Purpose：Dilated cardiomyopathy (DCM) is a most common cause of cardiac transplantation in children. However, little is known about the disease progression process of DCM in children. In this study, we explored the disease progression of DCM during early postnatal stages, using a knock-in mouse model for human DCM caused by ΔK210 mutation in the cardiac troponin T gene.

Methods：Cardiac functions were evaluated at 15 and 30 days old, using M-mode echocardiography and color Doppler.

Results：BW of mice at 30 days old was about 2-fold greater than at 15 days old. At 15 days old, HW of HM mice were already greater than WT. At 30 days old, HW of heterozygous (HT) mice also became greater than WT. LVIDd of DCM mice was significantly greater than WT both at 15 and 30 days old. LV wall thickness was not different between DCM and WT mice at 15 days old, but thinner in DCM mice at 30 days old. EF and FS declined in HM but maintained in HT at both ages. Fibrosis was observed only in 30 days old HM mice.

Conclusions：LV dilation and systolic dysfunction occur at a very early postnatal stage before weaning in this DCM mouse model, suggesting that this model is useful for the exploration of pathogenic mechanisms and therapeutics for very early onset DCM.

Today, exercise is regarded as one of therapies for heart failure (HF). However, the effects of exercise on patients with dilated cardiomyopathy (DCM) have not been established. A knock-in mouse model of human inherited DCM, TNNT2 ΔK210, shows similar characteristics to DCM patients. We aimed to examine how the frequency of voluntary exercise influence progression of heart failure using the DCM model mice.Homozygous ΔK210 (DCM) mice showed enlarged heart and frequent sudden death with t_1/2 of ～70 days. DCM mice were divided into 3 groups based on the frequency of voluntary exercise: no exercise control, every 2 days (2D) and daily exercise (ED). The 2D and ED groups started running at 1 month of age. At the 2 months of age, mice were sacrificed after an investigation with echocardiography, and their heart, lung, lower extremity muscles and body weights were measured. Gene expressions of HF- and arrhythmia-related genes in myocardium were quantified by qPCR analysis. The ejection fraction was significantly improved in ED group compared with 2D and control. ED group showed attenuated electrical remodeling in the hearts. These result indicated that daily voluntary exercise prevents progression of HF in DCM mice.

[Background] Our previous data demonstrated that hydrogen (H₂) inhalation improved cardiac function in a rat model of cardiac arrest. In this study, we examined the genes expression of post-cardiac arrest treated with H₂ using a DNA microarray based comprehensive approach. [Methods and results] Rats were subjected to 6 minutes of ventricular fibrillation cardiac arrest followed by cardiopulmonary resuscitation. Resuscitated rats were mechanically ventilated with 26% O₂ with or without 1.3% H₂. Sham-operated rats had the same operative procedure without cardiac arrest. Animal survival rate on day 7 was 38.4% (control group) vs. 71.4% (H₂ treated group). We isolated hearts on 7 days after resuscitation, and then analyzed gene expression using a DNA microarray. One hundred fifty-one genes were up-regulated while 109 were down-regulated genes in cardiac arrest rats. In addition, 511 genes were increased and 461 genes were decreased in H₂ treated group than those of the untreated group. Hierarchical clustering algorithm also showed  obvious differences in comprehensive gene expressions between the untreated and H₂ treated group. [Conclusion] Our data demonstrated that H₂ inhalation on post-cardiac arrest syndrome.

Heart failure caused by right ventricular hypertrophy and the fibrosis is a major cause of death in pulmonary hypertension. It has been reported that certain angiotensin AT₁ receptor blocker possesses inhibitory effect on cardiovascular remodeling. Thus, in the present study, we investigated whether olmesartan, an angiotensin AT₁ receptor blocker, could attenuate right ventricular hypertrophy and/or fibrosis using rats with monocrotaline-induced pulmonary hypertension.

Male Sprague-Dawley rats (5-week-old) were administered single subcutaneous injection of monocrotaline (60 mg/kg) and induced pulmonary hypertension. Olmesartan continuously infused subcutaneously for 4 weeks by use of an osmotic mini pump. The estimated dosage of olmesartan during the experimental period was around 3 mg/kg/day.

Treatment with olmesartan failed to improve right venricular hypertrophy, whereas it reduced right ventricular fibrosis in the monocrotaline-treated rats. Moreover, enhanced expression of fibrosis-related proteins including IL-6, IL-1beta, GDF15, CTGF, and MMP9 the rats was not observed in the olmesartan-treated rat right ventricles. These results suggest that olmesartan has a potential to be a therapeutic agent against pulmonary hypertension.

Eucommia ulmoides Oliver is known as an herbal medicine. Recently, Eucommia ulmoides oliver leaf extract (ELE) has been shown to improve vascular function and vascular hypertrophy in SHR. Pulmonary arterial hypertension (PAH) is a severe and progressive disease that causes right heart failure. The pathogenesis of PAH is generally characterized by persistent high pulmonary arterial resistance and pulmonary arterial remodeling. In the present study, we investigated the effects of ELE on hypoxia-induced PAH in mice. 10-weeks-old male C57BL/6J mice were orally administered a 5% ELE during exposure to hypoxia for 4 weeks. ELE significantly suppressed the elevation of right ventricular systolic pressure in hypoxia-induced PAH mice. In addition, hypoxia-induced pulmonary arterial muscularization was tended to be attenuated in ELE-treated mice. Our findings suggest that ELE may effectively improve the development of hypoxia-induced PAH by preventing the hypercontraction and/or vascular remodeling of pulmonary artery.

Due to the limited treatment options for refractory hypotension and vascular hyporeactivity to vasoconstrictors, the new drug development is needed. The effect of the naturally-occurring flavonoids, oroxylin-A, wogonin and baicalein, in the presence of serum on the tensions of rat tail arteries was examined by using in vitro blood-vessel myography. We found that oroxylin-A (1-300 μM) dissolved in rat serum (OroA/RS), promoted RS-induced but endothelium-independent vasoconstrictions in a concentration-dependent manner. On the other hand, baicalein and wogonin (structurally close to OroA) did not affect or attenuated RS-induced constrictions in tail arterial rings. Repeated applications of OroA (300 μM)/RS induced reproducible and long-lasting constrictions without tachyphylaxis in the arterial rings. OroA/RS-induced vasoconstrictions were blocked by 5-HT2A receptor antagonist (ketanserin, 0.3 μM) and GRK2 antagonist (CMPD101, 10 μM), but not by the inhibitors of ET-1 receptor, AT-1 receptor, or EP1/2 receptor. These results suggest that OroA with its special structure may potentiate the endothelium-independent contractions induced by RS via the 5-HT/GRK2 pathway in tail arteries. The intervention of OroA may be beneficial for the acute management of endotoxemic hypotensive shock.

