Analysis of serum levels and DNA methylation of fibroblast growth factor 21 using peripheral blood-derived genomes in patients with obesity.

Fibroblast growth factor (FGF) 21, a hormone produced by the liver, improves glucose and lipid metabolism. We recently demonstrated that the FGF21 gene (Fgf21) underwent DNA demethylation in the mouse liver via peroxisome proliferator-activated receptor (PPAR) α during the fetal to lactation periods. Furthermore, we found that the DNA methylation state of Fgf21 was involved in obesity in adult animals. In the present study, we analyzed the DNA methylation state of the FGF21 gene (FGF21) in obese patients using genomic DNA extracted from human monocytes and macrophages and investigated the pathophysiological significance of the FGF21 expression response to pemafibrate (PM), a PPARα ligand. We examined 67 patients with obesity stratified into in- and outpatient cohorts. A positive correlation was observed between serum FGF21 levels and triglyceride (TG) levels before PM administration. However, changes in serum FGF21 levels following PM administration did not correlate with the FGF21 DNA methylation rate, except at one CpG site. The body mass index (BMI) and serum TG levels positively correlated with the FGF21 DNA methylation rate, particularly at different CpG positions. A negative correlation was observed between absolute changes in serum FGF21 levels and the ratio of change in serum TG levels after PM administration. Collectively, these results indicate the potential of FGF21 DNA methylation as a surrogate indicator of BMI and serum TG levels, while absolute changes in serum FGF21 levels after PM administration may offer prognostic insights into the efficacy of reducing serum TG levels through PM administration.

Aberrant cytokeratin expression as a possible prognostic predictor in poorly differentiated colorectal carcinoma.

BACKGROUND AND AIM: The cytokeratin (CK)7(-) /CK20(+) immunoprofile is characteristic of colorectal carcinoma (CRC), although CK7(+) or CK20(-) phenotypes are occasionally encountered, particularly in histologically variant CRCs. We analyzed CK7/CK20 profiles in variant CRCs in association with clinicopathologic parameters and prognosis. METHODS: CK expression in well- and moderately differentiated adenocarcinoma (WMDA) (n = 63), poorly differentiated adenocarcinoma (PDA) (n = 91), mucinous adenocarcinoma (MUA) (n = 81), signet-ring cell carcinoma (SRCC) (n = 15), undifferentiated carcinoma (UDC) (n = 12), and adenosquamous carcinoma (n = 2) was analyzed using immunohistochemistry. Cut-off scores were set at 1% for CK7 and 25% for CK20 using the receiver operating characteristic curve analysis of PDA. Association between CK20(-) and better prognosis in PDA was validated in the second cohort (n = 66). RESULTS: CK7/CK20 immunoprofiling revealed a predominant CK7(-) /CK20(+) profile in WMDA, MUA, and SRCC, while the majority of UDC was characterized by a CK7(-) /CK20(-) profile. The CK7/CK20 profile in PDA was variable. Contingency table analysis revealed that CK expression was not significantly associated with any clinicopathologic parameters in WMDA, PDA, and MUA. However, survival analysis demonstrated that CK20(-) was significantly associated with better prognosis in PDA. Although CK20(-) was significantly associated with mismatch repair deficiency in PDA, it was an independent prognostic factor in multivariate analysis. Finally, we confirmed that CK20 status, determined using a 25% cut-off score, was a significant prognostic parameter in the second PDA cohort. CONCLUSIONS: CK20 status may be used as a prognostic predictor of PDA.

Three-dimensional micro-X-ray topography using focused sheet-shaped X-ray beam.

