Suppressive Effect and Molecular Mechanism of Houttuynia cordata Thunb. Extract against Prostate Carcinogenesis and Castration-Resistant Prostate Cancer.

Houttuynia cordata Thunb. (HCT) is a well-known Asian medicinal plant with biological activities used in the treatment of many diseases including cancer. This study investigated the effects of HCT extract and its ethyl acetate fraction (EA) on prostate carcinogenesis and castration-resistant prostate cancer (CRPC). HCT and EA induced apoptosis in androgen-sensitive prostate cancer cells (LNCaP) and CRPC cells (PCai1) through activation of caspases, down-regulation of androgen receptor, and inactivation of AKT/ERK/MAPK signaling. Rutin was found to be a major component in HCT (44.00 ± 5.61 mg/g) and EA (81.34 ± 5.21 mg/g) in a previous study. Rutin had similar effects to HCT/EA on LNCaP cells and was considered to be one of the active compounds. Moreover, HCT/EA inhibited cell migration and epithelial-mesenchymal transition phenotypes via STAT3/Snail/Twist pathways in LNCaP cells. The consumption of 1% HCT-mixed diet significantly decreased the incidence of adenocarcinoma in the lateral prostate lobe of the Transgenic rat for adenocarcinoma of prostate model. Similarly, tumor growth of PCai1 xenografts was significantly suppressed by 1% HCT treatment. HCT also induced caspase-dependent apoptosis via AKT inactivation in both in vivo models. Together, the results of in vitro and in vivo studies indicate that HCT has inhibitory effects against prostate carcinogenesis and CRPC. This plant therefore should receive more attention as a source for the future development of non-toxic chemopreventive agents against various cancers.

Anti-Allergic Drug Suppressed Pancreatic Carcinogenesis via Down-Regulation of Cellular Proliferation.

Pancreatic cancer is a fatal disease, and thus its chemoprevention is an important issue. Based on the recent report that patients with allergic diseases have a low risk for pancreatic cancer, we examined the potential chemopreventive effect of anti-allergic agents using a hamster pancreatic carcinogenesis model. Among the three anti-allergic drugs administered, montelukast showed a tendency to suppress the incidence of pancreatic cancer. Further animal study revealed a significantly decreased incidence of pancreatic cancer in the high-dose montelukast group compared with controls. The development of the pancreatic intraepithelial neoplasia lesions was also significantly suppressed. The Ki-67 labeling index was significantly lower in pancreatic carcinomas in the high-dose montelukast group than in controls. In vitro experiments revealed that montelukast suppressed proliferation of pancreatic cancer cells in a dose-dependent manner with decreased expression of phospho-ERK1/2. Montelukast induced G1 phase arrest. Conversely, leukotriene D4 (LTD4), an agonist of CYSLTR1, increased cellular proliferation of pancreatic cancer cells with an accumulation of phospho-ERK1/2. In our cohort, pancreatic ductal adenocarcinoma patients with high CYSLTR1 expression showed a significantly unfavorable clinical outcome compared with those with low expression. Our results indicate that montelukast exerts a chemopreventive effect on pancreatic cancer via the LTD4-CYSLTR1 axis and has potential for treatment of pancreatic carcinogenesis.

PBK expression predicts favorable survival in colorectal cancer patients.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide with high morbidity and mortality rates. The discovery of small molecule anticancer reagents has significantly affected cancer therapy. However, the anticancer effects of these therapies are not sufficient to completely cure CRC. PDZ-binding kinase (PBK) was initially identified as a mitotic kinase for mitogen-activated protein kinase and is involved in cytokinesis and spermatogenesis. Aberrant expression of PBK has been reported to be closely associated with malignant phenotypes of many cancers and/or patient survival. However, the expression of PBK and its association to patient survival in CRC have not been fully elucidated. In the present study, 269 primary CRCs were evaluated immunohistochemically for PBK expression to assess its ability as a prognostic factor. CRC tumor cells variably expressed PBK (range, 0-100%; median, 32%) in the nucleus and cytoplasm. Univariate analyses identified a significant inverse correlation between PBK expression and pT stage (P<0.0001). Furthermore, patients carrying CRC with higher PBK expression showed significantly favorable survival (P=0.0094). Multivariate Cox proportional hazards regression analysis revealed high PBK expression (HR, 0.52; P=0.015) as one of the potential favorable factors for CRC patients. PBK expression showed significant correlation to Ki-67 labeling indices (ρ=0.488, P<0.0001). In vitro, the PBK inhibitor OTS514 suppressed cellular proliferation of CRC cells with PBK expression through downregulation of P-ERK and induction of apoptosis. These results suggest that PBK-targeting therapeutics may be useful for the treatment of PBK-expressing CRC patients.

