Effect of cyclooxygenase and nitric oxide synthase inhibitors on tumor growth in mouse tumor models with and without cancer cachexia related to prostanoids.

The potential interaction between cyclooxygenase (Cox) and NO metabolic pathways in the control of local tumor growth was evaluated. Mice bearing either a sarcoma-derived tumor (C57B1; MCG 101) or a malignant melanoma (C3H/HeN; K1735-M2) were used. These models were principally different because they demonstrate, in tumor hosts, conditions with and without cancer cachexia, seemingly related to high and low production of prostanoids, respectively. Cox inhibitors (Cox-1 and Cox-2) decreased tumor growth by 35-40% in MCG 101-bearing mice but had no such effect on melanoma-bearing mice, despite the expression of the Cox-2 protein in melanoma cells. Indomethacin reduced prostanoid production in both tumor (MCG 101) and host tissues and reduced tumor cell proliferation, mainly in vivo. Nitric oxide synthase (NOS) inhibitors (N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine) reduced tumor growth in vivo by approximately 50% in both tumor models. Tumor growth reduction, related to NOS inhibition, was unrelated to prostanoid production and was an in vivo phenomenon in both tumor models. Specific inhibitors of inducible NOS activity, unexpectedly, had no effect in any tumor model, although inducible NOS protein was present in tumor tissues in large amounts. A combination of Cox and NOS inhibitors had no additive effect on tumor growth (MCG 101). Cox inhibition increased tumor tissue (MCG 101) expression of cNOS mRNA but had no significant effect on tumor tissue expression of the transferrin receptor, vascular endothelial growth factor, or basic fibroblast growth factor. NOS inhibition increased tumor tissue content of cNOS mRNA but showed as well a trend to increase mRNA content of the transferrin receptor and vascular endothelial growth factor. Our results suggest that NOS inhibitors can decrease the local growth of tumors that are either responsive or unresponsive to Cox inhibition. This effect may reflect cross-talk between Cox and NOS pathways within or among tumor cells, or it may represent unrelated effects on tumor and host cells. Whether NO inhibition may be used therapeutically in clinical tumors that are unresponsive to eicosanoid intervention remains to be evaluated.

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.

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.

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.

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.

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 cell's 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.

Modification of swine serum-induced bile duct lesion in BALB/c mice by cyclophosphamide.

Effects of cyclophosphamide (CY) on the antibody titer level and incidence and severity of swine serum (SS)-induced bile duct lesion (BDL) were examined. BDL induced by 0.2 ml of SS per head twice a week for 2 weeks was characterized by hyperplasia of biliary epithelial cells, proliferation of mucous glands, and periductal infiltration of eosinophils with mild fibrosis. CY showed no significant influence on the above-mentioned parameters at the dose levels of 140 and 210 mg/kg. On the other hand, CY lowered the antibody titer level and decreased the severity of BDL at the dose level of 280 mg/kg, and it suppressed the antibody response and BDL at the dose level of 280 x 2 mg/kg. Thus the antibody titer level and the severity of BDL were closely related each other.

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.

Nicotine improves cognitive disturbance in senescence-accelerated mice.

Senescence-accelerated mice (SAM), a murine model of age-related deterioration in learning ability, were studied as to the acetylcholine (ACh) contents in the brain tissues and the effect of nicotine administration. We found that the ACh content of SAM-P/8 (accelerated senescence-prone) mice was lower than that of SAM-R/1 (accelerated senescence-resistant) mice in the midbrain thalamus and the hypothalamus. In addition, an IP administration of nicotine was found to improve learning ability of SAM-P/8 as shown by performance of a passive avoidance task. Nicotine may potentiate cognitive function in SAM-P/8.

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.

Gastrectomized patients are in a state of chronic protein malnutrition analyses of 23 amino acids.

