Showing papers in "Japanese Journal of Cancer Research in 1996"

Intracellular Hyperthermia for Cancer Using Magnetite Cationic Liposomes: Ex vivo Study

Abstract: The effect of hyperthermia on solid glioma tissue formed subcutaneously in the left femoral region of female F344 rats was investigated. Magnetite cationic liposomes (MCLs), which have a positive surface charge, were used as heating mediators for intracellular hyperthermia. MCLs were injected into the solid tumors, which were then subjected to irradiation by an alternating magnetic field (118 kHz, 384 Oe). The rats were divided into four groups, which received no irradiation (control: group I), or irradiation for 30 min given once (group II), twice (group III) or three times (group IV), and the hyperthermic effect on tumor growth was evaluated. Complete tumor regression was observed in 87.5% of the rats in group IV. In the other groups, tumors completely regressed in 20 and 60% of the rats in groups II and III, respectively. Histological observations showed that in group I tumors, MCLs were localized only around the point where they were injected, while in group II tumors they were a little more dispersed. In the cases of group III and IV tumors, however, the distribution of the MCLs was found to be widespread, and necrotic cells were observed throughout almost the entire tumor tissue. The high percentage of complete regression of group IV is considered to be due to this wide distribution of the MCLs. Furthermore, the treated rats showed no severe side-effects. These results suggest that our magnetic particles are potentially effective tools for the treatment of solid tumors.

Expression of c-met/HGF Receptor in Human Non-small Cell Lung Carcinomas in vitro and in vivo and Its Prognostic Significance

Abstract: The expression of c-met/HGF receptor was evaluated in non-small cell lung cancers (NSCLC) by western blot analysis of 11 established cell lines and 104 surgically resected tissues. All cancer cell lines (eight adenocarcinomas, two squamous cell carcinomas and a large cell carcinoma) showed strong c-met protein bands of 145 kDa and 170 kDa. Moreover, c-met protein was demonstrated in 34 (72.3%) of 47 surgically resected adenocarcinomas, 20 (38.5%) of 52 squamous cell carcinomas and 3 of 5 others, and the results were mostly confirmed immunohistochemically in formalin-fixed and paraffin-embedded tumors of the same case. Although squamous cell carcinomas showed relatively high c-met protein expression in established cell lines, more adenocarcinomas than squamous cell carcinomas showed c-met protein expression in the original cancers. Furthermore, two cell lines used in this study originated from primary cancers negative for c-met protein expression, suggesting that c-met protein expression might be influenced by cultivation. Furthermore, clinicopathological study revealed that NSCLC with c-met protein expression tended to be in a higher pathological tumor stage and to have a worse outcome than those without such expression. In conclusion, c-met protein is expressed in cell lines and primary tumors of NSCLC, and this phenomenon is probably closely related to the aggressive behavior or progression of NSCLC, especially of adenocarcinomas.

A Prospective Randomized Trial of the Preventive Effect of Pre-operative Transcatheter Arterial Embolization against Recurrence of Hepatocellular Carcinoma

Abstract: To clarify whether pre-operative transcatheter arterial embolization (TAE) improves survival after hepatectomy, a prospective randomized comparative study was done. Of a total of 115 registered patients having solitary hepatocellular carcinoma (HCC) 2 to 5 cm in diameter, 18 (15.7%) were excluded after randomization. As a result, 97 patients were chosen as subjects and divided into two groups: hepatectomy with (group A: n=50) and without (group B: n=47) pre-operative TAE. The period of observation of the patients who survived the surgery was between 4.0 and 6.6 years. The randomization appeared to have provided well-balanced groups of patients and the clinico-pathological characteristics of the two groups were quite similar. The necrotic part of the cancerous lesions, as confirmed by operative specimens, amounted to 74.8+/-33.4% (mean +/-SD) in group A and 6.8+/-7.2% in group B (P<0.01). However, the cancer-free survival rates after hepatectomy in both groups showed little difference (39.1+/-7.0 (%+/-SE) and 31.1+/-0.1, respectively). We speculate that TAE is not effective against such HCC accessory lesions as minute intrahepatic metastasis and tumor thrombus and that pre-operative TAE does not improve post-operative survival.

Production of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human breast carcinomas.

Abstract: We examined production and tissue localization of matrix metalloproteinase (MMP)-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-9 (gelatinase B), tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in human breast carcinomas. In more than half of the cases, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 were immunolocalized in carcinoma cells and MMP-2 was on the carcinoma cell membranes as well, whereas MMP-3 was positively stained in less than 15% of the cases. MMP-1 staining in carcinoma cells was significantly higher in scirrhous carcinoma than in other types of carcinoma. MMP-9 expression was remarkably higher in the carcinoma cases with lymphnode metastasis than in the non-metastatic cases. MMP-3 was mainly expressed in T-lymphocytes infiltrated in the tumor stroma. Stromal fibroblasts were positive for all these MMPs except for MMP-3. The TIMP-1 levels released into the culture media by carcinoma tissues were significantly lower than those by fibroadenoma tissues, although there were no significant differences in the levels of MMP-1, MMP-2, MMP-9 and TIMP-2. Gelatin zymographical analyses showed that the activation rate of the zymogen of MMP-2 (proMMP-2) is significantly higher in the more advanced carcinoma group with lymphnode metastasis than in the metastasis-negative and fibroadenoma groups. These data indicate that MMP-1, MMP-2 and MMP-9 are highly expressed in human breast carcinoma tissue and suggest that activation of proMMP-2 may be an indicator of lymphnode metastasis of the breast carcinoma.