X-ray topography is a powerful method for analyzing crystal defects and strain in crystalline materials non-destructively. However, conventional X-ray topography uses simple X-ray diffraction images, which means depth information on defects and dislocations cannot be obtained. We have therefor developed a novel three-dimensional micro-X-ray topography technique (3D µ-XRT) that combines Bragg-case section topography with focused sheet-shaped X-rays. The depth resolution of the 3D µ-XRT depends mainly on the focused X-ray beam size and enables non-destructive observation of internal defects and dislocations with an accuracy on the order of 1 µm. The demonstrative observation of SiC power device chips showed that stacking faults, threading screw, threading edge, and basal plane dislocations were clearly visualized three-dimensionally with a depth accuracy of 1.3 µm. 3D µ-XRT is a promising new approach for highly sensitive and non-destructive analysis of material crystallinity in a three-dimensional manner.

Effects of Curcumin Combined With the 5-alpha Reductase Inhibitor Dutasteride on LNCaP Prostate Cancer Cells.

BACKGROUND/AIM: Curcumin is a natural compound of turmeric, which inhibits prostate cancer cell proliferation. This study examined whether treatment of LNCaP prostate cancer cells with the combination of curcumin and dutasteride, a 5-alpha reductase inhibitor, affect proliferation and the amount of testosterone and dihydrotestosterone. MATERIALS AND METHODS: LNCaP Cells were incubated with curcumin or the combination of curcumin and dutasteride and cell proliferation was measured at 72 h. LC-MS/MS was used to determine testosterone and dihydrotestosterone concentrations in prostate cancer cells. RESULTS: Curcumin combined with dutasteride suppressed proliferation and affected apoptosis of LNCaP cells. The combination of curcumin and dutasteride also reduced the amount of testosterone and dihydrotestosterone in LNCaP cells. The secretion of prostate-specific antigen was inhibited by the combination treatment in a dose-dependent manner. CONCLUSION: Treatment with the combination of curcumin and dutasteride may interfere with the intra-tumoral androgen activity.

Breast cancer resistance protein/ABCG2 is differentially regulated downstream of extracellular signal-regulated kinase.

Breast cancer resistance protein (BCRP)/ABCG2 is a drug efflux pump responsible for multidrug resistance in cancer cells. We report that dephosphorylation of extracellular signal-regulated kinase (ERK) by treatment with mitogen-activated protein kinase/ERK kinase (MEK) inhibitors causes two opposing effects, transcriptional upregulation and prompted protein degradation of endogenous BCRP in breast cancer MCF-7 cells. Endogenous BCRP was eventually found to be upregulated. Conversely, treatment with epidermal growth factor was associated with its downregulation in the cells. MEK inhibitors also caused prompted degradation of exogenous BCRP in MCF-7 and gastric cancer NCI-N87 cells that express exogenous BCRP without affecting its transcriptional levels, and potentiated anticancer agents in the cells. A lysosomal inhibitor abolished this prompted degradation of exogenous BCRP, but a proteasome inhibitor did not. Inhibition of p90 ribosomal protein S6 kinase (RSK), one of the downstream effectors of ERK, resulted in transcriptional upregulation of endogenous BCRP but did not affect the protein degradation of exogenous BCRP. The data suggest that BCRP expression is differentially regulated downstream of the MEK-ERK pathway, transcriptionally upregulated through the inhibition of the MEK-ERK-RSK pathway, and posttranscriptionally downregulated through the inhibition of the MEK-ERK-non-RSK pathway. Although the immediate downstream effector of ERK remains to be elucidated, the data provide new insights into regulatory mechanisms of BCRP activity and may assist the development of BCRP-specific expression modulators.

Immunolocation of Aquaporin 8 in Human Cataractous Lenticular Epithelial Cells.

PURPOSE/AIM: Aquaporin 8 (AQP8) is a diffusion facilitator of hydrogen peroxide (H2O2) through cell membranes. The purpose of this study was to confirm and localize AQP8 in human lenticular epithelial cells (LECs). MATERIALS AND METHODS: Lenticular anterior capsule samples, including LECs, were collected during cataract surgery of cataract patients after informed consent. The localization of AQP8 was detected by immunohistochemical staining using an antibody to AQP8. Real-time polymerase chain reaction (RT-PCR) was also used to determine the AQP8 mRNA expression levels. The PCR products were analyzed by gel electrophoresis following analyses of band density. RESULTS: Immunohistochemical staining showed AQP8 was distributed throughout the whole area of the anterior capsulotomy. AQP8 labeling was observed surrounding and within the cytoplasm of LECs. RT-PCR and gel electrophoresis also revealed the presence of AQP8 mRNA in the lenticular anterior capsule. The results of immunohistochemical staining were comparable to those of RT-PCR and gel electrophoresis. CONCLUSIONS: The results of this study indicate the distribution of AQP8 in human LECs. This is the first investigation confirming the presence of AQP8 in human LECs.

