Snail knockdown reverses stemness and inhibits tumour growth in ovarian cancer.

To develop effective therapies for advanced high grade serous ovarian cancer (HGSOC), understanding mechanisms of recurrence and metastasis is necessary. In this study, we define the epithelial/mesenchymal status of cell lines that accurately model HGSOC, and evaluate the therapeutic potential of targeting Snai1 (Snail), a master regulator of the epithelial/mesenchymal transition (EMT) in vitro and in vivo. The ratio of Snail to E-cadherin (S/E index) at RNA and protein levels was correlated with mesenchymal morphology in four cell lines. The cell lines with high S/E index (OVCAR8 and COV318) showed more CSC-like, motile, and chemoresistant phenotypes than those with low S/E index (OVSAHO and Kuramochi). We tested the role of Snail in regulation of malignant phenotypes including stemness, cell motility, and chemotherapy resistance: shRNA-mediated knockdown of Snail reversed these malignant phenotypes. Interestingly, the expression of let-7 tumour suppressor miRNA was upregulated in Snail knockdown cells. Furthermore, knockdown of Snail decreased tumour burden in an orthotopic xenograft mouse model. We conclude that Snail is important in controlling HGSOC malignant phenotypes and suggest that the Snail/Let-7 axis may be an attractive target for HGSOC treatment.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Effect of temperature on fatty acid composition in each lipid fraction of Spirometra erinaceieuropaei plerocercoids.

The effects of temperature and host fatty acids on the fatty acid contents of Spirometra erinaceieuropaei plerocercoids were investigated to clarify their role in sparganosis. After 24 hr incubation at 18 C in host snake serum, omega6 series fatty acids, especially arachidonic acid in the phospholipid fraction of the plerocercoids, increased compared with those of plerocercoids incubated at 37 C. The changes in the ratio of polyunsaturated to saturated fatty acids in the phospholipid fraction of plerocercoids incubated in physiological saline for 6 hr at 10 C were almost the same as the changes at 37 C. The ratio of polyunsaturated to saturated fatty acids of the triglyceride fraction showed almost opposite change versus the phospholipid fraction. The percentage of arachidonic acid in the phospholipid fraction of plerocercoids increased during the first 3 hr of incubation and then decreased, regardless of temperature. At 37 C, the percentage of arachidonic acid in the free fatty acid fraction fell for the first 3 hr of incubation and was significantly elevated at the end of the 6-hr incubation. At 10 C, however, arachidonic acid in the free fatty acid fraction decreased for the first hour of incubation, increased at 3 hr of incubation, then decreased again. These results suggest that fatty acids of the plerocercoids are frequently exchanged between fractions. Plerocercoids can mobilize arachidonic acid to the free fatty acid fraction more quickly at lower temperature than at higher temperature. They may utilize mobilized arachidonic acid early in the infection stage to produce prostaglandins. Alternatively, they can incorporate arachidonic acid into the phospholipid fraction again when arachidonic acid is readily available in the environment.

[Primary health care in China: problems and challenges].

The World Health Organization (WHO) put forward a global strategy goal of "Health for All by year 2000" and pointed out the task of achieving this based on the primary health care (PHC) during the Alma-Ate Conference in 1978. The government of China have endorsed the WHO agreement and performed much work toward it. However, the gap between urban areas and rural areas is widening. The accomplishment of the goal of "Health for All by the year 2000" is still a critical task in China. In this study, main health problems, government policies and measures on the framework of quality of life, health status, lifestyle and health-supporting environment, medical, health and welfare system and health policies in PHC of China were diagnosed and evaluated according to the "diagnosis and evaluation for health promotion" by Green and Kreuter (1991). Japanese experiences in a combined medical, health and welfare system, elderly care, environmental protection and health education are very helpful to strengthen PHC in China. A new partnership for "Health for All" between Japan and China to achieve the goal of "Health for All by the year 2000" is imperative.

Low levels of serum cholesterol and systolic blood pressure in Japanese with the apolipoprotein E3/2 genotype.