Inhibition of in vitro tumor cell invasion by ginsenoside Rg3.

Abstract: The effect of plant glycosides on tumor cell invasion was examined. Among the glycosides tested, ginsenoside Rg3 was found to be a potent inhibitor of invasion by rat ascites hepatoma cells (MM1), B16FE7 melanoma cells, human small cell lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN-1) cells, when examined in a cell monolayer invasion model. Structurally analogous ginsenosides, Rb2, 20(R)-ginsenoside Rg2 and 20(S)-ginsenoside Rg3 (a stereoisomer of Rg3), showed little inhibitory activity. Neither Rh1, Rh2, 20(R)-ginsenosides Rh1, Rb1, Rc nor Re had any effect. The effective ginsenoside, Rg3, tended to inhibit experimental pulmonary metastasis by highly metastatic mouse melanoma B16FE7 cells as well. Taking account of our previous finding that 1-oleoyl-lysophosphatidic add (LPA) induced invasion by MM1 cells in the monolayer invasion model, the effect of Rg3 on molecular events associated with the invasion induced by LPA was analyzed in order to understand the mechanism of the inhibition. Rg3, which suppressed the invasion induced by LPA, dose-dependently inhibited the LPA-triggered rise of intracellular Ca2+. Protein tyrosine phosphorylation triggered by LPA was not inhibited by Rg3.

E‐Cadherin Gene Mutations in Signet Ring Cell Carcinoma of the Stomach

Abstract: To clarify the significance of E-cadherin gene alterations in the development of diffuse-type adenocarcinoma of the stomach, we analyzed mutations of the E-cadherin gene using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method followed by direct sequencing. Twenty-two signet ring cell carcinomas of the stomach (10 intramucosal and 12 advanced cancers) were examined. Genomic DNA was extracted separately from cancerous and non-cancerous tissues and PCR-SSCP analysis was performed on exons 5 to 9 and the adjacent 30- to 40-base-pair intron sequences of the E-cadherin gene. Mobility shifts were found in 2 of the 10 intramucosal cancers. In 2 of the 12 advanced cancers, abnormalities of the E-cadherin gene were observed in intramucosal lesions as well as in deeply invaded areas. These results indicated that E-cadherin gene mutations are an early event in the development of signet ring cell carcinoma of the stomach. Direct sequencing revealed that the locations of mutations of the E-cadherin gene included the branch point sequence in the intron which is responsible for RNA splicing. Reverse transcriptase-PCR demonstrated aberrant RNA splicing in a case with a branch point mutation, suggesting that branch point mutations play an important role in functional modifications of E-cadherin in signet ring cell carcinoma of the stomach.

The relationship between aryl hydrocarbon hydroxylase and polymorphisms of the CYP1A1 gene

Abstract: We examined the relationship between aryl hydrocarbon hydroxylase (AHH) and the frequency of a Msp I mutation in the 3′-flanking region of cytochrome P450 (CYP) 1A1 (Mspl polymorphism) and another mutation in exon 7 (Ile-Val polymorphism) in 84 healthy male subjects in Fukuoka, Japan. AHH inducibility (3-methylcholanthrene (MC)-induced AHH activity/non-induced AHH activity) was correlated with the MspI polymorphism (P<0.0001) and age class (P=0.015), whereas no correlation was found for the Ile-Val polymorphism (P=0.509). Age-adjusted AHH inducibility (mean±SE) of the predominant homozygote (genotype A), the heterozygote (genotype B) and a homozygote rare allele (genotype C) genotypes was 4.89±0.36, 4.82±0.29 and 13.61±1.44, respectively. The genotype C showed much higher AHH inducibility than genotypes A and B (P<0.001), while no significant difference was observed between genotypes A and B. Non-induced AHH activity was also correlated with these polymorphisms. The AHH activity of a homozygous mutant Val/Val genotype (0.076±0.010 pmol/min/106 cells) was significantly higher (P<0.05) than that of the wild-type homozygous Ile/Ile (0.044±0.004 pmol/min/106 cells) and heterozygous Ile/Val (0.047±0.007 pmol/min/106 cells) genotypes. Our study suggests that the genotypes C and Val/Val, which are more frequent in smoking-related lung cancer, are closely related with high AHH inducibility and high non-induced AHH activity, respectively. Thus, the positive relationship between AHH inducibility and lung cancer is supported by our study. If our results are confirmed and the assessment of genotype becomes feasible on a population basis, identification of smokers who have genetically high susceptibility to lung cancer (genotype C or Val/Val) may become important for the prevention of lung cancer.

