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.

DNA Methylation Aberrations in Dimethylarsinic Acid-Induced Bladder Carcinogenesis.

Arsenic is a known human urinary bladder carcinogen. While arsenic is known to cause aberrant DNA methylation, the mechanism of arsenic-triggered bladder carcinogenesis is not fully understood. The goal of this study was to identify aberrant DNA methylation in rat bladder urothelial carcinoma (UC) induced by dimethylarsinic acid (DMAV), a major organic metabolite of arsenic. We performed genome-wide DNA methylation and microarray gene expression analyses of DMAV-induced rat UCs and the urothelium of rats treated for 4 weeks with DMAV. We identified 40 genes that were both hypermethylated and downregulated in DMAV-induced rat UCs. Notably, four genes (CPXM1, OPCML, TBX20, and KCND3) also showed reduced expression in the bladder urothelium after 4 weeks of exposure to DMAV. We also found that CPXM1 is aberrantly methylated and downregulated in human bladder cancers and human bladder cancer cells. Genes with aberrant DNA methylation and downregulated expression in DMAV-exposed bladder urothelium and in DMAV-induced UCs in rats, suggest that these alterations occurred in the early stages of arsenic-induced bladder carcinogenesis. Further study to evaluate the functions of these genes will advance our understanding of the role of aberrant DNA methylation in arsenic bladder carcinogenesis, and will also facilitate the identification of new therapeutic targets for arsenic-related bladder cancers.

Recent Insights into the Biomarkers, Molecular Targets and Mechanisms of Non-Alcoholic Steatohepatitis-Driven Hepatocarcinogenesis.

Non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (NASH) are chronic hepatic conditions leading to hepatocellular carcinoma (HCC) development. According to the recent "multiple-parallel-hits hypothesis", NASH could be caused by abnormal metabolism, accumulation of lipids, mitochondrial dysfunction, and oxidative and endoplasmic reticulum stresses and is found in obese and non-obese patients. Recent translational research studies have discovered new proteins and signaling pathways that are involved not only in the development of NAFLD but also in its progression to NASH, cirrhosis, and HCC. Nevertheless, the mechanisms of HCC developing from precancerous lesions have not yet been fully elucidated. Now, it is of particular importance to start research focusing on the discovery of novel molecular pathways that mediate alterations in glucose and lipid metabolism, which leads to the development of liver steatosis. The role of mTOR signaling in NASH progression to HCC has recently attracted attention. The goals of this review are (1) to highlight recent research on novel genetic and protein contributions to NAFLD/NASH; (2) to investigate how recent scientific findings might outline the process that causes NASH-associated HCC; and (3) to explore the reliable biomarkers/targets of NAFLD/NASH-associated hepatocarcinogenesis.

Dimethylarsinic acid induces bladder carcinogenesis via the amphiregulin pathway.

Dimethylarsinic acid (DMA) is a major metabolite in the urine of humans and rats exposed to inorganic arsenicals, and is reported to induce rat bladder carcinogenesis. In the present study, we focused on early pathways of carcinogenesis triggered by DMA that were also active in tumors. RNA expression in the bladder urothelium of rats treated with 0 and 200 ppm DMA in the drinking water for 4 weeks and in bladder tumors of rats treated with 200 ppm DMA for 2 years was initially examined using microarray analysis and Ingenuity Pathway Analysis (IPA). Expression of 160 genes was altered in both the urothelium of rats treated for 4 weeks with DMA and in DMA-induced tumors. IPA associated 36 of these genes with liver tumor diseases. IPA identified the amphiregulin (Areg)-regulated pathway as a Top Regulator Effects Network. Therefore, we focused on Areg and 6 of its target genes: cyclin A2, centromere protein F, marker of proliferation Ki-67, protein regulator of cytokinesis 1, ribonucleotide reductase M2, and topoisomerase II alpha. We confirmed high mRNA expression of Areg and its 6 target genes in both the urothelium of rats treated for 4 weeks with DMA and in DMA-induced tumors. RNA interference of human amphiregulin (AREG) expression in human urinary bladder cell lines T24 and UMUC3 decreased expression of AREG and its 6 target genes and decreased cell proliferation. These data suggest that Areg has an important role in DMA-induced rat bladder carcinogenesis.

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.

o-Toluidine metabolism and effects in the urinary bladder of humanized-liver mice.