Apolipoprotein E (apoE) plays an important role in lipoprotein and cholesterol metabolism. An association between serum cholesterol and blood pressure has been suggested by epidemiological and experimental studies. But it is still not clear whether the apoE polymorphism plays a role in regulating blood pressure. The present study was undertaken to determine the association among apoE genotype, serum cholesterol and blood pressure in 303 healthy Japanese workers. Amplified fragments of DNA by the polymerase chain reaction were digested with HhaI and analyzed by 3% agarose-gel electrophoresis. Individuals with the apoE3/2 genotype had significantly lower levels of total cholesterol and systolic blood pressure than either the apoE3/3 individuals or the apoE3/4 + 4/4 individuals (P <0.05). The hypothesis that apoE indirectly influences systolic blood pressure through total serum cholesterol was supported by a covariance analysis of linear structural equations.

Food intake, serum lipids and amino acids of school children in agricultural communities in Japan.

OBJECTIVE: To investigate the correlation among serum lipids, amino acids and diet of children in Japan where the mortality of ischemic heart disease is still very low. DESIGN: Cross-sectional study. SETTING: Two towns in Shimane prefecture, typical mountainous agricultural communities in the western part of the mainland of Japan. SUBJECTS: 514 children (10-15 y) in the communities were recruited. RESULTS: The mean cholesterol levels ranged from 3.9 to 4.4 mmol/l for boys and from 4.3 to 4.5 mmol/l for girls, and serum cholesterol level fell with age in boys. Serum cholesterol level of girls rose once between 11 and 13 y and fell gradually. Fish intake was positively correlated with serum omega-3 series fatty acids. Milk intake was negatively and soybean intake was positively correlated with omega-3/omega-6 series fatty acids ratio. Serum branched-chain amino acids were correlated negatively with serum polyunsaturated/saturated fatty acids ratio. CONCLUSIONS: Serum cholesterol level of children in agricultural communities in Japan has risen, and is probably affected by the change of food intake. Promoting the intake of soybean and fish, which are traditional Japanese foods, will be important in preventing atherosclerosis and ischemic heart disease. Attention should be paid to the relationship between branched-chain amino acids level in blood and fatty acids metabolism to verify the mechanism of the progress of atherosclerosis.

Effect of serum fatty acid composition on coronary atherosclerosis in Japan.

The serum lipid profiles of patient's with or without significant coronary stenosis diagnosed by coronary angiography and of control subjects were compared. The level of high-density-lipoprotein cholesterol and eicosapentaenoic acid were significantly lower in the patients with significant coronary stenosis than in the control subjects. It suggests that high-density-lipoprotein cholesterol and omega3 fatty acids may have a protective effect on the progress of coronary atherosclerosis. The frequency of eating dark-meat fish was positively associated with serum eicosapentaenoic acid and docosahexaenoic acid, omega3 fatty acids, and inversely associated with serum stearic acid and linoleic acid. The frequency of eating soybean products was positively associated with serum docosahexaenoic acid and inversely associated with serum linoleic acid. It is necessary to discuss a way to popularise a diet of dark-meat fish and soybean products as a means of preventing coronary heart disease.

Arachidonic acid metabolism to prostaglandin E2, D2, F2alpha, and I2 in the plerocercoid of Spirometra erinaceieuropaei.

Changes in the fatty acid composition of phospholipid and triglyceride fractions in Spirometra erinaceieuropaei plerocercoids were investigated after 0, 0.5, 1, 3, and 6 hr incubation at 10 C and 37 C with physiological saline containing 5 mM arachidonic acid and 10 mg/ml bovine serum albumin, pH 7.0. At 37 C, arachidonic acid was absorbed and incorporated rapidly into the triglyceride fraction (over 14.4% in composition), and decreased after 2-3 hr; at 10 C, the amount of triglyceride increased slowly and continued to a maximum of 12.9% during 6 hr of incubation. We used a simplified method to extract and purify prostaglandins from the plerocercoid of S. erinaceieuropaei. Prostaglandins were quantified using gas chromatography-mass spectrometry. Prostaglandin E2, PGD2, PGF2alpha, and 6-keto-PGF1alpha were detected under different incubation conditions. In the dose-dependent experiment, PGD2 was detected in plerocercoids incubated with 0.5, 1, 2, and 5 mM arachidonic acid, pH 7.0, at 25 C; PGE2 was detected with 2 and 5 mM arachidonic acid. In the time-dependent experiment, where plerocercoids were incubated with 5 mM arachidonic acid, pH 7.0 at 25 C, PGF2alpha was first detected at 15 min; thereafter, 6-keto-PGF1alpha was detected at 30 min and PGD2 and PGE2 were detected at 1 hr. Thromboxane B2 was not detected in either the dose-dependent or time-dependent experiments, and only PGE2 was detected in the incubation medium with 5 mM arachidonic acid at 1 hr. These results reveal that when plerocercoids change from reptilian to mammalian hosts, they are able to absorb and modify arachidonic acid bound to albumin and generate prostaglandins under suitable conditions. Prostaglandins exhibit potent biological functions for immunoresponses that may be relevant to parasitism and the success of larva migrans in S. erinaceieuropaei.

