Chiharu Doi

Suppression of facilitative glucose transporter 1 mRNA can suppress tumor growth

We attempted to suppress glucose transporter 1 (GLUT1) expression by transfecting MKN45 cells with cDNA for antisense GLUT1. Glucose transport was significantly decreased in cells with antisense GLUT1 compared with wild-type cells or cells with vector alone. Suppression of GLUT1 mRNA resulted in a decreased number of cells in the S phase. This was accompanied by overexpression of p21 protein. Tumorigenicity in the nude mice injected with antisense GLUT1 expressing cells was significantly slower than in those with wild-type MKN45 cells. These results suggest that antisense GLUT1 mRNA inhibits tumor growth through a G(1) arrest and that expression of antisense GLUT1 mRNA via gene therapy can be used as a tool in the treatment of cancer.

Insulin resistance in patients with cancer: relationships with tumor site, tumor stage, body-weight loss, acute-phase response, and energy expenditure.

We examined peripheral insulin sensitivity in 32 patients with cancer (17 with stomach cancer, 7 with colorectal cancer, and 8 with lung cancer) and 6 normal control subjects by the euglycemic hyperinsulinemic glucose clamp technique. The relationships between insulin resistance and tumor factors (type and stage), malnutrition, and inflammatory reaction were evaluated. Insulin sensitivity often was reduced in patients with cancer; however, the amount of glucose metabolized was not related to tumor site or stage. The decreased glucose uptake was negatively correlated with the acute-phase response but was not correlated with body-weight loss, serum albumin, or resting energy expenditure. Our results suggest that insulin resistance in cancer patients was not induced by malnutrition. Although the qualitative presence of tumor might be the major factor inducing insulin resistance, other factors such as inflammatory reactions might be involved in the development of insulin resistance.

Decreased Tissue Inhibitor of Metalloproteinase-2/Matrix Metalloproteinase Ratio in the Acute Phase of Aortic Dissection

PurposeAortic dissection is characterized by fragility of the tunica media, and matrix metalloproteinases (MMPs) are enzymes that degrade the extracellular matrix of the aorta. This study examines MMPs in patients with acute aortic dissection (AAD) in an attempt to elucidate the mechanisms of their actions.MethodsEnzyme-linked immunosorbent assays were used to measure the quantification of MMP-2, MMP-9, and the tissue inhibitor of metalloproteinase (TIMP)-2 in 30 patients with AAD, 12 patients with abdominal aortic aneurysm (AAA), and 16 control (CON) patients who underwent coronary artery bypass grafting.ResultsMMP-2 and TIMP-2 were significantly lower in the AAD group than in the CON group, at 36 ± 19 vs 58 ± 30 (P ≪ 0.01) and at 21 ± 25 vs 216 ± 130 (P ≪ 0.001), respectively. The TIMP-2/MMP-2 ratio was 3.7 ± 1.7 in the CON group and 0.9 ± 0.8 in the AAD group (P ≪ 0.001 vs CON), and the TIMP-2/MMP-9 ratio was 200 ± 170 in the CON group and 37 ± 80 in the AAD group (P ≪ 0.001 vs CON).ConclusionLow TIMP-2/MMP-2 and TIMP-2/MMP-9 ratios might play an important role in the onset of aortic dissection, when the tunica media becomes fragile with chronic breakage and degradation of the extracellular matrix.

Expression of nitric oxide synthase in gastric cancer

The expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in samples of normal gastric mucosa and gastric cancer were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-quantitative Western blot. In normal gastric mucosa, eNOS protein was found in all samples examined (mean, 70.2 +/- 60.1), relative to a standard protein. In gastric cancer specimens, eNOS protein was also detected in all samples, but the quantity (86.5 +/- 76.6) was not different from that found in samples of normal mucosa. The quantity of eNOS in gastric cancer tissues was negatively correlated with serosal invasion. iNbS mRNA, detected in nine of 18 cases, was slightly related to massive lymph node metastasis (n1-3 vs. n4). Neither tumor necrosis factor alpha (TNF-alpha) mRNA nor interleukin-6 (IL-6) mRNA was related to the expression of iNOS mRNA. These results suggest that iNOS not eNOS plays a role in gastric cancer tumor extension, but iNOS mRNA appears not to be induced by either TNF-alpha or IL-6.

Expression of facilitative glucose transporter 1 mRNA in colon cancer was not regulated by k-ras

The expression of facilitative glucose transporter isoforms in colon adenocarcinoma and the possible role of k-ras in inducing GLUT (glucose transporter) mRNA were studied. RT-PCR demonstrated GLUT2 and GLUT3 expression in 100% of the ten normal colon mucosa samples but detected no GLUT1 mRNA. By contrast, GLUT1 mRNA was detected in all 20 (100%) colon cancer samples examined. GLUT4 mRNA was not detected in either normal mucosa or colon cancer tissues. Semiquantitative PCR demonstrated equal amounts of GLUT2 and GLUT3 mRNA in both normal mucosa and colon cancer samples. A point mutation in codon 12 of k-ras was detected in only six of the 20 (30%) colon cancer samples. Thus, a major difference between normal colon epithelia and colon cancer was the acquisition of GLUT1 expression, which was unlikely to have been induced by a point mutation in codon 12 of k-ras.

