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.

Overexpression of miR-199/214 is a distinctive feature of iron-induced and asbestos-induced sarcomatoid mesothelioma in rats.

Malignant mesothelioma (MM) is one of the most lethal tumors in humans. The onset of MM is linked to exposure to asbestos, which generates reactive oxygen species (ROS). ROS are believed to be derived from the frustrated phagocytosis and the iron in asbestos. To explore the pathogenesis of MM, peritoneal MM was induced in rats by the repeated intraperitoneal injection of iron saccharate and nitrilotriacetate. In the present study, we used microarray techniques to screen the microRNA (miR) expression profiles of these MM. We observed that the histological subtype impacted the hierarchical clustering of miR expression profiles and determined that miR-199/214 is a distinctive feature of iron saccharate-induced sarcomatoid mesothelioma (SM). Twist1, a transcriptional regulator of the epithelial-mesenchymal transition, has been shown to activate miR-199/214 transcription; thus, the expression level of Twist1 was examined in iron-induced and asbestos-induced mesotheliomas in rats. Twist1 was exclusively expressed in iron saccharate-induced SM but not in the epithelioid subtype. The Twist1-miR-199/214 axis is activated in iron saccharate-induced and asbestos-induced SM. The expression levels of miR-214 and Twist1 were correlated in an asbestos-induced MM cell line, suggesting that the Twist1-miR-199/214 axis is preserved. MeT5A, an immortalized human mesothelial cell line, was used for the functional analysis of miR. The overexpression of miR-199/214 promoted cellular proliferation, mobility and phosphorylation of Akt and ERK in MeT5A cells. These results indicate that miR-199/214 may affect the aggressive biological behavior of SM.

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 may promote prostate carcinogenesis in rats.

Arsenic is a known human carcinogen, inducing tumors of the lung, urinary bladder, skin, liver and prostate. However, there are no reports of prostate tumors induced by arsenicals in in vivo animal models. In a previous study, we found that HMGB2 expression was a predictive marker for prostate carcinogens in the rat 4-week repeated dose test. In this study, six-week-old male F344 rats were orally treated with a total of six chemicals (2-acetylaminofluorene (2-AAF), p-cresidine, dimethylarsinic acid (DMA), glycidol, N-nitrosodiethylamine and acrylamide) for four weeks. Animals were sacrificed at the end of the study, and HMGB2 and Ki-67 immunohistochemistry was performed. The numbers of HMGB2- and Ki-67- positive cells in all prostate lobes were significantly increased by DMA, one of the arsenicals, compared with the controls. Meanwhile, the number of Ki-67-positive cells in lateral and dorsal prostate lobes was significantly decreased by 2-AAF with the reduction of body weight, but HMGB2 expression was not. The other chemicals did not change HMGB2 and Ki-67 expression. These data indicate that DMA may have an ability to enhance prostate carcinogenesis.

MET amplification in endometrial cancers with clear-cell carcinoma components.

Endometrial clear-cell carcinoma (ECC) is relatively rare. The expression of diagnostic markers in this disease is similar to that of clear-cell carcinoma, but the molecular carcinogenic events and therapeutic targets are mostly unknown. MET gene amplification has been reported in various cancers, including ovarian clear-cell carcinomas; however, the MET gene status has not previously been examined in ECC. We performed real-time quantitative PCR (QPCR) and fluorescence in situ hybridization (FISH) to analyze the MET gene statuses of 12 ECC cases. We found MET amplifications in two cases (2/12; 16.7%) by both methods. Of the 12 cases, 9 were pure clear-cell carcinomas, and 3 were mixed types that included mixes with endometrioid carcinomas in 2 cases, and the remaining case was a heterologous-type carcinosarcoma that primarily consisted of a clear-cell carcinoma component and a scarce chondrosarcoma component. Both of the MET amplification cases were mixed; one contained endometrioid features, and the other chondrosarcoma features. This is the first report to analyze the statuses of the MET gene in ECCs, and the two mixed cases exhibited amplifications that are shared with ovarian clear-cell carcinomas. Further studies with larger numbers of cases are necessary to reveal the relationship between ECC and MET amplification.

The NADPH Oxidase Inhibitor Apocynin Suppresses Preneoplastic Liver Foci of Rats.

Reactive oxygen species (ROS) have been revealed to be important factors for carcinogenesis and tumor progression. Therefore, we focused on an ROS-generating protein, nicotinamide adenine dinucleotide phosphate oxidase, and evaluated whether its inhibitor, apocynin, could suppress hepatocarcinogenesis in a medium-term rat liver bioassay. The number and size of glutathione S-transferase placental form (GST-P)-positive foci were significantly reduced by apocynin in a dose-dependent manner. The reduction of ROS generation by apocynin was confirmed by dihydroethidium staining. Apocynin treatment also significantly reduced Ki-67 positivity, downregulated cyclooxygenase 2, and suppressed the activation of the c-Myc pathway. Meanwhile, ROS generation was not different between GST-P-positive foci and surrounding GST-P-negative areas of the liver. In conclusion, the present data suggest that apocynin possesses a potential antihepatocarcinogenic property.

