A novel support vector machine-based 1-day, single-dose prediction model of genotoxic hepatocarcinogenicity in rats.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

Whole Genome Sequencing Analysis of Model Organisms Elucidates the Association Between Environmental Factors and Human Cancer Development.

Determining a novel etiology and mechanism of human cancer requires extraction of characteristic mutational signatures derived from chemical substances. This study explored the mutational signatures of N-nitroso bile acid conjugates using Salmonella strains. Exposing S. typhimurium TA1535 to N-nitroso-glycine/taurine bile acid conjugates induced a predominance of C:G to T:A transitions. Two mutational signatures, B1 and B2, were extracted. Signature B1 is associated with N-nitroso-glycine bile acid conjugates, while Signature B2 is linked to N-nitroso-taurine bile acid conjugates. Signature B1 revealed a strong transcribed strand bias with GCC and GCT contexts, and the mutation pattern of N-nitroso-glycine bile acid conjugates in YG7108, which lacks O6-methylguanine DNA methyltransferases, matched that of the wild-type strain TA1535, suggesting that O6-methyl-deoxyguanosine contributes to mutations in the relevant regions. COSMIC database-based similarity analysis revealed that Signature B1 closely resembled SBS42, which is associated with occupational cholangiocarcinoma caused by overexposure to 1,2-dichlolopropane (1,2-DCP) and/or dichloromethane (DCM). Moreover, the inflammatory response pathway was induced by 1,2-DCP exposure in a human cholangiocyte cell line, and iNOS expression was positive in occupational cholangiocarcinomas. These results suggest that 1,2-DCP triggers an inflammatory response in biliary epithelial cells by upregulating iNOS and N-nitroso-glycine bile acid conjugate production, resulting in cholangiocarcinoma via DNA adduct formation.

A carcinogenicity study of diphenylarsinic acid in C57BL/6J mice in drinking water for 78 weeks.

Diphenylarsinic acid (DPAA), a neurotoxic organic arsenical, is present in groundwater and soil in some regions of Japan owing to illegal dumping. The present study evaluated the potential carcinogenicity of DPAA, including investigating whether bile duct hyperplasia in the liver that was observed in a chronic study on 52 week mouse, develops into a tumor when administered to mice in their drinking water for 78 weeks. DPAA was administered to 4 groups of male and female C57BL/6J mice at concentrations of 0, 6.25, 12.5, and 25 ppm in drinking water for 78 weeks. A significant decrease in the survival rate was found for females in the 25 ppm DPAA group. Body weights of males in the 25 ppm and females in the 12.5 and 25 ppm DPAA groups were significantly lower than those of the controls. Histopathological evaluation of neoplasms in all tissues showed no significant increase in tumor incidence in any organ or tissue of 6.25, 12.5, or 25 ppm DPAA-treated male or female mice. In conclusion, the present study demonstrated that DPAA is not carcinogenic to male or female C57BL/6J mice. Taken together with the fact that the toxic effect of DPAA is predominantly restricted to the central nervous system in humans, and the finding that DPAA was not carcinogenic in a previous 104-week rat carcinogenicity study, our results suggest that DPAA is unlikely to be carcinogenic in humans.

The carbonic anhydrase inhibitor acetazolamide inhibits urinary bladder cancers via suppression of ß-catenin signaling.

Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and progression of urothelial carcinoma (UC) in humans. The purpose of the present study was to evaluate the effects of the CA inhibitor acetazolamide (Ace) on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder carcinogenesis in mice and explore the function of CA2 in muscle invasion by UC. Male mice were treated with 0.025% (experiment 1) or 0.05% BBN (experiment 2) in their drinking water for 10 weeks, then treated with cisplatin (Cis), Ace, or Cis plus Ace for 12 weeks. In experiment 1, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Ace and BBN→Cis+Ace groups. In experiment 2, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Cis+Ace group, and the incidence of muscle invasive UC was significantly decreased in both the BBN→Ace and the BBN→Cis+Ace groups. We also show that overexpression of CA2 by human UC cells T24 and UMUC3 significantly increased their migration and invasion capabilities, and that Ace significantly inhibited migration and invasion by CA2-overexpressing T24 and UMUC3 cells. These data demonstrate a functional association of CA2 with UC development and progression, confirming the association of CA2 with UC that we had shown previously by immunohistochemical analysis of human UC specimens and proteome analysis of BBN-induced UC in rats. Our finding that inhibition of CA2 inhibits UC development and muscle invasion also directly confirms that CA2 is a potential therapeutic target for bladder cancers.