A short form of gross motor function measure for Fukuyama congenital muscular dystrophy.

OBJECTIVES: The objective of this study was to confirm the validity of a short form of gross motor function measure for Fukuyama congenital muscular dystrophy (GMFM for FCMD). METHODS: This study is a case series and was conducted at the Tokyo Women's Medical University. Fifteen patients with FCMD were assessed using both the GMFM for FCMD with 68 items, which was created as a motor function measure for patients with FCMD on the basis of Rasch analysis, and the original GMFM with 88 items. The correlation between the GMFM for FCMD and the Ueda classification was assessed. Time required for each assessment was also evaluated. RESULTS: We found significant correlation between the GMFM for FCMD and the Ueda classification (r = 0.935); furthermore, the mean assessment time tended to decrease when using the GMFM for FCMD. CONCLUSIONS: GMFM for FCMD may be an appropriate motor function scale for patients with FCMD and might help decrease the assessment time.

Determination of the ratio between mercaptalbumin and nonmercaptalbumin by HPLC with fluorescence probe specifically binding to albumin.

A simple and selective HPLC-fluorescence (FL) method with FL probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM), for simultaneous determination of mercaptalbumin (HMA) and nonmercaptalbumin (HNA1) was developed. After HMA and HNA1 were separated on an ion-exchange column, they were on-line and post-column mixed with DAPIM. The DAPIM-albumin complex produces FL (λex 370nm and λem 510nm); however, DAPIM solution never gives the FL. Based on this mechanism, selective determination of HMA and HNA1 were achieved without any pretreatment and interfering peak. The proposed method was applied to the measurement of HMA and HNA1 in human serum of healthy volunteers and diabetes mellitus patients.

A novel lophine-based fluorescence probe and its binding to human serum albumin.

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510nm. The binding constant and binding number determined by Scatchard plot was 3.65×10(6)M(-1) and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.

Recurrent ventricular fibrillation related to hypokalemia in early repolarization syndrome.

We describe a case of early repolarization syndrome in which augmented J waves were documented during an electrical storm associated with hypokalemia. The patient was referred to our hospital for therapy to treat recurrent ventricular fibrillation (VF). The 12-lead electrocardiogram showed giant J waves associated with hypokalemia during multiple episodes of VF. Although antiarrhythmic agents or deep sedation were not effective for the VF, an intravenous supplementation of potassium completely suppressed the VF with a reduction in the J-wave amplitude. Our report discusses the possible relationship between hypokalemia and VF in early repolarization syndrome.

Expression of Bcl2l1, Clcf1, IL-28ra and Pias1 in the mouse heart after single and repeated administration of chlorpromazine.

Several chlorpromazine (CPZ)-related deaths have been suspected in forensic autopsies but these are difficult to identify precisely because only low concentrations of CPZ can usually be detected. Patients on CPZ therapy exhibit various cardiovascular diseases, such as arrhythmia and cardiomyopathy. As our previous study revealed that CPZ administration affects the expression of immediate early genes that are induced before any other genes, we expected that CPZ probably affects the heart and, in particular, the gene expression in heart. CPZ changes tumor necrosis factor (TNF) production. After stimulation of TNF, the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway is activated via TNF receptor (TNF-R), and the pathway participates in the regulation of cellular responses such as apoptosis [1]. We used semi-arrays to determine the JAK-STAT signaling pathway in a mouse cardiomyocyte cell line, HL-1, and real-time quantitative-PCR to determine whether the semi-array data applied in vivo in mouse heart after single and once-daily repeated (1-4weeks) low-dose (0.75mg/kg) or high-dose (7.5mg/kg) CPZ treatment. We found that expression of B cell lymphoma 2 like 1 (Bcl2l1), Cardiotrophin-like cytokine factor 1 (Clcf1), Interleukin-28 receptor alpha (IL-28ra) and Protein inhibitor of activated STAT-1 (Pias1) were significantly changed in vivo. All these genes are associated with apoptosis. The expression level of Bcl2l1 was elevated after a single high-dose CPZ treatment and after 1week of repeated high doses, but returned to baseline from week 2 to week 4. Clcf1 and IL-28ra expression increased from week 2 or 3 after low-dose CPZ treatment. Pias1 also increased from week 2 after low-dose CPZ treatment. Our results indicate that different doses of CPZ can induce distinct patterns of gene expression for preventing the apoptotic progression in mouse cardiomyocytes, suggesting that CPZ can affect cardiomyocytes via the JAK-STAT signaling pathway and that this might lead to cardiomyopathy. In addition, our data may help to clarify the pathophysiology of cardiomyopathy induced by CPZ and to diagnose cardiac sudden death following CPZ treatment.

