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Tumor-associated macrophages (TAM) in cancer stroma play important roles for cancer cell growth, invasion, angiogenesis, and metastases. We synthesized a novel photosensitizer, mannose-conjugated chlorin (M-chlorin), designed to bind mannose receptors highly expressed on TAMs. We evaluated the newly available photodynamic therapy (PDT) with M-chlorin against gastric and colon cancer. We evaluated PDT with M-chlorin for in vitro cytotoxicity and apoptosis induction in cancer cells compared with chlorin alone and glucose-conjugated chlorin (G-chlorin). The subcellular localization of M-chlorin was observed by confocal microscopy, and the M-chlorin PDT effects against TAMs including THP-1–induced M2-polarized macrophages were evaluated. Anticancer effects were also investigated in an allograft model where cytotoxic effects against TAMs in the cancer cell stroma were analyzed by immunohistochemistry. M-chlorin PDT strongly induced cell death in cancer cells to almost the same extent as G-chlorin PDT by inducing apoptosis. M-chlorin was incorporated into cancer cells where it localized mainly in lysosomes and endoplasmic reticula. M-chlorin PDT revealed strong cytotoxicity for M2 macrophages induced from THP-1 cell lines, and it induced stronger cytotoxicity than G-chlorin PDT in the allograft model through killing both cancer cells and TAMs in the cancer stroma. The M-chlorin PDT produced strong cytotoxicity against cancer tissue by inducing apoptosis of both cancer cells and TAMs in the cancer stroma. This novel PDT thus stands as a new candidate for very effective, next-generation PDT. Mol Cancer Ther; 14(2); 452–60. ©2014 AACR.

M-chlorin and G-chlorin accumulation in tumor regions.

Supplementary Figure Legends of Fig. S1 to Fig. S7.

Legends for supplemental figures

Targeting androgen receptor (AR) by pharmacologic intervention is one of the effective approaches for treatment of malignant prostate cancers. Histone deacetylase (HDAC) alters the epigenetic status of tumor-associated genes, including those for miRNAs (miRNA), and affects the behavior of cancers. Here, we examined the molecular effects of a HDAC inhibitor, OBP-801, on AR expression and tumor cell growth in prostate cancers. Treatment with OBP-801 efficiently suppressed cell growth of three prostate cancer lines (22Rv1, VCaP, and LNCaP), together with AR downregulation, regardless of their hormone sensitivity. Intriguingly, this effect by OBP-801 was not due to decreased transcriptional activity of the AR gene, but due to posttranscriptional regulation, namely by miRNA-mediated suppression. Among the upregulated miRNAs after OBP-801 treatment in the three prostate cancer cell lines, miR-320a, whose expression was significantly correlated with prognosis of prostate cancers (P = 0.0185), was the most closely associated with AR expression. An miR-320a mimic suppressed AR protein expression together with growth suppression, while anti-miR-320a oligonucleotide significantly abrogated the growth suppression by OBP-801 treatment. FISH analysis revealed that miR-320a was highly expressed in human normal prostate luminal cells, but was rarely expressed in prostate cancer cells. In an AR-dependent prostate tumorigenic rat model, OBP-801 treatment profoundly increased miR-320a expression and repressed prostate tumorigenesis. Our data demonstrated that OBP-801 effectively suppressed AR activity via epigenetic upregulation of miR-320a, which resulted in tumor cell growth suppression of prostate cancers. OBP-801 may be a potent AR-targeting therapeutic reagent in AR-positive prostate cancer regardless of androgen dependency. Cancer Res; 76(14); 4192–204. ©2016 AACR.

Supplementary Table S1. Background of Prostate Cancer Patients. Supplementary Table S2. List of the primers. Supplementary Table S3. 91 miRNAs upregulated in at least 1 of 3 cell lines.

