Yoshito Nakamura

Clinical Significance of High Mobility Group A2 in Human Gastric Cancer and Its Relationship to let-7 MicroRNA Family

Purpose: The high mobility group A2 (HMGA2) nonhistone chromosomal protein can modulate transcription by altering chromatin architecture. HMGA2 is highly expressed during embryogenesis and in various benign and malignant tumors. Recent studies report that HMGA2 is negatively regulated by the let-7 microRNA (miRNA) family. However, no studies have examined the clinical significance of HMGA2 and its relationship to the let-7 miRNA family in gastric cancer. Experimental Design: Using quantitative real-time reverse transcription–PCR, we analyzed HMGA2 expression with respect to various clinicopathologic factors in 110 patients with gastric cancer. We also did an association study comparing HMGA2 expression and let-7 miRNA family expression in gastric cancer. Results: Expression of HMGA2 in cancerous tissues was significantly higher than in noncancerous tissues (P < 0.05). Elevated HMGA2 expression was significantly correlated with serosal invasion (P < 0.05) and poor clinical prognosis (P < 0.05). A multivariate analysis showed that HMGA2 expression status was an independent prognostic factor (P < 0.05). An inverse correlation between HMGA2 and let-7a was found in gastric cancer cell lines (P = 0.08). The expressions of let-7a, let-7b, and let-7c in gastric cancer patients with low HMGA2 expression were significantly higher than those with high HMGA2 expression (P < 0.05). Conclusions: High expression of HMGA2 in gastric cancer correlates with tumor invasiveness and is an independent prognostic factor. Furthermore, our findings suggest that HMGA2 is negatively regulated by the let-7 miRNA family in human gastric cancer.

Inhibitory action of insulin-sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea-pig

The actions of troglitazone, pioglitazone, metformin and bezafibrate, agents that improve insulin‐resistance, on voltage‐dependent Ca2+ channels in arterial smooth muscle cells were examined by use of the conventional and nystatin‐perforated whole‐cell clamp methods. Single cells were freshly isolated from resistance mesenteric arteries of guinea‐pigs. The actions of these agents on 77 mM K+‐induced contraction of the isolated arteries were also examined with the use of isometric tension recording. The thiazolidinedione derivatives, troglitazone and pioglitazone, inhibited whole‐cell Ca2+ currents in a dose‐dependent manner with dissociation constants of 3.0 μM and 44.9 μM and Hill coefficients of 0.61 and 0.68, respectively. These two agents inhibited the 77 mM K+‐induced contraction with similar potencies as those inhibiting the Ca2+ currents. Metformin and bezafibrate had no apparent effects on the Ca2+ current or high K+‐induced contraction. The inhibitory action of troglitazone on Ca2+ currents was not affected by the command potential, the holding potential, or the stimulation frequency, suggesting that its mode of the action differs from that of known organic Ca2+ channel antagonists. The inhibitory action of troglitazone on Ca2+ currents was not affected by the addition of insulin to, or the removal of glucose from, the solutions. In conclusion, the thiazolidinedione derivatives directly inhibited the voltage‐dependent Ca2+ channels in a different manner from that of organic Ca2+ channel antagonists. This inhibitory action on Ca2+ channels was not a common feature of insulin‐sensitizing agents.

Troglitazone, an insulin sensitizer, increases forearm blood flow in humans

To test whether troglitazone, a thiazolidinedione insulin sensitizer, increases the peripheral blood flow, the changes in forearm blood flow (FBF) were evaluated by venous occlusion plethysmography in 11 lean healthy male volunteers (age range, 24 to 39 years) after a single oral dose of 200 mg of troglitazone. Forearm vascular resistance (FVR) was calculated from FBF and blood pressure. Two hours after the dose, FBF increased from 3.66+/-0.31 to 4.81+/-0.57 mL/100 mL/min (P < .01), and FVR decreased from 24.7+/-2.2 to 20.2+/-2.2 units (P < .01), whereas both these values did not change during the control recordings obtained without troglitazone. Blood pressure, blood glucose levels, and serum immunoreactive insulin levels did not change significantly during the observation period. Serum concentrations of nitrate ions decreased from 27.0+/-3.5 mmol/L to 23.1+/-2.7 mmol/L (P < .01) after the administration. These results suggest that troglitazone increases muscular blood flow through vasodilation induced by a mechanism other than the correction of hyperinsulinemia or the increase in nitric oxide. The present study provides the first evidence that troglitazone dilates the vasculature in humans.