Anti-N-methyl-d-aspartate receptor limbic encephalitis associated with mature cystic teratoma of the fallopian tube.

Anti-N-methyl-d-aspartate receptor (NMDAR) limbic encephalitis is the most common form of paraneoplastic encephalitis that is associated with teratomas. Because tumor removal leads to better clinical outcomes, it is essential to reveal the location of the teratomas. This is the first reported case of anti-NMDAR encephalitis associated with teratoma of the fallopian tube. Salpingo-oophorectomy improved neurological symptoms and immunohistochemical examinations indicated the expression of NMDAR on neuroglial cells within the fallopian tube teratoma. Teratomas of the fallopian tube cause anti-NMDAR encephalitis; the imaging analysis and exploratory laparoscopies of the fallopian tube as well as of the ovary should be considered. Surgical removal of both fallopian tubes and ovaries with a normal appearance should be considered for patients in whom immunotherapy is not effective.

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis.

Multi-wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50-nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.

Pain-reducing anesthesia prevents oxidative stress in human term placenta.

Anesthesia is sometimes used for the reduction of maternal pain in normal human term labor, but whether the drugs affect oxidative stress remains unclear. The placenta serves as an interface between the maternal and fetal vasculature. In this study, we immunohistochemically analyzed two markers for oxidative stress, namely 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal-modified proteins (HNE), using placentas from 21 cases of normal tansvaginal delivery (V group), 20 Caesarean sections (C group), and 17 normal transvaginal deliveries with epidural anesthesia (E group). 8-OHdG staining in the nuclei of trophoblasts lining the chorionic villi was significantly stronger in the V group either compared with the C or E group (p<0.001), without significant differences in the C and E groups (p = 0.792). Moderate to intense staining by HNE of the intravascular serum of chorionic villi vasculature was frequently observed in the placentas from the V group, but less frequently of those in either C or E groups (p<0.001), nor the p value comparing the C and E groups was significant (p = 0.128) for HNE staining. Our results suggest that although the role of oxidative stress and its influences on fetal state in the placenta in labor remains unclear, it seems to be lessened by epidural anesthesia.

Connexin 32 and luteolin play protective roles in non-alcoholic steatohepatitis development and its related hepatocarcinogenesis in rats.

Non-alcoholic steatohepatitis (NASH) has the potential to lead to the development of cirrhosis and hepatocellular carcinoma (HCC). Connexin (Cx) 32, a hepatocyte gap-junction protein, plays a preventive role in hepatocarcinogenesis. However, the precise contribution of Cx32 in the development of NASH has not been established. In this study, we aimed to clarify the role of Cx32 and the chemopreventive effect of luteolin, an antioxidant flavonoid, on the progression of NASH and NASH-related hepatocarcinogenesis. Cx32 dominant negative transgenic (Cx32ΔTg) and wild-type (Wt) rats at 10 weeks of age were given diethylnitrosamine and fed methionine-choline-deficient diet (MCDD) or MCDD with luteolin for 12 weeks. MCDD induced steatohepatitis and fibrosis along with increased inflammatory cytokine expression and reactive oxygen species in the liver. These effects were more severe in Cx32ΔTg rats as compared with Wt rats, and significantly suppressed by luteolin in both genotypes. Concerning NASH-related hepatocarcinogenesis, the number of glutathione S-transferase placental form (GST-P)-positive foci was greater in Cx32ΔTg versus Wt rats, and significantly reduced by luteolin in Cx32ΔTg rats. Microarray analysis identified brain expressed, X-linked 1 (Bex1) as an upregulated gene in Cx32ΔTg rat liver. Quantitative RT-PCR and in situ hybridization revealed that increased Bex1 mRNA was localized in GST-P-positive foci in Cx32ΔTg rats, and the expression level was significantly decreased by luteolin. Moreover, Bex1 knockdown resulted in significant growth inhibition of the rat HCC cell lines. These results show that Cx32 and luteolin have suppressive roles in inflammation, fibrosis and hepatocarcinogenesis during NASH progression, suggesting a potential therapeutic application for NASH.

Napsin A is a specific marker for ovarian clear cell adenocarcinoma.