Supplementary Fig. S1. Effect of OBP-801 and other HDAC inhibitors in prostate cancer cells. Supplementary Fig. S2. Western blot analysis of prostate cancer cells treated with OBP-801 or other HDAC inhibitors. Supplementary Fig. S3. Expression analysis of miR-320a, miR-320c, and miR-3065 in prostate cancer cells treated with OBP-801. Supplementary Fig. S4. Luciferase assay of AR 3'UTR with deletion or mutation in miR-320atarget sites. Supplementary Fig. S5. Inhibition of tumor growth and AR expression by OBP-801 in the 22Rv1 xenograft model. Supplementary Fig. S6. Predicted binding sites of rat miR-320aare indicated (arrowheads) in the rat Ar3'-UTR. Supplementary Fig. S7. Effects of combination therapies of OBP-801 and other conventional anti-androgen or chemotherapeutic agents in prostate cancer cell lines.

Additional information of Materials and Methods.

ACI/Seg (ACI) rats develop prostate cancers spontaneously with aging, similar to humans. Here, to identify genes involved in prostate cancer susceptibility, we did linkage analysis and oligonucleotide microarray analysis. Linkage analysis was done using 118 effective rats, and prostate cancer susceptibility 1 (Pcs1), whose ACI allele dominantly induced prostate cancers, was mapped on chromosome 19 [logarithm of odds (LOD) score of 5.0]. PC resistance 1 (Pcr1), whose ACI allele dominantly and paradoxically suppressed the size of prostate cancers, was mapped on chromosome 2 (LOD score of 5.0). When linkage analysis was done in 51 rats with single or no macroscopic testicular tumors, which had larger prostates and higher testosterone levels than those with bilateral testicular tumors, Pcs2 and Pcr2 were mapped on chromosomes 20 and 1, respectively. By oligonucleotide microarray analysis with 8,800 probe sets and confirmation by quantitative reverse transcription-PCR, only two genes within these four loci were found to be differentially expressed >1.8-fold. Membrane metalloendopeptidase (Mme), known to inhibit androgen-independent growth of prostate cancers, on Pcr1 was expressed 2.0- to 5.5-fold higher in the ACI prostate, in accordance with its paradoxical effect. Cdkn1a on Pcs2 was expressed 1.5- to 4.5-fold lower in the ACI prostate. Additionally, genes responsible for testicular tumors and unilateral renal agenesis were mapped on chromosomes 11 and 14, respectively. These results showed that prostate cancer susceptibility of ACI rats involves at least four loci, and suggested Mme and Cdkn1a as candidates for Pcr1 and Pcs2.

Supplementary Table 2 from Linkage and Microarray Analyses of Susceptibility Genes in ACI/Seg Rats: A Model for Prostate Cancers in the Aged

Supplementary Table 1 from Linkage and Microarray Analyses of Susceptibility Genes in ACI/Seg Rats: A Model for Prostate Cancers in the Aged

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-β 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.

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., Many CD103+ T cells were observed in the tumor epithelium accompanied by CD8+ T cells and were located around tertiary lymphoid structures (TLSs). Patients with a combination of CD103high and TLS‐rich had a good prognosis.

The 5-year survival rate of pancreatic ductal carcinoma (PDAC) patients is, This study describes the chemopreventive effects of luteolin on pancreatic carcinogenesis via STAT3 inactivation caused by DPYD down-regulation, resulting in suppression of cell proliferation. This is consistent with the association of high DPYD expression with the poor prognosis of pancreatic cancer.

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.

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.

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-β1, Il-1β, 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.

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‐3ʹ‐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‐3ʹ‐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., Luteolin suppresses cell proliferation through downregulation of mTOR signaling and upregulation of p21 in bladder cancers both in vitro and in vivo. mTOR activity is correlated with invasive ability in human bladder cancer cases. A metabolite of luteolin decreases cell viability and squamous differentiation in in vivo rat bladder cancer models.

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.

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.

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., AR-V7 has been highlighted as a major therapeutic resistance mechanism in castration-resistant prostate cancer (CRPC). This study describes a novel mechanism for downregulation of AR-V7 by miR-8080, which induces apoptosis and improves the therapeutic efficacy in CRPC.

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 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.