Clinical relevance of tertiary lymphoid structures in esophageal squamous cell carcinoma.

BACKGROUND: Tertiary lymphoid structures (TLSs) have been reported to be involved in immune responses in many carcinomas. This study investigated the significance of TLSs in esophageal squamous cell carcinoma, focusing on TLS maturation.  METHODS: The relationships of TLSs with clinicopathological features of 236 patients who underwent curative surgery for stage 0-IV esophageal squamous cell carcinoma were investigated. Mature TLSs, in which the germinal center formation was rich in CD23+ cells, were classified as TLSs containing a germinal center (GC-TLSs). GC-TLS densities were measured, and CD8+ cells were counted. The prognostic impact of GC-TLSs was assessed by Kaplan-Meier plots using the log-rank test for the relapse-free survival. A comparative study of GC-TLSs was performed using the Wilcoxon rank sum test. The relationship between GC-TLSs and CD8+ cells was examined by Spearman's rank correlation coefficient test. RESULTS: TLSs were located mainly at the invasive margin of the tumor in cases with esophageal squamous cell carcinoma. Among the patients treated with neoadjuvant chemotherapy, those with advanced disease had a better prognosis in the GC-TLS high-density group than did those in the GC-TLS low-density group. Patients in whom neoadjuvant chemotherapy was effective had more GC-TLSs than those in whom it was less effective. The density of GC-TLSs and the number of tumor-infiltrating CD8+ cells were higher in patients treated with neoadjuvant chemotherapy than in those without chemotherapy, and a weak correlation between the density of GC-TLSs and the number of tumor-infiltrating CD8+ cells was observed. Moreover, co-culturing of PBMCs with an anticancer drug-treated esophageal squamous cell carcinoma cell line increased the CD20 and CD23 expression in PBMCs in vitro. CONCLUSION: TLS maturation may be important for evaluating the local tumor immune response in patients treated with neoadjuvant chemotherapy for esophageal squamous cell carcinoma. The present results suggest that TLS maturation may be a useful target for predicting the efficacy of immunotherapy, including immune checkpoint inhibitor treatment for esophageal squamous cell carcinoma.

Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study.

BACKGROUND: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure. METHODS: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice. RESULTS: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans. CONCLUSIONS: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

Response biomarkers of inhalation exposure to cigarette smoke in the mouse lung.

Cigarette smoking is known to increase the risk of cancer and chronic obstructive pulmonary disease (COPD). In this study, we evaluated the effects of short-term nose-only inhalation exposure to cigarette smoke in mice. Male 10-week-old C57BL mice were exposed to clean air (control) or mainstream cigarette smoke for 1 h/day, 5 days/week, for 2 or 4 weeks. Exposure to cigarette smoke increased the number of inflammatory cells, especially neutrophils, in the bronchoalveolar lavage fluid, increased inflammatory cell infiltration foci, and caused an increase in the thickness of the peripheral bronchial epithelium. Microarray gene expression analysis indicated that smoke exposure induced inflammatory responses, including leukocyte migration and activation of phagocytes and myeloid cells, as early as two weeks after the initiation of exposure. Importantly, chemokine (C-C motif) ligand 17, resistin-like alpha, and lipocalin 2 were upregulated and may serve as useful markers of the toxic effects of exposure to cigarette smoke before pulmonary histological changes become evident.

DPYD, down-regulated by the potentially chemopreventive agent luteolin, interacts with STAT3 in pancreatic cancer.