BACKGROUND/AIMS: Malnutrition is one of the major postoperative complications of radical subtotal or total gastrectomy for gastric cancer. This study was conducted to clarify the nutritional consequences of radical gastrectomy with respect to protein metabolism. METHODOLOGY: To evaluate the nutritional status and the abnormalities in protein metabolism in such cases, serum concentrations of 23 amino acids were measured by high performance liquid chromatography in 40 patients who had undergone either subtotal (n = 20) or total (n = 20) gastrectomy more than 6 months prior to this analysis. RESULTS: Serum concentrations of total amino acids and nonessential amino acids were the same between gastrectomized patients and healthy controls (n = 50). However, concentrations of essential amino acids, essential amino acid/nonessential amino acid and branched-chain amino acid/total amino acid ratios were significantly lower in patient groups than in normal controls. Each essential amino acid was decreased and concentrations of glutamate and citrulline were increased in both patient groups compared with controls. The major differences between patients with subtotal and total gastrectomies included an increased ornithine and a decreased arginine concentration in patients with subtotal gastrectomy. CONCLUSIONS: These changes suggest that malabsorption of protein from the intestinal tract causes persistent proteolysis in the skeletal muscle for long periods of time after surgery in these patients and that changes in ornithine and citrulline levels may reflect more severe alterations in those with total gastrectomy.

A case of primary adenosquamous/squamous cell carcinoma of gallbladder directly invaded duodenum.

A rare case of primary gallbladder carcinoma is reported. A 67 year-old woman was admitted to our hospital for treatment of suspected duodenal carcinoma. A series of radiographic examinations demonstrated a giant tumor involving the duodenum, gallbladder, pancreatic head, and transverse colon. These extensions made it difficult to identify the primary origin of the carcinoma. Pancreatoduodenectomy, cholecystectomy, and resection of the transverse colon were performed. Macroscopically, ulcerative lesions were seen in both the gallbladder and the duodenum. Microscopic examination revealed adenosquamous cell carcinoma of the gallbladder, invasive of the adjacent organs, including circumferential invasion of the second portion of the duodenum. The patient tolerated the operation well and was discharged 28 days post-operatively, but died of liver metastasis 4 months after surgery. Local invasion of the surrounding tissues is characteristic of adenosquamous/squamous cell carcinoma of the gallbladder. Although surgery for cure is deemed possible, the rapid growth rate of this type of tumor may cast doubt on the value of extensive radical surgery.

Expression of facilitative glucose transporters in gastric tumors.

BACKGROUND/AIMS: Although cancer cells are known to have an increased rate of glucose metabolism, the complete mechanism for increased glucose uptake in tumor cells is unknown. METHODOLOGY: The presence of mRNA for 5 facilitative glucose transporter (GLUT) isoforms was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in paired samples of normal gastric mucosa and gastric tumor from 20 individuals. Expression of GLUT proteins was immunohistochemically determined in 70 resected gastric cancer specimens. RESULTS: By using RT-PCR, GLUT2 mRNA was detected in 80% of normal gastric mucosal samples, while GLUT4 mRNA was seen in only 40%, GLUT1 mRNA was not detected in normal gastric mucosa. In gastric carcinoma samples, GLUT1 mRNA was detected in 19 out of 20 cases (95%) and GLUT2, GLUT3, and GLUT4 mRNAs in all samples. By immunohistochemistry, GLUT1 protein was detected in 19% of the tumors. A majority of tumors (61%) expressed 1 or more transporter protein. The presence of GLUT1 protein in a tumor was positively correlated with the tumor's invasion into the gastric wall, lymphatics or blood vessels and with lymph node metastases. The post-operative survival of patients with tumor expressing GLUT1 protein was significantly worse than those with tumor without GLUT1 protein. CONCLUSIONS: Gastric cancer cells may acquire the ability to produce GLUT1 mRNA by malignant transformation. Increased expression of the high-affinity glucose transporters, GLUT1 and/or GLUT4, in tumor cells may drain glucose preferentially to the tumor at the expense of the tumor-bearing host.

Lectin histochemistry of dorsal skin of Wistar-derived hypotrichotic WBN/Ila-Ht rats.

