Shiyan Wang

Inhibitory role of peroxisome proliferator-activated receptor gamma in hepatocarcinogenesis in mice and in vitro†

Although peroxisome proliferator‐activated receptor gamma (PPARγ) agonist have been shown to inhibit hepatocellular carcinoma (HCC) development, the role of PPARγ in hepatocarcinogenesis remains unclear. We investigated the therapeutic efficacy of PPARγ against HCC. PPARγ‐deficient (PPARγ+/−) and wild‐type (PPARγ+/+) littermates were used in a diethylnitrosamine (DEN)‐induced HCC model and treated with PPARγ agonist (rosiglitazone) or the vehicle alone for 8 months. The effects of PPARγ on HCC cell growth and apoptosis were examined using PPARγ‐expressing adenovirus (Ad‐PPARγ). PPARγ+/− mice were more susceptible to DEN‐induced HCC than PPARγ+/+ mice (94% versus 62%, P < 0.05), and rosiglitazone significantly reduced the incidence of HCC in PPARγ+/+ mice (vehicle 62% versus treatment 24%, P < 0.01), but not in PPARγ+/− mice, indicating that PPARγ suppresses hepatocellular carcinogenesis. A pronounced expression of PPARγ was observed in a HCC cell line (Hep3B) infected with Ad‐PPARγ. Such induction markedly suppressed HCC cell viability (P < 0.01). Further, Hep3B infection with Ad‐PPARγ revealed a decreased proportion of cells in S‐phase (12.92% versus 11.58%, P < 0.05), with arrest at G2/M phase (38.2% versus 55.68%, P < 0.001), and there was concomitant phosphorylation of the key G2/M phase inhibitors cdc25C and cdc2. PPARγ overexpression increased cell apoptosis (21.47% versus 35.02%, P < 0.01), mediated by both extrinsic (Fas and tumor necrosis factor‐α) and intrinsic (caspase‐9, caspase‐3, caspase‐7, and poly[ADP‐ribose] polymerase) pathways. Moreover, PPARγ directly induced a putative tumor suppressor gene, growth differentiation factor‐15. Conclusion: Loss of one PPARγ allele is sufficient to enhance susceptibility to HCC. PPARγ suppresses tumor cell growth through reducing cell proliferation and inducing G2/M phase arrest, apoptosis, and up‐regulating growth differentiation factor‐15. Thus, PPARγ acts as a tumor‐suppressor gene in the liver. HEPATOLOGY 2010

Heme Oxygenase-1 Protects Against Steatohepatitis in Both Cultured Hepatocytes and Mice

BACKGROUND & AIMS Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced tissue injury. We investigated the role of HO-1 in nutritional steatohepatitis in vitro and in vivo. METHODS AML-12 hepatocytes were cultured in methionine- and choline-deficient (MCD) medium. Cells were transfected with an adenovirus vector that expressed HO-1 (Ad-HO-1) or incubated with the HO-1 inducer hemin or the HO-1 inhibitor stannic mesoporphyrin for 24 hours. C57BL6 mice and db/db mice were fed MCD or control diets, with or without hemin, for up to 4 weeks. RESULTS AML-12 cells exposed to MCD medium developed significant steatosis, increased release of alanine aminotransferase, and showed signs of oxidative injury. Incubation with hemin induced HO-1 protein, suppressed steatosis, and reduced levels of alanine aminotransferase and lipid peroxidation. A comparable effect was observed in cells transfected with Ad-HO-1, whereas incubation of these cells with stannic mesoporphyrin completely abolished the Ad-HO-1- or hemin-mediated protection of hepatocytes. Mice injected with hemin significantly attenuated MCD-induced steatohepatitis and increased HO-1 protein and activity. This effect was associated with up-regulation of antioxidant chaperones and enzymes, down-regulation of proinflammatory cytokines, and up-regulation of the anti-inflammatory interleukin-22. Moreover, the reduction in steatosis caused by hemin was affected by up-regulation of peroxisome proliferator-activated receptor-alpha and by down-regulation of sterol regulatory element binding protein-1c. CONCLUSIONS HO-1 can interrupt progression of nutritional steatohepatitis by inducing an antioxidant pathway, suppressing production of cytokines, and modifying fatty acid turnover. Induction of HO-1 might provide a new approach for treatment of steatohepatitis.

ADAMTS9 is a functional tumor suppressor through inhibiting AKT/mTOR pathway and associated with poor survival in gastric cancer.

Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is methylated in cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. ADAMTS9 was silenced in 6 out of 8 gastric cancer cell lines. The loss of ADAMTS9 expression was regulated by promoter hypermethylation and could be restored by demethylation agent. Ectopic expression of ADAMTS9 in gastric cancer cell lines (AGS, BGC823) inhibited cell growth curve in both the cell lines (P<0.0001), suppressed colony formation (P<0.01) and induced apoptosis (P<0.001 in AGS, P<0.01 in BGC823). Moreover, conditioned culture medium from ADAMTS9-transfected cell lines significantly disrupted the human umbilical vein endothelial cell tube formation capacity on Matrigel (P<0.01 in AGS, P<0.001 in BGC823). The in vivo growth of ADAMTS9 cells in nude mice was also markedly diminished after stable expression of ADAMTS9 (P<0.001). On the other hand, ADAMTS9 knockdown promoted cell proliferation (P<0.001). We further revealed that ADAMTS9 inhibited tumor growth by blocking activation of Akt and its downstream target the mammalian target of rapamycin (mTOR). ADAMTS9 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase, eIF4E-binding protein and downregulated hypoxia-inducible factor-1α. Therefore, this is the first demonstration that ADAMTS9 is a critical tumor suppressor of gastric cancer progression at least in part through suppression of oncogenic AKT/mTOR signaling. Moreover, promoter methylation of ADAMTS9 was detected in 29.2% (21/72) of primary gastric tumors. Multivariate analysis showed that patients with ADAMTS9 methylation had a poorer overall survival (relative risk (RR)=2.788; 95% confidence interval, 1.474–5.274; P=0.002). Kaplan–Meier survival curves showed that ADAMTS9 methylation was significantly associated with shortened survival in gastric cancer patients (P=0.001, log-rank test). In conclusion, ADAMTS9 acts as a functional tumor suppressor in gastric cancer through inhibiting oncogenic AKT/mTOR signaling pathway. Methylation of ADAMTS9 is an independent prognostic factor of gastric cancer.

Zinc-finger protein 545 is a novel tumour suppressor that acts by inhibiting ribosomal RNA transcription in gastric cancer

Objective Zinc-finger protein 545 (ZNF545) is a member of the family of Krüppel-associated box-containing zinc-finger proteins. The aim of this study was to clarify its biological function as a tumour suppressor in gastric cancer. Design The biological function of ZNF545 was determined by cell growth and apoptosis assays. The ZNF545 target signal pathway was identified by promoter luciferase assay, northern blot, run-on transcription assay, chromatin immunoprecipitation and coimmunoprecipitation assays. The clinical application of ZNF545 was assessed in primary gastric cancers. Results ZNF545 was silenced or reduced in 16 out of 18 gastric cancer cell lines by promoter hypermethylation. Restoration of ZNF545 expression in gastric cancer cell lines suppressed cell proliferation and induced apoptosis. These effects of ZNF545 were attributed to inhibition of ribosomal RNA (rRNA) transcription. Inhibition of rRNA transcription by ZNF545 was further revealed to be associated with direct ribosomal DNA (rDNA) promoter binding, recruitment of the corepressor, heterochromatin protein 1β, and reduction of trimethylated histone H3 at the Lys4 residue at the rDNA locus. ZNF545 methylation was detected in 51.9% (41/79) of gastric cancer tissues, 27.0% (20/74) of adjacent non-tumour gastric tissues (p=0.001), but none of 20 normal controls. Multivariate analysis revealed that patients with ZNF545 methylation had a significant decrease in overall survival. Kaplan–Meier survival curves showed that ZNF545 methylation was significantly associated with shortened survival in patients with stage I–II gastric cancer. Conclusions ZNF545 acts as a functional tumour suppressor in gastric cancer by inhibiting rRNA transcription. Its methylation at early stages of gastric carcinogenesis is an independent prognostic factor.

Phyllanthus urinaria ameliorates the severity of nutritional steatohepatitis both in vitro and in vivo

Hepatic oxidative stress plays a critical role in metabolic forms of steatohepatitis. Phyllanthus urinaria, an herbal medicine, has been reported to have potential antioxidant properties. We tested the effects of P. urinaria on nutritional steatohepatitis both in vitro and in vivo. Immortalized normal hepatocytes (AML‐12) or primary hepatocytes were exposed to control, the methionine‐and‐choline‐deficient (MCD) culture medium, in the presence or absence of P. urinaria for 24 hours. Hepatocyte triglyceride, release of alanine aminotransferase, lipoperoxides, and reactive oxygen species production were determined. Age‐matched C57BL/6 and db/db mice were fed control or MCD diet for 10 days with or without P. urinaria. Hepatic steatosis, necroinflammation, triglycerides, and lipid peroxide levels were determined. Hepatic expression of inflammatory factors and lipid regulatory mediators were assayed. P. urinaria reduced steatosis and alanine aminotransferase (ALT) levels in culture of hepatocytes in a dose‐dependent manner. Phyllanthus prevented MCD‐induced hepatic fat accumulation and steatohepatitis in mice. This effect was associated with repressed levels of hepatic lipid peroxides, reduced expression of cytochrome P450‐2E1, pro‐inflammatory tumor necrosis factor alpha, interleukin‐6, dampened activation of inflammatory c‐Jun N‐terminal kinase (JNK) and nuclear factor kappa B (NF‐κB), increased expression of lipolytic cytochrome P450 (Cyp4a10), and suppressed transcriptional activity of lipogenic CCAAT/enhancer binding protein β (C/EBPβ). Hepatic acyl co‐enzyme A oxidase that regulated hepatic beta‐oxidation of fatty acid and other lipid regulators were not affected by P. urinaria. In conclusion, P. urinaria effectively alleviated the steatohepatitis induced by the MCD, probably through dampening oxidative stress, ameliorating inflammation, and decreasing lipid accumulation. (HEPATOLOGY 2008.)

Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer.

Zinc-finger protein 331 (ZNF331), a Kruppel-associated box zinc-finger protein gene, was identified as a putative tumor suppressor in our previous study. However, the role of ZNF331 in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. ZNF331 was silenced or downregulated in 71% (12/17) gastric cancer cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancer tissues. In contrast, ZNF331 was readily expressed in various normal adult tissues. The downregulation of ZNF331 was closely linked to the promoter hypermethylation as evidenced by methylation-specific PCR, bisulfite genomic sequencing and reexpression by demethylation agent treatment. DNA sequencing showed no genetic mutation/deletion of ZNF331 in gastric cancer cell lines. Ectopic expression of ZNF331 in the silenced cancer cell lines MKN28 and HCT116 significantly reduced colony formation and cell viability, induced cell cycle arrests and repressed cell migration and invasive ability. Concordantly, knockdown of ZNF331 increased cell viability and colony formation ability of gastric cancer cell line MKN45. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach were applied to analyze the molecular basis of the biological functions of ZNF331. In all, 10 downstream targets of ZNF331 were identified to be associated with regulation of cell growth and metastasis. The tumor-suppressive effect of ZNF331 is mediated at least by downregulation of genes involved in cell growth promotion (DSTN, EIF5A, GARS, DDX5, STAM, UQCRFS1 and SET) and migration/invasion (DSTN and ACTR3), and upregulation of genome-stability gene (SSBP1) and cellular senescence gene (PNPT1). A novel target of ZNF331 (DSTN) was functionally validated. Overexpression of DSTN in BGC-823 cells increased colony formation and migration ability. In conclusion, our results suggest that ZNF331 possesses important functions for the suppression of gastric carcinogenesis as a novel functional tumor-suppressor gene.

Yin Yang 1 contributes to gastric carcinogenesis and its nuclear expression correlates with shorter survival in patients with early stage gastric adenocarcinoma

BackgroundYin Yang 1 (YY1) is a transcription factor that regulates diverse biological processes and increasing recognized to have important roles in carcinogenesis. The function and clinical significance of YY1 in gastric adenocarcinoma (GAC) have not been elucidated.MethodsIn this study, the functional role of YY1 in gastric cancer was investigated by MTT proliferation assays, monolayer colony formation, cell cycle analysis, signaling pathway analysis, Western blot analysis and in vivo study through YY1 knockdown or overexpression. Immunohistochemical study with YY1 antibody was performed on tissue microarray consisting of 247 clinical GAC samples. The clinical correlation and prognosis significance were evaluated.ResultsYY1 expression was up-regulated in gastric cancer cell lines and primary gastric cancers. Knocking down YY1 by siYY1 inhibited cell growth, inducing G1 phase accumulation and apoptosis. Ectopic YY1 expression enhanced cell proliferation in vitro and in vivo. Knocking down YY1 in gastric cancer cells suppressed proliferation by inhibiting Wnt/β-catenin pathway, whereas its overexpression exerted oncogenic property by activating Wnt/β-catenin pathway. In primary GAC samples, YY1 nuclear expression correlated with shorter survival and predicted poor prognosis in early stage GACs.ConclusionOur data demonstrated that YY1 contributes to gastric carcinogenesis in gastric cancer. In early stage GACs YY1 might serve as a poor prognostic marker and possibly as a potential therapeutic target.

Dapper homolog 1 is a novel tumor suppressor in gastric cancer through inhibiting the nuclear factor-κB signaling pathway.