High incidence of synchronous cancer of the oral cavity and the upper gastrointestinal tract

A high incidence of synchronous esophageal or gastric carcinoma in preoperative patients with carcinoma of the oral cavity was reported. Esophageal carcinoma was found in seven out of 56 patients (12.5%) and gastric cancer in five patients (8.9%) by videoendoscopy aided with lugol staining in the esophagus and indigocarmine solution in the stomach, although all patients were completely asymptomatic for these lesions. All patients were male, regular drinkers and heavy smokers. The depth of invasion of such tumors was limited to either mucosa or submucosa. Those esophageal and gastric lesions beside the primary oral cancers were positive for p53 protein by immunohistochemistry. Careful preoperative evaluation of not only the esophagus but also the stomach should be a routine procedure in patients with carcinoma of the oral cavity.

Glucose uptake in the human gastric cancer cell line, MKN28, is increased by insulin stimulation

The expression of the insulin-responsive glucose transporter (GLUT) 4 was studied in three histologically different human gastric cancer cell lines, MKN28, MKN45, and STSA. RT-PCR demonstrated GLUT1 and GLUT4 mRNA in all three cell lines. MKN28 cells expressed GLUT4 protein more than MKN45 and STSA cells by immunohistochemistry. Insulin stimulation of MKN28 cells resulted in a 22% increase in glucose uptake over that found under basal conditions (0.60 +/- 0.05 fmol/cell per min after insulin stimulation versus 0.53 +/- 0.07 fmol/cell per 3 min at basal). No increase in glucose uptake occurred with insulin stimulation in MKN45 or STSA cells. We conclude that the insulin responsive GLUT4 is expressed in MKN28, MKN45, and STKM1 human gastric cancer cell lines, albeit in different amounts. The greater expression of this transporter in MKN28 cells is likely responsible for the cells ability to increase glucose uptake with insulin stimulation. However, the role played by GLUT4 in regulating the amount of glucose uptake would not be large in those human gastric cancer cell lines.

Insulin resistance was connected with the alterations of substrate utilization in patients with cancer

To elucidate the contribution of insulin resistance to substrate utilization, insulin sensitivity and substrate oxidation were examined in 19 cancer patients and five normal controls using the euglycemic hyperinsulinemic glucose clamp technique combined with indirect calorimetry. Glucose uptake and storage were significantly decreased in cancer patients compared with that of controls. In cancer patients, both glucose storage and oxidation were positively correlated with metabolized glucose as measured by the M value in cancer patients. Decrease in glucose uptake was mainly a reflection of decreased glucose storage rather than glucose oxidation. Inversely fat oxidation was negatively correlated with both the M value and glucose oxidation in cancer patients. In cancer patients with insulin resistance, decreased glucose uptake was closely associated with a rapid decrease in glucose storage, an increase in fat oxidation and a mild decrease in glucose oxidation, suggesting that insulin resistance was connected with the alterations of substrate utilization which may induce host depletion.

Insulin Resistance and the Alterations of Glucose Transporter-4 in Adipose Cells From Cachectic Tumor-Bearing Rats

BACKGROUND Insulin resistance may play an important role in cancer cachexia; however, its mechanisms remain to be clarified. METHODS Cellular mechanisms of insulin resistance in tumor-bearing rats (TBR) were investigated in isolated adipose cells by measuring 3-O-[14C]methyl glucose transport activity and glucose transporter-4 (GLUT4) protein in low-density microsomes at a basal state and in the plasma membrane at an insulin-stimulated state. RESULTS The insulin-stimulated glucose transport activity in adipose cells from TBR was significantly lower than that of control rats (CTR) (0.51 +/- 0.25 and 2.27 +/- 0.11 fmol/cell/min, respectively). The amount of GLUT4 in low-density microsomes at a basal state and in plasma membrane at an insulin-stimulated state was less in TBR than in CTR. CONCLUSIONS These data suggest that the insulin resistance seen in the adipose cells of these tumor-bearing rats was caused in part by both a decreased amount of GLUT4 protein in a basal state and a decreased translocation of GLUT4 in response to insulin stimulation.

Alteration in immunoexpression of glucose transporter 2 in liver of tumour-bearing rats

To elucidate interactions between the glucose transport system and hepatic glucose production in the tumour‐bearing state, glycogen storage, expression of glucose transporter isoform 2 (Glut 2) and activities of glucose‐6‐phosphatase (G‐6‐Pase) and hexokinase were histochemically examined in hepatocytes of tumour‐bearing rats. Five male F344 rats, subcutaneously inoculated with methylcholanthrene(MCA)‐induced sarcoma cells were compared with five pair‐fed animals and four ad libitum fed controls. Glycogen storage was markedly decreased in liver cells of tumour‐bearing rats compared to in those of control animals. Glut 2 immunoreactivity was uniformly seen in the cellular membrane of hepatocytes from control animals. In rats bearing sarcoma, the staining intensity was significantly decreased, suggesting that Glut 2 with its bi‐directional transport capacity was down‐regulated in the tumour‐bearing state. Positive staining for hexokinase activity was located in the perivenous area in livers from control animals and was more diffusely located and more intense in livers from tumour‐bearing animals. G‐6‐Pase activity, limited to the peripheral area in livers from controls, extended to the intermediate area and had stronger reactivity in livers from tumour‐bearing animals. In the tumour‐bearing cachectic condition, glucose may be partially consumed by a futile cycle, hepatic metabolic zonation was disturbed, and the release of glucose from the liver may not be mediated by a facilitative glucose transporter‐2.