Simple Summary Protein canopy homolog 2 (CNPY2) controls the outgrowth of neurites through positive regulation of their outgrowth in neuroblastoma and pheochromocytoma due to stabilizing the myosin regulatory light chain (MRLC). Given the important role in endoplasmic reticulum (ER) stress and proteolysis, we focused on investigation of CNPY2 expression in mouse and human hepatocarcinogenesis and its clinical relevance in virus-associated liver cancer. CNPY2 elevation and coordinated accumulation of numerous cytoskeletal proteins and those involved in ER and mitochondrial stresses was found in mouse altered foci and tumors by proteome analysis of microdissected lesions. In Huh7 and HepG2 human liver cancer cells, CNPY2 increase was significantly associated with cell cycle progression, activated cell proliferation and invasion. CNPY2 expression was high in HCV-associated HCC patients, being positively associated with survival. To the best of our knowledge, this is the first report to demonstrate that CNPY2 may become a useful prognostic biomarker in HCV-associated HCC. Abstract 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.

Multi‐walled carbon nanotube‐7 (MWCNT‐7) fibers are biopersistent and have a structure similar to asbestos. MWCNT‐7 has been shown to induce malignant mesothelioma when administered by intrascrotal or intraperitoneal injection in rats and mice, and an inhalation study demonstrated that rats exposed to respirable MWCNT‐7 developed lung tumors. MWCNT‐N, which is similar to MWCNT‐7, was shown to induce both lung tumors and malignant mesothelioma in rats when administered by trans‐tracheal intrapulmonary spraying (TIPS). The present study was performed to investigate the carcinogenicity of MWCNT‐7 when administered by the TIPS method. Ten‐week‐old male F344/Crj rats were divided into 3 groups and administered 0.5 mL vehicle, 0.250 μg/mL MWCNT‐7 or 0.250 μg/mL crocidolite once a week for 12 weeks (total doses of 1.5 mg/rat) and then observed for up to 104 weeks. Rats in the MWCNT‐7 group began to die from pathologies associated with the development of malignant mesothelioma 35 weeks after the final TIPS administration. Overall, the incidence of malignant mesothelioma in the MWCNT‐7 group was significantly higher than in the vehicle or crocidolite groups.

DNA damage caused by Helicobacter pylori infection and chronic inflammation or exposure to genotoxic agents is considered an important risk factor of gastric carcinogenesis. In this study, we have evaluated a short-term technique to detect DNA damage response to various chemical carcinogens; it involves visualization of Ser 139-phosphorylated histone H2AX (γ-H2AX) foci by immunohistochemistry and expression analysis of other genes by quantitative RT-PCR. Six-week-old male rats were intragastrically administered N-methyl-N-nitrosourea (MNU), 3,2’-dimethyl-4-aminobiphenyl (DMAB), dimethylnitrosamine (DMN), and 1,2- dimethylhydrazine (DMH) for 5 days/week for 4 weeks, using corn oil as a vehicle. Animals were sacrificed at day 28, and their stomachs were excised. γ-H2AX foci formation, indicating DNA double-strand breaks, was observed in the proliferative zone of both fundic and pyloric glands. The number of positive cells per gland was significantly high in pyloric glands in the MNU group and in fundic glands in the MNU and DMAB groups. A significant increase in p21waf1 mRNA level was observed in the DMN group compared with the control, which was in contrast to the decreasing tendency of the h2afx mRNA level in the MNU and DMN groups. Apoptotic cells positive for γ-H2AX pan or peripheral nuclear staining were observed on the surface layer of the fundic mucosa in the MNU group. The fundic pepsinogen a5 (pga5) mRNA level showed a significant decrease, indicating gland damage. The pyloric pepsinogen c mRNA level showed no change. In conclusion, γ-H2AX in combination with other gene expression analyses could be a useful biomarker in a short-term experiment on gastric chemical genotoxicity.