The 5-year survival rate of pancreatic ductal carcinoma (PDAC) patients is <10% despite progress in clinical medicine. Strategies to prevent the development of PDAC are urgently required. The flavonoids Luteolin (Lut) and hesperetin (Hes) may be cancer-chemopreventive, but effects on pancreatic carcinogenesis in vivo have not been studied. Here, the chemopreventive effects of Lut and Hes on pancreatic carcinogenesis are assessed in the BOP-induced hamster PDAC model. Lut but not Hes suppressed proliferation of pancreatic intraepithelial neoplasia (PanIN) and reduced the incidence and multiplicity of PDAC in this model. Lut also inhibited the proliferation of hamster and human pancreatic cancer cells in vitro. Multi-blot and microarray assays revealed decreased phosphorylated STAT3 (pSTAT3) and dihydropyrimidine dehydrogenase (DPYD) on Lut exposure. To explore the relationship between DPYD and STAT3 activity, the former was silenced by RNAi or overexpressed using expression vectors, and the latter was inactivated by small molecule inhibitors or stimulated by IL6 in human PDAC cells. DPYD knock-down decreased, and overexpression increased, pSTAT3 and cell proliferation. DPYD expression was decreased by inactivation of STAT3 and increased by its activation. The frequency of pSTAT3-positive cells and DPYD expression was significantly correlated and was decreased in parallel by Lut in the hamster PDAC model. Finally, immunohistochemical analysis in 73 cases of human PDAC demonstrated that DPYD expression was positively correlated with the Ki-67 labeling index, and high expression was associated with poor prognosis. These results indicate that Lut is a promising chemopreventive agent for PDAC, targeting a novel STAT3-DPYD pathway.

Tertiary lymphoid structures show infiltration of effective tumor-resident T cells in gastric cancer.

Several studies have reported that tissue-resident memory T cells (TRM cells) or tertiary lymphoid structures (TLSs) are associated with a good prognosis. The aim of this study was to clarify the association of TRM cells and TLSs in the tumor immune microenvironment in gastric cancer (GC). We performed immunohistochemical and immunofluorescence staining to detect the presence of CD103+ T cells and to assess the association between CD103+ T cells and TLSs. CD103+ T cells were observed in the tumor epithelium accompanied by CD8+ T cells and were associated with a better prognosis in GC. Furthermore, CD103+ T cells were located around TLSs, and patients with CD103high had more rich TLSs. Patients who had both CD103high cells and who were TLS-rich had a better prognosis than patients with CD103low cells and who were TLS-poor. Moreover, for patients who received PD-1 blockade therapy, CD103high and TLS-rich predicted a good response. Flow cytometry was performed to confirm the characteristics of CD103+ CD8+ T cells and showed that CD103+ CD8+ T cells in GC expressed higher levels of PD-1, granzyme B, and interferon-γ than CD103- CD8+ T cells. Our results suggested that CD103+ CD8+ cells in GC are correlated with TLSs, resulting in enhanced antitumor immunity in GC.

Selective extension of cerebral vascular calcification in an autopsy case of Fahr's syndrome associated with asymptomatic hypoparathyroidism.

We report an autopsy case of Fahr's syndrome in an 85-year-old woman associated with asymptomatic hypoparathyroidism. The patient was diagnosed as having brain calcification at 65 years of age. She developed mild dementia at 75, parkinsonism at 76, and severe dementia at 82. Computed tomography revealed extensive, symmetric intracranial calcification, involving both sides of the basal ganglia and cerebellar dentate nuclei, and severe cerebral atrophy that developed afterwards. A neuropathological examination revealed intracranial calcification, particularly in the wall of the arterioles and capillaries having numerous calcium deposits. Severe vascular calcification and severe neuronal loss without α-synuclein accumulation were found in the substantia nigra. There were high-level neuropathological changes indicative of Alzheimer's disease. Although the colocalization of calcium and amyloid-ß deposits in the same arterial wall was rare, both of them were located in a similar layer of the arterial wall. The vascular calcification in the basal ganglia spread continuously through the corona radiata into the selective cerebral areas along the medullary arteries, but did not involve the corpus callosum or insular region. Stone formation was observed at the corona radiata adjacent to the superolateral angles of the lateral ventricles. We hypothesized that there would be a stereotypical extension pattern of vascular calcification related to the arrangement of penetrating arteries in Fahr's syndrome.

Recruitment of miR-8080 by luteolin inhibits androgen receptor splice variant 7 expression in castration-resistant prostate cancer.