Suppression of promoter-dependent transcriptional activity of inducible nitric oxide synthase by sodium butyrate in colon cancer cells.

Butyrate suppresses the growth of colon cancer cells, inducing differentiation and apoptosis in vitro. Increased expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) has been suggested to be closely involved in colon carcinogenesis. In this study, effects of sodium butyrate on the promoter-dependent transcriptional activity of iNOS and COX-2 genes were investigated in a colon cancer cell line, DLD-1, using a reporter gene assay system. Sodium butyrate significantly reduced promoter-dependent iNOS transcriptional activity dose-dependently at concentrations higher than 0.1 mM. COX-2 transcriptional activity was not suppressed, but slightly increased. While hyperacetylated histones appeared at concentrations of sodium butyrate suppressing iNOS gene promoter activity, promoter-dependent transcriptional activities of iNOS and COX-2 genes were both increased by the histone deacetylase inhibitor trichostatin A. These results suggested that sodium butyrate exhibits differential effects on iNOS and COX-2 genes, acting to suppress iNOS expression via mechanisms independent of histone acetylation.

The herbal medicine Shosaiko-to exerts different modulating effects on lung local immune responses among mouse strains.

Shosaiko-to (SST), a Chinese/Japanese traditional herbal medicine, has recently been demonstrated to increase lung interleukin-6 (IL-6) levels and to ameliorate pulmonary disorders in BALB/c mice (BALB). In the present study, we examined the effects of SST on lung cytokine levels and lipopolysaccharide (LPS)-induced lung injury in C57BL/6 mice (B6), which are known to show different immune responses from BALB due to the difference in genetic backgrounds. In B6, in contrast with BALB, SST decreased lung IL-6 levels and exacerbated LPS-induced lung injury. Investigation of the active components of SST suggested that multiple ingredients were supposed to be responsible for IL-6-attenuating activity in vivo. Further, we examined the effect of metabolites of major ingredients of SST on IL-6 production from lung immune cells in vitro. Saikogenin D and oroxylin A attenuated IL-6 production in LPS-stimulated alveolar macrophages of B6 more than in that of BALB. Liquiritigenin, which was previously reported to enhance IL-6 production in anti-CD3 monoclonal antibody-stimulated lung mononuclear cells of BALB, showed no effect on that of B6. These findings suggest that SST may have different, possibly even opposite, effects on lung immunity in hosts with different genetic backgrounds.

Lindera strychnifolia is protective against post-ischemic myocardial dysfunction through scavenging hydroxyl radicals and opening the mitochondrial KATP channels in isolated rat hearts.