Expression and roles of heat shock proteins in human breast cancer

Abstract: Heat shock proteins (hsps) are thought to play important roles in the cell cycle and various processes of carcinogenesis. Therefore, we evaluated the expression of hsps, mainly hsp90 and hsp70, in human breast cancer tissues. Hsp90alpha mRNA was expressed at much higher levels in the cancerous tissue than in the non-cancerous tissue. In addition, a close correlation between hsp90alpha mRNA expression and the proliferating-cell-nuclear-antigen labeling index (PCNA LI) was observed for the cancerous tissue. These findings suggest that increased expression of the hsp90alpha isoform may play a role in cell proliferation. On the other hand, hsp90beta mRNA expression was significantly higher in poorly differentiated carcinomas than in well differentiated carcinomas of the breast. The intracellular localization of hsp70 was consistent with that of ubiquitin. In specimens showing hsp70 in the nucleus, the PCNA LI was significantly high. Hsc73 mRNA, a member of the hsp70 family, was also expressed at higher levels in cancerous tissues associated with a high PCNA LI than in non-cancerous tissues. These results suggest that hsp90alpha may play a role in cancer cell proliferation and that hsp90beta may contribute to cell differentiation and structural constitution. In addition, hsp70, especially hsc73, is related to ubiquitin and seems to be a marker for cancer proliferation.

Inactivation of the E-Cadherin gene in primary gastric carcinomas and gastric carcinoma cell lines

Abstract: We investigated the E (epithelial)-cadherin gene for mutations and loss of heterozygosity (LOH) in 24 primary gastric carcinomas (12 differentiated and 12 undifferentiated types, including 3 signet-ring cell carcinomas), as well as 4 gastric carcinoma cell lines of the undifferentiated type (MKN-45, GCIY, HGC-27 and GT3TKB). We utilized PCR-SSCP and RT-PCR followed by direct sequencing to detect gene mutations and skipped exons, and RT-PCR-SSCP to examine LOH. In primary carcinomas, gene mutations or skipped exons were detected in 4 of 9 (44%) undifferentiated carcinomas of the scattered type, including 2 signet-ring cell carcinomas, and in none of the 3 undifferentiated carcinomas of the adherent type and 12 differentiated carcinomas. Demonstrated mutations of the E-cadherin gene included an 18 bp deletion (codon 418-423) and a 3 bp deletion (codon 400, calcium-binding domain), both located in exon 9. Skipping of exon 9 with a 1 bp insertion at codon 337, and skipping of exon 8 with a 1 bp deletion at codon 336, also were detected. LOH was confirmed in all of the carcinomas in which gene mutations or skipped exons (3/3 informative cases) were demonstrated. The MKN-45 cell line exhibited an 18 bp deletion at the exon 6-intron 6 boundary with loss of the wild-type allele, and 2 of the remaining 3 cell lines (HGC-27 and GT3TKB) had lost expression without detectable structural alteration of the E-cadherin gene. These data provide support for classic two-hit inactivation of the E-cadherin gene in a high percentage of undifferentiated carcinomas of the scattered type.

Cell Death Induced by Baicalein in Human Hepatocellular Carcinoma Cell Lines

Abstract: We examined the action of baicalein, a flavonoid contained in the herbal medicine sho-saiko-to (TJ-9), on three cell lines of human hepatocellular carcinoma (HCC). Treatment with baicalein strongly inhibited the activity of topoisomerase II and suppressed the proliferation of all three HCC cell lines. But the mode of cell death induced by baicalein differed according to the cell line. Baicalein induced apoptosis in a concentration-dependent mannner in only one cell line, and an increased concentration of baicalein produced cell death via necrosis in the other two lines. These results suggest that the inhibition of topoisomerase II is not by itself sufficient for induction of apoptosis, and that there is a more important mechanism which can account for the difference in susceptibility of cells to apoptosis induced by baicalein.

Expression of thymidine phosphorylase in human gastric carcinoma.

Abstract: The activity of thymidine phosphorylase (dThdPase) has heen reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet-derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immnnostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a × 400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA-1) against dThdPase and studied the expression of dThdPase using TMA-1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P<0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase-positive gastric carcinomas was higher (P<0.001) than that in dThdPase-negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P<0.001). Further, the average size of dThdPase-positive carcinomas was significantly larger (P<0.001) than that of negative carcinomas and the mean microvessel count in dThdPase-positive gastric carcinomas was also significantly higher (P<0.001) than that in dThdPase-negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P<0.001) or lymph node metastasis (P=0.013) by multivariate logistic analysis. Further, patients with dThdPase-positive carcinoma showed a significantly worse prognosis than those with dThdPase-negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease.