A need exists for seeking effective treatments for castration-resistant prostate cancer (CRPC) in response to its emergence following androgen deprivation therapy as a major clinical problem. In the present study, we investigated the chemopreventive and chemotherapeutic potential of luteolin, a flavonoid with antioxidative properties, on prostate cancer, including CRPC. Luteolin inhibited the progression of rat prostate carcinogenesis by induction of apoptosis in a transgenic rat for adenocarcinoma of prostate (TRAP) model. Luteolin decreased cell proliferation in a dose-dependent manner and induced apoptosis with the activation of caspases 3 and 7 in both rat (PCai1, established from a TRAP prostate tumor) and human (22Rv1) CRPC cells. Dietary luteolin also suppressed tumor growth via an increase in apoptosis and inhibition of angiogenesis in PCai1 and 22Rv1 xenografts implanted in castrated nude mice. We also focused on androgen receptor splice variant 7 (AR-V7), which contributes to cell proliferation and therapeutic resistance in CRPC. Luteolin dramatically suppressed AR-V7 protein expression in 22Rv1 cells in vitro and ex vivo. Microarray analysis identified MiR-8080, which contains a possible target sequence for AR-V7 3'-UTR, as a gene upregulated by luteolin. MiR-8080 transfection decreased the AR-V7 expression level and the induction of apoptosis in 22Rv1 cells. Furthermore, miR-8080 knockdown canceled luteolin decreasing AR-V7 and the cell growth of 22Rv1. MiR-8080 induced by luteolin intake enhanced the therapeutic effect of enzalutamide on 22Rv1 xenografts under castration conditions. These results indicate luteolin inhibits CRPC by AR-V7 suppression through miR-8080, highlighting luteolin and miR-8080 as promising therapeutic agents for this disease.

Luteolin suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway.

Luteolin is a natural flavonoid with strong anti-oxidative properties that is reported to have an anti-cancer effect in several malignancies other than bladder cancer. In this study, we describe the effect of luteolin on a human bladder cancer cell line, T24, in the context of the regulation of p21, thioredoxin-1 (TRX1) and the mechanistic target of rapamycin (mTOR) pathway. Luteolin inhibited cell survival and induced G2/M cell-cycle arrest, p21 upregulation and downregulation of phospho(p)-S6, which is downstream of mTOR signaling. Luteolin also upregulated TRX1 and reduced intracellular reactive oxygen species production. In a subcutaneous xenograft mouse model using the rat bladder cancer cell line, BC31, tumor volumes were significantly decreased in mice orally administered luteolin compared to control. Immunohistochemical analysis revealed that increased p21 and decreased p-S6 expression were induced in the luteolin treatment group. Moreover, in another in vivo N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat bladder cancer model, the oral administration of luteolin led to a trend of decreased bladder tumor dimension and significantly decreased the Ki67-labeling index and p-S6 expression. Furthermore, the major findings on the metabolism of luteolin suggest that both plasma and urine luteolin-3'-O-glucuronide concentrations are strongly associated with the inhibition of cell proliferation and mTOR signaling. Moreover, a significant decrease in the squamous differentiation of bladder cancer is attributed to plasma luteolin-3'-glucuronide concentration. In conclusion, luteolin, and in particular its metabolized product, may represent another natural product-derived therapeutic agent that acts against bladder cancer by upregulating p21 and inhibiting mTOR signaling.

The standard form of CD44 as a marker for invasion of encapsulated papillary carcinoma of the breast.

Encapsulated papillary carcinoma (EPC), a rare variant of papillary carcinoma of the breast, is regarded as a transition form between carcinoma in situ and invasive carcinoma. Here, we tried to identify differences in immunohistochemical phenotype between 10 EPCs with invasive properties (EPC with invasion) and 17 non-invasive EPCs (EPC). We immunohistochemically assessed the expression of hormone receptors, matrix metalloproteinase (MMP) 2 and MMP9, vascular endothelial growth factor (VEGF), CD31, and D2-40, markers of tumor-associated macrophages (CD163, CD206), Ki-67 and stem cell markers (CD44 and CD24). The frequency of MMP9-positive cases and the number of tumor-associated macrophages infiltrating into the fibrous capsule were significantly higher in EPC with invasion than in EPC. The expression of the standard form of CD44 (CD44s) was significantly stronger in EPC with invasion than in EPC (P = 0.0036) and was correlated with MMP2 expression and M2-like macrophage infiltration. A multivariate logistic model analysis showed that CD44s expression in tumor cell and infiltration of CD163 positive macrophage in EPC capsule showed an independent odds ratio for invasion of EPC. Thus, CD44s may be a potential marker predicting invasive potential of EPC and could play an important role in progression to the invasive phase of EPC.

