocarbon (PAH), is a known environmental carcinogen found in coal tar, tobacco smoke, barbecued food and incomplete combustion of auto fuel. BaP recruits cytochrome P450 to transform itself into benzo(a)pyrene-7,8-diol-9,10-epoxide (B(a)PDE), which covalently interacts with DNA causing tumorigenesis. BaP can be detoxified through GNMT and induces GNMT translocation into the cellular nucleus. GNMT translocation is accompanied by phosphorylation, but the role of phosphorylation in GNMT remains to be explored. Using liquid chromatography coupled with tandem mass spectrometry, this study identified serine 9 of GNMT as the phosphorylation site upon BaP treatment. When serine 9 was mutated and lost the capability to be phosphorylated, the occurrence of BaP-induced GNMT nuclear translocation was dramatically decreased. Also, this mutant from of GNMT lost the ability of phosphorylation and increased cytochrome P450 1A1 (Cyp1a) expression upon BaP treatment. In addition, protein kinase C (PKC) and c-Jun NH2-terminal kinase (JNK) may be required for such phosphorylation. Further characterization of phosphorylated GNMT for its link to BaP may bring new insights into chemical detoxification.

Somatic mutations of PREX2 gene in patients with hepatocellular carcinoma.

Characterized with a high recurrence rate and low detection rate, prevention is the best approach to reduce mortality in hepatocellular carcinoma (HCC). The overexpression of Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is observed in various tumors, including HCC; and the frequent PREX2 mutations in melanoma are associated with invasiveness. We sought to identify somatic mutations and the functional changes in mutational signatures of PREX2. Genomic DNA sequencing was performed in 68 HCC samples with three types of hepatitis viral infection status: HBs Ag-positive, anti-HCV Ab-positive, and negative for any hepatitis B or C markers. Stabilities and interactions of proteins as well as cell proliferation and migration were evaluated. Fourteen non-silent point mutations in PREX2 were detected, with 16 of 68 HCC patients harboring at least one non-silent mutation. All mutant forms of PREX2, except for K400f, had an extended half-life compared with wild-type PREX2. Moreover, only the half-life of S1113R was twice that of the wild-type. PREX2 mutant-S1113R also promoted migration and activated the AKT pathway as well as impaired HectH9-mediated ubiquitination. Our study identified a gain-of-function mutation of PREX2 - S1113R in HCC. Such mutation enhanced PREX2 protein stability, promoted cell proliferation, and was associated with aggressiveness of HCC.

Induction of GNMT by 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside through proteasome-independent MYC downregulation in hepatocellular carcinoma.

Glycine-N-methyl transferase (GNMT) a tumor suppressor for hepatocellular carcinoma (HCC) plays a crucial role in liver homeostasis. Its expression is downregulated in almost all the tumor tissues of HCC while the mechanism of this downregulation is not yet fully understood. Recently, we identified 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) as a GNMT promoter enhancer compound in HCC. In this study, we aimed to delineate the mechanism by which PGG enhances GNMT expression and to investigate its effect on GNMT suppression in HCC. Microarray and pathway enrichment analysis revealed that MYC was a major target of PGG. PGG suppressed MYC mRNA and protein expression in Huh7 and Hep G2 cells in a dose- and time-dependent fashion. Furthermore, MYC expression was also reduced in xenograft tumors in PGG treated mice. Moreover, shRNA-mediated knocked-down or pharmacological inhibition of MYC resulted in a significant induction of GNMT promoter activity and endogenous GNMT mRNA expression in Huh7 cells. In contrast, overexpression of MYC significantly inhibited GNMT promoter activity and endogenous GNMT protein expression. In addition, antibodies against MYC effectively precipitated the human GNMT promoter in a chromatin immunoprecipitation assay. Lastly, GNMT expression was negatively correlated with MYC expression in human HCC samples. Interestingly, PGG not only inhibited MYC gene expression but also promoted MYC protein degradation through proteasome-independent pathways. This work reveals a novel anticancer mechanism of PGG via downregulation of MYC expression and establishes a therapeutic rationale for treatment of MYC overexpressing cancers using PGG. Our data also provide a novel mechanistic understanding of GNMT regulation through MYC in the pathogenesis of HCC.

AAV serotype 8-mediated liver specific GNMT expression delays progression of hepatocellular carcinoma and prevents carbon tetrachloride-induced liver damage.