Subsite (cervix/endometrium)‐specific Risk and Protective Factors in Uterus Cancer

Abstract: In Japan the incidence of cervical cancer has been high, but has recently been decreasing gradually, while the incidence of endometrial cancer is running at lower levels but is gradually increasing. To clarify the common and/or specific risk and/or protective factors of cervical cancer(CC) in contrast with endometrial cancer (EC), a comparative case-control study was conducted at the Aichi Cancer Center Hospital, Japan. In total, 556 CC cases and 145 EC cases were included and 26,751 women, confirmed as free of cancer, were chosen as the common control group. Odds ratio and its 95% confidence interval (95%CI) for each exposure variable were estimated by using an unconditional logistic regression model adjusted for age and first-visit year. Habitual smoking and experience of pregnancy increased the risk of CC, while decreasing the risk of EC. Greater body mass index (>20), daily intake of fruit and more frequent intake of boiled or broiled fish (>1-2 times/week) decreased the risk of CC, whereas they increased the risk of EC. Daily intake of milk decreased the risk of CC. The results obtained from this study suggest that several EC-increasing risk factors are in fact CC-decreasing determinants. The observed risk reduction in both CC and EC by physical exercise and dietary control for health is noteworthy from the public health standpoint and warrants further investigation.

Tobacco, alcohol and dietary factors associated with the risk of oral cancer among Japanese.

Abstract: The mortality rate among Japanese men due to oral cancer is increasing, but risk factors among Japanese other than smoking and drinking have not been examined. To investigate the dietary factors involved in oral cancer, we conducted a hospital-based case-referent study in Aichi, Japan. Cases comprised 189 men and 77 women aged 20-79 years with one of the following cancers: tongue, mouth, oropharynx and hypopharynx. The reference group comprised 9,858 male and 26,669 female out-patients without cancer. Smoking and drinking were highly associated with an increased risk of oral cancer. Japanese sake showed a lower odds ratio (OR) than beer or hard liquor (OR= 3.6, 4.5 and 4.8, respectively). In the cross analysis between smoking and drinking, smoking combined with drinking increased the risk of oral cancer to three times that of smoking only (OR=6.2 vs. 2.2). Frequent intake of raw vegetables (OR = 0.5) and fruit (OR = 0.5) were inversely associated with the risk of oral cancer after adjustment for age, sex, smoking, drinking and year of visit. Western-style breakfast and salty food preference decreased the risk of oral cancer, and salty food preference was still statistically significant by multivariate analysis (OR= 0.7). In conclusion, smoking cessation, drinking control and frequent intake of raw vegetables and fruit among Japanese are likely to be effective preventive measures against oral cancer.

Aberrant DNA Methylation on Chromosome 16 Is an Early Event in Hepatocarcinogenesis

Abstract: In order to clarify the significance of DNA methylation in both earlier and later stages of hepatocarcinogenesis, the DNA methylation state on chromosome 16, on which loss of heterozygosity (LOH) has frequently been detected in human hepatocellular carcinomas (HCCs), was examined. DNA from primary HCCs and tissues showing chronic hepatitis and liver cirrhosis, which are considered to be precancerous conditions, was analyzed by digestion with methylation-sensitive and non-sensitive restriction enzymes. DNA hypermethylation at the D16S32, tyrosine aminotransferase (TAT) and D16S7 loci and hypomethylation at the D16S4 locus were detected in 18%, 58%, 20% and 48% of examined HCCs, respectively. Aberrant DNA methylation occurred more frequently in advanced HCCs than in early HCCs. Moreover, DNA hypermethylation at the D16S32, TAT and D16S7 loci was frequently observed in chronic hepatitis and liver cirrhosis. The incidence of DNA hypermethylation was higher than that of LOH (42% at the TAT locus). These data suggest that DNA hypermethylation might predispose the locus to allelic loss. Aberrant DNA methylation is a significant change which may participate in the early developmental stages of HCCs.

Inhibitory effects of tea catechins, black tea extract and oolong tea extract on hepatocarcinogenesis in rat

Abstract: Inhibitory effects of individual tea catechins ((--)-epicatechin, (--)-epigallocatechin, (--)-epicatechin gallate, (--)-epigallocatechin gallate), black tea extract and oolong tea extract on hepatocarcinogenesis were investigated. Male F344 rats received a single dose of diethylnitrosamine (200 mg/kg, i.p.), and thereafter phenobarbital (0.05%) was administered in the drinking water for a period of 6 weeks. Tea catechins, black tea extract or oolong tea extract were given during the entire experimental period, during only the initiation period or during only the promotion period. All four tea catechins, black tea extract and oolong tea extract (0.05 or 0.1%) significantly decreased the number and area of preneoplastic glutathione S-transferase placental form-positive foci in the liver. These results suggest that tea catechins, black tea extract and oolong tea extract have a chemopreventive action against hepatocarcinogenesis.

