Leukemia inhibitory factor (LIF) signaling regulates transcription factors to maintain the self-renewability and pluripotency of embryonic stem (ES) cells. Recently, we have proposed a network model that consists of transcription factors such as, Klf4, Sox2, Tbx3, Nanog, and Oct3/4, which form a parallel pathway downstream from LIF signaling (Nature, 460, 2009, Niwa et al.). In this parallel pathway, the transcription factors maintain the pluripotency of ES cells through mutual balance with some degree of redundancy and compensation. While self-renewability and pluripotency are maintained well under such seemingly stringent regulation, studies of single cells revealed heterogeneity among individual ES cells. This heterogeneity may underlie the mechanism that allows ES cells to exit self-renewal and enter into differentiation to exert pluripotency. Here we focus on recent studies on the heterogeneity of ES cells and discuss their inherent metastability.
Parkinson’s disease (PD) is one of the candidate diseases for cell transplantation therapy, since successful clinical experiments have accumulated using human fetal tissue grafting for PD patients. Although some grafted PD patients have shown drastic improvements, several issues still remain with regard to using human fetal tissue. This review highlights the recent advances in stem cell technology toward clinical applications using human pluripotent stem cells. In particular, pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPSCs), are the focus as a source of cell transplantation therapy that can be used instead of human fetal tissues. Additionally, efficient methods for stem cell maintenance and differentiation have been developed and improved toward the clinical transition. These advances in the basic technologies have helped accelerate the realization of regenerative medicine. We also review the current topics regarding disease modeling and drug screening using iPSC technology. Finally, we also describe the future prospects of these stem cell research fields toward clinical application.
Cell therapies for severe ischemic diseases such as limb ischemia, acute myocardial infarction, and cerebral ischemia have been developed through in vitro and in vivo animal and clinical studies. Active cells for angiogenic cell therapy are believed endothelial progenitor cells (EPCs). EPCs have been extensively investigated to clarify their origin and biology. Many sources of EPCs have been proposed, including mononuclear cells (MNCs) fraction containing CD34+ or CD133+ (AC133+), isolated CD34+ and AC133+ cells, and induction and differentiation of EPCs from hematopoietic stem cells (HSCs). However, in vivo mechanisms by which EPCs contribute to neovascularization should be clarified. Many in vitro, in vivo, and clinical studies have been performed using these cells; angiogenic cell therapy will become an important regimen for severe ischemic diseases.
Induced pluripotent stem cell (iPSC) technology has great potential to establish novel therapeutic approaches in regenerative medicine and disease analysis. Although cell therapy using iPSC-derived cells still has many hurdles to overcome before clinical applications, disease analysis using patient-specific iPSCs may be of practical use in the near future. There are several reports that patient-specific iPSC-derived cells have recapitulated the apparent cellular phenotypes of a wide variety of diseases. Moreover, some studies revealed that it could be possible to discover effective new drugs and to clarify disease pathogenesis by examination of patient-specific iPSC-derived cells in vitro. We have recently reported that iPSCs can be a diagnostic tool in a patient with a novel mutation. For definitive diagnosis in a patient with long QT syndrome who had an uncharacterized genetic mutation, we succeeded in clarifying the patient’s cellular electrophysiologic characteristics and the molecular mechanism underlying the disease phenotype through the multifaceted analyses of patient-specific iPSC-derived cardiomyocytes. In this review, we focus on the conceptual and practical issues in disease modeling using patient-specific iPSCs and discuss future directions in this research field.
Human pluripotent stem cells (hPSCs), i.e. human embryonic stem cells and human induced pluripotent stem cells, are able to self-renew and differentiate into multiple cell types. Because of these abilities, numerous attempts have been made to utilize hPSCs in regenerative medicine/cell therapy. hPSCs are, however, also tumorigenic, that is, they can give rise to the progressive growth of tumor nodules in immunologically unresponsive animals. Therefore, assessing and managing the tumorigenicity of all final products is essential in order to prevent ectopic tissue formation, tumor development, and/or malignant transformation elicited by residual pluripotent stem cells after implantation. No detailed guideline for the tumorigenicity testing of hPSC-derived products has yet been issued for regenerative medicine/cell therapy, despite the urgent necessity. Here, we describe the current situations and issues related to the tumorigenicity testing of hPSC-derived products and we review the advantages and disadvantages of several types of tumorigenicity-associated tests. We also refer to important considerations in the execution and design of specific studies to monitor the tumorigenicity of hPSC-derived products.
