Search

Additional file 4. T, B, NK cell analysis in NIG(NSIG) mice compared to NSG mice. (A) Analysis of T, B and NK cell composition in blood from C57BL/6, NIG(NSIG) and NSG male mice. (B) Analysis of T, B and NK cell composition in spleen from NIG(NSIG) and NSG male mice. (C) Graph quantifying results of (A) and (B).

Additional file 2. Genotyping of F4 generation mice. The mouse numbers, from F4 #1–6, are shown above each lane. Wild type mouse genomic DNA serves as negative control. F4 mice were genotyped by PCR products sequencing with PCR amplificon. All mice are represented by a single band at 633 bp. (B) Sequence analysis of the mutated IL2Rg alleles. The sequencing peak of the wild type mouse is shown in upper case and that of F4 homozygous mutant mouse in lower case. The target sequence of sgRNAs are shown in bold letters. The mutant alleles of each mouse are labeled with the mouse ID number. The deleted sequences are marked in dash.

Additional file 1. Genotyping and sequencing of F1, F2 and F3 heterozygous mice. (A) Genotyping results of F1 generation mice. F1 mice were genotyped by the T7E1 assay with PCR amplificon, and resultant products were electrophoresed in 2.4% agarose gel. Wild type allele is represented by a single band at 633 bp, while the heterozygous alleles (red) are obtained as two bands at 453 bp and 633 bp. The sequence analysis of F1 heterozygous mice is shown in lower case. The target sites of sgRNAs are shown in bold letters. (B) PCR genotyping results of F2 and F3 generation mice. Wild type mouse genomic DNA serves as negative control. Mice were genotyped by PCR product sequencing with PCR amplificon. All mice are represented by a single band at 633 bp. (C) Sequence analysis of the heterozygous mutant alleles. The sequencing peak of the wild type mouse is shown in upper case and that of heterozygous mutant mouse in lower case. The mutant alleles of each mouse are labeled with the mouse ID number. The target sites of sgRNAs are shown in bold letters. The deleted sequences are marked in dash.

Additional file 3. Expression analysis of IL2Rg mRNA in lymphoid organs of NSIG mice. IL2Rg mRNA expression level results of NOD/SCID and NIG(NSIG) mice. The mRNA levels of IL2Rg were determined by qRT-PCR analysis in the three regions (5′, middle and 3′) of IL2Rg gene and RNAs were extracted from thymus and spleen tissues of NOD/SCID and NIG(NSIG) mice.

// Bing Han 1, 2 , Hye-Jun Shin 1 , In Seon Bak 1, 3 , Yesol Bak 1, 4 , Ye-Lin Jeong 1, 5 , Taeho Kwon 1 , Young-Ho Park 1 , Hu-Nan Sun 6 , Cheol-Hee Kim 2 , Dae-Yeul Yu 1 1 Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Korea 2 Department of Biology, Chungnam National University, Daejeon, 305-764, Korea 3 Department of Toxicology Evaluation, Graduate School of Preclinical Laboratory Science, Konyang University, Daejeon, 363-700, Korea 4 Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, 143-701, Korea 5 Department of Animal Biosystem Sciences, Chungnam National University, Daejeon, 305-764, Korea 6 College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing, 163319, China Correspondence to: Dae-Yeul Yu, email: dyyu10@kribb.re.kr Keywords: peroxiredoxin I, H-ras G12V , hepatic tumorigenesis, reactive oxygen species, gene regulation Received: February 11, 2016 Accepted: July 27, 2016 Published: August 10, 2016 ABSTRACT Peroxiredoxin I (Prx I), an antioxidant enzyme, has multiple functions in human cancer. However, the role of Prx I in hepatic tumorigenesis has not been characterized. Here we investigated the relevance and underlying mechanism of Prx I in hepatic tumorigenesis. Prx I increased in tumors of hepatocellular carcinoma (HCC) patients that aligned with overexpression of oncogenic H-ras. Prx I also increased in H-ras G12V transfected HCC cells and liver tumors of H-ras G12V transgenic (Tg) mice, indicating that Prx I may be involved in Ras-induced hepatic tumorigenesis. When Prx I was knocked down or deleted in HCC-H-ras G12V cells or H-ras G12V Tg mice, cell colony or tumor formation was significantly reduced that was associated with downregulation of pERK pathway as well as increased intracellular reactive oxygen species (ROS) induced DNA damage and cell death. Overexpressing Prx I markedly increased Ras downstream pERK/FoxM1/Nrf2 signaling pathway and inhibited oxidative damage in HCC cells and H-ras G12V Tg mice. In this study, we found Nrf2 was transcriptionally activated by FoxM1, and Prx I was activated by the H-ras G12V /pERK/FoxM1/Nrf2 pathway and suppressed ROS-induced hepatic cancer-cell death along with formation of a positive feedback loop with Ras/ERK/FoxM1/Nrf2 to promote hepatic tumorigenesis.