Detection of Gastric Cancer Micrometastases in Lymph Nodes by Amplification of Keratin 19 mRNA with Reverse Transcriptase‐Polymerase Chain Reaction

Abstract: A sensitive method for the detection of gastric cancer micrometastases in lymph nodes was developed. The method was based on amplification of keratin 19 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Keratin 19 RT-PCR showed that keratin 19 mRNA was expressed in all 12 gastric cancers, but not in any of 20 normal control lymph nodes, indicating that keratin 19 mRNA is a good target of RT-PCR for the detection of gastric cancer micrometastases in lymph nodes. Serial dilution studies of RNA extracted from gastric cancers against RNA extracted from control lymph nodes demonstrated that the detection sensitivity of the keratin 19 RT-PCR method was one cancer cell in 10 3 -10 5 lymph node cells. Detectability of lymph node metastases was compared between keratin 19 RT-PCR and conventional histological examination, using 100 lymph nodes obtained from 12 gastric cancer patients. Keratin 19 mRNA was detected in all of the seven lymph nodes which were histologically metastasis-positive. Of the 93 lymph nodes which were histologically metastasis-negative, 79 were found not to express keratin 19 mRNA but 14 were found to express keratin 19 mRNA, indicating that these lymph nodes contained micrometastases which could not be detected by histological examination. These results demonstrate that keratin 19 RT-PCR is a more sensitive method than histological examination for the detection of gastric micrometastases in lymph nodes.

Replication of Hepatitis C Virus in Cultured Non‐neoplastic Human Hepatocytes

Abstract: We established a replication system for hepatitis C virus (HCV) using the PH5CH non-neoplastic human hepatocyte line that had been immortalized with simian virus 40 large T antigen. In cells inoculated with sera derived from two HCV-positive blood donors, positive-stranded HCV RNA was detected up to 30 days postinoculation (p.i.). Semi-quantitative analysis of HCV RNA revealed that HCV multiplied during the period of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) in both cases indicated that HVR1 populations from the cells at 8 days p.i. were apparently different from those of the original inocula. HVR1 populations in infected cells became homogeneous or just a few species were selected over time. These results suggest that HCV is replicating in the human hepatocyte PH5CH cells. This culture system will be useful for detailed studies of the biological effects of HCV in human hepatocytes.

Sonodynamically induced antitumor effect of a gallium-porphyrin complex, ATX-70.

Abstract: The sonodynamically induced antitumor effect of a gallium-porphyrin complex, ATX-70, was evaluated in mice bearing colon 26. In order to find the optimum timing for the ultrasonic exposure after the administration of ATX-70, the ATX-70 concentrations in the plasma, skin, and tumor were measured and analyzed. Antitumor effect was estimated by measuring the tumor size. When used alone, ultrasound showed a slight antitumor effect, which became increasingly significant as the dose of ATX-70 was increased, while use of ATX-70 alone had no significant effect. At an ATX-70 dose of 2.5 mg/kg or higher, the average tumor size decreased to smaller than a half by three days after the ultrasonic exposure. This was smaller than a third of the size of the untreated tumors on the same day. From these results, it is concluded that ATX-70 significantly sensitizes tumors to ultrasound, demonstrating a synergistic antitumor effect.

Suppression of Human Pancreatic Cancer Growth in BALB/c Nude Mice by Manumycin, a Farnesyl:protein Transferase Inhibitor

Abstract: Activating mutations of Ki-ras have been detected in most human pancreatic adenocarcinomas. Since Ras protein requires farnesylation to function, we investigated the effects of manumycin, a potent farnesyl:protein transferase inhibitor, on the growth in nude mice of a human pancreatic cancer cell line, MIA PaCa-2, with a point mutation in the Ki-ras gene. Tumor-bearing mice received intraperitoneal injection of 1 or 5mg/kg manumycin daily for 5 days, or 2 mg/kg manumycin daily for 2 weeks. Growth of inoculated tumors was significantly inhibited by the treatment. The treatment significantly (P<0.05) lowered the numbers of bromodeoxyuridine-incorporating tumor cells. Manumycin did not have apparent hepatotoxicity in vivo. Farnesyl:protein transferase inhibitors could offer a new approach for cancer chemotherapy.

Mutational Analysis of Mismatch Repair Genes, hMLH1 and hMSH2, in Sporadic Endometrial Carcinomas with Microsatellite Instability

Abstract: Microsatellite instability, monitored by replication error (RER), has been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch-repair system. We previously reported that nearly one-quarter of sporadic endometrial carcinomas examined revealed an RER-positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch-repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2. Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer ; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch-repair genes, such as hPMS1 and hPMS2, or of unknown genes related to the mismatch-repair system.