Purpose: Aortic aneurysm (AA) and aortic dissection (AD) are common diseases among the elderly people and potentially lethal. However, there is currently no management to prevent these diseases. Quercetin, an abundant polyphenol in onions, is reported to improve vascular function. In this study, we investigated the effects of quercetin on AAs or AD in mice. Methods: In C57BL/6J male mice, angiotensin II (Ang II) and β-aminopropionitrile (BAPN) were administered to induce hypertension and degeneration of elastic lamina respectively, leading to AAs. If L-NAME were added, mice shows endothelial dysfunction and more AD incidence. We call these AA model as "AB" and AD model as "LAB". Results: Quercetin treatment reduced the incidence of AAs and the death from aortic rupture in AB mice, and the incidence of AD and rupture in LAB mice. The activity of MMP-2/9 were upregulated in aortae, but suppressed by quercetin treatment. The expressions of vascular endothelial cell adhesion molecule-1 and F4/80, a marker of macrophages, are also suppressed in quercetin treated group. Conclusions: These findings suggest that quercetin prevents AA and AD via its protective effects against endothelial dysfunction, elastin degeneration and inflammation.

Extracellular matrix (ECM), mainly composed of elastin and collagens, is a fundamental structural component participating in vascular function. Previously, our group demonstrated that LTBP-4 and Fibulin-5 are key molecules in regulating elastic fiber formation. Here, we analysed quantitative changes of LTBP-4 and Fibulin-5 with aging in mice. Thoracic aortas were resected from wild-type (WT) mice from two- to 48-weeks of age and Ltbp4S-ko or Fbln5-ko mice at four-months after birth. Expression of LTBP-4 or Fibulin-5 was evaluated by Western blot. Three-dimentional structures of elastin and collagen were visualized using two-photon microscopy. In WT mice, LTBP-4- and Fibulin-5 protein levels in aorta decreased with aging. Compared to young WT mice, higher degree of elastin break and collagen accumulation were found in ascending aortic wall of old WT, Ltbp4- and Fbln5-ko mice, suggesting a similar pathophysiology of increased aortic stiffness in these mice. In accordance, pulse pressure in old WT- and ko mice was higher than that in WT young mice. Further, higher incidence of aortic disease (aneurysm or dissection) induced by angiotensin Ⅱ infusion was found in Fbln5-ko than WT mice. Thus, ECM remodeling caused by progressive decrease in intrinsic LTBP-4 and Fibulin-5 levels may induce age-related atrial stiffening and vascular disease such as dissection and aneurysm.

Aortic valve stenosis (AS) is a common heart valve disease in elderly people, and is mostly accompanied by ectopic valve calcification. We recently demonstrated that tumor necrosis factor-α (TNF-α) induces calcification of human aortic valve interstitial cells (HAVICs) obtained from AS patients. In this study, we investigated the role of matrix Gla protein (MGP), a known calcification inhibitor that antagonizes bone morphogenetic protein 2 (BMP2) in TNF-α-induced calcification of HAVICs. HAVICs isolated from aortic valves were cultured, and calcification was induced with 30 ng/mL TNF-α. Gene expression of the calcigenic marker, BMP2, was significantly increased in response to TNF-α, while the gene and protein expression of MGP was strongly decreased. To confirm the role of MGP, MGP-knockdown HAVICs and HAVICs overexpressing MGP were generated. In HAVICs, in which MGP expression was inhibited by small interfering RNA, calcification and BMP2 gene expression were induced following long-term culture for 32 days. In contrast, HAVICs overexpressing MGP had significantly decreased TNF-α-induced calcification. These results suggest that MGP acts as a negative regulator of HAVIC calcification.

Cyclic mechanical stretch (CMS) leads to vascular smooth muscle cells proliferation, cell death and migration, resulting in vascular remodeling and subsequent vascular failure. However, the effect of CMS on gene induction in cardiovascular diseases remains to be determined. We have revealed that CMS caused cell death in rat aortic smooth muscle cells (RASMCs) in JNK and p38-dependent manners. To explore the causal role of CMS in initiating cell death signaling and MAPKs events, we compared transcript profiles of CMS-induced RASMCs death using cDNA microarrays. Inducible nitric oxide synthase (iNOS) gene was identified as having significantly differential expression in response to CMS. We further identified using qPCR analysis that CMS induced iNOS expression in a p38-dependent manner in RASMCs. The result also showed that NO production was increased, implying that NO was synthesized by CMS-induced iNOS. In contrast, NO production was inhibited by p38 inhibitor. Moreover, a iNOS inhibitor strongly increased CMS-induced cell death; whereas a NO donor significantly inhibited CMS-induced cell death in RASMCs, indicating that iNOS protects RASMCs from CMS-stimulated cell death via the p38 signal pathway.

Smoking is known to be accompanied with a decrease in nitric oxide (NO) bioavailability in the vascular system. This study investigated whether chronic administration of cigarette smoke extract (CSE) influences on soluble guanylate cyclase (sGC) redox state, a determinant of NO bioavailability. Rats were subcutaneously administered phosphate saline buffer (PBS), gas phase-CSE (gp-CSE) or whole phase-CSE (wp-CSE) for 4 weeks, and vascular reactivity was examined in organ chamber experiments. In both the aorta and pulmonary artery, the relaxant response to acetylcholine was attenuated to a similar extent by administration of gp-CSE or wp-CSE. On the other hand, regardless of vessel type, sodium nitroprusside (reduced sGC stimulant)-induced and BAY 60-2770 (oxidized/heme-free sGC stimulant)-induced relaxation were identical in the three groups. These findings suggest that chronic CSE administration induces endothelial dysfunction but has no significant impact on vascular sGC redox state.

M-1 is a major metabolite of sarpogrelate, a selective inhibitor of 5-hydroxytryptamine (5-HT) _2A receptor. Our aim was to evaluate the effect of M-1 on the 5-HT-induced vasoconstriction in isolated endothelium denuded human internal thoracic artery (ITA) obtained from patients undergoing coronary bypass surgery. We investigated the effects of M-1, sarpogrelate and SB224289, a selective antagonist of the 5-HT_1B receptor, on the 5-HT-induced vasoconstriction. The vasoconstriction induced by 5-HT was significantly inhibited by M-1 in a concentration-dependent manner. Supramaximum concentrations of sarpogrelate or SB224289 significantly, but not completely, inhibited the 5-HT-induced vasoconstriction. In addition, simultaneous pretreatment with both sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. M-1 also significantly almost completely inhibited the 5-HT-induced vasoconstriction, which mimics the effects of the simultaneous pretreatment with sarpogrelate and SB224289. These results demonstrate that M-1 inhibits 5-HT-induced vasoconstriction via the blocking activity of not only 5-HT_2A receptors but also 5-HT_1B receptors in human ITA.

Magnesium ion (Mg²⁺) plays an essential role in various cellular functions. Mg²⁺ deficiency or abnormal Mg²⁺ metabolism is related to various cardiovascular diseases, such as ischemic heart disease and arrhythmias. Recently, various candidate genes of Mg²⁺ transporters are reported, but their functional roles are still unknown. We first treated mice with three kinds of magnesium diets (low-magnesium diet, normal-magnesium diet, or high-magnesium diet) for 4 weeks, and found that the tissue expression levels of several Mg²⁺ transporters were dependent on magnesium intake. We also found that phenylephrine-induced contraction was attenuated in isolated aorta from low-magnesium-fed mice. Furthermore, to investigate the functional roles of these Mg²⁺ transporters, we generated several genetically altered mice targeting their Mg²⁺ transporters. Interestingly, phenylephrine-induced contraction was reduced in isolated aorta from these genetically altered mice. On the other hand, when these genetically altered mice were fed with a high-magnesium diet, the phenylephrine-induced contraction was recovered to normal level. These results suggest that these Mg²⁺ transporters may play important roles in the maintenance of Mg²⁺ homeostasis and vascular functions.