Lindera strychnifolia (tendai-uyaku), a medicinal plant, has long been used for the treatment of cardiac, renal and rheumatic diseases in Japan. We aim to clarify (1) whether L. strychnifolia is protective against post-ischemic myocardial dysfunction, and (2) whether its effect is related to scavenging hydroxyl radicals and opening the mitochondrial KATP channels in isolated rat hearts. Male Sprague-Dawley rats were orally given 1 ml/day of L. strychnifolia, which was extracted from 0.75 and 1.5 g/kg of roots of L. strychnifolia for 4 days. The rat hearts were excised and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. The hearts were paced at 320 beats/min except during ischemia. Left ventricular developed pressure (LVDP, mmHg), +/- dP/dt (mmHg/sec) and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 minutes consisting of a 30-minute pre-ischemic period followed by 30 minutes of global ischemia and 60 minutes of reperfusion with or without 5-HD, a mitochondrial KATP channel blocker. The levels of lactate, LDH and 2,5-DHBA, an indicator of hydroxyl radicals, in the perfusate during reperfusion period were also measured. Treatment with L. strychnifolia significantly improved LVDP and +/- dP/dt without altering coronary flow during reperfusion. The 100 microM of 5-HD in Krebs-Henseleit solution was perfused during the 10 minutes of pre-ischemic periods. Pretreatment with 5-HD abolished the improvement of LVDP and +/- dP/dt by L. strychnifolia. L. strychnifolia significantly attenuated the levels of lactate, LDH and 2,5-DHBA during reperfusion, and which were restored by pretreatment with 5-HD. In conclusion, L. strychnifolia is protective against post-ischemic left ventricular dysfunction through scavenging hydroxyl radicals and opening the KATP channels in the isolated rat heart.

Extracts from the roots of Lindera strychifolia induces apoptosis in lung cancer cells and prolongs survival of tumor-bearing mice.

Lindera strychifolia, a scandent shrub Lauraceous medicinal plant, has been used in Chinese traditional medicine as a palliative and an anti-spasmodic. It also shows cytotoxic effects against several tumor cell lines and inhibits marcromolecule biosynthesis. This study investigated the anti-tumor effects of L. strychifolia extract against lung cancer cells using in vitro and in vivo models. Two human lung cancer cell lines A549 (adenocarcinoma) and SBC-3 (small cell carcinoma), and a non-tumor cell line 3T3-L1 (mice fibroblasts) were subjected to L. strychifolia extract treatment. On lung cancer cells, L. strychifolia induced cell growth inhibition in a dose-dependent manner. Conversely, the extract did not show any significant cytotoxic effect on 3T3-L1 cells. Therefore, the extract is specific for tumor cells. Tumor cells treated with L. strychifolia extract showed typical morphological appearance of apoptosis including nuclei fragmentation and cell condensation. The in vivo effects of L. strychifolia extract were investigated in C57BL/6 mice transplanted with Lewis lung cancer (LL-2) cells, and in BALB/c nude mice transplanted with A549 or SBC-3 human lung cancer cells. Oral administration of L. strychifolia extract prolonged survival time and inhibited tumor growth in a dose-dependent manner by inducing apoptosis in the LL-2 cell mice model. Furthermore, in A549 or SBC-3 cell nude mice models, oral administration of L. strychifolia extract also significantly inhibited tumor growth at the 5.0 mg/ml concentration. These findings suggested that the components of L. strychifolia have anticancer activity and may contribute to clinical applications in the prevention and treatment of lung cancer.

Expression of cytokeratin 20 indicates invasive histological phenotype in poorly differentiated colorectal adenocarcinoma.

BACKGROUND/AIM: Cytokeratin (CK) 20 expression is an independent prognostic factor of poorly differentiated adenocarcinoma (PDA) of the colon and rectum. We aimed to investigate the mechanism of its involvement through a clinicopathological study. PATIENTS AND METHODS: We analyzed 156 surgically resected PDAs, which were sub-classified as solid type (Por1) showing expansive growth, or non-solid type (Por2) showing infiltrative growth. Associations of CK20 expression with morphological features and molecular markers were analyzed. RESULTS: CK20(+) PDA (n=91) was associated with more advanced disease stage and unfavorable prognosis compared with CK20(-) PDA (n=65). Pathologically, CK20(+) PDA was significantly associated with p53 overexpression, Por2, abundant fibrous stroma, and stepwise de-differentiation, while CK20(-) PDA was significantly associated with mismatch repair deficiency, Por1, sparse fibrous stroma, and de novo histogenesis. CONCLUSION: CK20 expression in PDA is closely associated with invasive histological features, providing prognostic significance, and may also point to a specific histogenetic pathway.

Differential mucin phenotypes and their significance in a variation of colorectal carcinoma.