Chemoprevention by Pravastatin, a 3‐Hydroxy‐3‐methylglutaryl‐coenzyme A Reductase Inhibitor, of N‐Methyl‐N‐nitrosourea‐induced Colon Carcinogenesis in F344 Rats

Abstract: A potential chemopreventive action of pravastatin (Pr), a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on colon carcinogenesis was evaluated in F344 rats. All rats at 7 weeks of age received an intrarectal dose of 2 mg of N-methyl-N-nitrosourea 3 times weekly for 2 weeks in experiment I (2 groups of 16 rats each), and for 3 weeks in experiment II (4 groups of 30 rats each). They were given drinking water containing 0 ppm (control) or 200 ppm Pr during weeks 1 to 40 in experiment I, and containing 0 ppm (control), 25 ppm, 5 ppm and 1 ppm Pr during weeks 4 to 40 in experiment II. The body weight gains, and food and water intakes were similar in all the groups. The incidence of colon carcinomas at termination of the experiment at week 40 was not different in the 200 ppm Pr and control groups in experiment I (63% vs. 69%), while it was significantly lower in the 25 ppm and 5 ppm groups, but not in the 1 ppm Pr group, compared with the control group in experiment II (50%, 48%, and 77% vs. 80%). This inhibitory effect of Pr against colon carcinogenesis was not related to the cholesterol-lowering effect of this agent. We postulate that Pr inhibits the promotion stage of colon carcinogenesis, perhaps through modulation of cholesterol synthesis in situ in the colonic mucosa, thereby suppressing farnesyl isoprenylation of growth-regulating proteins such as p21 ras.

Reduced activity of anabolizing enzymes in 5-fluorouracil-resistant human stomach cancer cells.

Abstract: The mechanism of resistance to 5-fluorouracil (5-FU) was studied with NUGC-3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5-day exposures to stepwise-increasing concentrations of 5-FU in vitro. NUGC-3/5FU/L was 200-fold and over 16-fold resistant to 96-h and 1-h exposures to 5-FU, respectively. NUGC-3/5FU/L incorporated less 5-FU into RNA, indicating resistance to the RNA-directed action of 5-FU. On the other hand, NUGC-3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5-FU. Polymerase chain reaction-single-strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5-FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC-3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl-transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC-3/5FU/L than in the parent NUGC-3. These results indicated that resistance to 5-FU in NUGC-3/5FU/L is due to reduced activities of 5-FU-anabolizing enzymes, but not to an alteration of TS. 2′-Deoxyinosine effectively enhanced TS inhibition by 5-FU in the resistant cells, thus markedly sensitizing them to 5-FU.

Chemopreventive Effect of a Xanthine Oxidase Inhibitor, 1′‐Acetoxychavicol Acetate, on Rat Oral Carcinogenesis

Abstract: The effect of a xanthine oxidase inhibitor, 1'-acetoxychavicol acetate (ACA), on 4-nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis was investigated in male F344 rats. All rats except those in the ACA-alone and untreated groups were given 4-NQO (20 ppm) In the drinking water for 8 weeks to induce oral cancer. Starting 1 week before the 4-NQO exposure, animals were fed diet containing 100 ppm or 500 ppm ACA for 10 weeks, followed by the basal diet without ACA for 22 weeks. Other groups were fed the diet containing ACA at 100 ppm or 500 ppm for 22 weeks, starting 1 week after the cessation of 4-NQO exposure. The remaining groups consisted of rats given 500 ppm ACA alone or untreated rats. At the termination of the experiment (32 weeks), the incidences of tongue neoplasms and preneoplastic lesions, polyamine levels in the tongue tissue, and cell proliferation activity estimated in terms of 5-bromodeoxyuridine (BrdU)-labeling index and by morphometric analysis of silver-stained nucleolar organizer regions' protein (AgNORs) were compared among the groups. Feeding of ACA at the two doses during initiation or postinitiation significantly decreased the development of tongue carcinoma (93-100% reduction, P < 0.001) and preneoplasia (43-50% reduction for hyperplasia and 34-48% reduction for dysplasia, P < 0.05). There were no such lesions in rats fed ACA alone or those in the untreated control group. The number of AgNORs per cell nucleus was significantly decreased by feeding of ACA at a high dose (500 ppm) (29% inhibition, P < 0.05). The BrdU-labeling index was also reduced by dietary administration of ACA (23-32% inhibition, P < 0.01). In addition, ACA feeding reduced tongue polyamine levels (35-40% inhibition, P < 0.05). These results indicate that ACA inhibited rat oral carcinogenesis, and such inhibition might be related to suppression of cell proliferation in the oral mucosa by the xanthine oxidase inhibitor.

Effect of MUC1 mucin, an anti-adhesion molecule, on tumor cell growth.