Occupational exposure to aromatic amines is one of the most important risk factors for urinary bladder cancer. When considering the carcinogenesis of aromatic amines, metabolism of aromatic amines in the liver is an important factor. In the present study, we administered ortho-toluidine (OTD) in the diet to mice for 4 weeks. We used NOG-TKm30 mice (control) and humanized-liver mice, established via human hepatocyte transplantation, to compare differences in OTD-induced expression of metabolic enzymes in human and mouse liver cells. We also investigated OTD-urinary metabolites and proliferative effects on the urinary bladder epithelium. RNA and immunohistochemical analyses revealed that expression of N-acetyltransferases mRNA in the liver tended to be lower than that of the P450 enzymes, and that OTD administration had little effect on N-acetyltransferase mRNA expression levels. However, expression of CYP3A4 was increased in the livers of humanized-liver mice, and expression of Cyp2c29 (human CYP2C9/19) was increased in the livers of NOG-TKm30 mice. OTD metabolites in the urine and cell proliferation activities in the bladder urothelium of NOG-TKm30 and humanized-liver mice were similar. However, the concentration of OTD in the urine of NOG-TKm30 mice was markedly higher than in the urine of humanized-liver mice. These data demonstrate differences in hepatic metabolic enzyme expression induced by OTD in human and mouse liver cells, and consequent differences in the metabolism of OTD by human and mouse liver cells. This type of difference could have a profound impact on the carcinogenicity of compounds that are metabolized by the liver, and consequently, would be important in the extrapolation of data from animals to humans.

Evaluation of the Mechanisms Involved in the Development of Bladder Toxicity following Exposure to Occupational Bladder Cancer Causative Chemicals Using DNA Adductome Analysis.

Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the carcinogenic mechanisms in rat bladder by comprehensive analysis of DNA adducts (DNA adductome). DNA was extracted from the bladder epithelia of rats treated with AAs, including acetoacet-o-toluidine (AAOT) and o-toluidine (OTD), and adductome analysis was performed. Principal component analysis-discriminant analysis revealed that OTD and AAOT observed in urinary bladder hyperplasia could be clearly separated from the controls and other AAs. After confirming the intensity of each adduct, four adducts were screened as having characteristics of the OTD/AAOT treatment. Comparing with the in-house DNA adduct database, three of four candidates were identified as oxidative DNA adducts, including 8-OH-dG, based on mass fragmentation together with high-resolution accurate mass (HRAM) spectrometry data. Therefore, findings suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium exposed to AAs. Consequently, the administration of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, in six-week-old rats fed with 0.6% OTD in their diet resulted in simple hyperplastic lesions in the bladder that were suppressed by apocynin. The labeling indices of Ki67, γ-H2AX, and 8-OHdG were significantly decreased in an apocynin concentration-dependent manner. These findings indicate that oxidative stress may have contributed to the development of urinary cancer induced by OTD.

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.

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.

Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation.

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs' production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy.

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.

Interplay between protein acetylation and ubiquitination controls MCL1 protein stability.

The anti-apoptotic myeloid cell leukemia 1 (MCL1) protein belongs to the pro-survival BCL2 family and is frequently amplified or elevated in human cancers. MCL1 is highly unstable, with its stability being regulated by phosphorylation and ubiquitination. Here, we identify acetylation as another critical post-translational modification regulating MCL1 protein stability. We demonstrate that the lysine acetyltransferase p300 targets MCL1 at K40 for acetylation, which is counteracted by the deacetylase sirtuin 3 (SIRT3). Mechanistically, acetylation enhances MCL1 interaction with USP9X, resulting in deubiquitination and subsequent MCL1 stabilization. Therefore, ectopic expression of acetylation-mimetic MCL1 promotes apoptosis evasion of cancer cells, enhances colony formation potential, and facilitates xenografted tumor progression. We further demonstrate that elevated MCL1 acetylation sensitizes multiple cancer cells to pharmacological inhibition of USP9X. These findings reveal that acetylation of MCL1 is a critical post-translational modification enhancing its oncogenic function and provide a rationale for developing innovative therapeutic strategies for MCL1-dependent tumors.

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.

Canopy Homolog 2 as a Novel Molecular Target in Hepatocarcinogenesis.