AIM: To investigate mucin expression profiles in colorectal carcinoma (CRC) histological subtypes with regard to clinicopathologic variables and prognosis. METHODS: Mucin (MUC)2 and MUC5AC expressions were assessed by immunohistochemistry for a total of 250 CRC cases that underwent surgical resection. CRCs included 63 well-to-moderately differentiated adenocarcinomas (WMDAs), 91 poorly differentiated adenocarcinomas (PDAs), 81 mucinous adenocarcinoma (MUAs), and 15 signet-ring cell carcinomas (SRCCs). MUC2 and MUC5AC were scored as positive when ≥ 25% and ≥ 1% of cancer cells were stained positive, respectively. The human mutL homolog 1 and human mutS homolog 2 expressions were assessed by immunohistochemistry in PDAs to investigate mismatch-repair (MMR) status. Tumors that did not express either of these two were considered MMR-deficient. Results were analyzed for associations with clinicopathologic variables and the prognosis in individual histological CRC subtypes. RESULTS: MUC2-positive and MUC5AC-positive WMDA percentages were 49.2% and 30.2%, respectively. In contrast, MUC2-positive and MUC5AC-positive PDA percentages were 9.5% and 51.6%, respectively. MUC2 levels tended to decrease and MUC5AC levels tended to increase from WMDA to PDA. In 21 tumors comprising both adenoma and adenocarcinoma components in a single tumor (4 WMDAs, 7 PDAs, and 10 MUAs), MUC2 was significantly downregulated in PDA and MUC5AC was downregulated in PDA and MUA in the adenoma-carcinoma sequence. These results suggested that MUC2 levels might be associated with malignant potential and that MUC5AC expression was an early event in tumorigenesis. Despite worse prognoses than WMDA, high MUC2 expression levels were maintained in MUA (95.1%) and SRCC (71.5%), which suggested a pathogenesis for these subtypes distinct from that of WMDA. No significant associations were found between MUC2 expression and any clinicopathologic variables in any histological subtype. MUC5AC expression in PDA was closely associated with right-sided location (P = 0.017), absence of nodal metastasis (P = 0.010), low tumor node metastasis stage (P = 0.010), and MMR deficiency (P = 0.003). MUC2 expression in WMDA was a marginal prognostic factor for recurrence/metastasis-free survival (RFS) by univariate Cox analysis (P = 0.077) but not by multivariate Cox analysis (P = 0.161). MUC5AC expression in PDA was a significant prognostic factor for RFS by univariate Cox analysis (P = 0.007) but not by multivariate Cox analysis (P = 0.104). Kaplan-Meier curves and log-rank tests revealed that MUC2 expression was marginally associated with a better WMDA prognosis [P = 0.064 for RFS and P = 0.172 for overall survival (OS)] but not for PDA. In contrast, MUC5AC expression was significantly and marginally associated with a better PDA prognosis in terms of RFS and OS, respectively (P = 0.004 for RFS and P = 0.100 for OS), but not for WMDA and MUA. CONCLUSION: Mucin core protein expression profiles and clinical significance differ according to histological CRC subtypes. This may reflect different pathogeneses for these tumors.

The PI3K/Akt inhibitor LY294002 reverses BCRP-mediated drug resistance without affecting BCRP translocation.