Magnesium ion (Mg²⁺) is an essential divalent cation, and intracellular Mg²⁺ concentration is tightly controlled by various Mg²⁺ transporters. Therefore, Mg²⁺ transporter dysfunction may consequently lead to a variety of diseases, such as cardiovascular, neuronal, and muscular diseases. Recently, several candidate genes for Mg²⁺ transporters have been identified. However, the regulation mechanisms of Mg²⁺ homeostasis are mostly unknown. To clarify these issues, we focused on Mg²⁺-permeable non-selective cation channel TRPM7, and generated kidney-specific transgenic mouse model overexpressing the dominant negative TRPM7 mutant (M7DN-Tg), as an experimental tool. We confirmed that TRPM7 currents in HEK293 cells were almost completely inhibited by co-expression of the M7DN construct. We found that M7DN-Tg exhibited dysregulation of serum Mg²⁺ level and urinary Mg²⁺ excretion. Interestingly, vascular contractile responses in M7DN-Tg were significantly attenuated compared to the responses in wild-type mice. In M7DN-Tg, Mg²⁺-enriched diet recovered the abnormal responses to the normal level. These results suggest that TRPM7 is involved in the　regulation of Mg²⁺ homeostasis. M7DN-Tg will be a useful animal model for studying magnesium disorders.

Vascular smooth muscle cells (VSMCs) play critical roles in vascular homeostasis regarding stability and tonic regulation. VSMCs can switch their phenotype back and forth between highly proliferative synthetic and fully differentiated contractile in response to changes of vessel environment. Although critical for vascular homeostasis, this so-called phenotype switching is a cause of vascular diseases such as atherosclerosis and hypertension. In pathological conditions, proliferation of VSMCs are accelerated, which adversely affects prognosis. Therefore, the mechanism underlying transition from active synthetic to quiescent contractile phenotype has attracted attention but is largely unknown. In this study, we investigated the importance of canonical transient receptor potential 6 (TRPC6) in VSMCs phenotype switching. TRPC6 deficient (TRPC6(-/-)) VSMCs was more sensitive to the differentiation stimuli. We revealed that TRPC6(-/-) VSMCs have more polarized membrane potential and higher Akt activity than wild type cells under the differentiation pressure. TRPC6 physically and functionally coupled with lipid phosphatase PTEN, a negative regulator of Akt activation. These findings indicate suppression of TRPC6 can facilitate VSMCs differentiation and novel therapeutic strategy for several vascular diseases.

Recently, we reported that drinking of Citrus unshiu juice (CU) or Citrus iyo juice (CI) drinking attenuated vascular remodeling in vascular injury mouse model. This effect was more marked with CI. Therefore, we focused on flavanone, hesperidin, which is more abundantly contained in CI compared with CU, and investigated the effect of hesperidin in vascular injury in mice. Eight-week-old male C57BL/6 mice were administrated 100 mg/kg/day hesperidin orally by gavage. Two weeks after administration, vascular injury was induced by polyethylene cuff placement on the femoral artery. Neointima formation was determined 14 days after cuff placement by evaluating intima/media ratio. One week after cuff placement, mRNA levels were measured by quantitative real-time RT-PCR. Treatment with hesperidin did not change systolic blood pressure and body weight compared with that in control mice. Neointima formation in the injured artery was significantly increased 2 weeks after cuff placement. Treatment with hesperidin significantly decreased neointimal formation. Expression of mRNA of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 were increased by cuff placement. These increases tended to decrease in treatment with hesperidin. These results suggest that the intake of hesperidin in citrus fruits juice should prevent vascular injury.

Human iPSC cardiomyocytes (hiPS-CMs) have been used for the risk assessment of drug-induced QT prolongation and ventricular tachycardia called torsade de pointes. We have evaluated 12 compounds using CardioECR. The 12 compounds include high (azimilide, bepridil, dofetilide, ibutilide), intermediate (chlorpromazine, cisapride, clarithromycin, clozapine) and low (diltiazem, loratidine, metoprolol, mexiletine) risk classes. The effects of these compounds on field potential (FP) and impedance signals were evaluated with iCell cardiomycytes². In the high risk group, azimilide, dofetilide and ibutilide prolonged the field potential duration (FPD) and induced EADs, but bepridil stopped the beating at the highest concentration. In low risk group compounds, diltiazem, metoprolol and mexiletine stopped beating, and loratidine showed no apparent change in FPD. The intermediate compounds stopped beating or induced EADs at higher concentrations. These results suggested that CardioECR can be used as a platform to assess the QT risk with hiPS-CMs. As CardioECR can utilize the impedance data in addition to the FP signals, an integrated analysis using both signals is useful for more accurate interpretation of compound nature.

Although impedance has been increasingly used to detect the proarrhythmic and contractile abnormalities in iPSC cardiomyocytes (CM), there are still ambiguities in interpreting impedance signals to explore the mechanisms of drug actions. In the present study, we have measured the impedance signals with xCELLigence CardioECR and sequential live imaging with CQ1 to examine the correlation of impedance with Ca²⁺ transients, action potentials, and muscle motion in the same preparation of iCell CM². Beat duration of impedance signals showed the frequency dependent nature as with those of action potential, and had good correlation with Ca²⁺ signals in the change of signal duration induced by CiPA II non-core site compounds. Early afterdepolarization induced by cisapride could be identified more clearly in the Ca²⁺ or action potential signals than impedance ones. The peak amplitude was reduced by the calcium channel blockade in any of impedance, Ca²⁺ and motion signals, but the correlation among these signals was not likely to be clear compared with those of duration parameters. The combination of impedance signals and imaging data can be a strong tool to elucidate mechanisms of drug action on iPSC CM.

[Purpose] We examined electrophysiological indices of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) sheets in order to quantitatively estimate multichannel blocking actions of bepridil and amiodarone using MEA system in comparison with that of E-4031. [Methods] We analyzed the field potential duration(FPD), effective refractory period, current threshold and conduction property using a programmed electrical stimulation protocol to obtain the post repolarization refractoriness(PRR) and coefficient a of the relationship between the pacing cycle length and FPD. [Results & Conclusions] Electropharmacological profiles of drugs were successfully characterized; namely, 1) the changes in the current threshold and conduction property provided important information of Na⁺ channel blocking kinetics, 2) the change of coefficient a reflected drug-induced inhibition of hERG K⁺ channel, 3) the PRR indicated the relative contribution of these drugs to Na⁺ and K⁺ channel blockade, and 4) L-type Ca²⁺ channel blocking action was obvious in the field potential waveform of the hiPSC-CMs sheets, which will help to predict whether the net balance of Ca²⁺ and K⁺ channel blockade of a drug is proarrhythmic or antiarrhythmic.

