Miyako Baba

Diagnosis of pancreatic cancer by detecting telomerase activity in pancreatic juice: comparison with K-ras mutations

OBJECTIVE:Telomerase activity is reported to be specific and very frequent in human malignancy. K-ras mutations are also very frequently detected in pancreatic cancer, but their specificity for pancreatic cancer is controversial. We examined the telomerase activity and K-ras mutations in pancreatic juice from patients with pancreatic disease.METHODS:Pancreatic juice was obtained endoscopically at endoscopic retrograde pancreatography from 10 patients with pancreatic cancer, three with chronic pancreatitis, and three with a normal pancreas. The telomerase activity in pancreatic juice was assayed by telomeric repeat amplification protocol. K-ras mutations in exon 1 codon 12 were examined by the two-step polymerase chain reaction combined with restriction enzyme digestion, followed by single-strand conformation polymorphism analysis and direct sequencing.RESULTS:Telomerase activity of >5.0 was detected in eight of 10 (80%) subjects with pancreatic cancer, but in none with chronic pancreatitis or normal pancreas. K-ras mutations were detected not only in eight of 10 (80%) subjects with pancreatic cancer but also in two of three with chronic pancreatitis and in one of three with a normal pancreas.CONCLUSIONS:It was shown that the detection of telomerase activity in pancreatic juice is a more useful diagnostic tool for pancreatic cancer than that of K-ras mutations.

Inhibition by ginsenoside Rg3 of bombesin-enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane in Wistar rats

The effects of concomitant use of bombesin and ginsenoside Rg3 on the incidence of peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane were investigated in male inbred Wistar rats. From the start of the experiment, rats were given weekly s.c. injections of azoxymethane (7.4mg/kg body weight) for 10 weeks and s.c. injection of bombesin (40μg/kg body weight) every other day, and from week 20, s.c. injections of ginsenoside Rg3 (2.5 or 5.0mg/kg body weight) every other day until the end of the experiment in week 45. Bombesin significantly increased the incidence of intestinal tumors and cancer metastasis to the peritoneum in week 45. It also significantly increased the labeling index of intestinal cancers. Although administration of a higher dose of ginsenoside Rg3 with bombesin had little or no effect on the enhancement of intestinal carcinogenesis by bombesin, the location, histologic type, depth of involvement, infiltrating growth pattern, labeling and apoptotic indices and tumor vascularity of intestinal cancers, it significantly decreased the incidence of cancer metastasis. These findings indicate that ginsenoside Rg3 inhibits cancer metastasis through activities that do not affect the growth or vascularity of intestinal cancers.

Attenuation by genistein of sodium‐chloride‐enhanced gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in Wistar rats

The effects of prolonged administration of genistein, a tyrosine‐kinase inhibitor, on sodium‐chloride‐enhanced induction of gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, and the labeling and apoptotic indices and vessel counts in the gastric mucosa and gastric cancers, were investigated in Wistar rats. After 25 weeks of the carcinogen treatment, rats were fed chow pellets containing 10% sodium chloride and were given s.c. injections of genistein at dosages of 15 mg/kg or 30 mg/kg body weight every other day. In week 52, the incidence of gastric cancers was significantly greater in rats fed sodium chloride than in untreated control rats. Prolonged administration of genistein at a dosage of 30 mg/kg, but not 15 mg/kg, body weight significantly reduced the incidence of gastric cancers, which was increased by oral treatment with sodium chloride. Genistein at the higher dose significantly decreased the labeling index and vessel counts of the antral mucosa and the gastric cancers (which were increased by treatment with sodium chloride) and significantly increased the apoptotic index of the antral mucosa and the cancers (which was lowered by the treatment with sodium chloride). These findings suggest that genistein attenuates gastric carcinogenesis promoted by sodium chloride, by inducing increased apoptosis and lower cell proliferation and angiogenesis of antral mucosa and gastric cancers. Int. J. Cancer 80:396–399, 1999.

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.

Inhibition by D-limonene of experimental hepatocarcinogenesis in Sprague-Dawley rats does not involve p21ras plasma membrane association

The effects of d‐limonene on hepatocarcinogenesis induced by N‐nitrosomorpholine (NNM) and on membrane‐associated p21ras and labeling and apoptotic indices of the liver were investigated in male Sprague‐Dawley rats. Rats were given drinking water containing NNM for 8 weeks, and from the beginning of experimental week 9, they received chow pellets containing 1% or 2% limonene. The preneoplastic and neoplastic liver lesions (cellular alteration foci, neoplastic nodules and hepatocellular carcinomas), and hepatic foci staining positive for glutathione‐S‐transferase, placental type (GST‐P) were examined microscopically and histochemically. At week 16, quantitative histologic analysis showed that oral administration of 1% or 2% limonene resulted in significant reductions in the number and mean area of GST‐P‐positive hepatic foci and the number of cellular alteration foci, neoplastic nodules and hepatocellular carcinomas. Limonene, at both doses, also caused significant decreases in the labeling indices and significant increases in the apoptotic indices of cellular alteration foci, neoplastic nodules, hepatocellular carcinomas and adjacent liver. However, limonene, at both doses, had no significant influence on the production of membrane‐associated p21ras in the visible liver white nodules. These findings indicate that limonene inhibits hepatocarcinogenesis and suggest that this effect may be clearly related to its effect in inhibiting cell proliferation and in enhancing apoptosis, but not through ras oncoprotein plasma membrane association.

In vivo electroporetic transfer of bcl-2 antisense oligonucleotide inhibits the development of hepatocellular carcinoma in rats.