2-Methoxy-5-(2-(3,4,5-trimethoxyphenyl)thiophen-3-yl) aniline (DAT-230) is a novel synthesized compound of combretastatin-A-4 derivative with more stability. The present study is to investigate its anti-tumor activity and molecular mechanisms in human gastric adenocarcinoma SGC-7901 cells. DAT-230 inhibited SGC-7901 cells growth. The treatment of DAT-230 resulted in microtubule de-polymerization and G2/M phase arrest. Besides the accumulation and translocation of Cyclin B1, reduction of p-14/15-cdc2 and mitosis delay denoted the Cyclin B1-cdc2 complex active and M phase arrest in SGC-7901 cells treated with DAT-230. Mitochondria pathway participated in apoptosis after G2/M arrest in SGC-7901 cells treated with DAT-230, which was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP), activation of caspase-3 and caspase-9, changes of Bcl-2 and Bax expression, decrease of mitochondrial membrane potential and release of cytochrome c from mitochondria. In vivo, DAT-230 delayed tumor growth in BALB/c nude mice with human gastric adenocarcinoma xenografts. Besides apoptosis was detected with terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay in tumor tissue. In conclusion, DAT-230 is a promising microtubule inhibitor with great anti-tumor activity to SGC-7901, in vitro and in vivo. Its potential to be a candidate of anti-cancer agent is worth of being further investigated.
The present study was planned to explore the effect of Lactobacillus (L.) acidophilus on the T helper-17 (Th17) immune response in a mouse model of β-lactoglobulin (β-lg) allergy. Bovine β-lg sensitised BALB/c mice were orally administered with different doses of heat-killed L. acidophilus (low, 5×107 colony forming unit (CFU); medium, 5×108 CFU; high, 5×109 CFU) in 200 µL of phosphate buffered saline (PBS) three times a week, starting from 1 week before β-lg sensitisation for 4 weeks. After the allergen challenge, the numbers of blood eosinophils and neutrophils were examined by light microscope; the levels of cytokine (interleukin (IL)-12, IL-4, tumor growth factor (TGF)-β, IL-10, IL-6 and IL-17A), total immunoglobulin E (IgE) and β-lg-specific IgE contents in the serum were measured with enzyme-linked immunosorbent assay (ELISA); The mRNA expression levels of TGF-β, IL-17A,CD25, Foxp3, retinoic acid-related orphan receptor γt (RORγt) and IL-10 were analyzed using real-time polymerase chain reaction (PCR). The results showed that oral administration of L. acidophilus suppressed hypersensitivity responses, attenuated the numbers of inﬂammatory cells and inhibited IgE production. We found up-regulation of TGF-β and down-regulation of IL-17A in the serum of L. acidophilus-treated group, along with IL-6 levels was significantly decreased than that of the allergy group (p<0.05). Moreover, the mRNA expression levels of CD25, forkhead box P3 and TGF-β were significantly higher in the spleen of L. acidophilus-treated group, while the mRNA expression levels of IL-17A, RORγt and IL-10 were significantly lower than that in the allergy group (p<0.05). In conclusion, the suppression of major allergic symptoms by oral administration of L.acidophilus was probably due to improve the regulatory T (Treg)/Th17 balance and inhibit the IL-6 production.
Aim of this study was to identify the molecular mechanisms of tanshinone IIA-induced apoptosis in chronic myelogenous leukemia (CML) cells. Cytotoxicity of tanshinone IIA was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our data demonstrate that tanshinone IIA induced apoptosis by increasing the sub-G1 DNA contents and DNA fragmentation in KBM-5 CML cell line. In addition, tanshinone IIA significantly reduced mitochondrial membrane potential (MMP), mediated cytochrome c release from mitochondria and activated caspase-3 and 9, indicating mitochondria-dependent apoptosis by tanshinone IIA. Tanshinone IIA attenuated expression of several apoptosis-related proteins such as c-inhibitor of apoptosis protein (IAP) 2, Mcl-1L and Bcl-2. Interestingly, although tanshinone IIA notably enhanced the phosphorylation of both c-Jun N-terminal protein kinase (JNK) and p38, JNK inhibitor, but not p38 inhibitor, reversed tanshinone IIA-induced apoptosis. Our findings suggest that tanshinone IIA induces mitochondria-dependent apoptosis via activation of JNK in KBM 5 cells as a potent anti-cancer agent for CML therapy.
