Epcam regulates intrahepatic bile duct reconstruction in zebrafish, providing a potential model for primary cholangitis model.

Epithelial cell adhesion molecules (EpCAMs) have been identified as surface markers of proliferating ductal cells, which are referred to as liver progenitor cells (LPCs), during liver regeneration and correspond to malignancies. These cells can differentiate into hepatocytes and biliary epithelial cells (BECs) in vitro. EpCAM-positive LPCs are involved in liver regeneration following severe liver injury; however, the in vivo function of EpCAMs in the regenerating liver remains unclear. In the present study, we used a zebrafish model of LPC-driven liver regeneration to elucidate the function of EpCAMs in the regenerating liver in vivo. Proliferating ductal cells were observed after severe hepatocyte loss in the zebrafish model. Analyses of the liver size as well as hepatocyte and BEC markers revealed successful conversion of LPCs to hepatocytes and BECs in epcam mutants. Notably, epcam mutants exhibited severe defects in intrahepatic duct maturation and bile acid secretion in regenerating hepatocytes, suggesting that epcam plays a critical role in intrahepatic duct reconstruction during LPC-driven liver regeneration. Our findings provide insights into human diseases involving non-parenchymal cells, such as primary biliary cholangitis, by highlighting the regulatory effect of epcam on intrahepatic duct reconstruction.

Opposing roles of HDAC6 in liver regeneration and hepatocarcinogenesis.

Histone deacetylase 6 (HDAC6), a deacetylase of p53, has emerged as a privileged inhibitory target for cancer therapy because of its deacetylating activity for p53 at K120 and K373/382. However, intricate roles of HDAC6 in hepatocellular carcinogenesis have been suggested by recent evidence, namely that HDAC6 ablation suppresses innate immunity, which plays critical roles in tumor immunosurveillance and antitumor immune responses. Therefore, it is valuable to determine whether HDAC6 ablation inhibits hepatocellular carcinogenesis using in vivo animal models. Here, we firstly showed that HDAC6 ablation increased K320 acetylation of p53, known as pro-survival acetylation, in all tested animal models but did not always increase K120 and K373/382 acetylation of p53, known as pro-apoptotic acetylation. HDAC6 ablation induced cellular senescence in primary MEFs and inhibited cell proliferation in HepG2 cells and liver regeneration after two-thirds partial hepatectomy. However, the genetic ablation of HDAC6 did not inhibit hepatocarcinogenesis, but instead slightly enhanced it in two independent mouse models (DEN + HFD and DEN + TAA). Notably, HDAC6 ablation significantly promoted hepatocarcinogenesis in a multiple DEN treatment hepatocellular carcinoma (HCC) mouse model, mimicking chronic DNA damage in the liver, which correlated with hyperacetylation at K320 of p53 and a decrease in inflammatory cytokines and chemokines. Our data from three independent in vivo animal HCC models emphasize the importance of the complex roles of HDAC6 ablation in hepatocellular carcinogenesis, highlighting its immunosuppressive effects.

Anti-Inflammatory Effect of Phytoncide in an Animal Model of Gastrointestinal Inflammation.

Background: Phytoncide is known to have antimicrobial and anti-inflammatory properties. Purpose: This study was carried out to confirm the anti-inflammatory activity of two types of phytoncide extracts from pinecone waste. Methods: We made two types of animal models to evaluate the efficacy, an indomethacin-induced gastroenteritis rat model and a dextran sulfate sodium-induced colitis mouse model. Result: In the gastroenteritis experiment, the expression of induced-nitric oxide synthase (iNOS), a marker for inflammation, decreased in the phytoncide-supplemented groups, and gastric ulcer development was significantly inhibited (p < 0.05). In the colitis experiment, the shortening of the colon length and the iNOS expression were significantly suppressed in the phytoncide-supplemented group (p < 0.05). Conclusions: Through this study, we confirmed that phytoncide can directly inhibit inflammation in digestive organs. Although further research is needed, we conclude that phytoncide has potential anti-inflammatory properties in the digestive tract and can be developed as a functional agent.

The basal transcriptional activity of the murine Klf10 gene is regulated by the transcriptional factor JunB.

BACKGROUND: Krüppel-like factor 10 (KLF10) belongs to the Sp1-like transcription factor family, which plays an important role in many directions, e.g., cell proliferation, apoptosis, and differentiation. Its 5' upstream regions are conserved across mammalian species. However, the regulatory mechanism has not been elucidated yet. OBJECTIVE: Nonetheless the basal transcriptional regulation mechanisms of these regions are unknown. Here, we characterized it which is indispensable for the basal transcription of the Klf10 gene. METHODS: Seven deletions of 5' upstream DNA fragments from the 10 kb mKlf10 genomic DNA were produced by PCR and cloned into the upstream of the luciferase (Luc) reporter gene in the pGL3 basic plasmid. RESULT: The luciferase reporter assay showed that the DNA sequence at positions from -101 to +68 was required for a principle activity in the promoter of mKlf10 gene, in which transcriptional factor binding motifs, one JunB and two Sp1 sites, are included. Mutations at the sequence of JunB motif, but not at the two Sp1, abrogated the promoter activity completely, suggesting the indispensable role of JunB site for basal transcription of mKlf10 gene. Moreover, electrophoretic mobility and supershift assays (EMSA) uncovered that JunB protein bound to this region specifically. CONCLUSION: Taken together, our study revealed that the JunB but not Sp1 at mKlf10 promoter functions as a positive basic factor for the transcriptional activity of the gene.