Identification of overexpressed genes in hepatocellular carcinoma, with special reference to ubiquitin-conjugating enzyme E2C gene expression

This study consisted of 2 aims: (i) to determine genes associated with hepatocellular carcinoma (HCC) by microarray analysis; and (ii) to evaluate the clinicopathological significance of human ubiquitin‐conjugating enzyme E2C (Ube2c) found to be overexpressed in HCC from microarray analysis. Laser microdissection and cDNA‐microarray were performed to identify genes associated with HCC. We then focused on the Ube2c gene. Using real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), Ube2c expression status and clinicopathological significance were studied in 65 clinical HCC samples. A number of genes upregulated in HCC cells compared to noncancerous liver cells were identified, one of which was the Ube2c gene. Ube2c gene expression in the cancer tissue was higher than in the corresponding noncancerous tissue in 62 of the 65 cases (95.4%, p < 0.01). Tumors with high Ube2c expression showed higher frequencies of tumor invasion to capsular formation (fc‐inf), invasion to portal vein (vp) and tumor de‐differentiation (p < 0.05). Patients with high Ube2c expression also showed significantly worse disease‐free survival rates than those with low Ube2c expression (p < 0.01). In addition, Ube2c expression was found to be an independent prognostic factor for disease‐free survival rate in multivariate analysis. We identified differentially expressed genes between HCC and normal liver tissues. Of those, the Ube2c gene appeared to be associated with HCC progression, and may be useful as a prognostic indicator for HCC patients.

Clinicopathological and biological significance of mitotic centromere-associated kinesin overexpression in human gastric cancer

Mitotic centromere-associated kinesin (MCAK) is a microtubule (MT) depolymerase necessary for ensuring proper kinetochore MT attachment during spindle formation. To determine MCAK expression status and its clinicopathological significance, real-time reverse transcriptase–polymerase chain reaction was used in 65 cases of gastric cancer. MCAK gene expression in cancer tissue was significantly higher than expression in non-malignant tissue (P<0.05). Elevated MCAK expression was significantly associated with lymphatic invasion (P=0.01) and lymph node metastasis (P=0.04). Furthermore, patients with high MCAK expression had a significantly poorer survival rate than those with low MCAK expression (P=0.008). Immunohistochemical study revealed that expression of MCAK was primarily observed in cancer cells. Additionally, a gastric cancer cell line (AZ521) that stably expressed MCAK was established and used to investigate the biological effects of the MCAK gene. In vitro results showed that cells transfected with MCAK had a high rate of proliferation (P<0.001) and increased migratory ability (P<0.001) compared to mock-transfected cells. This study demonstrated that elevated expression of MCAK may be associated with lymphatic invasion, lymph node metastasis, and poor prognosis. These characteristics may be due in part to the increased proliferative and migratory ability of cells expressing MCAK.

Stretch-Activated Channels in Arterial Smooth Muscle of Genetic Hypertensive Rats

Electrical and contractile responses of small arteries to mechanical stress are reportedly enhanced in spontaneously hypertensive rats (SHR), compared with those in Wistar Kyoto rats (WKY). We have previously shown that stretch-activated cation channels exist in arterial smooth muscle membrane, of which opening causes Na+ and Ca2+ influx and membrane depolarization. We thus hypothesize that activation of stretch-activated channels is enhanced in arterial smooth muscle of SHR compared with WKY. To test this hypothesis, stretch-activated currents were recorded in single smooth muscle cells of resistance mesenteric arteries from SHR and WKY (16 to 24 weeks of age). In the whole-cell recording, membrane stretch was applied by inflating the cell with positive pressure to the recording pipette. Cell-inflation evoked Gd3+-sensitive cation currents. This current appeared with less stretch stimulation and its amplitude was larger in SHR cells compared with WKY cells. In the cell-attached recording, suction to the recording pipette evoked single stretch-activated channel currents (conductance of 32 pS with 150 mmol/L Na+), which were blocked by Gd3+. Channels were activated with less negative pressure and their availability was greater in SHR cells than in WKY cells. Results suggest that the activation of stretch-activated channels is enhanced in smooth muscle of resistance arteries from SHR compared with WKY, which may contribute to the enhanced vascular responses to mechanical stress in SHR.

Comparative actions of insulin sensitizers on ion channels in vascular smooth muscle.

Thiazolidinedione and isoxazolidinedione insulin sensitizers activate peroxisome proliferator-activated receptor gamma (PPAR gamma). Some thiazolidinediones modify ion channels in smooth muscles; however, the mechanism by which their actions occur has not been clarified. We, thus, examined the effects of three thiazolidinediones (troglitazone, pioglitazone, and rosiglitazone) and isoxazolidinedione (JTT-501), as well as an intrinsic ligand for PPAR gamma, 15-deoxy-Delta(12,14) prostaglandin J(2) (prostaglandin J(2)), on voltage-operated Ca(2+) currents (I(Ca)), voltage-dependent K(+) currents (I(Kv)), and Ca(2+)-activated K(+) currents (I(Kca)), to clarify whether a thiazolidinedione structure or PPAR gamma activation is related to their actions on ion channels. The whole-cell patch clamp method was used to record currents in smooth muscle cells from guinea-pig mesenteric arteries. Thiazolidinediones inhibited I(Ca) in a dose-dependent manner (troglitazone>pioglitazone=rosiglitazone). Troglitazone (> or =1 microM) and rosiglitazone (100 microM), but not pioglitazone, inhibited I(Kv). Rosiglitazone (> or =10 microM) enhanced, troglitazone (> or =1 microM) inhibited, and pioglitazone did not affect I(Kca). A high concentration of JTT-501 (100 microM) inhibited I(Ca), I(Kv), and I(Kca) to a similar extent. Prostaglandin J(2) enhanced I(Kca), but affected neither I(Ca) nor I(Kv). In summary, the three thiazolidinediones and isoxazolidinedione act differently on Ca(2+) and K(+) channels in vascular smooth muscle. The action of thiazolidinediones on I(Ca) could be attributed to specific regions of the molecules and not to activation of PPAR gamma. Involvement of PPAR gamma activation in the stimulation of I(Kca) is possible but should be tested further.