Photodynamic therapy (PDT) induces selective cell death of neoplastic tissue and connecting vasculature by combining photosensitizers with light. Here we clarified the types of cell death induced by PDT in combination with the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) in order to evaluate the potential of this therapy as a treatment for glioma. PDT with NPe6 (NPe6-PDT) induces dose-dependent cell death in human glioblastoma T98G cells. Specifically, cell death modalities were observed in NPe6-PDT treated T98G cells, including signs of apoptosis (activation of caspase-3, expression of phosphatidylserine, and DNA fragmentation) and necrosis (stainability of propidium iodide). In addition, high doses of NPe6-PDT decreased the proportion of apoptotic cell death, while increasing necrosis. Closer examination of apoptotic characteristics revealed release of cytochrome-c from mitochondria as well as activation of both caspse-9 and caspase-3 in cells treated with low doses of NPe6-PDT. Benziloxycarbonyl-Leu-Gln(OMe)-His-Asp(OMe)-fluoromethyl-ketone (Z-LEHD-fmk), a caspase-9 specific inhibitor, and benziloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe)-fluoromethyl-ketone (Z-DQMD-fmk), a caspase-3 specific inhibitor, showed dose-dependent prevention of cell death in NPe6-PDT treated cells, indicating that mitochondrial apoptotic pathway was a factor in the observed cell death. Further, the cell morphology was observed after PDT. Time- and NPe6-dose dependent necrotic features were increased in NPe6-PDT treated cells. These results suggest that NPe6-PDT could be an effective treatment for glioma if used in mild doses to avoid the increased necrosis that may induce undesirable obstacles.
Antithrombotic drugs have been increasingly used for treating ischemic cardiovascular diseases among the elderly in Japan. However, antithrombotic drugs are known to be risk factors for gastrointestinal injury. Therefore, we conducted a pharmacoepidemiologic study on patients receiving antithrombotic drugs to identify the risk factors for gastrointestinal injury. This retrospective case-control study included patients who were prescribed antithrombotic drugs at the Ehime University Hospital between April and September 2010. Of the 3271 patients who received antithrombotic drug therapy, 172 (5.3%) developed gastrointestinal injuries, including gastric ulcers, duodenal ulcers, and hemorrhagic gastrointestinal injuries. Further, the incidence of gastrointestinal injury was higher in patients with hypertension than in those without (p<0.0001). Multivariate adjusted odds ratios and 95% confidence intervals were calculated using stepwise logistic regression. The adjusted odds ratios for gastrointestinal injury were 1.56 (95% confidence interval 1.07–2.36) for hypertension, 1.70 (1.17–2.50) for low-dose aspirin, 2.77 (1.70–4.49) for clopidogrel, 1.95 (1.23–3.08) for warfarin, and 4.13 (2.88–5.95) for nonsteroidal anti-inflammatory drugs. On the other hand, the non-adjusted odds ratio for drug-associated gastrointestinal injury was 0.43 (0.20–0.84) for eicosapentaenoic acid (EPA). In addition, we found that patients aged 70 years or older were at increased risk of drug-associated gastrointestinal injury. These findings suggest that while many antithrombotic drugs are risk factors for gastrointestinal injury, EPA may be a safe option for suppressing or preventing gastrointestinal injury.