Cellular responses toward cytotoxic drugs are influenced by crosstalk between oncogenic signals and resistance mechanisms. Inhibition of the PI3K/Akt pathway is effective in sensitizing cancer cells of various organs, although the mechanisms largely remain to be elucidated. Breast cancer resistance protein (BCRP)/ABCG2, a drug efflux pump, confers resistance to multiple anticancer agents such as SN-38 and topotecan. Previous studies reported that inhibition of the PI3K/Akt pathway, by gene knockout or PI3K inhibitors, modulated BCRP-mediated drug transport via BCRP translocation in hematopoietic stem cells, renal polarized cells and glioma stem-like cells of mammals. In this study, we assessed the effects of PI3K inhibitors, LY294002 and wortmannin, on BCRP-mediated anticancer drug resistance of human cancer MCF-7 and A431 cells. LY294002, but not wortmannin, reversed the BCRP-mediated SN-38 and topotecan resistance. LY294002 treatment did not affect total or cell surface BCRP levels as determined by western blotting and flow cytometry but blocked BCRP-mediated topotecan efflux in a dose-dependent manner. Immunohistochemical analyses also demonstrated unchanged cellular BCRP distribution. BCRP overexpression in MCF-7 and A431 cells did not confer LY294002 resistance, suggesting that LY294002 is not a transported substrate of BCRP. LY294002 is a derivative of quercetin, a member of flavonoids. Taken together, these results suggest that LY294002 inhibits BCRP-mediated drug transport not by BCRP translocation through the PI3K/Akt signal but putatively as a competitive inhibitor in a major subset of cancer cells. Due to its dual effects, LY294002 could be a lead compound for developing more effective and tolerable reagents for cancer treatment.

Anti-CD3 induces bi-phasic apoptosis in murine intestinal epithelial cells: possible involvement of the Fas/Fas ligand system in different T cell compartments.

Recent studies have suggested that Fas-mediated apoptosis is involved in the pathogenesis of intestinal injury. In this study, we determined the role of Fas/Fas ligand (FasL) interactions in different T cell compartments using a murine model of small intestinal injury. An intraperitoneal injection of 145-2C11 (anti-CD3) antibody into C3H/HeN, BALB/c and MRL mice induced mucosal flattening and rapid, bi-phasic intestinal epithelial cell (IEC) apoptosis, which was detected by conventional light and electron microscopy and by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. In the first, early phase, villous apoptosis was observed up to 4 h after injection, and in the second, later phase, apoptotic crypt cells gradually accumulated for up to 24 h. The early and later phases of apoptosis were reduced in lpr/lpr and nude mice compared with those in control strains. In addition, the kinetics of Fas-mediated killer activity induced by the antibody injection were different between intestinal intraepithelial lymphocytes (IEL) and splenocytes (SPL) and seemed to correlate with the bi-phasic occurrence of the apoptosis. Finally, the transfer of intestinal IEL from euthymic to nude mice induced both phases of apoptosis, whereas SPL induced the second phase's crypt apoptosis only by the antibody injection. Together, these results suggest the involvement of Fas-mediated killer activity of thymus-derived T cells in different compartments. Namely, T cell populations in different compartments are differentially involved in the induction of IEC apoptosis and contribute to the complex pathogenesis of immune-mediated intestinal injury in which Fas/FasL interactions may play a critical role.

Sheng-Mai-San is protective against post-ischemic myocardial dysfunction in rats through its opening of the mitochondrial KATP channels.

The present study used isolated rat hearts to investigate whether (1) Sheng-Mei-San (SMS), a traditional Chinese formulation comprising Radix Ginseng, Radix Ophiopogonis and Fructus Schisandrae, is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effect of SMS is related to scavenging of hydroxyl radicals and opening the mitochondrial KATP channels. The excised hearts of male Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s) and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min consisting of a 30-min pre-ischemic period followed by a 30-min global ischemia and 60-min reperfusion. Lactate, lactate dehydrogenase (LDH) and 2,5-dihydroxybenzoic acid (2,5-DHBA) concentrations in the effluent were measured during reperfusion. Three days' treatment with SMS (1.67 ml/kg per day) inhibited the rise in LVEDP and improved the post-ischemic LVDP and +/-dP/dt significantly better than in the untreated control hearts during reperfusion. SMS increased the coronary flow at baseline, and during reperfusion. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial KATP channel blocker, abolished the inhibition of the rise in LVEDP, the increase in coronary flow and the improvement in LVDP and +/-dP/dt induced by SMS. SMS significantly attenuated the concentrations of lactate, LDH and 2,5-DHBA during reperfusion, but the pretreatment with 5-HD restored them; 5-HD alone did not affect the concentrations. SMS improved the post-ischemic myocardial dysfunction through opening the mitochondrial KATP channels.