Many prostate cancer patients develop resistance to treatment called castration-resistant prostate cancer (CRPC) which is the major cause of recurrence and death. In the present study, four cyclohexanone curcumin analogs were synthesized. Additionally, their anticancer progression activity on CRPC cell lines, PC3 and PLS10 cells, was examined. We first determined their anti-metastasis properties and found that 2,6-bis-(4-hydroxy-3-methoxy-benzylidene)-cyclohexanone (2A) and 2,6-bis-(3,4-dihydroxy-benzylidene)-cyclohexanone (2F) showed higher anti-invasion properties against CRPC cells than curcumin. Analog 2A inhibited both MMP-2 and MMP-9 secretions and activities, whereas analog 2F reduced only MMP activities. These findings suggest that the compounds may inhibit CRPC cell metastasis by decreased extracellular matrix degradation. Analog 2A, the most potent analog, was then subjected to an in vivo study. Similar to curcumin, analog 2A was detectable in the serum of mice at 30 and 60 minutes after i.p. injections. Analog 2A and curcumin (30 mg/kg bodyweight) showed a similar ability to reduce tumor area in lungs of mice that were i.v. injected with PLS10 cells. Additionally, analog 2A showed superior growth inhibitory effect on PLS10 cells than that of curcumin both in vitro and in vivo. The compound inhibited PLS10 cells growth by induction of G1 phase arrest and apoptosis in vitro. Interestingly, analog 2A significantly decreased tumor growth with downregulation of cell proliferation and angiogenesis in PLS10-bearing mice. Taken together, we could summarize that analog 2A showed promising activities in inhibiting CRPC progression both in vitro and in vivo.

Simple Summary The aim of the present study was to discover novel early molecular biomarkers of liver neoplasms which arise in non-alcoholic steatohepatitis (NASH) Stelic Animal Model (STAM) mice. Significant increase of lipid deposits, hepatocyte ballooning, fibrosis, and incidences and multiplicities of hepatocellular adenomas and carcinomas were detected in the livers of 18-week-old STAM mice. From the results of proteome analysis of STAM mice hepatocellular carcinomas, significant elevation of a novel protein, cache domain-containing 1 (CACHD1) was found. Furthermore, we observed CACHD1-positive foci in STAM mice livers, which number, area, and cell proliferation index within the foci were significantly elevated. Results of immunohistochemical and in vitro functional analysis indicated that CACHD1 may become a useful early biomarker and potential molecular target in NASH-associated hepatocarcinogenesis, which is involved in control of cell proliferation, autophagy and apoptosis. Abstract 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.

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.

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.

INTRODUCTION AND OBJECTIVE:There is a need for seeking effective treatments for CRPC, because its emergence following ADT is a major clinical problem. In the present study, we investigated chemopre...

P-REX2a is a PTEN inhibitor that also activates Rac 1. No associations with P-REX2a and human endometrial cancers have been reported to date. In this study, we immunohistochemically analyzed 155 uterine endometrial malignancies for P-REX2a expression. The P-REX2a-positive tumors displayed worse prognosis independent of PTEN expression. Then, we transduced either P-REX2a expression vector or short hairpin RNAs targeting P-REX2a into 2 uterine endometrioid carcinoma cell lines, OMC-2 and JHUEM-14. Ectopic expression of P-REX2a led to increased cell proliferation only in the PTEN-expressing OMC-2 cells but did not show any change in the PTEN-negative JHUEM-14 cells or the P-REX2a-knockdown cells. Induction of P-REX2a increased and knockdown of P-REX2a decreased cell migration in both cell lines. Then, we performed expression microarray analysis using these cells, and pathway analysis revealed that the expression of members of the GPCR downstream pathway displayed the most significant changes induced by the knockdown of P-REX2a. Immunohistochemical analysis revealed that Vav1, a member of the GPCR downstream pathway, was expressed in 139 of the 155 endometrial tumors, and the expression levels of Vav1 and P-REX2a showed a positive correlation (r = 0.44, p < 0.001). In conclusion, P-REX2a enhanced cell motility via the GPCR downstream pathway independently of PTEN leading to progression of uterine endometrioid malignancies and poor prognosis of the patients.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. Cigarette smoke inhalation in mice and orally administered nicotine in rats and mice increased urothelial cell proliferation. Nicotine, a major component of smoke, induced cell proliferation in multiple cell types in vitro. In the present study, the enhancing effects of nicotine on F344 rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Nicotine administered in drinking water for 32 weeks following 4 weeks of BBN treatment significantly increased the incidence and number of urothelial carcinomas dose-dependently. Ki67 and pSTAT3 labeling indices and expression of nicotinic acetylcholine receptor alpha 7 (nAChRα7) in non-tumor bladder urothelial lesions were significantly increased by nicotine, but the TUNEL assay for apoptosis showed no increase. In a 4 week study, inhibitors of nicotinic acetylcholine receptor decreased nicotine-induced urothelial simple hyperplasia and Ki67 labeling index in the bladder and kidney pelvis at a single cytotoxic dose of nicotine (40 ppm). Urothelial cytotoxicity with regenerative proliferation was observed by light and scanning electron microscopy. In vitro, nicotine was not cytotoxic to rat or human immortalized urothelial cells (do not express nicotine receptors) below millimolar concentrations, nor in human RT4, T24 or UMUC3 urothelial carcinoma cells (express nicotine receptors). However, nicotine slightly, but statistically significantly, increased cell proliferation at micromolar concentrations in human urothelial carcinoma cells. These data suggest that nicotine enhances urinary bladder carcinogenesis by inducing cytotoxicity with regenerative proliferation. The possible role of direct mitogenesis, involving nAChR and STAT3 signaling and of nicotine receptors requires further investigation at non-cytotoxic doses of nicotine.