Modulating steroid hormone levels is a curative and preventive measure for Cushing’s syndrome, aldosteronism, and various stress-triggered symptoms. Polyphenols have been reported to inhibit steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase. However, evidence for their inhibitory effects is fragmentary because it has been determined in studies with small groups of steroid hormones. To investigate the effects of steroids on complete steroidogenic pathways, comprehensive analysis of steroid hormones is necessary. Here we cultured forskolin-stimulated NCI-H295R, a human adrenocortical carcinoma cell line, in the presence of a polyphenol and employed GC-MS to simultaneously determine the levels of nine steroid hormones (pregnenolone, progesterone, deoxycorticosterone, aldosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and estradiol) in cell culture supernatant. We found that daidzein, genistein, apigenin, hesperetin, naringenin, and eriodictyol significantly reduced deoxycorticosterone and androstenedione levels (p<0.05), suggesting inhibition of 3β-HSD by these polyphenols. Apigenin was more potent than other polyphenols in increasing the levels of pregnenolone and 17α-hydroxyprogesterone, suggesting that it inhibits cytochrome P450 (CYP) 17 and CYP21, as well as 3β-HSD. Real-time reverse transcription polymerase chain reaction showed that apigenin significantly downregulated the expression levels of 3β-HSD, CYP17, and CYP21 mRNA (p<0.05). This is the first study to demonstrate the inhibitory effects of apigenin on CYP17 and CYP21.
3,4,5-Trimethoxycinnamic acid (TMCA), methyl 3,4,5-trimethoxycinnamate (M-TMCA) and p-methoxycinnamic acid (PMCA) have been identified as the major bioactive components in the serum collected from rats treated with oral administration of Polygalae Radix (“YuanZhi,” the roots of Polygala tenuifolia WILLD.), a traditional Chinese medicine used to relieve insomnia, anxiety and heart palpitation. The present study was designed to investigate its direct electrophysiological effects on isolated ventricular myocytes from rabbits. Whole-cell configuration of the patch-clamp technique was used to measure action potential (AP) and membrane currents in single ventricular myocytes enzymatically isolated from adult rabbit hearts. Ca2+ transients were recorded in myocytes loaded with the Ca2+ indicator Fluo-4AM. Among three bioactive substances of Polygala metabolites, only M-TMCA (15–30 µM) significantly shortened action potential duration at 50% and 90% repolarization (APD50 and APD90) in cardiomyocytes in a concentration-dependent and a reversible manner. M-TMCA also inhibited L-type calcium current (ICa,L), but showed effect on neither transient outward potassium current (Ito) nor steady-state potassium current (IK,SS). Furthermore, M-TMCA abolished isoprenaline plus BayK8644-induced early afterdepolarizations (EADs) and suppressed delayed afterdepolarizations (DADs) and triggered activities (TAs). This potential anti-arrhythmic effects were likely attributed by the inhibition of ICa,L and the suppression of intracellular Ca2+ transients, which consequently suppress the generation of transient inward current (Iti). These findings suggest that M-TMCA may protect the heart from arrhythmias via its inhibitory effect on calcium channel.
Pioglitazone, a peroxisome proliferator-activated receptor-γ agonist, is indicated for the treatment of type 2 diabetes. Pioglitazone undergoes hepatic metabolism by cytochrome P450 2C8 (CYP2C8) and interindividual variability exists in pioglitazone disposition and response. In previous analyses, it has been shown that the CYP2C8*3 polymorphism significantly impacts pioglitazone pharmacokinetics in humans. The purpose of this investigation was to develop a population pharmacokinetic model using nonlinear mixed effects analysis to evaluate and quantify the effect of CYP2C8*3, demographic, and clinical variables on interindividual variability in pioglitazone pharmacokinetics in nondiabetic adults. Data were obtained from 31 healthy volunteers (n=16 CYP2C8*1/*1, n=15 CYP2C8*3 carriers) who had previously participated in the monotherapy phase of a pioglitazone drug–drug interaction study. Participants received a single 15 mg dose of pioglitazone followed by a 48-h sampling period. A two-compartment model with first order absorption and elimination (Akaike Information Criteria (AIC)=2889) showed a better fit for pioglitazone than a one-compartment model (AIC=3008). Covariate analysis revealed that CYP2C8*3 had a significant effect on pioglitazone central compartment clearance (CL/F; p=0.0005) and intercompartmental clearance (Q/F; p=0.004). Pioglitazone CL/F and Q/F were 52% and 286% higher, respectively, in carriers of the CYP2C8*3 allele than in CYP2C8*1 homozygotes. Furthermore, inclusion of CYP2C8*3 as a covariate on CL/F and Q/F decreased interindividual variability in these parameters by 5.2% and 14%, respectively. Other variables (e.g., sex, body weight) were not significant covariates on pioglitazone pharmacokinetics in the model. In summary, CYP2C8*3 significantly affected pioglitazone CL/F, Q/F, and interindividual variability in these parameters in this healthy volunteer cohort.