Glycine N-methyltransferase (GNMT) is abundantly expressed in normal livers and plays a protective role against tumor formation. GNMT depletion leads to progression of hepatocellular carcinoma (HCC). In this study, we investigated the activity of ectopic GNMT delivered using recombinant adeno-associated virus (AAV) gene therapy in mouse models of liver cirrhosis and HCC. Injection of AAV serotype 8 (AAV8) vector carrying the GNMT gene (AAV8-GNMT) in Gnmt-/- mice increased GNMT expression and downregulated pro-inflammatory responses, resulting in reduced liver damage and incidence of liver tumors. Moreover, AAV8-GNMT resulted in the amelioration of carbon tetrachloride (CCl4)-induced liver fibrosis in BALB/c mice. We showed that AAV8-GNMT protected hepatocytes from CCl4-induced liver damage.  AAV8-GNMT significantly attenuated the levels of pro-fibrotic markers and increased efficiency of hepatocyte proliferation. These results suggest that correction of hepatic GNMT by gene therapy of AAV8-mediated gene enhancement may provide a potential strategy for preventing and delaying development of liver diseases.

MicroRNA-224 down-regulates Glycine N-methyltransferase gene expression in Hepatocellular Carcinoma.

Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.

Celecoxib enhances the therapeutic efficacy of epirubicin for Novikoff hepatoma in rats.

Epirubicin is a chemotherapy agent for hepatocellular carcinoma (HCC). However, the outcome of HCC patients receiving epirubicin remains unsatisfactory. Moreover, our previous study indicated that celecoxib suppresses HCC progression and liver cancer stemness. This study evaluated the potential of celecoxib to serve as a complementary therapy during epirubicin treatment. Cell proliferation, apoptosis, invasiveness, and anchorage-independent growth were analyzed in hepatoma cells. Therapeutic efficacy was validated in rat orthotopic Novikoff hepatoma. After animal sacrifice, the antitumor mechanism of celecoxib and epirubicin combined therapy was investigated by histological analysis. Celecoxib enhanced the cytotoxic activity of epirubicin in HCC cells by promoting apoptosis. Besides, celecoxib potentiated the antineoplastic function of epirubicin in inhibiting the invasiveness and anchorage-independent growth of HCC cells. Ultrasound monitoring showed that combined therapy was more potent than either therapy alone in perturbing HCC progression. Consistently, the size and weight of dissected HCC tissues from rats receiving combined therapy were smallest among all groups. HCC treated with combined therapy exhibited the highest prevalence of apoptotic cells, which was accompanied by reduced proliferating and angiogenic activities in tumor tissues. Moreover, the expression levels of cancer stemness markers (CD44 and CD133) and drug transporter MDR-1 were significantly diminished in rats receiving combined therapy. Besides, celecoxib treatment increased the infiltration of cytotoxic T lymphocytes (CTLs) and reduced the number of regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and the expression of immune checkpoint PD-L1 in HCC tissues during epirubicin therapy. Celecoxib augmented the therapeutic efficacy while modulated cancer stemness and antitumor immunity. Thus, celecoxib may serve as complementary therapy to improve the outcome of patients with advanced HCC during epirubicin treatment.

Bioactive Triterpenoids from the Leaves and Twigs of Lithocarpus litseifolius and L. corneus.

Phytochemical investigation of the leaves and twigs of Lithocarpus litseifolius and Lithocarpus corneus resulted in the isolation of four new triterpenoids (1: -4: ), three triterpenoids (5: -7: ) isolated from a natural source for the first time, and six known compounds (8: -13: ). In addition, four known triterpenoids (14: -17: ) were isolated from L. corneus. Compound 1: is a 3,4-seco-lupane-type triterpenoid, and compounds 2: -4: are lupane-type triterpenoids in different oxidation states. The structures of all isolated compounds were identified by spectroscopic methods, especially NMR and mass spectrometry data. The absolute configuration of 2: and 3: was confirmed by X-ray single crystallographic analysis. The anti-inflammatory activities of 1: -17: and anti-HIV activities of 2: -17: were evaluated. Among them, 3-epi-betulinic acid (8: ) showed a strong anti-HIV activity comparable to abacavir, a drug used for treating HIV/AIDS. 3,4-seco-4(23),20(29)-lupadiene-3,28-dioic acid (5: ) exhibited potent inhibition of superoxide-anion generation with 86.9 ± 2.8% inhibition at 1 µM.

Characterization of a new murine cell line of sarcomatoid hepatocellular carcinoma and its application for biomarker/therapy development.