Detection of neurotransmitters is one of the most efficient methods for monitoring neurodegenerative diseases. Several electrochemical methods have been developed for neurotransmitter detection with conductive materials. In this study, gold-crumpled graphene (Au-crGr) composite modified electrodes were used to improve electrode conductivity and affinity with neurotransmitters. Au-crGr composites were synthesized at various component ratios and the structures were confirmed by electron microscopy. After the fabrication of Au-crGr composite modified electrodes, electrochemical methods and surface-enhanced Raman spectroscopy (SERS) were used to validate the spectroelectrochemical properties of various electrodes. The highest electrochemical and SERS signal intensity from a dopamine solution was obtained with Au-crGr composite modified electrodes. Moreover, secreted neurotransmitters from human neuroblastoma cells were successfully detected using the new material in a spectroelectrochemical method, and these results were confirmed by ultra-performance liquid chromatography and mass spectrometry. Our newly developed Au-crGr composite modified electrodes can be used as a catecholamine sensor and to study diagnostic methods for neurodegenerative diseases.

Background & Aims: Hepatic steatosis occurs frequently in patients with chronic hepatitis B virus (HBV) or chronic hepatitis C virus (HCV) infection. Recently, several studies suggested that steatosis plays an important role as a cofactor in other liver diseases such as hepatic fibrosis, hepatitis, and liver cancer. In contrast to HCV, however, the molecular mechanism by which HBV mediates hepatic steatosis has not been clearly studied. Here, we show the molecular mechanism by which hepatitis B virus X protein (HBx) induces hepatic steatosis. Methods: Lipid accumulation and the expression of various lipid metabolic genes were investigated in HBx-transfected Chang liver cells, HepG2-HBx stable cells, and HBx-transgenic mice. Results: Overexpression of HBx induced hepatic lipid accumulation in HepG2-HBx stable cells and HBx-transgenic mice. It also up-regulated the messenger RNA and protein levels of sterol regulatory element binding protein 1, but not peroxisome proliferator-activated receptor alpha (PPARα). Moreover, we also determined that the expression of HBx increases PPARγ gene expression as well as its transcriptional activity in hepatic cells, mediated by CCAAT enhancer binding protein α activation. Finally, we showed that HBx expression is able to up-regulate the gene expressions of various lipogenic and adipogenic enzymes in hepatic cells. Conclusions: We showed that the increased HBx expression causes lipid accumulation in hepatic cells mediated by sterol regulatory element binding protein 1 and PPARγ, which could be a putative molecular mechanism mediating the pathophysiology of HBV infection.

It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway.

Aims: Coupled responses of mutated K-ras and oxidative stress are often an important etiological factor in non–small-cell lung cancer (NSCLC). However, relatively few studies have examined the control mechanism of oxidative stress in oncogenic K-ras-driven NSCLC progression. Here, we studied whether the redox signaling pathway governed by peroxiredoxin I (Prx I) is involved in K-rasG12D-mediated lung adenocarcinogenesis. Results: Using human-lung adenocarcinoma tissues and lung-specific K-rasG12D-transgenic mice, we found that Prx I was significantly up-regulated in the tumor regions via activation of nuclear erythroid 2-related factor 2 (Nrf2) transcription. Interestingly, the increased reactive oxygen species (ROS) by null mutation of Prx I greatly promoted K-rasG12D-driven lung tumorigenesis in number and size, which appeared to require the activation of the ROS-dependent extracellular signal-regulated kinase (ERK)/cyclin D1 pathway. Innovation: Taken together, these results suggest that Prx I functions as an Nrf2-dependently inducible tumor suppressant in K-ras-driven lung adenocarcinogenesis by opposing ROS/ERK/cyclin D1 pathway activation. Conclusion: These findings provide a better understanding of oxidative stress-mediated lung tumorigenesis. Antioxid. Redox Signal. 19, 482–496.