In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an effective derivatization method followed by high-performance liquid chromatography (HPLC) coupled to electrochemical ionization mass spectrometry was used. The derivatization reaction of biological samples with ethyl chloroformate occurred rapidly at room temperature in aqueous conditions, and the resulting derivatives were isocratically separated with good selectivity using a C18 reversed-phase column within 30 min. The study results showed that the new derivatization procedure offers an excellent means of simultaneous determination of 5-HT, DA and their metabolites in mouse brain homogenates, which are important in a number of physiological and behavioral functions.
Stearoyl-CoA desaturase-1 (SCD-1) catalyzes the biosynthesis of monounsaturated fatty acids, and their abnormality is possibly responsible for obesity, insulin resistance, hepatic steatosis and nonalcoholic steatohepatitis (NASH). A novel SCD-1 inhibitor, N-(2-hydroxy-2-phenylethyl)-6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxamide, has been obtained. The compound inhibited liver SCD-1 activity and increased liver triglyceride accumulation in mice fed with non-fat, high-sucrose diets. In order to evaluate the effects of the SCD-1 inhibitor on NASH development, rats were fed with lipogenic methionine and choline-deficient (MCD) diets for 8 weeks. The SCD-1 inhibitor was administered once-daily at a dose of 30 or 100 mg/kg/d by oral gavage. Administration of a high dose of the SCD-1 inhibitor decreased triglyceride accumulation in the liver of NASH rats by 80%. Administration of a high dose of the SCD-1 inhibitor attenuated the increase of aspartate aminotransferase (AST) and alanine transaminase (ALT) by 86% and 78%, respectively. Hepatic steatosis, hepatocellular degeneration and inflammatory cell infiltration were histologically observed in the liver of NASH rats, and administration of the SCD-1 inhibitor ameliorated these crucial observations in NASH. In summary, an SCD-1 inhibitor ameliorated hepatic triglyceride accumulation, liver injury, hepatocellular degeneration and inflammation in experimental NASH models. These results suggest that SCD-1 maybe a promising target for the treatment of NASH.
Imatinib mesylate (IM), a widely prescribed powerful tyrosine kinase inhibitor, has been associated with increased risk of heart failure and is known to induce cell apoptosis and death in isolated cardiomyocytes. In addition to acquired long QT syndrome, pharmacological inhibition of human ether-à-go-go-related gene (HERG) channel has been reported to involve in apoptosis. The present study was undertaken to characterize the biophysical properties of IM on HERG and the molecular determinants of HERG blockade using mutant channels (Y652A and F656A). Wild type (WT) and mutant HERG channels were expressed in HEK-293 cells and Xenopus oocytes and the currents (IHERG) were measured using patch-clamp and two-microelectrode voltage-clamp techniques. IM inhibited WT IHERG in a concentration-dependent manner with an IC50 of 19.51±2.50 µmol/L and 44.76±1.54 µmol/L in HEK-293 cells and Xenopus oocytes, respectively. The IM-induced inhibition of WT IHERG followed a voltage- and time-dependent manner. The blockade was enhanced by further activation of currents, which were in accordance with an open-channel blockade. The V1/2 for steady-state activation shifted from −15.48±1.21 to –26.66±2.98 mV (p<0.05, n=6). The inactivation kinetics and voltage dependence of steady-state inactivation of the WT HERG channel were not significantly altered by IM. Two S6 domain mutants, F652A and Y656A, attenuated IM-induced inhibition of WT IHERG. Therefore, IM preferentially blocked the open HERG channel through F652 and Y656, providing a molecular mechanism for the cardiac side effects during the clinical administration of IM.