Background: The present study is to estimate the geometrical and constituent heterogeneity effects on electrical conduction on in vitro monolayer consisting of atrial-like cardiomyocytes (ALCMs) derived from human induced pluripotent stem cells (hiPSCs) and human atrial fibroblasts (HAFbs) under HF field stimuli (HFFS).

Method: We induced hiPSCs into ALCMs by adding all-trans retinoic acid (ATRA). The ALCMs and HAFbs were transferred in defined ratios on manually fabricated plate with geometrical transition. HFFS were delivered, and the electrical propagation was assessed by optical mapping.

Results: ATRA-treated CMs showed atrial specific properties compared to untreated CMs. HFFS preferentially induced impaired conduction on ALCMs with an abrupt geometrical transition, but not on ALCMs with uniform geometry. In addition, the co-culture of HAFbs with the ALCMs deteriorated the stability of electrical conduction than in mono-culture of ALCMs.

Conclusion: Geometrical heterogeneity under HFFS jeopardizes electrical conduction on in vitro ALCMs monolayer. Constituent heterogeneity represented by HAFbs contributes to the deterioration of the electrical conduction stability.

GPR120/FFAR4 has been recognized as a functional fatty acid receptor and an attractive therapeutic target for metabolic diseases. Previously, we have demonstrated that GPR120/FFAR4 deficit (GPR120KO) mice facilitate an inflammatory response of the nonalcoholic fatty liver diseases (NAFLD) after short-term a 0.1% methionine and choline deficient high-fat (CDAHF) feeding compared to WT mice. In this study, we investigated whether GPR120KO mice after long-term CDAHF feedings induce the progression of nonalcoholic steatohepatitis (NASH). Mice fed with CDAHF diet for 6 weeks showed a significant increase in plasma aspartate transaminase and alanine transaminase levels, fatty deposition, inflammatory cell infiltration, and sever fibrosis. Both WT mice and GPR120KO mice fed CDAHF diet showed increment of the number of crown like structures and the immunoreactivity for F4/80 positive cells. However, GPR120KO mice significantly increased TGF-b mRNA collagen type 1 α mRNA in the liver compared to WT mice fed CDAHF diet, indicating that GPR120KO mice fed CDAHF diet showed more severe liver fibrosis than that of WT mice fed CDAHF diet. Therefore, our findings suggest that GPR120 signaling could be helpful as a regulatory factor of NAFLD/NASH progression.

Low-grade inflammation persists in many patients with clinically quiescent inflammatory bowel disease　(IBD). The current study aimed to establish low-grade IBD model mice. In addition, the anti-inflammatory　effect of the cholinesterase inhibitor neostigmine was also investigated in this model. C57BL/6J mice were used. Colitis was induced by the addition of 0.1–3%(w/v) dextran sulfate sodium (DSS) to drinking water for 7 days. Following 3% DSS treatment, weight loss, appearance of bloody stool and changes in stool quality were evident by day 4 and peaked at day 7. DSS at 1% elicited low-grade inflammation in the colonic　mucosa and increased myeloperoxidase (MPO) activity. In immunohistochemical study, increased MPO-immunopositive neutrophils were observed in the colonic mucosa of low-grade colitis model. Neostigmine dose-dependently inhibited the increase of MPO activity. The α7-nicotinic receptor antagonist partly　reversed anti-inflammatory effect of neostigmine. In conclusion, we suggest that low-grade IBD model mice are established by using 1% DSS-containing drinking water, and neostigmine provides anti-inflammatory　effect through the stimulation of α7-nicotinic receptors in this model.

TRPV1 expressing on sensory neurons in mouse lower esophageal sphincter (LES) is considered as association with heartburn symptoms in patients with acid reflux esophagitis (RE). In the present study, we examined changes in TRPV1 and TRPM8 expressions in LES in RE model rats. RE was produced by wrapping duodenum with Nelaton catheter, and ligating the transitional zone between the forestomach and the glandular portion with silk thread under anesthesia. TRP channel and neuronal markers in rat LES were detected by using immunohistochemical staining. In normal rats, numerous TRPV1 nerve fibers were detected in mucosal and submucosal layers. TRPM8-expressing cell bodies were observed in myenteric plexus. In double labeling studies, TRPV1 partly colocalized with calcitonin gene related peptide (CGRP), substance P and neuronal nitric oxide synthase (nNOS) on nerve fibers. In RE model rats, TRPV1-expressing nerve fibers were increased mainly in muscle and mucosal layers. Moreover, TRPM8-expressing cell bodies were increased in myenteric plexus. In conclusion, we suggest that increased TRPV1-expressing nerve fibers and TRPM8-expressing cell bodies were found in myenteric plexus in RE model rats, which might be responsible for heartburn in RE.

The bromodomain and extra-terminal (BET) inhibitor have emerged as promising new cancer agents via regulation of epigenetic mechanism. Recent studies further demonstrate that BET inhibitors exhibit anti-inflammatory effects in animal models of various inflammatory diseases. In the present study, we examined the effect of BET inhibitor on inflammatory bowel disease (IBD) in experimentally-induced murine Crohn's disease (CD)-like ileitis models. Ileitis was induced in male C57BL/6 mice by subcutaneous administration of indomethacin and the ileum was examined 48 h later. CN210 was given orally 30 min before and 24 h after indomethacin administration. Further, the effect of CN210 on LPS-stimulated cytokine expression in cultured RAW264.7 cells. The administration of CN210 reduced the severity of indomethacin-induced ileitis in a dose-dependent manner. Indomethacin-induced upregulation of inflammatory cytokines such asTNF-α, IL-1β and IL-6was also significantly attenuated by administration of CN210. In RAW264.7 cells, LPS upregulated the expression of inflammatory cytokines, and this response was potently abrogated by CN210. These findings suggest that CN210 ameliorates indomethacin-induced ileitis via inhibition of inflammatory cytokine expression. Thus, CN210 is a novel candidate for the treatment with IBD including CD.

BLT2, a low-affinity leukotriene B4 (LTB4) receptor, is highly expressed in intestinal epithelial cells. Recently, 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) was identified as an endogenous ligand for BLT2. However, the precise role of this receptor has not been fully understood. The present study investigated the role of BLT2 in the healing of intestinal injury using BLT2-deficient (BLT2KO) mice, transgenic mice with intestinal epithelium-specific overexpression of BLT2 (villin-BLT2-Tg), and murine epithelial cell line (YAMC). The intestinal injury was induced in mice by subcutaneous administration of indomethacin and the healing of injury was determined 48, 72 and 96 h later. The wound (diameter: 1 mm) was inflicted on monolayer of YAMC and wound closure was evaluated 6 h later. Further, cell proliferation was determined by WST-1 assay. The healing of indomethacin-induced intestinal injury was significantly delayed in BLT2KO mice when compared with wild-type (WT) mice. In contrast, villin-BLT2-Tg mice exhibited healing-promoting properties when compared with WT mice. In YAMC, CAY10583, a BLT2 agonist, concentration-dependently promoted wound repair and cell proliferation. The similar effect was observed by 12-HHT. These findings suggest that BLT2 accelerates the healing of intestinal injury. This effect is at least partly mediated via promotion of epithelial cell proliferation.