Dapper homolog 1 (DACT1) is a disheveled partner in the planar cell polarity pathway. By using genome-wide promoter methylation screening, dapper homolog 1 (DACT1) was found to be frequently methylated in gastric cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. We demonstrated that DACT1 was silenced in 7 of 10 gastric cancer cell lines and in primary gastric cancers. Transcriptional gene silence of DACT1 was mainly regulated by promoter hypermethylation. Ectopic expression of DACT1 in silenced gastric cancer cell lines (AGS, BGC823 and MGC803) by stable transfection suppressed colony formation (P < 0.001), induced cell apoptosis (P < 0.01) and retarded tumorigenesis in nude mice (P < 0.001). The tumor suppressive effect of DACT1 was further confirmed by loss of DACT1 function experiment. The proapoptotic and antiproliferative effect by DACT1 was associated with inhibition of nuclear factor (NF)-κB activation and its downstream factors, including B-cell CLL/lymphoma-2, Bcl-X, interleukin-8 and tumor necrosis factor-α. Moreover, promoter methylation of DACT1 was detected in 29.3% (60/205) of primary gastric tumors. DACT1 methylation was significantly associated with tumor metastasis (P < 0.05), invasion (P < 0.05) and advanced tumor stage (P < 0.0005). These findings provided insight into the role of DACT1 as a novel functional tumor suppressor in gastric cancer through inhibiting NF-κB signaling pathway. Promoter methylation of DACT1 is associated with tumor aggressiveness.

DACT2 is a functional tumor suppressor through inhibiting Wnt/β-catenin pathway and associated with poor survival in colon cancer

Dapper homolog (DACT) 2 is one of the Dact gene family members, which are important modulators of Wnt signaling pathway. We aim to clarify its epigenetic inactivation, biological function and clinical implication in colon cancer. DACT2 was silenced in five out of eight colon cancer cell lines, but robustly expressed in normal colon tissues. The loss of DACT2 expression was regulated by promoter hypermethylation. Restoring DACT2 expression in colon cancer cell lines suppressed tumor cell growth by inducing cell apoptosis and inhibiting cell proliferation both in vitro and in vivo. Moreover, DACT2 overexpression effectively reduced lung metastasis of colon cancer cells in nude mice. These effects by DACT2 were attributed to inhibition of Wnt/β-catenin signaling. Reexpression of DACT2 significantly suppressed the transcriptional activity of both wild-type β-catenin and degradation-resistant form mutant β-catenin (S33Y). DACT2 could actively shuttle into and out of nuclei, with its predominant steady-state localization in the cytoplasm dependent on its nuclear export signal. Co-immunoprecipitation results indicated that DACT2 strongly associated β-catenin as well as lymphoid enhancer-binding factor 1 (LEF1) and directly disrupted the formation of the β-catenin–LEF1 complex in the nucleus. Whereas in the cytoplasm, DACT2 restored junctional localization of E-cadherin–β-catenin complexes and prevented β-catenin nuclear translocation through direct interaction with β-catenin. DACT2 methylation was detected in 43.3% (29/67) of colon cancer tissues, but none in normal controls. Multivariate analysis revealed that patients with DACT2 methylation had a significant decrease in overall survival (P=0.006). Kaplan–Meier survival curves showed that DACT2 methylation was significantly associated with shortened survival in stage I–III colon cancer patients. In conclusion, DACT2 acts as a functional tumor suppressor in colon cancer through inhibiting Wnt/β-catenin signaling. Its methylation at early stages of colon carcinogenesis is an independent prognostic factor.

IL-1B-511 Polymorphism Is Associated With Increased Risk of Certain Subtypes of Gastric Cancer in Chinese: A Case-Control Study

OBJECTIVES:The association of interleukin-1B (IL-1B)-511 polymorphism with gastric cancer is still controversial, and the association of IL-1B-511 polymorphism with subtypes of gastric cancer is still largely unknown. We investigated whether the association between IL-1B-511 polymorphism and gastric cancer risk varies by clinically important tumor characteristics and the prognostic value of this polymorphism in a large population-based case–control study among Chinese.METHODS:A population-based case–control study was conducted between 1999 and 2006 in Guangdong Province, China. A total of 1,010 gastric cancer patients and 1,500 healthy controls were enrolled in this study. Polymorphism in IL-1B-511 was analyzed by PCR–restriction fragment length polymorphism on 501 gastric cancers and 500 healthy controls.RESULTS:Compared with the CC genotype, carriers of IL-1B-511 TT genotype had an increased gastric cancer risk (odds ratio (OR)=1.97, 95% confidence interval (CI)=1.29–3.01, P=0.0016). TT genotype was significantly associated with intestinal type of gastric cancer (OR=3.16, 95% CI=1.74–5.71, P=0.0001) but not with diffuse or mixed-type gastric cancer. The test for OR heterogeneity between the intestinal-type and non-intestinal-type gastric cancers was statistically significant (P=0.02). In subgroup analyses, TT genotype was found to be associated with poorly differentiated gastric cancer (OR=3.31, 95% CI=1.43–3.60, P<0.0001), but not with moderately or well-differentiated gastric cancer. IL-1B-511 genotypes were not associated with the prognosis of gastric cancer patients.CONCLUSIONS:IL-1B polymorphism influences certain subtypes of gastric cancer according to the clinical and pathological features. Understanding the etiologic heterogeneity of gastric cancer may result in improvements in controlling this disorder.