Ovarian clear cell adenocarcinoma has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Differential diagnosis of clear cell adenocarcinoma from other ovarian surface epithelial tumors is important for its treatment. Napsin A is a known diagnostic marker for lung adenocarcinoma, and expression of napsin A is reported in a certain portion of thyroid and renal carcinomas. However, napsin A expression in ovarian surface epithelial tumors has not previously been examined. In this study, immunohistochemical analysis revealed that in 71 of 86 ovarian clear cell adenocarcinoma patients (83%) and all of the 13 patients with ovarian clear cell adenofibroma, positive napsin A staining was evident. No expression was observed in 30 serous adenocarcinomas, 11 serous adenomas or borderline tumors, 19 endometrioid adenocarcinomas, 22 mucinous adenomas or borderline tumors, 10 mucinous adenocarcinomas, or 3 yolk sac tumors of the ovary. Furthermore, expression of napsin A was not observed in the normal surface epithelium of the ovary, epithelia of the fallopian tubes, squamous epithelium, endocervical epithelium, or the endometrium of the uterus. Therefore, we propose that napsin A is another sensitive and specific marker for distinguishing ovarian clear cell tumors (especially adenocarcinomas) from other ovarian tumors.

Connective tissue growth factor and ß-catenin constitute an autocrine loop for activation in rat sarcomatoid mesothelioma.

Due to the formerly widespread use of asbestos, malignant mesothelioma (MM) is increasingly frequent worldwide. MM is classified into epithelioid (EM), sarcomatoid (SM), and biphasic subtypes. SM is less common than EM but is recognized as the most aggressive type of MM, and these patients have a poor prognosis. To identify genes responsible for the aggressiveness of SM, we induced EM and SM in rats, using asbestos, and compared their transcriptomes. Based on the results, we focused on connective tissue growth factor (Ctgf), whose expression was significantly increased in SM compared with EM; EM itself exhibited an increased expression of Ctgf compared with normal mesothelium. Particularly in SM, Ctgf was a major regulator of MM proliferation and invasion through activation of the ß-catenin-TCF-LEF signalling pathway, which is autocrine and formed a positive feedback loop via LRP6 as a receptor for secreted Ctgf. High Ctgf expression also played a role in the epithelial-mesenchymal transition in MM. Furthermore, Ctgf is a novel serum biomarker for both early diagnosis and determining the MM prognosis in rats. These data link Ctgf to SM through the LRP6-GSK3ß-ß-catenin-TCF-Ctgf autocrine axis and suggest Ctgf as a therapeutic target.

Ovarian cancer: new developments in clear cell carcinoma and hopes for targeted therapy.

Until recently, ovarian clear cell carcinoma was recognized by its unique morphology and unfavorable patient outcome primarily due to tumor chemoresistance. Recently, specific molecular characteristics of ovarian clear cell carcinoma, such as PI3CA mutation, ARID1a mutation and MET amplification, have been elucidated. In addition, an association between endometriosis and the tumor has also been a focus of research in recent years. The aim of this review is to discuss the specificity and importance of molecular changes and various intriguing points that are not solved until today. Finally, future aspects, including hopes for the development of novel therapies, are discussed.

Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production.

Like many other human cancers, the development of malignant mesothelioma is closely associated with a chronic inflammatory condition. Both macrophages and mesothelial cells play crucial roles in the inflammatory response caused by asbestos exposure. Here, we show that adipocytes can also contribute to asbestos-induced inflammation through dysregulated adipocytokine production. 3T3-L1 preadipocytes were differentiated into mature adipocytes prior to use. These cells took up asbestos fibers (chrysotile, crocidolite and amosite) but were more resistant to asbestos-induced injury than macrophages and mesothelial cells. Expression microarray analysis followed by reverse transcription-PCR revealed that adipocytes respond directly to asbestos exposure with an increased production of proinflammatory adipocytokines [e.g. monocyte chemoattractant protein-1 (MCP-1)], whereas the production of anti-inflammatory adipocytokines (e.g. adiponectin) is suppressed. This was confirmed in epididymal fat pad of mice after intraperitoneal injection of asbestos fibers. Such dysregulated adipocytokine production favors the establishment of a proinflammatory environment. Furthermore, MCP-1 marginally promoted the growth of MeT-5A mesothelial cells and significantly enhanced the wound healing of Y-MESO-8A and Y-MESO-8D human mesothelioma cells. Our results suggest that increased levels of adipocytokines, such as MCP-1, can potentially contribute to the promotion of mesothelial carcinogenesis through the enhanced recruitment of inflammatory cells as well as a direct growth and migration stimulatory effect on mesothelial and mesothelioma cells. Taken together, our findings support a potential cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis.

Expression of chromobox homolog 7 (CBX7) is associated with poor prognosis in ovarian clear cell adenocarcinoma via TRAIL-induced apoptotic pathway regulation.