Although irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder, the pathophysiology has poorly understood. Several studies demonstrated that the traumatic stress in early childhood increases the risk of IBS. The present study tried to construct a novel mouse model for IBS induced by early childhood social defeat stress. Four weeks old of male C57BL/6 mice were exposed to a trained aggressor mouse for 5-10 min daily for 10 days. After exposure to stress, these mice were maintained under normal conditions for 5 weeks. The state of early childhood social defeat stress was continued until 5 weeks after the exposure to the stress. The number of 5-HT- and CGRP-positive nerve fibers were significantly increased while the number of CD4-positive cells were significantly decreased in the colonic mucosa with early childhood social defeat stress. The visceromotor response to colorectal distention was significantly increased in stress mice compared with normal mice, indicating the development of colonic visceral hyperalgesia. The severity of visceral hypersensitivity was attenuated to the control level by TRPV1 antagonist BCTC. Taken together, these results suggest that early childhood social defeat stress induces IBS-associated visceral hyperalgesia in adulthood, probably via activation of TRPV1. Thus, this model may be useful for studies on the pathophysiology of stress-associated IBS.

Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. We have originally established an advanced imaging system for visualizing in vivo behavior of osteoclasts with an intravital two-photon excitation microscope. We also developed pH-activatable fluorescent probes to detect low pH areas on the bone surface and succeeded in visualizing areas where osteoclasts actually resorb bones in vivo. However, the spatiotemporal dynamics of acidification by osteoclasts remains unclear. In this study, we developed a novel image analysis system to evaluate the relationship between acidic regions and osteoclast dynamics. By means of this system, we found that the acidification by osteoclasts on the bone surface shows a characteristic pattern that is dependent on motility of osteoclasts. This approach would be beneficial for understanding the mechanism of osteoclastic bone resorption in vivo and would thus be useful for evaluating the efficacy of novel anti-bone-resorptive drugs.

Disodium dihydrogen-4-[(methylthio) phenylthio] methanebisphosphonate (MPMBP) is a novel, non-nitrogen-containing BP with an antioxidant side chain. In this study, we compared the effects of MPMBP with those of zoledronate on bone remodeling in rats. MPMBP or zoledronate was subcutaneously injected every three days in neonatal or growing rats. The animals were euthanized after sequential labeling with tetracycline and calcein, and their tibias, femurs, jaw bones were harvested and examined. Bone morphometric analyses revealed that MPMBP increased the bone mass of the distal femur and proximal tibia. The zoledronate-treated neonatal rats showed reduced weight gain, suppression of longitudinal growth in the hind limbs, and a marked delay in tooth eruption, whereas the MPMBP-treated rats showed normal weight gain and eruption of teeth at the appropriate developmental stage. Further, the MPMBP-treated rats showed higher fluorescence intensity of calcein in their trabeculae, revealing that new mineralization occurred following treatment with MPMBP. In conclusion, our results show that MPMBP has a potent anabolic effect on bones, whereas zoledronate has severe adverse effects due to excessive inhibition of bone resorption and/or bone remodeling.

Purpose: b-series gangliosides are involved in the regulation of cell growth, neurite extension, and apoptosis. However, little is known about their roles in bone metabolism. In this study, we investigated effects of deletion of b-series gangliosides in bone metabolism. 

Material & Methods: We examined expression levels of b-series gangliosides (GD3, GD2, GD1b, and GT1b) in MC3T3E1 osteoblast-like cells, RAW264.7 pre-osteoclast, and primary bone marrow cells using flow cytometry. To determine whether b-series gangliosides are involved in bone metabolism, we analyzed bone phenotype of GD3 synthase-knockout (GD3S KO) mice lacking all b-series gangliosides using mCT.

Results & Conclusion: b-series gangliosides were not detected in MC3T3E1 cells. On the other hand, they were detectable in both RAW264.7 cells and primary bone marrow cells. However their expression was reduced after induction of osteoclastogenesis. No differences in bone phenotype between GD3S KO and wild type mice at the age of 15 weeks were detected. However, bone volume (BV/TV) in GD3S KO mice at the age of 40 weeks was higher than that in wild type mice. Correctively, these results suggest that b-series gangliosides may prevent age-related bone loss.

The number of patients suffering from osteoporosis increases year by year. Osteoporosis is characterized by decreased bone density and increases the risk of fracture. The femoral neck is the most possible part of the fracture. In this study, bone strength of ovariectomized osteopenia (OVX) model mouse was measured using a newly improved stage device for femoral neck fracture. METHODS Female ICR mice were divided into the sham and OVX groups. Eight weeks after surgery, right and left femurs were taken out. The right femur neck was set on a bone strength tester and the maximum breaking load (N) was measured. The left femur was measured for bone density using a CT. All results were regression analyzed with Stat View analysis software and their correlation was obtained. RESULTS In the right femur, N value of the OVX group showed a significant decrease compared to that of the sham group. The value of bone density of OVX group was also lower. Regression analysis of N value and bone density revealed a significant positive correlation coefficient in total bone density and cancellous bone density. CONCLUSION The present study indicates that the novel strength test of the femoral neck using the femoral neck fracture stage is useful for an evaluation of OVX model mice.

Protease-activated receptors (PARs) have a classical heptahelical structure within the plasma membrane and stimulate phosphoinositide turnover by coupling to G-protein. There are four known members of the receptor family of PARs: PAR-1, 3 and 4, which are activated by thrombin and gingipain, and PAR-2, which is activated by trypsin and mast cell tryptase. We previously demonstrated that IL-1α increased ESE-3 mRNA expression through MEK1/2 pathway in human oral epithelial cells (HO-1-N-1 cells). The present study was undertaken to investigate the interaction between interleukin-1α (IL-1α) and PAR-2 expressions in HO-1-N-1 cells. The cells were cultured to semi-confluence and treated with IL-1α or PAR-2 agonist for 6 - 24 hours. RNA was isolated from the cells, and IL-1α and PARs expressions were analyzed by RT-PCR. To measure the amount of IL-1α in cell culture supernatant, ELISA was performed according to the manufacturer's protocol using the Human IL-1α Quantikine ELISA Kit (R&D systems, MN, USA). HO-1-N-1 cells showed PAR-1 and PAR-2 mRNA expression. PAR-2 agonist increased IL-1α mRNA expression, and IL-1α increased PAR-2 mRNA expression in the dose dependent manner at 6 hours in HO-1-N-1 cells. PAR-2 agonist increased extracellular IL-1α level in HO-1-N-1 cells. These results suggest that IL-1α and PAR-2 may play an important role in inflammatory oral mucosal disease.