The aim of this study was to develop lipid nanocarriers that combine tretinoin and tetracycline for the efficient topical delivery to treat acne vulgaris. Two different nanocarriers, nanoemulsions (NEs) and nanostructured lipid carriers (NLCs), were prepared, and we examined their average size, zeta potential, drug encapsulation percentage, and drug permeation via the skin. The antibacterial activities of the nanosystems against Staphylococcus aureus, Pseudomonas aeruginosa, and Propionibacterium acnes were evaluated by an agar diffusion assay and the amount of total protein. A ca. 200-nm particle size was achieved with the prepared nanoparticles. The size increased when incorporating a cationic surfactant. Dual-drug loading did not largely affect the size of negatively charged nanoparticles, but significantly reduced the particle size of positively charged nanocarriers. NEs and NLCs exhibited high entrapment of tretinoin which ranged 60–100%. Tetracycline mainly resided in the aqueous phase, with ca. 10% of molecules located at the particulate interface. An in vitro skin permeation study showed that NLCs enhanced tetracycline flux by about 2-times over the control solution. Tretinoin permeation was generally unaffected after nanoparticulate encapsulation. There was no significant difference in tretinoin delivery before or after tetracycline incorporation, while tetracycline permeation significantly decreased by 2-fold in the dual-drug system. Nanoparticulate loading mostly maintained the antibacterial activity of tetracycline. Negatively charged NEs and NLCs even strengthened the antibacterial ability against S. aureus compared to the control solution. This is the first report examining skin permeation and antibacterial activities of dual-drug nanocarriers for acne treatment.
Novel polycation liposomes decorated with cyclic(Cys-Arg-Gly-Asp-D-Phe) peptide (cyclicRGD)-polyethylene glycol (PEG) (RGD-PEG-polycation liposomes (PCL)) were previously developed for cancer therapy based on RNA interference. Here, we demonstrate the in vivo delivery of small interfering RNA (siRNA) to tumors by use of RGD-PEG-PCL in B16F10 melanoma-bearing mice. Pharmacokinetic data obtained by positron emission tomography showed that cholesterol-conjugated siRNA formulated in RGD-PEG-PCL markedly accumulated in the tumors. Delivered by RGD-PEG-PCL, a therapeutic cocktail of siRNAs composed of cholesterol-conjugated siRNAs for c-myc, MDM2, and vascular endothelial growth factor (VEGF) were able to significantly inhibit the growth of B16F10 melanoma both in vitro and in vivo. These data suggest that targeted delivery of siRNAs by use of RGD-PEG-PCL has considerable potential for cancer treatment.
We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.
Recent epidemiological studies have shown that moderate coffee consumption is associated with a lower risk of certain types of cancers, particularly colon cancer in postmenopausal women. To elucidate the molecular basis for the preventive action of coffee, we investigated the effect of coffee on estrogen sulfotransferase (SULT) because sulfation is the major pathway involved in the inactivation of estrogens. We found that coffee reduced SULT1E1 gene expression in human colon carcinoma Caco-2 cells. Treatment with 2.5% (v/v) coffee for 24 h resulted in a 60% reduction of the expression of the SULT1E1 gene in Caco-2 cells. Corresponding to reduced SULT1E1 gene expression, cytosolic estrogen SULT activity toward E2 (20 nM) decreased by 25%. In addition, an accumulation of E2 sulfates in the medium, which reflects cellular activity of estrogen SULT, decreased after the cells were treated with coffee. Major bioactive constituents in coffee such as caffeine, chlorogenic acid and caffeic acid did not show any effect. The inhibitory activity was extractable by using ethyl acetate. We also found that the inhibitory activity was produced by roasting the coffee beans. Mithramycin, an inhibitor of the transcription factor stimulating protein 1 (Sp1), was able to restore coffee-reduced SULT1E1 gene expression. Our data suggest that daily coffee consumption may reduce estrogen SULT activity, thereby enhancing estrogenic activity in the colon.