Ovarian cancer is the most lethal gynecologic malignancy, and clear cell adenocarcinoma of the ovary (OCCA), in particular, has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Chromobox (CBX) 7 is a polycomb repressive complex 1 component that prolongs the lifespan of normal human cells by downregulating the INK4a/ARF expression which promotes cell-cycle progression. However, recent reports studying the relationship between CBX7 expression and patient survival have differed regarding the tumor cell origins, and the precise role of CBX7 in human carcinomas remains obscure. In this study, we analyzed CBX7 expression by immunohistochemistry in 81 OCCA patients and evaluated its association with their clinical outcomes. Both the overall and progression-free survival rates of the CBX7-positive patients were significantly shorter than those of the CBX7-negative patients (p < 0.05). CBX7 knockdown experiments using two OCCA cell lines, TOV21G and KOC-7C, revealed that cell viability was significantly reduced compared to the control cells (p < 0.001). Expression microarray analysis revealed that apoptosis-related genes, particularly tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were significantly upregulated in CBX7 knockdown cells (p < 0.01). We further confirmed that CBX7 knockdown resulted in TRAIL-induced apoptosis in the OCCA cells. Thus, in this study, we showed for the first time that CBX7 was associated with a decreased OCCA prognosis. We also successfully demonstrated that the TRAIL pathway is a novel target for CBX7 expression modulation in these cells, and therapeutic agents utilizing the TRAIL pathway may be particularly effective for targeted OCCA therapy.

Lack of presence of the human cytomegalovirus in human glioblastoma.

Recent reports have indicated human cytomegalovirus (HCMV) to be associated with human glioblastoma carcinogenesis. In established examples of viral carcinogenesis, viral DNA and one or more of its products have been detected in most tumor cells of biopsies in the majority of cases. To test whether HCMV is associated with human glioblastoma based on this criterion, we measured the number of viral DNA molecules per cell in both frozen and paraffin-embedded tumor biopsies from 58 patients using real-time quantitative PCR (QPCR). Immunohistochemical and fluorescence in situ hybridization (FISH) to detect HCMV proteins and genome was performed in 10 cases using formalin-fixed paraffin-embedded glioblastoma tissues. Southern blotting using DNA extracted from four glioblastoma cell lines together with immunoblotting using the four cell lines and five glioblastoma tissue samples were also performed. We further confirmed the immunoblot bands using liquid chromatography-tandem mass spectrometry assay. As a result, HCMV DNA was not detected in the tumor cells from any of the glioblastoma cases by QPCR detecting two different HCMV genes, in clear contrast to samples from patients with HCMV infection. Southern blotting and immunoblotting of cell lines and FISH using paraffin sections were all negative. However, immunoblotting and immunohistochemistry using tissue samples were partly positive, but HCMV proteins were not detected by proteomic analysis, suggesting false positivity of the analyses. As our QPCR analysis could detect 10 copies of HCMV DNA mixed with DNA extracted from 10(4) HCMV-negative cells, we conclude that HCMV is not persistent, at least in the tumor cells, of developed human glioblastoma.

Diameter and rigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis.

Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameter-dependent piercing of the cell membrane. Thin MWCNTs (diameter ∼ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter ∼ 150 nm) or tangled (diameter ∼ 2-20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health.

Establishment of an invasive prostate cancer model in transgenic rats by intermittent testosterone administration.

We have established a transgenic rat for adenocarcinoma of the prostate (TRAP) model that features uniform adenocarcinoma development in prostatic lobes at high incidence within a short experimental period. However, no invasive carcinomas with reactive stroma characteristics similar to those in man were observed. We therefore have focused on a new model for invasive carcinoma of the prostate using TRAP rats. In experiment 1, male TRAP rats in groups 1 and 2 were treated with orchiectomy at day 0 of the experiment. Rats in groups 1-3 underwent testosterone propionate (TP) implantation from weeks 1 to 4 and from weeks 6 to 16. Rats in groups 1 and 3 were given 3,2'-dimethyl-4-aminobiphenyl (DMAB) after TP implantation. The rats of group 4 served as controls. In experiment 2, the rats were divided into three groups, none of which received DMAB or orchiectomy, treated with TP continuously or with the treatment withdrawn once or twice. In experiment 1, invasive adenocarcinomas with abundant collagenous stroma were found in the dorsolateral and anterior prostate, some of which showed perineural space invasion at week 16. The number of invasive carcinoma foci was most frequent in group 3. In experiment 2, invasive adenocarcinoma development in the lateral prostates was correlated with the number of TP administration/withdrawal cycles. In conclusion, our newly established rat model for invasive adenocarcinoma of the prostate could serve as a useful preclinical model for evaluating the in vivo efficacy of preventive and therapeutic agents targeting of the tumor microenvironment.

ß4GalT6 is involved in the synthesis of lactosylceramide with less intensity than ß4GalT5.

Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, ß1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of ß4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that ß4GalT6 plays a role as a LacCer synthase, whereas ß4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.

Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis.

Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.