Dimethylarsinic acid (DMA) enhanced lung carcinogenesis via histone H3K9 modification in a transplacental mouse model.

Pregnant CD-1 mice received 200 ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8-18, and tumor formation was assessed in offspring at the age of 84 weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

A novel model of non-alcoholic steatohepatitis with fibrosis and carcinogenesis in connexin 32 dominant-negative transgenic rats.

Non-alcoholic steatohepatitis (NASH) is a recognized risk factor for liver fibrosis and malignancies, and is associated with features of metabolic syndrome, such as obesity and insulin resistance (IR). We previously demonstrated that the disturbance of connexin 32 (Cx32), a gap junctional protein of hepatocytes, exacerbated NASH in Cx32 dominant-negative transgenic (Cx32ΔTg) rats fed methionine choline-deficient diet (MCDD). MCDD is well-established means of inducing NASH in rodents; however, the Cx32ΔTg-MCDD NASH model does not reproduce obesity and IR. In this study, we aimed to establish an improved NASH model. Eight-week-old male Cx32ΔTg and wild-type (Wt) rats received a high-fat diet (HFD) with dimethylnitrosamine (DMN) for 12 weeks. The HFD with DMN led to gains in body, liver, and visceral fat weights in both genotypes. IR was significantly greater in Cx32ΔTg than in Wt rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions (Tnfα, Il-6, Tgf-ß1, Il-1ß, Timp2, and Col1a1), steatohepatitis, and fibrosis were significantly greater in Cx32ΔTg as compared with Wt rats. Regarding carcinogenesis, the number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic hepatic foci were significantly increased in Cx32ΔTg versus Wt rats. Moreover, activation of NF-κB and JNK contributed to the progression of NASH in Cx32ΔTg rats. These results suggest that Cx32 dysfunction promoted the progression of NASH, metabolic syndrome, and carcinogenesis. Therefore, the novel Cx32ΔTg-HFD-DMN NASH model may be a rapid and useful tool for evaluating the progression of NASH.

Low reduction of prostate volume is a significant predictor of prostate cancer at subsequent biopsy in patients with dutasteride: A retrospective study.

Appropriate decision of prostate biopsy in men with 5α-reductase inhibitor (5AR inhibitor) is still unclear to avoid unnecessary biopsy. We retrospectively investigated patients with initial PSA 4.0 ng/ml or more and underwent subsequent prostate biopsy following dutasteride treatment. From September 2009 to August 2018, 399 cases of benign prostate hyperplasia (BPH) were treated with dutasteride in our department. Of the total, 36 cases with elevated pre-treatment PSA (4.0 ng/ml or more) and underwent subsequent prostate biopsy were included into this study. We evaluated PSA kinetics and changing prostate volumes (PV), and detection of prostate cancer. Overall, average PSA reduced by half at 6 months from dosing. Pre-treatment biopsy was performed in 17 of 36 cases, and all were diagnosed as having no malignancy. After treatment, prostate cancer was detected in 15 cases by subsequent biopsy. Fourteen of 15 cases were clinically significant cancer (Gleason score 7 or more). Logistic regression analysis detected a nominal association between prostate cancer detection and three variants, PSAD, PV reduction (1-Before/After PV) and abnormal MRI findings. In addition to abnormal MRI findings and pre-treatment of high PSAD, the case with low reduction of PV after treatment should consider performing prostate biopsy.

Accumulation of 8-hydroxydeoxyguanosine, L-arginine and Glucose Metabolites by Liver Tumor Cells Are the Important Characteristic Features of Metabolic Syndrome and Non-Alcoholic Steatohepatitis-Associated Hepatocarcinogenesis.