A Modified Protocol of Diethylnitrosamine Administration in Mice to Model Hepatocellular Carcinoma.

We aimed to create an animal model for hepatocellular carcinoma (HCC) with a short time, a high survival rate, as well as a high incidence of HCC in both males and females than previously reported. The Diethylnitrosamine (DEN) model has an age-related effect. A single dose of DEN treatment is not enough in young mice up to 50 weeks. The same pattern is shown in an adult with multiple-dose trials whether or not there is some promotion agent. In this study, two-week old C57BL6 mice were given a total of eight doses of DEN, initially 20mg/kg body weight, and then 30mg/kg in the third week, followed by 50mg/kg for the last six weeks. The first group is DEN treatment only and the other two groups received thioacetamide (TAA) treatment for four or eight weeks after one week of rest from the last DEN treatment. An autopsy was performed after 24 weeks of the initial dose of DEN in each group. The cellular arrangement of HCC in the entire group was well-differentiated carcinoma and tumor presence with no significant impact on the survival of mice. Increased levels of the biochemical markers in serum, loss of tissue architecture, hepatocyte death, and proliferation were highly activated in all tumor-induced groups. This finding demonstrates an improved strategy to generate an animal model with a high occurrence of tumors combined with cirrhosis in a short time regardless of sex for researchers who want to investigate liver cancer-related.

Gastroprotective effect of phytoncide extract from Pinus koraiensis pinecone in Helicobacter pylori infection.

For centuries, herbs have been used by traditional therapists around the world to treat gastrointestinal tract disorders, such as gastritis. We hypothesized that the anti-Helicobacter pylori properties of phytoncide, which is extracted from pinecone waste, would facilitate use as a natural gastroprotective product to treat gastrointestinal tract disorders. Thus, we investigated in vitro antibacterial efficacy against H. pylori by agar diffusion assay. To determine the gastroprotective properties of phytoncide, we conducted hematoxylin and eosin staining, performed assays for the detection of the cytotoxin gene, and evaluated pro-inflammatory cytokine expression in H. pylori-infected C57BL/6 mice. Phytoncide significantly inhibited the survival of H. pylori in the gastrointestinal system of C57BL/6 mice. Reduction of gastric severity in H. pylori-infected mice was associated with reductions in the expression levels of pro-inflammatory cytokines in the gastric mucosa, and of the cytotoxin CagA gene in phytoncide treated groups (P < 0.05 and P < 0.01). In conclusion, phytoncide significantly inhibited the growth of H. pylori in gastro tissue, possibly due to the abundant α-pinene present in the phytoncide as detected by HPLC analysis. Further studies are needed to validate our findings, but we suggest that phytoncide has the potential to be used as a natural ingredient in anti-H. pylori products.

KLF10 as a Tumor Suppressor Gene and Its TGF-ß Signaling.

Krüppel-like factor 10 (KLF10), originally named TGF-ß (Transforming growth factor beta) inducible early gene 1 (TIEG1), is a DNA-binding transcriptional regulator containing a triple C2H2 zinc finger domain. By binding to Sp1 (specificity protein 1) sites on the DNA and interactions with other regulatory transcription factors, KLF10 encourages and suppresses the expression of multiple genes in many cell types. Many studies have investigated its signaling cascade, but other than the TGF-ß/Smad signaling pathway, these are still not clear. KLF10 plays a role in proliferation, differentiation as well as apoptosis, just like other members of the SP (specificity proteins)/KLF (Krüppel-like Factors). Recently, several studies reported that KLF10 KO (Knock out) is associated with defects in cell and organs such as osteopenia, abnormal tendon or cardiac hypertrophy. Since KLF10 was first discovered, several studies have defined its role in cancer as a tumor suppressor. KLF10 demonstrate anti-proliferative effects and induce apoptosis in various carcinoma cells including pancreatic cancer, leukemia, and osteoporosis. Collectively, these data indicate that KLF10 plays a significant role in various biological processes and diseases, but its role in cancer is still unclear. Therefore, this review was conducted to describe and discuss the role and function of KLF10 in diseases, including cancer, with a special emphasis on its signaling with TGF-ß.

Characterization of the porcine Nanog 5'-flanking region.

OBJECTIVE: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. METHODS: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. RESULTS: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. CONCLUSION: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.

Minimal Pleural Effusion in Small Cell Lung Cancer: Proportion, Mechanisms, and Prognostic Effect.