Inducible nitric oxide (iNOS) is closely correlated with chronic inflammation in hepatitis B virus X protein (HBx)-induced hepatocellular carcinoma (HCC). However, the molecular mechanisms through which iNOS contribute to hepatocarcinogenesis remain poorly understood. Therefore, we investigated the role of iNOS in signaling pathways underlying HBx-induced liver tumorigenesis. iNOS deletion showed a marked decrease in the hepatic tumor size and stage of HBx transgenic (Tg) mice, indicating a strong contribution of iNOS signaling pathways to hepatocarcinogenesis. In addition, we found that nitric oxide (NO) increased HBx mRNA by recruiting CREB to the CRE site of HBV enhancer in HepG2 cells, suggesting a positive feedback loop between HBx and iNOS signaling pathway. Moreover, iNOS-modulated JNK activation was associated with sustained upregulation of Cyclin D1 in HBxTg mice and HepG2-HBx cells. These results imply that iNOS may play a key role in HBx-associated HCC development. Taken together, our findings demonstrate that iNOS aligns with HBx to promote tumor progression. These findings provide a better understating of the mechanism involving HBx-mediated hepatic tumorigenesis and selective inhibition of iNOS may have therapeutic applications in HBx-associated HCC.

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

Long term hepatitis B virus (HBV) infection is a major risk factor in pathogenesis of chronic liver diseases, including hepatocellular carcinoma (HCC). The HBV encoded proteins, hepatitis B virus X protein and preS, appear to contribute importantly to the pathogenesis of HCC. Both are associated with oxidative stress, which can damage cellular molecules like lipids, proteins, and DNA during chronic infection. Chronic alcohol use is another important factor that contributes to oxidative stress in the liver. Previous studies reported that treatment with antioxidants, such as curcumin, silymarin, green tea, and vitamins C and E, can protect DNA from damage and regulate liver pathogenesis-related cascades by reducing reactive oxygen species. This review summarizes some of the relationships between oxidative stress and liver pathogenesis, focusing upon HBV and alcohol, and suggests antioxidant therapeutic approaches.

The hepatitis B virus X-protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx-transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2-DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC-MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3-ketoacyl-CoA thiolase, intestinal fatty acid-binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx-mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.

Aim To investigate the effect of methylsulfonylmethane (MSM), recently reported to have anti-cancer effects, in liver cancer cells and transgenic mice. Methods Three liver cancer cell lines, HepG2, Huh7-Mock and Huh7-H-ras (G12V), were used. Cell growth was measured by Cell Counting Kit-8 and soft agar assay. Western blot analysis was used to detect caspases, poly (ADP-ribose) polymerase (PARP), and B-cell lymphoma 2 (Bcl-2) expressions. For in vivo study, we administered MSM to H-ras (12V) transgenic mice for 3 mo. Results MSM decreased the growth of HepG2, Huh7-Mock and Huh7-H-ras (G12V) cells in a dose-dependent manner. That was correlated with significantly increased apoptosis and reduced cell numbers in MSM treated cells. Cleaved caspase-8, cleaved caspase-3 and cleaved PARP were remarkably increased in the liver cancer cells treated with 500 mmol/L of MSM; however, Bcl-2 was slightly decreased in 500 mmol/L. Liver tumor development was greatly inhibited in the H-ras (12V) transgenic mice treated with MSM, compared to control, by showing reduced tumor size and number. Cleaved PARP was significantly increased in non-tumor treated with MSM compared to control. Conclusion Liver injury was also significantly attenuated in the mice treated with MSM. Taken together, all the results suggest that MSM has anti-cancer effects through inducing apoptosis in liver cancer.