Purpose Programmed cell death 1 (PD1) and PD ligand 1 (PDL1) are speculated to have an important role in maintaining a normal pregnancy and there are also a few reports of an association between the single‐nucleotide polymorphisms (SNPs) of PD1 or PDL1 and diseases in humans. The association was examined between the polymorphisms of PD1, PDL1, CTLA4 and recurrent pregnancy loss (RPL). Methods The study group consisted of 243 women with two or more pregnancy losses and 176 parous women. The frequency of three SNPs of PD1, two of PDL1, and four of CTLA4 were compared between the patients and controls. Next, the subsequent live birth rates in the patients with RPL with and without risk alleles were examined. Results Two SNPs (rs36084323 and rs3481962) of PD1 were found to occur at significantly higher frequencies in the patient group than in the control group. The subsequent live birth rate of the patients with vs. without the risk alleles of rs36084323 and rs3481962 were 83.3% vs. 83.3% and 81.6% vs. 84.0%, respectively. Conclusion Variations of the PD1 gene were identified as risk factors for RPL. However, the presence or absence of the PD1 risk alleles had no predictive effect on the subsequent pregnancy outcome.

Screening prostatic carcinogens is time-consuming due to the time needed to induce preneoplastic and neoplastic lesions. To overcome this, we investigated alternative molecular markers for detection of prostatic carcinogens in a short period in rats. After treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), expression of high-mobility group protein B2 (HMGB2) was up-regulated in rat ventral prostate. To evaluate the applicability of HMGB2 in the early detection of carcinogenicity of chemicals using animal models, we examined HMGB2 expression in prostate of rats. Six-week-old male F344 rats were gavaged for four weeks with a total of eight individual chemicals, divided into two categories based on prostate carcinogenicity. Animals were sacrificed at the end of the study and HMGB2 immunohistochemistry was performed. HMGB2 expression in least one prostate lobe was significantly increased by all four prostate carcinogens compared with the controls. In contrast, the four chemicals that were not carcinogenic in the prostate did not cause HMGB2 up-regulation. Additionally, high HMGB2 expression in neoplastic lesions in both rat and human was detected. Therefore HMGB2 expression may be a good screening tool for the identification of potential of prostate carcinogens.

We study the structural, electronic, and optical properties of the Pt-based vacancy-ordered double perovskites A2PtX6 (A = K, Rb, Cs; X = Cl, Br, I) in the tetragonal P4/mnc polymorph using the relativistic all-electron calculations based on the density functional theory. It is found that the rotation angles θ of the PtX6 octahedra are non-zero for all the compounds. For a given A-site cation, θ increases from Cl to Br to I while for a given halide anion, θ decreases from K to Rb to Cs. As a result, the largest θ is found for K2PtI6. Taking the three iodides, K2PtI6, Rb2PtI6 and Cs2PtI6, as representatives, it is found that K2PtI6 is a direct-band-gap semiconductor and that Rb2PtI6 and Cs2PtI6 are quasi-direct-band-gap semiconductors with the small differences between the indirect and direct band gaps. The band gap in the tetragonal P4/mnc polymorph is larger than the corresponding one in the cubic F m 3 m polymorph. Employing the spectroscopically limited maximum efficiency as a metric for quantifying the photovoltaic performance, K2PtI6, Rb2PtI6, and Cs2PtI6 are found to be promising absorber materials for thin-film solar cells.