Dental implant therapy is a highly effective treatment for recovering occlusion after tooth loss. An important factor in the success of dental implants is establishing strong osseointegration. If more epithelial cells migrate to the implant bone interface than mesenchymal stem cells, effective osseointegration may fail. Therefore, controlling epithelial cell adhesion and motility would be an effective strategy to increase the success rate of osseointegration. Laminin-332 is a major component of the basement membrane and is composed of three chains (α3, β3 and γ2). It is well-known that laminin-332 regulates cellular functions such as adhesion, proliferation, apoptosis and differentiation. These biological functions depend on changes in the structural arrangement of laminin-332 by proteolytic cleavage. We focused on cleavage site of α3 and developed antibodies that target the cleavage site. To investigate the influence of the monoclonal antibody on the cell adhesion function of epithelial cells for the α3 chain of laminin-332, we compared it with the cell adhesion function of human epithelial cells from the Cas9-22 cell line. The monoclonal antibody significantly decreased cell adhesion for the laminin-332 α3 chain when compared with no monoclonal antibody in both laminin-332 doses, 1 and 10μg/mL. We could propose that it would be possible that we change the biological function of laminin-332 to control cell adhesion for the purpose of regulating dental implant therapy.

Back ground: Pathogenic oral bacteria induces not only peridontitis and dental caries, but also contribute to causing systemic diseases. However, large-scale etiological study for characterizing regional distribution pattern of pathogenic bacteria is lacking.

Aim: To obtain data of distribution pattern of pathogenic bacteria in shimane prefecture.

Method: Saliva samples were collected from local residents of 7 area in shimane prefecture (1588 samples). Bacterial genome DNA was extracted. Then, following bacteria was detected by PCR; P.gingivalis (P.g), T.denticola (T.d), T. forsythia (T.f), P. intermedia (P.i), A. actinomycetemcomitans (A.a), S. mutans (S.m).

Results: Infection percentage of T.f and A.a was 90% and more in all area. In contrast, frequency of T.d inection was very low every area. There are regional differences in infection of P.g, P.i, S.m. Especially inland residents frequently had P.g compared to people in other area. One area showed outstanding low infection percentage in S.m. Number of people who had both P.g and S.m were almost same with number of S.m-infected people in each area.

Conclusion: Each area showed distinct infections pattern, suggesting that living environment affects the infection of oral pathogenic bacteria.

Cancer immunotherapy opened the new way of cancer therapy and is noticed as the fourth therapy. Consequently, we need a system that can evaluate effectiveness of immune checkpoint inhibitors.

In this study, we measured tumor infiltrating lymphocytes (TILs) and cytokine/chemokine productions which were triggered by anti-PD-1 antibody treatment in CT26WT (colon), LLC (lung) or B16F10 (melanoma)-bearing mice syngeneic models. Anti-PD-1 or control antibody were administered intraperitoneally twice a week for two weeks. The anti-tumor effect was evaluated by tumor volume. Furthermore, after isolation of TILs from tumor, the TILs population was analyzed by flowcytometer. The productions of cytokine/chemokine including IFN-γ, TNF-α and CCL5 were measured by Cytometric Bead Array (CBA) or AlphaLISA analysis.

As a result, changes in the response against antibody, lymphocyte distribution and cytokine/chemokine productions were confirmed in the anti-PD-1 antibody treatment group. The differences in immune function were observed in the three mouse tumor cell lines. The method for evaluating the effectiveness of immune checkpoint inhibitors was established by analyzing TILs and inflammatory mediators.

Recently, extracellular vesicles—particularly exosomes—have been recognized as intercellular communicators in the tumor microenvironment. As exosomic cargo, deregulated microRNAs (miRNAs) can shape the surrounding microenvironment in a cancer-dependent manner. Previous studies have shown inconsistent results regarding miR-200c-3p expression levels in OSCC cell lines, tissues, or serum—likely because of the heterogeneous characters of the specimen materials. For this reason, single-cell clone analyses are necessary to effectively assess the role of exosome-derived miRNAs on cells within the tumor microenvironment. In this study, we performed integrated microarray profiling to compare exosome-derived miRNA and exosome-treated cell-derived mRNA expression. Data were acquired from noninvasive SQUU-A and highly invasive SQUU-B tongue cancer cell clones derived from a single patient to determine candidate miRNAs that promote OSCC invasion. Matrigel invasion assays confirmed that hsa-miR-200c-3p was a key pro-invasion factor among six miRNA candidates. Consistently, silencing of the miR-200c-3p targets, CHD9 and WRN, significantly accelerated the invasive potential of SQUU-A cells. Thus, our data indicate that miR-200c-3p in exosomes derived from a highly invasive OSCC line can induce a similar phenotype in non-invasive counterparts.

Immune checkpoint inhibitors such as anti-PD-1 antibody and anti-CTLA-4 antibody have recently been approved for the treatment of melanoma or non-small cell lung cancer. In this study, we examined an evaluation method for tumor infiltrating lymphocytes (TILs), which is one of the evaluation items of immune checkpoint inhibitors.

BALB/c mice were subcutaneously inoculated with CT26WT(mouse colorectal cancer cell line). They were allocated into anti-PD-1, anti-CTLA-4, or their combination treatment and the control groups. The drugs were administered intraperitoneally twice a week for two weeks. The tumor diameters were measured and tumor volumes were calculated. Observation and measurement were performed up to 14 days after the initiation of administration. Then, the tumor was excised and dispersed. TILs were isolated using CD45 microbeads, stained by fluorescently-conjugated antibodies and analyzed using flow cytometer.

As a result, the drugs changed the proportion of TILs subsets including regulatory T cells (Treg), CD8+ T cells, dendritic cells (DC) and myeloid-derived suppressor cells (MDSC). It is suggested that the evaluation system described above is useful for identification of TILs subsets in the CT26WT-bearing mouse model.

Tumor tissue environment is generally exposed to low oxygen, nutrition depletion and high interstitial pressure condition. These circumstances are caused by vascular hyper-permeability, irregular vascularization and immature vessels. We previously reported that prolyl hydroxylase inhibitor (PHDi) induced tumor blood vessel normalization and improved tumor microenvironment (TME) in tumor bearing mouse. In this study, we examined whether improvement of TME by PHDi elicit phenotypic alteration of tumor infiltrated immune cells, especially macrophage (Mf). Lewis lung carcinoma cells were transplanted subcutaneously. Mice were treated with PHDi intraperitoneally at day10 after tumor transplantation. Then tumor tissues were collected at day16 and analyzed immune cells by flowcytometry and immunofluorescence staining. we performed phagocytosis assay using sorted Mf from tumor tissue and bone derived Mf. Mf ratio in total leukocyte were significantly increased in PHDi treated tumor in both immunohistochemical and flowcytometric analysis. Lymphocyte ratio didn't change in PHDi treated tumor. Both in vivo and ex vivo experiments showed that phagocytosis ability of Mf increased about 1.5 folds in PHDi treated Mf. these Mfs may affect tumor progression.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is an important molecule for the progression of cytokinesis and accumulates the cleavage furrow during cytokinesis. Here, we investigated whether phospholipase C (PLC)-related catalytically inactive protein (PRIP), a metabolic modulator of PI(4,5)P₂, regulates PI(4,5)P₂-mediated cytokinesis. PRIP localized to the cleavage furrow during cytokinesis, and PRIP-knockdown HeLa cells displayed abnormal cytokinesis. Importantly, PI(4,5)P₂ accumulation at, and the localization of RhoA and phospho-myosin II regulatory light chain to, the cleavage furrow were reduced in the PRIP-knockdown cells. The overexpression of oculocerebrorenal syndrome of Lowe-1 (OCRL1), a phosphatidylinositol-5-phosphatase, in cells decreased PI(4,5)P₂ levels during early cytokinesis and showed cytokinesis abnormalities. However, these abnormal cytokinesis phenotypes were ameliorated by the co-expression of PRIP but not by a PI(4,5)P₂-unbound PRIP mutant. Collectively, PRIP is a component at the cleavage furrow to maintain PI(4,5)P₂ metabolism and regulates RhoA-dependent progression of cytokinesis.