Expression of C-fos, Fos-B, Fosl-1, Fosl-2, Dusp-1 and C-jun in the mouse heart after single and repeated chlorpromazine administrations.

Many cases of sudden chlorpromazine (Chl)-related deaths have been identified in forensic autopsies. Because Chl concentration detected in such cases is often low, identifying the cause of death can be difficult. Patients on Chl therapy exhibit arrhythmia and cardiomyopathy. Thus, Chl may affect the heart, particularly, gene expression there. Immediate early genes (IEGs) are expressed following stimulation. Using real-time quantitative-PCR, we investigated the mRNA expression of IEGs, including C-fos, Fos-B, Fosl-1, Fosl-2, Dusp-1 and C-jun, in the mouse heart after once-daily high-dose (7.5 mg/kg) or low-dose (0.75 mg/kg) of Chl single and repeated (1-4 weeks) injections. We showed that single high-dose Chl administration induced IEGs except C-jun. This induction was not observed after the repeated administration, and thus; suggested that the transcriptome is altered after repeated administration and tolerance is developed to Chl. Moreover, C-jun expression decreases after repeated administration. These results reflect that C-jun is down-regulated to avoid cardiomyopathy caused by the over stimulation of C-jun. In future, we intend to clarify the Chl-induced IEG cascade via IEGs in the mouse heart. Chl treatment can result in cardiovascular diseases. Investigation of the transcriptome in the heart after repeated Chl administration will aid in elucidating the patho-physiology of Chl-related cardiovascular diseases.

C-fos, fos-B, c-jun and dusp-1 expression in the mouse heart after single and repeated methamphetamine administration.

In forensic autopsy, there are numerous sudden methamphetamine (MA)-related deaths. The concentration of MA in the blood is measured to determine the cause of death in case of MA-related death. As a low concentration of MA is detected in MA-related death cases, it is sometimes difficult to identify to the cause of death. MA abusers often exhibit various cardiovascular diseases. MA induces arrhythmia and morphological change in cultured cardiomyocytes. Therefore, MA might affect heart cells, especially in terms of gene expression. Immediate early genes (IEGs) are expressed before some specific gene expressions following certain stimuli. We investigated the expression of IEGs, including c-fos, fos-B, c-jun and dusp-1 mRNA, in the mouse heart after a once-daily MA injection for 1day, 2 or 4weeks using real-time quantitative PCR. We showed that high-dose (10mg/kg) MA administration on day 1 induced mRNA expression of the four IEGs. In contrast, low-dose (1mg/kg) administration on day 1 did not induce any c-fos expression. These findings were characteristic only of the heart, since c-fos increased after treatment at any dose in the brain, suggesting that the intracellular signal cascade differs in these two organs. Nevertheless, we confirmed the transcriptional tolerance in the heart as well as the brain on chronic administration by investigating IEG expression. We were unable to explain why the expressions of IEGs were similar between both doses of MA after chronic administration, although these differed after the single treatment. Additionally, these results strongly suggest that the transcriptome must be altered after long-term treatment. As MA abuse results in various cardiovascular diseases, investigation of the transcriptome in the heart after chronic MA administration will aid in elucidating the patho-physiology of MA-related cardiovascular disease.

The time-course analysis of gene expression during wound healing in mouse skin.

RNA analysis has been applied to forensic work to determine wound age. We investigated mRNA expression using quantitative RT-PCR of ten genes, including c-fos, fosB, mitogen-activated protein kinase phosphatase-1 (MKP-1), CD14, chemokine (C-C motif) ligand 9 (CCL9), placenta growth factor (PlGF), mast cell protease-5 (MCP-5), growth arrest specific 5 (Gas5), beta-2 microglobulin (B2M) and major urinary protein-1 (MUP-1), in terms of repair response in adult mice. The expression level of c-fos, fosB and MKP-1 transcripts increased drastically, peaked within 1h, and that of the CD14 and CCL9 transcripts peaked from 12 to 24h. An increase in PlGF and MCP-5 mRNA appeared on about day 5. Gas5, B2M and MUP-1 transcripts showed no significant change. Each gene had differentially expressional patterns with time-course. Our result implied that the observation of the 7 genes in wounded skin could serve to aid in the accurate diagnosis of wound age.

Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses.

TLR22 occurs exclusively in aquatic animals and its role is unknown. Herein we show that the fugu (Takifugu rubripes) (fg)TLR3 and fgTLR22 link the IFN-inducing pathway via the fg Toll-IL-1R homology domain-containing adaptor protein 1(fgTICAM-1, or TRIF) adaptor in fish cells. fgTLR3 resides in endoplasmic reticulum and recognizes relatively short-sized dsRNA, whereas fgTLR22 recognizes long-sized dsRNA on the cell surface. On poly(I:C)-stimulated fish cells, both recruit fgTICAM-1, which in turn moves from the TLR to a cytoplasmic signalosome region. Thus, fgTICAM-1 acts as a shuttling platform for IFN signaling. When fish cells expressing fgTLR22 are exposed to dsRNA or aquatic dsRNA viruses, cells induce IFN responses to acquire resistance to virus infection. Thus, fish have a novel TICAM-1-coupling TLR that is distinct from the mammalian TLR3 in cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute of human cell-surface TLR3 and serve as a surveillant for infection with dsRNA virus to alert the immune system for antiviral protection in fish.

Pan-vertebrate toll-like receptors during evolution.

Human toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) to raise innate immune responses. The human TLR family was discovered because of its sequence similarity to fruit fly (Drosophila) Toll, which is involved in an anti-fungal response. In this review, we focus on the origin of the vertebrate TLR family highlighted through functional and phylogenetic analyses of TLRs in non-mammalian vertebrates. Recent extensive genome projects revealed that teleosts contain almost all subsets of TLRs that correspond to human TLRs (TLR1, 2, 3, 4, 5, 7, 8, and 9), whereas the urochordate Cionaintestinalis contains only a few TLR genes. Therefore, mammals likely obtained almost all TLR family members at the beginning of vertebrate evolution. This premise is further supported by several functional analyses of non-mammalian TLRs. We have summarized several teleost TLRs with unique properties distinct from mammalian TLRs to outline their specific roles. According to Takifugu rubripes genome project, the puffer fish possesses fish-specific TLR21 and 22. Surprisingly, phylogenetic analyses indicate that TLR21 and 22 emerged during an early period of vertebrate evolution in parallel with other TLRs and that the mammalian ancestor lost TLR21 and 22 during evolution. Our laboratory recently revealed that TLR22 recognizes double-strand RNA and induces interferon production through the TICAM-1 adaptor, as in TLR3, but unlike TLR3, TLR22 localizes to the cell surface. Therefore, differential expression of TLR3 and TLR22, rather than simple redundancy of RNA sensors, may explain the effective protection of fish from RNA virus infection in the water. In this review, we summarize the similarities and differences of the TLR family in various vertebrates and introduce these unique TLRs for a possible application to the field of clinical practices for cancer or virus infection.

Combinational recognition of bacterial lipoproteins and peptidoglycan by chicken Toll-like receptor 2 subfamily.

Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappaB reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pam3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappaB in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappaB. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events.

Inhibition of lipid A-mediated type I interferon induction by bactericidal/permeability-increasing protein (BPI).

Lipopolysaccharide (LPS), a major constituent of the outer membrane of gram-negative bacteria, consists of polysaccharides and a lipid structure named lipid A. Lipid A is a typical microbial pattern molecule that serves as a ligand for Toll-like receptor 4 (TLR4). TLR4 signals the presence of lipid A to recruit adaptor molecules and induces cytokines and type I interferon (IFN) by activating transcription factor, NF-kappaB or IRF-3. Here we showed that chemically synthesized TLR4-agonistic lipid A analogues but not antagonistic lipid A activate IFN-beta promoter in TLR4-expressing HEK293 cells. The amplitude of IFN-beta promoter activation was in parallel with that of NF-kappaB. LPS-binding protein (LBP) was required for efficient IFN-beta induction in this system, and this LBP activity was antagonized by bactericidal/permeability-increasing protein (BPI). Thus, we first show that BPI blocks the TLR4 responses by exogenous administration of BPI to lipid A-sensitive cells. Although the functional mechanism whereby extra-cellular BPI modulates the intra-cellular signal pathways selected by the TLR adaptors, MyD88 and TICAM-1 (TRIF), remains unknown, we infer that the lipid A portion of LPS participates in LBP-amplified IFN-beta induction and that BPI binding to LPS leads to inhibition of the activation of NF-kappaB and IFN-beta by LPS or agonistic lipid A via TLR4 in an extrinsic mode. BPI may serve as a therapeutic potential against endotoxin shock by acting as a regulator for the MyD88- and TICAM-1 pathways in the LPS-TLR4 signaling.