Abstract: MUC1 mucin is expressed in a wide variety of tumors and is considered to function as an anti-adhesion molecule which inhibits cell-to-cell interactions. To reveal the biological significance of this activity in tumor cells, MUC1 cDNA was transfected into EJNIH3T3 cells and human colon cancer cell lines, CHCY1 and DLD1. The in vivo growth rate of MUC1+ (MUC1-transfected) EJNIH3T3, CHCY1 and DLD1 cells in SCID mice was clearly lower than that of MUC1- (mock transfectant) cells. Several in vitro experiments using MUC1+ EJNIH3T3 cells were performed to analyze the mechanisms for the decreased in vivo tumor growth. It was found that (i) the in vitro growth rate of MUC1+ EJNIH3T3 cells was also decreased compared to that of MUC1- cells, (ii)the DNA synthesis of MUC1+ EJNIH3T3 cells after stimulation with either growth factor (fetal calf serum or bombesin) or extracellular matrix (collagen or fibronectin) was lower than that of MUC1- cells, and (iii) MUC1+ EJNIH3T3 cells grew more slowly than MUC1- cells on both collagen- and fibronectin-coated dishes. These data suggest that MUC1 mucin may regulate tumor cell growth through inhibition of cell-to-cell, growth factor-to-receptor and cell-to-matrix interactions.

Expression of Mucin Carbohydrates and Core Proteins in Carcinomas of the Ampulla of Vater: Their Relationship to Prognosis

Abstract: We examined the expression of carbohydrate antigens which are associated with the earliest steps in mucin glycosylation (Tn and sialosyl-Tn) and the expression of the mucin core protein antigens associated with MUC1 gene product (DF3 antigen) as well as MUC2 gene product (intestinal-MRP antigen) in tissues from 38 patients with carcinoma of the ampulla of Vater, in order to determine whether these mucin antigens are available as tumor markers or not, and to evaluate whether their expression is correlated with the biological behavior of the carcinomas or not. DF3 antigen showed a relatively high expression rate (61%) in the carcinoma tissues, but was rarely expressed in the non-neoplastic epithelium around the carcinomas in the region of the ampulla of Vater. Tn and sialosyl-Tn antigens showed high expression rates in the carcinoma tissues (86% and 84% each), whereas they showed rare or no expression in the non-neoplastic epithelium around the carcinomas, except for highly restricted expression in the duodenal villous epithelium. The patients with positive DF3 expression in the carcinoma showed significantly poorer survival than those with negative DF3 expression (P < 0.05), whereas the patients with positive intestinal-MRP expression in the carcinoma showed significantly more favorable survival than those with negative intestinal-MRP expression (P < 0.05). The expression rate of DF3 antigen was significantly higher in the cases with deep invasion into the pancreas (89%) than in those with no or minimal invasion (52%) (P < 0.05). In contrast, the expression rate of intestinal-MRP antigen was significantly higher in the cases with no or minimal invasion into the pancreas (38%) than in those with deep invasion (0%) (P<0.05). In conclusion, the expression of DF3, To and sialosyl-Tn antigens is an effective histopathological indicator for carcinomas in the area of the ampulla of Vater, and the expression of DF3 and intestinal-MRP antigens is a useful indicator of the prognosis of the patients.

Acquisition of Invasive Phenotype in Gallbladder Cancer Cells via Mutual Interaction of Stromal Fibroblasts and Cancer Cells as Mediated by Hepatocyte Growth Factor

Abstract: Growth and motility of carcinoma cells are regulated through their interactions with host stromal cells, i.e., tumor-stromal interactions. Hepatocyte growth factor (HGF), a ligand for c-Met tyrosine kinase, is a stromal-derived regulator of growth, motility, and morphogenesis. HGF stimulated proliferation and motility of GB-d1 gallbladder carcinoma cells from a patient with gallbladder cancer. HGF induced in vitro invasion of GB-d1 cells into a collagen gel matrix, and this potent, invasive effect was not seen with epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, or platelet-derived growth factor. Although GB-d1 did not produce HGF, the cells did produce a factor which enhances HGF production in human skin fibroblasts, and this factor proved to be interleukin-1 beta (IL-1 beta). When GB-d1 cells were co-cultured with fibroblasts such that a collagen gel matrix was layered between the GB-d1 cells and fibroblasts, GB-d1 cells invaded the gel, but invasion of the cells in the co-culture system was inhibited by antibodies against HGF and partially inhibited by antibodies against IL-1 beta. Thus, GB-d1 cell-derived IL-1 beta stimulates HGF production in stromal fibroblasts and HGF up-regulated in the fibroblasts induces invasion of GB-d1 cells. The looped interaction of carcinoma cells and stromal fibroblasts mediated by HGF and a HGF-inducer such as IL-1 beta may be one mechanism which would explain the acquisition of malignant phenotype through tumor-stromal interactions.

Fibrotic focus in invasive ductal carcinoma: an indicator of high tumor aggressiveness.