PURPOSE: To determine the frequency and investigate possible mechanisms and prognostic relevance of minimal (<10-mm thickness) pleural effusion in patients with small cell lung cancer (SCLC). MATERIALS AND METHODS: The single-center retrospective study was approved by the institutional review board of the hospital, and informed consent was waived by the patients. A cohort of 360 consecutive patients diagnosed with SCLC by using histologic analysis was enrolled in this study. Based on the status of pleural effusion on chest computed tomographic (CT) scans at diagnosis, patients were classified into three groups: no pleural effusion, minimal pleural effusion, and malignant pleural effusion. Eighteen variables related to patient, environment, stage, and treatment were included in the final model as potential confounders. RESULTS: Minimal pleural effusion was present in 74 patients (20.6%) and malignant pleural effusion in 83 patients (23.0%). Median survival was significantly different in patients with no, minimal, or malignant pleural effusion (median survival, 11.2, 5.93, and 4.83 months, respectively; P < .001, log-rank test). In the fully adjusted final model, patients with minimal pleural effusion had a significantly increased risk of death compared with those with no pleural effusion (adjusted hazard ratio, 1.454 [95% confidence interval: 1.012, 2.090]; P = .001). The prognostic effect was significant in patients with stage I-III disease (adjusted hazard ratio, 2.751 [95% confidence interval: 1.586, 4.773]; P < .001), but it disappeared in stage IV disease. An indirect mechanism representing mediastinal lymphadenopathy was responsible for the accumulation in all but one patient with minimal pleural effusion. CONCLUSIONS: Minimal pleural effusion is a common clinical finding in staging SCLC. Its presence is associated with worse survival in patients and should be considered when CT scans are interpreted.

Interpretation of a heterogeneous radiological response as tumor heterogeneity or a non-tumor diagnosis: A case report.

Heterogeneous radiological responses (HRRs) among tumor lesions are usually observed following chemotherapy or radiation treatment in cancer patients. When HRR is observed after chemotherapy or radiation treatment, a change in anticancer treatment is recommended due to the clinically high suspicion of resistance in the majority of cases. The present study reports the case report of a patient with limited-stage small cell lung cancer, diagnosed by bronchoscopy, who received concurrent chemoradiation therapy. Upon response evaluation, the majority of lesions irradiated had nearly completely disappeared following treatment, but one lesion had apparently increased in size. For histological confirmation, a percutaneous needle biopsy for the lesion was performed, however, non-specific necrosis was found and the results were inconclusive for the differentiation of other causes from tumor necrosis. Several acid-fast bacilli were identified on Ziehl-Neelsen staining for the differential diagnosis. This case suggests that a non-tumor diagnosis should be considered when HRR presents after treatment that is expected to result in a higher response rate, particularly in tuberculosis endemic areas.

ERCC1 and personalized medicine in lung cancer.

Excision repair cross-complementing group 1 (ERCC1) is known to be a key player in nucleotide excision repair (NER) pathway. Its prognostic or predictive relevance has been extensively investigated in cancer patients including non-small-cell lung cancer. However, several questions should be addressed before its clinical application as biomarker for patient classification or guiding platinum treatment.

Multiple hypercoagulability disorders at presentation of non-small-cell lung cancer.

Hypercoagulability disorders are commonly encountered in clinical situations in patients with a variety of cancers. However, several hypercoagulability disorders presenting as first symptoms or signs in cancer patients have rarely been reported. We herein described a case of a woman with adenocarcinoma of the lung presenting with deep vein thrombosis, nonbacterial thrombotic endocarditis, recurrent cerebral embolic infarction, and heart failure.

Prognostic impact of minimal pleural effusion in non-small-cell lung cancer.

PURPOSE: Minimal (< 10 mm thick) pleural effusion (PE) may represent an early phase of malignant PE, but its clinical relevance has rarely been studied. Therefore, we examined the proportion of minimal PE in patients with non-small-cell lung cancer (NSCLC) and its impact on survival. We also considered possible accumulation mechanisms in our data set. PATIENTS AND METHODS: On the basis of PE status from chest computed tomography scans at diagnosis, 2,061 patients were classified into three groups: no PE, minimal PE, and malignant PE. Twenty-one variables associated with four factors-patient, stage migration, tumor, and treatment-were investigated for correlation with survival. RESULTS: Minimal PE presented in 272 patients (13.2%). Of 2,061 patients, the proportion of each stage was the following: 5.2% stage I, 10.9% stage II, 13.2% stage IIIA, 23.8% stage IIIB, and 13.9% stage IV. Minimal PE correlated significantly with shorter survival time than did no PE (median survival time, 7.7 v 17.7 months; log-rank P < .001), even after full adjustment with all variables (adjusted hazard ratio, 1.40; 95% CI, 1.21 to 1.62). Prognostic impact of minimal PE was higher in early versus advanced stages (Pinteraction = .001). In 237 patients (87.8%) with minimal PE, pleural invasion or attachment as a direct mechanism was observed, and it was an independent factor predicting worse survival (P = .03). CONCLUSION: Minimal PE is a commonly encountered clinical concern in staging NSCLCs. Its presence is an important prognostic factor of worse survival, especially in early-stage disease.