O-GlcNAcylation is a post-translational modifications regulating dynamic intracellular signaling by two enzymes, OGT and OGA, which add and remove the modification, respectively. In many types of cancer cells, O-GlcNAcylation is elevated and contributes to the transformed phenotypes, but the molecular mechanisms is not fully understood. In this study, we examined O-GlcNAcylation-mediated cancer cell proliferation focusing on FOXM1 oncogenic transcription factor to regulate cell cycle. Elevated O-GlcNAcylation promoted cell proliferation in MKN45 gastric cancer cells, accompanying with increasing FOXM1 nuclear localization. FOXM1 was not O-GlcNAcylated, but was polyubiquitinated, which was reduced by elevated O-GlcNAcylation. We found some molecules involved in FOXM1 proteasomal degradation which are regulated by O-GlcNAcylation. One is GSK-3β Ser/Thr kinase mediating FOXM1 phosphorylation to induce the ubiquitination (Ub). Elevated O-GlcNAcylation reduced GSK-3β activity following increased FOXM1 protein. The other is FBXL2 ubiquitin E3 ligase mediating FOXM1 Ub. Elevated O-GlcNAcylation reduced FBXL2 protein via increased its Ub. These data suggest that O-GlcNAcylation-mediated FOXM1 stabilization could promote cancer progression.

Excessive phosphorylation of intracellular proteins is one of the causes of the development and malignant progression of cancer. Not a few molecular-targeted anticancer drugs have been developed to inhibit abnormal activation of limited number of kinases. Therefore, novel drugs development from this point of view is now facing difficulty. On the other hand, inovative drug discovery that targets "phosphatase activation", as a different angle of "kinase inhibition", has not been realized.Protein phosphatase 2A (PP2A) is an essential holoenzyme that is implicated as an important tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl-esterase (PME-1) catalyzes specifically the demethylation of PP2A catalytic subunit (PP2Ac). PME-1 also inhibits　PP2A activity by directly binding to its phosphatase active site; the role as PP2A inhibitory protein. We revealed that PP2A inhibitory function, but not methyl-esterase activity, is important for tumor-promoting function of PME-1. We also found that PME-1 inhibition and p53 activation synergistically exert anti-cancer effects on human lung cancer cell line A549.

Cancer is one of the most serious diseases all over the world, especially metastasis and drug resistance are leading causes of death. There is an urgent need to establish new strategies for drug discovery. Success in the drug discovery depends on the development of appropriate tumor models that correspond closely to native tumor situation. Matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis and are regulators of tumor milieu. The cancer stem cell model fits well with tumorigenesis, metastasis and drug resistance. We have shown that cancer cell aggregation led to hypoxic tumoroids with marked upregulation of reprogramming and stemness genes as increased cancer stem cell using a 3D culture system. In the present study, we established a novel MMP9 promoter-driven cell-based reporter system using a rapidly metastatic colon cancer cell in the 3D culture system that evaluates cancer stemness and invasiveness. We used a concept of drug repositioning-using known molecules for new indications. We selected several compounds with inhibition to both tumoroid formation and MMP9 promoter activity. One of the compounds inhibited primary tumor formation, invasion and metastasis.

We previously reported that inflammatory stimulation reduced the tumor suppressor gene Programmed cell death 4 (Pdcd4) protein expression level and enhanced the metastatic abilityof human colon cancer cells. In this study, we investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulation on the expression levels of Pdcd4 protein and migratory ability in mouse Colon-26 cancer cells and B16-BL6 melanoma cells.

The expression level of Pdcd4 protein in Colon-26 cells treated with TPA significantly decreased in a concentration-dependent manner. On the other hand, Pdcd4 protein in B16-BL6 cells treated with TPA significantly increased in a concentration-dependent manner. In addition, the migratory ability of Colon-26 cells treated with TPA were significantly increased, but the migration of B16-BL6 cells treated with TPA were significantly decreased. Furthermore, we measured the melanin content of the cells and tyrosinase activity as indices of activation of Protein Kinase C (PKC). The melanin content and tyrosinase activity were significantly decreased in B16-BL6 cells treated with TPA.

These results indicated that Pdcd4 might be a negative regulator in the migratory ability of Colon-26 and B16-BL6 cells. Although the activation of PKC promoted degradation of Pdcd4 in Colon-26 cells, it did not function in B16-BL6 cells.

Menthol has a cooling effect via TRPM8 activation resulting in a relaxation, an anti-inflammation and an analgesic by the inhalation and the topical application. Previously, we demonstrated that menthol induced cytotoxicity in different ways dependent on its concentrations in lung cancer cell line, A549. However, the detail mechanisms of the cytotoxic action are unclear. The expression of molecules related to cytotoxicity was detected by using western blotting. Flow cytometrical analyses were performed to detect apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential. The expressions of some cyclins and cyclin-dependent kinases were suppressed by menthol (1 mM). At 2 mM, menthol evoked apoptosis Ca²⁺-independently concomitant with an increase of intracellular levels of ROS. N-acetyl cysteine failed to inhibit the menthol-induced apoptosis. Menthol rapidly decreased the mitochondrial membrane potential. These results suggest that menthol induces ROS-independent cytotoxicity despite the ROS production. Dysfunction of mitochondria may be involved in apoptotic action of menthol in A549.

Ceramides are bioactive lipids that mediate cell death in cancer cells and ceramide-based therapy is now being tested in dose-escalating phase 1 clinical trials as a cancer treatment. The most representative ceramide formulation is ceramide nanoliposomes (CNL) that have been preclinically studied. However, the effect of CNL in ovarian cancer still remains an open question. We now investigate the therapeutic efficacy and signaling mechanisms of CNL in ovarian cancer. Treatment of ovarian cancer cells with CNL decreased cell viability in a dose-dependent manner. Importantly, CNL-treated cancer cells died with programmed necrosis (necroptosis), but not apoptosis. Mechanistically, dying SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like (MLKL) as evidenced by oligomerization in necroptosis. In addition, inhibition of MLKL, but not upstream RIP kinases, abolished CNL-induced cell death. In a cell-free system, ceramide was revealed to interact recombinant MLKL. Those results suggest CNL exhibited a cytotoxic effect by inducing MLKL-dependent necroptosis. In clinical studies, relapse-free survival was significantly extended in high MLKL mRNA expression group of patients with breast cancer, demonstrating correlation of MLKL expression with good prognosis. Taken together, our studies give insight into pharmacotherapeutic significance of necroptosis-inducing reagents in cancer treatment.