Lamprey TLRs with properties distinct from those of the variable lymphocyte receptors.

Fish express mammalian-type (M-type) TLRs consisting of leucine-rich repeats (LRRs) and Toll-IL-1R (TIR) homology domain for immunity, whereas invertebrates in deuterostomes appear to have no orthologs of M-type TLRs. Lampetra japonica (lamprey) belongs to the lowest class of vertebrates with little information about its TLRs. We have identified two cDNA sequences of putative TLRs in the lamprey (laTLRs) that contain LRRs and TIR domains. The two laTLRs were 56% homologous to each other, and their TIRs were similar to those of members of the human TLR2 subfamily, most likely orthologs of fish TLR14. We named them laTLR14a and laTLR14b. We raised a rabbit polyclonal Ab against laTLR14b and identified a 85-kDa protein in a human HEK293 transfectant by immunoblotting using the Ab. FACS, histochemical, and confocal analyses showed that laTLR14b is expressed intracellularly in lamprey gill cells and that the overexpressed protein resides in the endoplasmic reticulum of human and fish (medaka) cell lines. Because natural agonists of TLR14 remained unidentified, we made a chimera construct of extracellular CD4 and the cytoplasmic domain of laTLR14. The chimera molecule of laTLR14b, when expressed in HEK293 cells, elicited activation of NF-kappaB and, consequently, weak activation of the IFN-beta promoter. laTLR14b mRNA was observed in various organs and leukocytes. This lamprey species expressed a variable lymphocyte receptor structurally independent of laTLR14 in leukocytes. Thus, the jawless vertebrate lamprey possesses two LRR-based recognition systems, the variable lymphocyte receptor and TLR, and the M-type TLRs are conserved across humans, fish, and lampreys.

Effect of dietary conjugated linoleic acid on liver regeneration after a partial hepatectomy in rats.

We examined the effect of dietary conjugated linoleic acid (CLA) on liver regeneration after a partial hepatectomy (PH) in Sprague-Dawley rats. PH was performed on rats fed a 0 or 1 wt.% CLA diet for 3 wk. Average liver weight in the CLA fed rat population was heavier than the control rat population at the time of PH and 1-d after PH. Conversely. CLA fed rats' liver weight was significantly lower than control rats at 7-d after PH. This suggests that dietary CLA reduced liver weight gain after PH. Dietary CLA did not affect serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) activities. However. CLA significantly reduced serum albumin levels at 1-d but not at 7-d after PH. 5-Bromo- and 5-iododeoxyuridine incorporation into hepatocytes 1-d post PH was lower in the CLA group. In conclusion, the data suggests that dietary CLA inhibits DNA synthesis after PH, which results in hepatocyte proliferation inhibition.

Recombinant perchloric acid-soluble protein suppresses the immunoglobulin production of human-human hybridoma HB4C5 cells.

Because perchloric acid-soluble protein (PSP) has been conserved evolutionally in various species from Escherichia coli to humans, it may reflect an involvement in basic cellular regulation. However, the precise function of PSP is currently unknown. In this study, we examined the direct effect of PSP on the production of immunoglobulin (Ig) using human B, HB4C5, NAT-30, and U266 cells because it has been reported that subcutaneous administration of PSP affects rodent immune systems. Suppression of Ig productivity and decrement of the cell viability was recognized only in HB4C5 cells by the addition of PSP into the medium. On the other hand, PSP had no effect on Ig productivity and cell viability in NAT-30 and U266 cells. In addition, PSP was clearly incorporated by HB4C5 but not by the other cells. These results suggest that the Ig production suppressed by PSP, which has been previously reported to inhibit protein synthesis, contributed to the incorporation of PSP into the HB4C5 cells.

Repair of oxidative cytosine damage by DNA glycosylases.

5-Hydroxyuracil (HOU) and 5-hydroxycytosine (HOC) are major oxidative lesions of cytosine with mutagenic potentials. Therefore, HOU and HOC need to be removed from DNA to avoid mutation. In this study, oligonucleotide substrates containing HOU and HOC were synthesized by DNA polymerase reactions and tested for DNA glycosylases. Ung exhibited an extremely low activity for HOU as compared to uracil (U). In contrast, hSMUG1 excised HOU and U with a comparable efficiency. Ung and hSMUG1 did not excise HOC.