To investigate the potential use of a bcl‐2 antisense oligonucleotide for therapy against hepatocellular carcinoma, we examined the effects of the electroporetic transfer of a bcl‐2 antisense oligonucleotide on rat hepatocarcinogenesis induced by N‐nitrosomorpholine (NNM). Sprague–Dawley rats were given water containing 175 mg/l NNM for 8 weeks and received intraperitoneal injections of a bcl‐2 antisense phosphorothioate oligonucleotide, a sense oligonucleotide or a scrambled sequence oligonucleotide encapsulated in empty liposomes, at a dose of 150 μg oligonucleotide/kg body weight, every 4 weeks. One hour after injection, in vivo electroporation was performed on the liver to achieve selective transfer of the oligonucleotides. By week 16, the rats that had received the bcl‐2 antisense oligonucleotide had significantly fewer and smaller precancerous liver lesions positive for glutathione‐S‐transferase (placental type), and a significantly lower incidence of hepatocellular carcinoma in the electroporation zone than rats that had received the sense or the scrambled oligonucleotides. Moreover, the bcl‐2 antisense oligonucleotide significantly increased the apoptotic indices in foci, neoplastic nodules and in hepatocellular carcinomas. The expression of bcl‐2 mRNA also decreased, and 3′‐fragments of bcl‐2mRNA produced by cleavage at the antisense target site were detected in rat liver. Mean cellular fluorescence in the liver increased with higher doses of fluorescein‐isothiocyanate‐labeled antisense or sense oligonucleotides. Our results show that the electroporetic transfer of bcl‐2 antisense oligonucleotide can inhibit rat hepatocarcinogenesis. Int. J. Cancer 85:260–266, 2000. ©2000 Wiley‐Liss, Inc.

Low Prevalence of Helicobacter pylori Infection in Patients with Hamartomatous Fundic Polyps

Helicobacter pylori infection of the gastricmucosal surface was investigated in patients withhamartomatous fundic polyps or hyperplastic polyps andin patients without endoscopic evidence of disease(healthy subjects). Presence of H. pylori infection wasdetermined by culture, histologic examination, and theendoscopic phenol red test. Adherence of H. pylori wasevaluated with scanning electron microscopic examination of antral biopsy specimens. Bothprevalence of H. pylori infection (P < 0.001) and H.pylori adherence (P < 0.05) were less in patientswith hamartomatous fundic polyps than in healthy subjects and patients with hyperplastic polyps.However, the percentages of plasma cells in gastricmucosa that contained IgA and of gastric epithelialcells that expressed Lewis b did not differsignificantly among the three groups. These findings suggestthat defense mechanisms against the attachment of H.pylori other than IgA or Lewis b antigen are present inpatients with hamartomarous fundic polyps.

Bile Reflux: A Possible Cause of Stomach Ulcer in Nontreated Mutant Mice of W/Wv, Genotype

The chronic antral ulcer developed spontaneously in (WB X C57BL/6)F1-W/WV mice. Natural history of the ulcer was investigated to make clear the mechanisms involved in the development of ulcer in this mutant mouse. Although no histopathologic abnormalities of the stomach were detected at the fifth day after birth, erosions developed at the tenth day and the severity of the ulcerative lesions advanced with age. Antral pH of the W/WV mice was higher than that of the congenic +/+ mice, and the content of gastrin in the glandular stomach of the former was lower than that of the latter. Therefore, hyperacidity cannot be the cause of the ulcer in the W/WV mice. Bilirubin was demonstrable in the stomach contents of the suckling W/WV mice. After subcutaneous injection of [35S]sulfobromophthalein, the 35S radio-activity in the stomach contents of the 10-day-old W/WV mice was about 40 times as great as that observed in the +/+ littermates. As the peak of the bile reflux occurred shortly before the development of obvious ulcer that penetrated beyond the muscularis mucosae, the bile reflux could be a cause rather than a result of the ulcer.

d-Limonene inhibits N-nitrosobis(2-oxopropyl)amine induced hamster pancreatic carcinogenesis

The effect of d-limonene on pancreatic carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine (BOP) was investigated in Syrian golden hamsters. During and after 5 weekly injections of BOP, each hamster was fed diet containing d-limonene. In week 26, quantitative histological analysis showed that prolonged treatment with d-limonene significantly reduced the number of pancreatic carcinomas. Administration of d-limonene did not cause a significant increase in the apoptotic index, but caused a significant decrease in the BrdU labeling index of carcinoma. These findings indicate that d-limonene inhibits the development of pancreatic carcinoma not by enhancing tumor cell loss through apoptosis, but rather by inhibiting cell proliferation.

Inhibition by Xiao‐chai‐hu‐tang (TJ‐9) of Development of Hepatic Foci Induced by N‐Nitrosomorpholine in Sprague‐Dawley Rats

The effect of Xiao‐chai‐hu‐tang (TJ‐9) on hepatocarcinogenesis induced by N‐nitrosomorpholine (NNM) was investigated in male Sprague‐Dawley rats. Rats were given normal chow pellets containing 0.5% or 1.0% TJ‐9 until the end of the experiment, and drinking water containing NNM for 8 weeks. Pre‐neoplastic and neoplastic lesions staining for γ‐glutamyl transpeptidase (GGT) or the placenta] type of glutathione‐S‐transferase (GST‐P) were examined histochemically. In Week 15, quantitative histological analysis showed that prolonged treatment with 0.5% TJ‐9 significantly reduced the number and volume of GGT‐positive and GST‐P‐positive hepatic lesions. Treatment with 1.0% TJ‐9 inhibited the development of GGT‐positive and GST‐P‐positive lesions, but was less effective than 0.5% TJ‐9. Administration of 0.5% TJ‐9 also caused a significant increase in the proportion of helper T lymphocytes and a significant decrease in the labeling index of pre‐neoplastic lesions. These findings indicate that TJ‐9 inhibits the development of hepatic foci.