Malignant mesothelioma (MM) is a fatal tumor caused by past exposure to asbestos. The absence of highly specific markers for MM has served an obstacle for its diagnosis and therapy. We previously produced a monoclonal antibody (mAb) against MM, SKM9-2. We also discovered that SKM9-2 specifically binds to protein HEG homolog 1 (HEG1) that is a novel mucin-like membrane protein. This specificity of SKM9-2 to MM was due to the recognition of HEG1 glycopeptide containing a sialylated glycan. However, there was no mAb that bound to HEG1 glycosylation-independently. We produced new mAbs against HEG1 in this study. Purified partial HEG1expressed in mammalian cells was immunized in mice or rats. After antigen-immunized spleen cells were isolated from mice or rats,the cells were fused with myeloma cells by electrical cell fusion. Hybridomas were screened by ELISA, western blotting orflow cytometry.Through the screening of about 10,000 clones, we obtained more than 10 clones of anti-HEG1 mAb. These antibodies could bind to HEG1 glycosylation-independently. We also obtained two mAbs that can be used in immunohistochemistry. We will investigate the non-glycosylated HEG1expressionin normal tissues.

To improve the success rate of anti-cancer drug development, clinically relevant tumor models are needed. Patient-derived xenograft (PDX) tumor, which is generated by direct implantation of human tumor into immunodeficient mice, has been well-accepted as a more clinically relevant model than cell line-derived xenograft (CDX) tumor. 

We have been developing both in vivo and in vitro screening systems for anti-cancer drugs using PDX tumors that had been established in the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN). In this study, we confirmed anti-cancer drug efficacy using PDX in vivo and in vitro.

In the in vivo study, the PDX tumor was subcutaneously transplanted into immunodeficient mice, and a control group and drug group were set.  Gemcitabine or 5-FU was administered once or twice a week, and observation was carried out until 28 days after starting administration. Anti-cancer effect was evaluated by tumor volume.

In the in vitro study, three-dimensional culture was performed using dispersed cells derived from PDX tumor.  Drugs were added to the medium and the cell viability was confirmed by ATP assay.

In both studies, the effect of anti-cancer drug was confirmed, and an evaluation system in vivo and in vitro using PDX could be established.

OBJECTIVE: Cisplatin (CDDP) is known to frequently cause nausea and vomiting, renal injury as a side effect. There are many kinds of 5-HT3 receptor antagonists, which are one of the basic antiemetic drugs for nausea and vomiting by cancer chemotherapy, such as the first generation ondansetron, granisetron, the second generation palonosetron. It is suggested that ondansetron may be a risk factor for the onset of CDDP-induced acute kidney injury (AKI). Therefore, in this study, the effect of various 5-HT3 receptor antagonists on CDDP-induced renal injury was examined.

METHODS: C57BL/6 mice were intraperitoneally administered with CDDP. Renal function was evaluated by serum creatinine and blood urea nitrogen. Histological damage in the cortex of HE‐stained kidney sections was scored. Various 5-HT3 receptor antagonists were administered 30 minutes before administration of CDDP.

RESULTS: CDDP-induced renal injury got significantly worse by pre-administration of ondansetron, but not by pretreatment of palonosetron compared with cisplatin alone group.

CONCLUSIONS: These results suggest that the second generation 5-HT3 receptor antagonist may have less effect on CDDP‐induced AKI than the first generation.

Glial L-glutamate (L-Glu) transporter EAAT2 removes L-Glu at the synaptic cleft, thereby maintaining an efficient neurotransmission. Docosahexaenoic acid (DHA) is a major constituent of astrocyte membrane phospholipids and is released after L-Glu stimulation, however, the effects of DHA on L-Glu transporter have not been fully clarified. We studied the effects of DHA on EAAT2 electrophysiologically. Bath-applied DHA increased the amplitude of EAAT2 currents, but had no effects on EAAT1, another glial EAAT subtype. DHA has a negative charged carboxyl group that is deprotonated in a pH dependent manner. When the extracellular pH was decreased, the enhancement of EAAT2 by DHA was disappeared. No chargeable DHA analogue had no effects on EAAT2 currents, suggesting the negative charge is important. We identified which part of EAAT2 is important for the effects of DHA using EAAT1/2 chimeras. By substituting the transport domain of EAAT1 by that of EAAT2, the effects of DHA on EAAT1 were turned out to be enhancement, suggesting this region is important for the enhancement of EAAT2 current by DHA. Currently, we are identifying the essential region for the effect of DHA.

L-Theanine is an amino acid ingredient in green tea with a structural analogy to glutamine and is suggested to be taken up into the cells via an L-glutamine transporter Slc38a1. Recently, the oral intake of L-theanine is expected to suppress anxiety, sleep disturbance, and cognitive impairment. Some reports showed that L-theanine possesses anticancer activities against some cancers. Here, it was investigated whether L-theanine inhibits cell proliferation and its mechanism is via Slc38a1. L-Theanine inhibited cell proliferation in mouse motor neuron cell line (NSC-34), mouse neuroblastoma cell line (Neuro 2A) and human neuroblastoma cell line (SH-SY5Y) in a dose- and time-dependent manner. However, it had little effect in human brain glioblastoma cell line (U-251 MG), mouse astrocyte cell line (C8-D1A), mouse brain endothelial cell line (bEnd3) and human umbilical vein endothelial cells (HUVEC). There was a positive correlation between the L-theanine-dependent inhibition of cell proliferation and the expression level of Slc38a1 mRNA (r² = ～0.6). Therefore, it was suggested that in these cell lines, the suppressive effect on cell proliferation was caused by L-theanine which was taken up into the cells via Slc38a1.

BACKGROUND/AIM: JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a key role in essential amino acid uptake in cancer cells. While the co-incubation inhibitory effect of JPH203 has been characterized in conventional uptake assay, its pre-incubation inhibitory effects remain undetermined. Therefore, we aimed to characterize pre-incubation inhibitory effects of JPH203 on LAT1 function. METHODS: Pre-incubation effects were examined by leucine uptake assay using LAT1-positive human colon cancer HT-29 cells. RESULTS: In time-dependency analysis, pre-incubation of HT-29 cells with 10 uM JPH203 for 30, 60, and 120 min resulted in a significant decrease of the leucine uptake activity (42%, 32%, and 28% of those obtained from the control cells, respectively). Similarly, in concentration-dependency analysis, pre-incubation of the cells with JPH203 (1, 10, and 30 uM for 120 min) decreased the activity level to 68%, 25%, and 3% of those of the control cells, respectively. CONCLUSION: We have identified potent pre-incubation inhibitory effects of JPH203 on LAT1 function. Combination effects of pre- and co-incubation inhibitory effects are currently under examination.