Abstract: Histological examination of invasive ductal carcinoma of the breast often demonstrates the presence of an extensive central fibrotic focus (FF). The clinicopathological significance of the FF, or scar, in primary invasive ductal carcinoma is still ambiguous. One hundred and fifty-three cases of invasive ductal carcinoma (IDC) were classified into two groups, those with and those without FF. The differences in frequency of immunohistochemically detected overexpression of c-erbB-2 protein and nuclear accumulation of p53 protein, and the labeling index of proliferating cell nuclear antigen (PCNA), as well as histopathological parameters were compared between these two groups. IDCs smaller than 50 mm with FF showed a higher frequency of high-grade tumors, a higher frequency of lymph node metastasis, and a significantly higher frequency of c-erbB-2 protein overexpression than those without FF. In tumors of 20 mm or less, the incidence of nuclear accumulation of p53 protein was significantly higher in tumors with than those without FF. Tumors with FF showed a significantly higher PCNA labeling index than those without FF, regardless of tumor size. The present results indicate that the presence of FF is an important clinicopathological parameter associated with a higher degree of malignancy in IDCs, especially those smaller than 50 mm. Therefore, dividing IDCs into those with and those without FF appears to be meaningful clinicopathologically.

Prevention of mammary tumorigenesis in acatalasemic mice by vitamin E supplementation.

Abstract: Adult male and female acatalasemic (C3H/AnLCs b Cs b ), hypocatalasemic (C3H/AnLCS c CS c ) and normal mice of C3H strain fed on regular laboratory chow for 15 months showed an increased incidence of spontaneous mammary tumor in the decreasing order of female acatalasemic, male acatalasemic, female hypocatalasemic and male hypocatalasemic mice. Normal mice did not develop mammary tumor. We conducted a prospective study with female acatalasemic mice, which showed the highest incidence of mammary tumor, to examine the preventive effect of vitamin E on mammary tumor. Female acatalasemic mice were fed on vitamin E-deficient (28 animals) and vitamin E-supplemented diet (25 animals) for 29 months. The incidence of mammary tumor in mice given the vitamin E-supplemented diet was 47%, while that in mice given vitamin E-deficient diet was 82% (P <0.002). Mammary tumors were apparent after 9 months of vitamin E deprivation and after 14 months of vitamin E supplementation. Female normal mice did not develop mammary tumor during a comparable period of time. The mean catalase activity of mammary gland in acatalasemic mice was 18.8% of that in normal mice. The results indicate that vitamin E protects acatalasemic mice against the development of mammary tumor.

Screening for Colorectal Cancer by Immunochemical Fecal Occult Blood Testing

Abstract: Screening for colorectal cancer using the conventional Hemoccult test has been shown to reduce mortality associated with cancer by 33% through a randomized controlled trial. However, the magnitude of effectiveness is small in terms of cost-effectiveness. The recently developed immunochemical fecal occult blood test (IFOBT) provides a potential replacement for the Hemoccult test as a screening test, due to its superior performance characteristics such as higher sensitivity shown in preliminary studies and the fact that it does not require any dietary restriction. The IFOBT method is reviewed, especially in relation to its specificity. In known colorectal cancer subjects, IFOBTs have shown both higher sensitivity and specificity than the Hemoccult test. Similarly, IFOBT has demonstrated a higher sensitivity than Hemoccult for colorectal cancer in an asymptomatic population. A nationwide screening program in Japan has demonstrated the feasibility of this approach for large population screening. However, the positivity rate varied according to the conditions at each screening facility. Therefore, technical factors that influence the positivity rate of IFOBTs in the screening program are discussed. Case-control studies have strongly suggested that screening using IFOBT would reduce mortality from colorectal cancer by 60% or more. Several observational studies have provided support for this estimate. The feasibility and effectiveness of population-based screening by IFOBT are discussed.

Establishment and Characterization of Human Gastric Carcinoma Lines with High Metastatic Potential in the Liver: Changes in Integrin Expression Associated with the Ability to Metastasize in the Liver of Nude Mice

Abstract: There is a need to establish animal models which are suitable for investigation of human gastric cancer metastasis to the liver. To this end, a human gastric carcinoma line, AZ521 was injected into the spleens of nude mice. Cells from the few liver metastatic foci of injected AZ521 were expanded "in vitro" and subsequently injected into the spleens of nude mice. By repeating these procedures three times, we were able to obtain a cell line, designated as AZ-H3c, with high metastatic potential in nude mice. Liver metastasis developed in 15 of 21 (71%) animals injected with AZ-H3c, but only in 14% of those injected with parental AZ521. Further, AZ-H3c caused faster tumor development than did AZ521. However, the primary AZ-H3c tumors and liver metastatic AZ-H3c tumors showed essentially the same histological appearance. We also analyzed the cell surface expression of adhesion molecules. The data showed that the expression of VLA-1, VLA-2, VLA-3, VLA-4, VLA-5 was enhanced in AZ-H3c. In contrast, the expression of VLA-6, (alpha(v)beta3), E-cadherin, ICAM-1 and LFA-1 was reduced in this high-metastatic line. These results suggest that (beta1) integrins play an important role in the liver metastasis of human gastric carcinoma cells. Our high-metastatic line should be useful for studies aimed at the prevention of liver metastasis.