A lectin histochemical study was carried out on the dorsal skin of Wistar-derived hypotrichotic WBN/Ila-Ht rats (HtRs) and Wistar rats (WRs) at 3, 7 and 24 weeks of age to clarify the lectinhistochemical characteristics of the skin during their development. The lectins examined were Concanavalia ensiformis (Con A), Dolichos biflorus agglutinin (DBA), Griffonia simpliciolia (GS-I), Helix pomatia agglutinin (HPA), Arachis hypogaea (PNA), Glycine maximus agglutinin (SBA), Ulex europeus agglutinin (UEA-I) and Triticum vulgaris agglutinin (WGA). None of the nucleated cell layers of the epidermis had DBA-binding sites, but they were all stained intensely with HPA and weakly with Con A irrespective of the strain and age of the rats. As to the other 5 lectins, the intensity of binding activity was generally weaker in HtRs than in WRs and at 3 weeks of age than at 7 or 24 weeks of age, respectively. Among them, UEA-I mainly bound to the spinous cell layer but not to the basal cell layer, suggesting that alpha-L-fucose would be expressed on the cell surface according to the differentiation of keratinocytes. In addition, GS-I, HPA and UEA-I bound to the hair follicle epithelium and many lectins stained sebaceous gland epithelial cells. In conclusion, except for the binding intensity of some lectins, there were no specific differences between HtRs and Wrs in the lectinhistochemical characteristics of the dorsal skin epidermis. The present data on the rat skin would be useful from the viewpoint of comparative lectinhistochemistry.

Glomerular lesions in unilateral nephrectomized and diabetic (UN-D) mice.

Experimental diabetes was induced in both control and unilaterally nephrectomized male mice by injecting streptozotocin (SZ) (50 mg/kg x 5 days) one week after nephrectomy. The time course changes in the glomerular lesions were examined for up to 12 weeks after completion of the SZ-injection (12WAI). In unilateral nephrectomized and diabetic mice, mild segmental expansion of the mesangial area developed at 4WAI, and it progressed to prominent segmental glomerulosclerosis at 12WAI. In the electron microscopic examination at 12WAI, marked expansion of the mesangial area, segmental thickening of the glomerular basement membrane, fusion of the foot processes of podocytes and a prominent increase in the number of microvilli of capillary endothelial cells were observed. On the other hand, mild to moderate expansion of the glomerular mesangial area was only sporadically found in unnephrectomized diabetic mice at 12WAI. Interestingly, Bowman's capsules of diabetic mice were generally lined with flattened epithelia but those of non-diabetic mice with cuboidal or low columnar epithelia.

Histopathology of streptozotocin-induced diabetic DBA/2N and CD-1 mice.

Histopathological examinations were carried out on female DBA/2N and CD-1 mice which were autopsied 4 and 12 weeks after six daily intraperitoneal injections of streptozotocin (SZ). Histopathological changes related to SZ treatment were found in the pancreas, liver and kidneys. Little difference was observed between the two strains in the histological changes of the pancreas (a decrease in size of the islets, and degranulation and a decrease in the number of B cells) and liver (hypertrophy of hepatocytes and cytoplasmic invagination into hepatocyte nuclei). With regard to the changes in the kidneys, DBA/2N mice showed characteristic inclusions positive to periodic acid-Schiff reagent in the distal tubule epithelial cells, while CD-1 mice showed remarkable luminal dilatation and epithelial cell deformation of distal tubules. SZ-induced diabetes had no influence on the development of spontaneous cardiovascular lesions in DBA/2N mice under the present experimental conditions.

Transmissibility of the D variant of encephalomyocarditis virus (EMC-D) in mice.

Transmissibility of the D variant of encephalomyocarditis virus (EMC-D) was examined. Eight-week-old ICR:CD-1 male mice inoculated with 10(5) plaque forming units (PFU)/animal of EMC-D intranasally, orally or intraperitoneally showed marked viraemia and prominent pancreatic lesions at 2 days after inoculation (2 DAI), and excreted virus in faeces from 2 to 8 DAI (virus titre: 10(3)-10(5) PFU/g). Only a small proportion of control mice housed with EMC-D-inoculated mice for 10 days developed viraemia and pancreatic lesions.

Age-related non-neoplastic lesions in the heart and kidneys of Syrian hamsters of the APA strain.

Spontaneous cardiac and renal lesions in APA hamsters were examined histopathologically. Myocardial degeneration, valvular thickening, coronary arterial degeneration and increase in heart weight were common in old hamsters. These changes, which suggest cardiac failure, seem to be related to cardiac thrombosis which predominantly affected the left atrium and was found in over 40% of each sex over 16 months of age. Neither glomerular amyloidosis nor arteriolar nephrosclerosis was detected. In general the histopathology of renal lesions in APA hamsters resembled that of the condition known as glomerulonephrosis in rats. Renal lesions occurred more frequently and more severely and developed more rapidly in females than in males. There was no apparent correlation between cardiac thrombosis and renal disease.