Sodium‐potassium pump current in smooth muscle cells from mesenteric resistance arteries of the guinea‐pig

1 The Na+‐K+ pump current was studied in smooth muscle cells from mesenteric resistance arteries of guinea‐pigs by the use of the perforated patch‐clamp technique in the presence of blockers for various ion channels and exchangers. 2 When the Na+ concentration in the pipette solution ([Na+]i) was 50 mM, an increase in the extracellular K+ concentration ([K+]o) from 0 to 10 mM caused an outward current. Both the removal of K+ from the bath solution and the application of 10 μM ouabain abolished this current. Thus, this K+‐induced and ouabain‐sensitive current was considered to be the Na+‐K+ pump current. 3 The amplitude of the Na+‐K+ pump current increased as the membrane potential was made more positive until around 0 mV, while the amplitude saturated at more positive potentials than 0 mV. 4 An increase in [K+]o or [Na+]i amplified the Na+‐K+ pump current. For [K+]o, the binding constant (Kd) was 1.6 ± 0.3 mM and the Hill coefficient (nH) was 1.1 ± 0.2 (n= 6). For [Na+]i, Kd was 22 ± 5 mM and nH was 1.7 ± 0.5 (n= 4–19). 5 The presence of various monovalent cations other than Na+ in the bath solution also evoked the Na+‐K+ pump current. The order of potency was K+≥ Rb+ > Cs+≫ Li+. 6 Ouabain inhibited the Na+‐K+ pump current in a dose‐dependent manner with a Kd of 0.35 ± 0.03 μM and an nH of 1.2 ± 0.1 (n= 6–8). 7 The Na+‐K+ pump current increased as temperature increased. The temperature coefficient (Q10; 26–36 °C) was 1.87 (n= 9). 8 In summary the present study characterized for the first time the Na+‐K+ pump current in vascular smooth muscle cells by the use of the voltage‐clamp method. The use of this method should provide essential information for Na+,K+‐ATPase‐mediated changes in the cell functions of vascular smooth muscle cells.

Opa Interacting Protein 5 (OIP5) Is a Novel Cancer-testis Specific Gene in Gastric Cancer

BackgroundIdentification of novel cancer-specific antigens is important for the advancement of immunotherapy. Our aim was to identify cancer-specific genes in gastric cancer.MethodsUsing cDNA microarray analysis, we detected genes overexpressed specifically in gastric cancer cells. The expression levels of selected genes, including OIP5, was confirmed by real time RT-PCR analysis in tumor/normal paired bulk samples of 58 clinical cases. The expression levels of selected genes in normal tissues were also determined with a human total RNA master panel. We also compared the expression status of OIP5 with that of the other known cancer-testis specific genes.ResultsTwenty-two genes were determined to be upregulated in gastric cancer cells. Among these, three genes (CDC6, Exo1, and OIP5) were selected and confirmed to be upregulated in the tumor tissue compared to normal tissue. A human total RNA master panel demonstrated that OIP5, but not Exo1 or CDC6, showed high specificity in testis. Thus OIP5 may be considered a cancer-testis specific gene. In 58 clinical cases of gastric cancer examined, we found OIP5 gene expression in 27 cases (47%). Thirteen of these 27 cases showed no expression of the known cancer specific genes such as MAGE-1, MAGE-3 or NY-ESO-1.ConclusionsUsing a combination of LMD and microarray, we identified OIP5 as a cancer-testis specific gene. Further expression analysis in a set of clinical cases revealed that OIP5 may be a novel immunotherapy target for patients with gastric cancer.

Circumferential spontaneous coronary artery dissection in an elderly man : A case report

An 82-year-old man developed acute inferior myocardial infarction. Emergent coronary angiography demonstrated thrombotic occlusion in the right coronary artery. Intracoronary thrombolytic therapy was performed with successful recanalization. However, circumferential dissection with luminal stenosis was revealed at the point of thrombus formation. Repeat angiography 6 months later showed resolution of the dissec tion. This is the first description of circumferential spontaneous coronary artery dissec tion. In addition, the present patient is older than any previous patients.