Doping is one of the most serious problems for athletes, and it is important that pharmacists have more interaction with athletes to ensure safer drug usage. Education is one of the most important roles of sports pharmacists, who are specialists regarding drug usage for athletes. We investigated pharmacy students’ interests and comprehension regarding drug usage, doping and supplement intake by using the form of a questionnaire, since it is important to know how they understand these subjects as part of their greater educational program. The subjects were sophomore and junior pharmacy students at three universities. It was revealed that most of the students have negative images regarding doping violation, and they answered that they are familiar with doping. However, only sixteen percent of the students had attended lectures by specialists on doping. In addition, one third of pharmacy students did not know that some over-the-counter (OTC) drugs might contain doping substances. With regard to supplement intake, approximately two thirds of the respondents had an interest in and positive image of supplement intake. However, it was revealed that only one third of them recognized supplements as food, and their information regarding supplements was obtained from uncertain media. It was suggested that it is important for pharmacy students to have more opportunities to learn about what doping is. More education and enlightenment by sports pharmacists would be effective for pharmacy students as well as athletes, and it would help us to broaden the scope of what we can do for athletes and society.
We compared effects of the antiarrhythmic drug bepridil on the electrophysiological parameters in the isolated pulmonary vein preparation from guinea pigs with those in the left atrium. Three pairs of bipolar electrodes were attached to the left atrium, pulmonary vein and junctional region of left atrium and pulmonary vein to measure intra-atrial and intra-pulmonary vein conduction velocity and effective refractory period. Bepridil at 10 µM prolonged the effective refractory period with little effect on the conduction velocity in the pulmonary vein, whereas the drug failed to affect the electrophysiological parameters in the left atrium. Using the conventional microelectrode technique, action potential of the isolated pulmonary vein preparation and left atrium were measured. Bepridil prolonged the action potential duration of the pulmonary vein more potently than that of the left atrium. These results suggest that antiarrhythmic effects of bepridil on reentry within the pulmonary vein are estimated to be greater than within the left atrium, which may be one of the key considerations to understand its antiarrhythmic mechanisms.
Pristimerin is a naturally occurring triterpenoid that causes cytotoxicity in several cancer cell lines. However, the mechanism of action for the cytotoxic effect of pristimerin has not been unexplored. The purpose of this study was to investigate the effect of pristimerin on cytotoxicity using the epidermal growth factor receptor 2 (HER2)-positive SKBR3 human breast cancer cell line. Pristimerin inhibited proliferation in dose- and time-dependent manners in cells. We found it to be effective for suppressing HER2 protein and mRNA expression. Fatty acid synthase (FASN) expression and FASN activity were downregulated by pristimerin. Adding of exogenous palmitate, the end product of de novo fatty acid synthesis, reduced the proliferation activity of pristimerin. The changes in HER2 and FASN expression induced by pristimerin altered the levels of Akt and mitogen-activated protein kinase (MAPK) phosphorylation (Erk1/2, p38, and c-Jun N-terminal kinase (JNK)). Pristimerin lowered the levels of phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets such as phosphoprotein 70 ribosomal protein S6 kinase and 4E binding protein1. Pristimerin inhibited migration and invasion of cells, and co-treatment with the mTOR inhibitor rapamycin additionally suppressed these activities. Pristimerin-induced apoptosis was evaluated using Western blotting for caspase-3, -8, -9, and poly (ADP-ribose) polymerase expression and flow cytometric analysis for propidium iodide labeling. These results suggest that pristimerin is a novel HER2-downregulated compound that is able to decrease fatty acid synthase and modulate the Akt, MAPK, and mTOR signaling pathways to influence metastasis and apoptosis. Pristimerin may be further evaluated as a chemotherapeutic agent for HER2-positive breast cancers.
Oxaliplatin is commonly used anti-cancer drugs, but it frequently causes peripheral neuropathic pain. Recently, we reported that elcatonin, a synthetic analog of eel calcitonin, attenuated the oxaliplatin- and paclitaxel-induced cold and mechanical allodynia in rats. In the present study, we determined whether salmon calcitonin also had anti-allodynic effects on oxaliplatin-induced neuropathy in rats. The rats were treated with a single dose of oxaliplatin (6 mg/kg, intraperitoneally (i.p.)). Oxaliplatin resulted in cold and mechanical allodynia. We assessed the anti-allodynic effects of subcutaneously administered salmon calcitonin (20 U/kg/d) by cold stimulation (8°C) directly to the hind paw of the rats and by using the von Frey test. Salmon calcitonin almost completely reversed the effects of both cold and mechanical allodynia. These results suggest that salmon calcitonin is also useful for treatment of oxaliplatin-induced neuropathy clinically.