In the present study, the role of a novel protein involved in neurite development and endoplasmic reticulum (ER) stress, canopy homolog 2 (CNPY2), was investigated in mouse and human hepatocarcinogenesis. Firstly, a sensitive quantitative and qualitative detection of protein expression using QSTAR Elite LC-Ms/Ms was performed for the analysis of lysates of microdissected hepatocellular altered foci (AF), adenomas (HCAs), carcinomas (HCCs) and peri-tumoral livers from C57Bl/6J mice treated with diethylnitrosamine (DEN) and then maintained for 27 or 38 weeks on basal diet. Significant overexpression of 18.5 kDa CNPY2 processed form was demonstrated in AF, HCAs and HCCs, while low expression was observed in the livers of DEN-treated and control mice. Furthermore, CNPY2 elevation in AF and tumors was coordinated with accumulation of numerous cytoskeletal proteins, including cytokeratins 8 and 18, actin, non-muscle myosin and septin 9 and those involved in ER and mitochondrial stresses such as calreticulin, prohibitins 1 and 2 and YME1-like-1. Knockdown of CNPY2 in Huh7 and HepG2 human liver cancer cells resulted in significant suppression of cell survival and invasive potential, inhibition of cyclin D1, induction of p21Waf1/Cip1 and suppression of the apoptosis inhibitor Bcl2. In contrast, transfection of a mouse CNPY2 (mCNPY2-Ds-Red) vector plasmid in Huh7 and HepG2 cancer cells, with subsequent accumulation of CNPY2 in the ER, resulted in significant increase in cancer cells survival. Clinicopathological analysis in 90 HCV-positive HCC patients, revealed significant association of CNPY2 overexpression with poor overall (p = 0.041) survival. Furthermore, CNPY2 increase was associated with vessel invasion (p = 0.038), poor histological differentiation (p = 0.035) and advanced clinical stage (p = 0.016). In conclusion, CNPY2 is a promising molecular target elevated early in hepatocarcinogenesis and prognostic marker for human HCV-associated HCC. CNPY2 is involved in the processes of ER stress, cell cycle progression, proliferation, survival and invasion of liver tumor cells.

Cache Domain Containing 1 Is a Novel Marker of Non-Alcoholic Steatohepatitis-Associated Hepatocarcinogenesis.

In the present study, potential molecular biomarkers of NASH hepatocarcinogenesis were investigated using the STAM mice NASH model, characterized by impaired insulin secretion and development of insulin resistance. In this model, 2-days-old C57BL/6N mice were subjected to a single subcutaneous (s.c.) injection of 200 µg streptozotocin (STZ) to induce diabetes mellitus (DM). Four weeks later, mice were administered high-fat diet (HFD) HFD-60 for 14 weeks (STAM group), or fed control diet (STZ group). Eighteen-week-old mice were euthanized to allow macroscopic, microscopic, histopathological, immunohistochemical and proteome analyses. The administration of HFD to STZ-treated mice induced significant fat accumulation and fibrosis development in the liver, which progressed to NASH, and rise of hepatocellular adenomas (HCAs) and carcinomas (HCCs). In 18-week-old animals, a significant increase in the incidence and multiplicity of HCAs and HCCs was found. On the basis of results of proteome analysis of STAM mice HCCs, a novel highly elevated protein in HCCs, cache domain-containing 1 (CACHD1), was chosen as a potential NASH-HCC biomarker candidate. Immunohistochemical assessment demonstrated that STAM mice liver basophilic, eosinophilic and mixed-type altered foci, HCAs and HCCs were strongly positive for CACHD1. The number and area of CACHD1-positive foci, and cell proliferation index in the area of foci in mice of the STAM group were significantly increased compared to that of STZ group. In vitro siRNA knockdown of CACHD1 in human Huh7 and HepG2 liver cancer cell lines resulted in significant inhibition of cell survival and proliferation. Analysis of the proteome of knockdown cells indicated that apoptosis and autophagy processes could be activated. From these results, CACHD1 is an early NASH-associated biomarker of liver preneoplastic and neoplastic lesions, and a potential target protein in DM/NASH-associated hepatocarcinogenesis.

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.

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.

Cell proliferation of rat bladder urothelium induced by nicotine is suppressed by the NADPH oxidase inhibitor, apocynin.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis in a two-stage carcinogenesis model. Nicotine also induced cytotoxicity in the bladder urothelium in a short-term study. In the present study, male rats were treated with nicotine (40 ppm) in drinking water co-administered with the NADPH oxidase inhibitor, apocynin (0, 250 or 750 mg/kg) in diet for 4 weeks. The apocynin treatment induced no clinical toxic effects. Reduction of reactive oxygen species (ROS) by apocynin was confirmed by immunohistochemistry of 8-OHdG in the bladder urothelium. Incidences of simple hyperplasia, cell proliferation and apoptosis were reduced by apocynin treatment in the bladder urothelium. However, despite reduction of cell proliferation (labeling index), apocynin did not affect the incidence of simple hyperplasia, apoptosis, or ROS generation in the kidney pelvis urothelium, in addition to 8-OHdG positivity induced by nicotine being lower. In vitro, apocynin (500 µM) reduced ROS generation, but induced cell proliferation in bladder cancer cell lines (T24 and UMUC3 cells). These data suggest that oxidative stress may play a role in the cell proliferation of the bladder urothelium induced by nicotine.