Sarcomatoid hepatocellular carcinoma (SHC) is a rare type of HCC with significantly poorer survival than ordinary HCC. Little is known about the mechanism associated with SHC and its biomarkers and therapy. Here, we established a mouse liver cancer cell line and designated as Ymac-1. A sarcomatous appearance was observed in the allograft tumor arose from Ymac-1. Liver-secreted plasma proteins were found in Ymac-1 cultured supernatant by proteomics analysis. The positive staining of CK7, CK8, Vimentin and the suppressed expression of AFP indicated that Ymac-1 is a SHC cell line. Compared to its original tumor, an elevated level of EMT markers, N-cadherin and Vimentin, was found in Ymac-1. Ymac-1 displayed a higher migration rate and side population percentage than a mouse ordinary HCC cell line-Hepa1-6. Microarray analysis was performed to identify potential biomarkers/therapeutic targets for SHC. G6pd, a vital enzyme in pentose phosphate pathway, is highly expressed in Ymac-1. Depletion of G6pd in Ymac-1 reduced CD133 expression and sphere formation. Positive correlations between G6PD and CD133 were observed in human specimen. Higher expression of both G6PD and CD133 in tumor were associated with poor survival. In summary Ymac-1 can be a useful SHC cell model for novel biomarker and therapy development.

Glycine N-methyltransferase deficiency in female mice impairs insulin signaling and promotes gluconeogenesis by modulating the PI3K/Akt pathway in the liver.

BACKGROUND: Glycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt-/-) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice. METHODS: Eleven-week-old Gnmt-/- and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling. RESULTS: Gnmt-/- mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt-/- mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt-/- mice. CONCLUSIONS: Our data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.

Identification of Phosphorylated Cyclin-Dependent Kinase 1 Associated with Colorectal Cancer Survival Using Label-Free Quantitative Analyses.

Colorectal cancer is the most common form of cancer in the world, and the five-year survival rate is estimated to be almost 90% in the early stages. Therefore, the identification of potential biomarkers to assess the prognosis of early stage colorectal cancer patients is critical for further clinical treatment. Dysregulated tyrosine phosphorylation has been found in several diseases that play a significant regulator of signaling in cellular pathways. In this study, this strategy was used to characterize the tyrosine phosphoproteome of colorectal cell lines with different progression abilities (SW480 and SW620). We identified a total of 280 phosphotyrosine (pTyr) peptides comprising 287 pTyr sites from 261 proteins. Label-free quantitative analysis revealed the differential level of a total of 103 pTyr peptides between SW480 and SW620 cells. We showed that cyclin-dependent kinase I (CDK1) pTyr15 level in SW480 cells was 3.3-fold greater than in SW620 cells, and these data corresponded with the label-free mass spectrometry-based proteomic quantification analysis. High level CDK1 pTyr15 was associated with prolonged disease-free survival for stage II colorectal cancer patients (n = 79). Taken together, our results suggest that the CDK1 pTyr15 protein is a potential indicator of the progression of colorectal cancer.

Patients infected by tuberculosis and human immunodeficiency virus facing their disease, their reactions to disease diagnosis and its implication about their families and communities, in Burkina Faso: a mixed focus group and cross sectional study.

BACKGROUND: Patients facing tuberculosis (TB) and human immunodeficiency virus (HIV) infection receive particular care. Despite efforts in the care, misconceptions about TB and HIV still heavily impact patients, their families and communities. This situation severely limits achievement of TB and HIV programs goals. This study reports current situation of TB patients and patients living with HIV/AIDS (PLWHA) facing their disease and its implications, by comparing results from both qualitative and quantitative study design. METHODS: Cross sectional study using mixed methods was used and excluded patients co-infected by TB and HIV. Focus group included 96 patients (6 patients per group) stratified by setting, disease profile and gender; from rural (Orodara Health District) and urban (Bobo Dioulasso) areas, all from Hauts-Bassins region in Burkina Faso. Quantitative study included 862 patients (309 TB patients and 553 PLWHA) attending TB and HIV care facilities in two main regions (Hauts-Bassins and Centre) of Burkina Faso. RESULTS: A content analysis of reports found TB patients and PLWHA felt discriminated and stigmatized because of misconceptions with its aftermaths (rejection, emotional and financial problems), mainly among PLWHA and women patients. PLWHA go to healers when facing limited solutions in health system. There are fewer associations for TB patients, and less education and sensitization sessions to give them opportunity for sharing disease status and learning from other TB patients. TB patients and PLWHA still need to better understand their disease and its implication. Access to care (diagnosis and treatment) remains one of the key issues in health system, especially for PLWHA. Individual counseling is centered among PLWHA but not for TB patients. With research progress and experiences sharing, TB patients and PLWHA have some hope to implement their life project, and to receive psychosocial and nutritional support. CONCLUSION: Despite international aid, TB patients and PLWHA are facing misconceptions effects. There is a need to reinforce health education towards patients and healers, inside community, health centers and associations, and for specific settings. International aid must be adapted to specific targets and strategies implementing programs. Maintaining psychosocial and nutritional support is crucial for better outcomes of medication adherence. Individual counseling has to be centered among TB patients and PLWHA.