To uncover mechanisms and explore novel biomarkers of obesity, type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH)-associated hepatocarcinogenesis, cellular and molecular alterations in the liver, and hepatocellular carcinomas (HCCs) were investigated in NASH model 60-week-old Tsumura, Suzuki, Obese Diabetic (TSOD) mice and NASH HCC patients. Markedly elevated lipid deposition, inflammation, fibrosis, and peroxisome proliferation in the liver, preneoplastic lesions, and HCCs of TSOD mice were accompanied by accumulation of polysaccharides in the cellular cytoplasm and nuclei and increase of oxidative DNA damage marker, 8-hydroxydeoxyguanosine (8-OHdG) formation in the liver and altered foci. Metabolomics of TSOD mice HCCs demonstrated significant elevation of the concentration of amino acid L-arginine, phosphocreatine, S-adenosylmethionine/S-adenosylhomocysteine ratio, adenylate, and guanylate energy charges in coordination with tremendous rise of glucose metabolites, mostly fructose 1,6-diphosphate. L-arginine accumulation in HCCs was associated with significant under-expression of arginase 1 (ARG1), suppression of the urea cycle, methionine and putrescine degradation pathways, activation of Ser and Thr kinase Akt AKT, phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, ß-catenin, mammalian target of rapamycin (mTOR), and cell proliferation. Furthermore, clinicopathological analysis in 20 metabolic syndrome/NASH and 80 HCV-positive HCC patients demonstrated significant correlation of negative ARG1 expression with poor tumor differentiation, higher pathological stage, and significant decrease of survival in metabolic syndrome/NASH-associated HCC patients, thus indicating that ARG1 could become a potential marker for NASH HCC. From these results, formation of oxidative stress and 8-OHdG in the DNA and elevation of glucose metabolites and L-arginine due to ARG1 suppression in mice liver cells are the important characteristics of T2DM/NASH-associated hepatocarcinogenesis, which may take part in activating oxidative stress resistance, synthesis of phosphocreatine, cell signaling, methylation, and proliferation.

Dehydrozingerone, a Curcumin Analog, as a Potential Anti-Prostate Cancer Inhibitor In Vitro and In Vivo.

Curcumin (Cur) exhibits biological activities that support its candidacy for cancer treatment. However, there are limitations to its pharmacological effects, such as poor solubility and bioavailability. Notably, the use of Cur analogs has potential for addressing these limitations. Dehydrozingerone (DZG) is a representative of the half-chemical structure of Cur, and many reports have indicated that it is anticancer in vitro. We, therefore, have hypothesized that DZG could inhibit prostate cancer progression both in vitro and in vivo. Results revealed that DZG decreased cell proliferation of rat castration-resistant prostate cancer, PLS10 cells, via induction of the cell cycle arrest in the G1 phase in vitro. In the PLS10 xenograft model, DZG significantly decreased the growth of subcutaneous tumors when compared to the control via the inhibition of cell proliferation and angiogenesis. To prove that DZG could improve the limitations of Cur, an in vivo pharmacokinetic was determined. DZG was detected in the serum at higher concentrations and remained up to 3 h after intraperitoneal injections, which was longer than Cur. DZG also showed superior in vivo tissue distribution than Cur. The results suggest that DZG could be a candidate of the Cur analog that can potentially exert anticancer capabilities in vivo and thereby improve its bioavailability.

Role of γ-H2AX as a biomarker for detection of bladder carcinogens in F344 rats.

Phosphorylation of histone H2AX at serine 139 (γ-H2AX) is known to be induced by direct DNA damage or cellular metabolic imbalances and malfunctions. Previous studies have reported that γ-H2AX is a useful biomarker for early detection of genotoxic bladder carcinogens in rats. The purpose of the present study was to determine the role of γ-H2AX as a biomarker for detection of non-genotoxic bladder carcinogens in rats. Six-week-old male F344 rats were treated with 15 different chemicals for 4 weeks. Immunohistochemical analyses revealed that all three genotoxic bladder carcinogens and six out of seven non-genotoxic bladder carcinogens significantly increased γ-H2AX formation in the bladder urothelium of rats. In addition, four out of five rat bladder noncarcinogens did not increase γ-H2AX formation in the bladder urothelium regardless of genotoxicity. These results suggest that γ-H2AX is a useful biomarker for detection of both genotoxic and non-genotoxic bladder carcinogens in rats.