[Participatory action-research in the development of health policies for Izumo City, Japan].

The method of participatory action-research has been introduced in planning and implementing health and welfare projects for the elderly in Izumo City since 1993. Public health researchers have participated in the projects with other related administrative sectors, citizen and non-profit organizations. This study analyzes the establishment and implementation of health policies of Izumo from the following five aspects: policy-making, priority, education, intersectoral cooperation and community development. The participatory action-research method was useful for developing the ability of health-staff to work with citizens; for integrating community care and community development; for using community resources effectively for primary health care; and for developing community-based education of citizens, medical students and health staff. This method can be and should be adopted for strengthening the policy-making ability of public health researchers who learn through cooperation with citizens and non-profit organizations.

Phase I study of sequentially administered topoisomerase I inhibitor (irinotecan) and topoisomerase II inhibitor (etoposide) for metastatic non-small-cell lung cancer.

We conducted a phase I study of irinotecan (CPT-11) and etoposide (VP-16) given sequentially to untreated patients with metastatic non-small-cell lung cancer. Arm A: CPT-11 was given over 90 min on days 1-3 and VP-16 was given over 60 min on days 4-6. Arm B: VP-16 was given on days 1-3 and CPT-11 on days 4-6. G-CSF was given to all patients daily on days 7-17. Twenty-seven patients were entered randomly at the two arms. The major dose-limiting toxicities in arms A and B were granulocytopenia and diarrhoea. Transient elevations of transaminases and bilirubin were observed in both arms. The degree of the toxicities did not differ between the two arms. The maximum tolerated doses (MTDs) were 60 mg m-2 CPT-11 and 60 mg m-2 VP-16 in both arms. Of the 13 patients who received more than two cycles, two out of five achieved partial response (PR) at the first level of arm A and one out of four achieved PR at the second level of arm B. We conclude that these schedules of sequential CPT-11 and VP-16 administration were inappropriate because of severe toxicities.

Inhibitory effect of nicotinamide to paraquat toxicity and the reaction site on complex I.

The inhibitory effect and mechanism of action of nicotinamide to paraquat toxicity were studied in male Sprague-Dawley rats. Proteins of submitochondrial particles (SMP), especially of mol. wt. 25-30 kDa, in rat lungs were destroyed by paraquat radicals, and aggregated protein bands approximately 100 kDa were observed by polyacrylamide electrophoresis. The competitive inhibition effects were observed of nicotinamide on NADH oxidation by paraquat via SMP in rat lungs and the Ki was 9.3 mM. The inhibitory effects of nicotinamide on lipid peroxidation by paraquat with rat lung and liver SMP were verified. The times of occurrence of dyspnea and death in rats after paraquat exposure were delayed by nicotinamide administration. The activity of NADH: ubiquinone reaction of NADH:ubiquinone oxidoreductase (complex I) in rat lung was reduced 24 h after paraquat exposure, and was protected by nicotinamide. The activity of NADH:ferricyanide reaction of complex I was, however, reduced by administration not only of paraquat but also nicotinamide. These results imply that nicotinamide is inhibitory to paraquat toxicity. Nicotinamide, paraquat, and ferricyanide may react at overlapping sites on complex I.

Effects of organophosphorous compounds on fatty acid compositions and oxidative phosphorylation system in the brain of rats.

Effects of organophosphorous compounds, O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate (MEP), O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN) and O-(4-cyanophenyl) O-ethyl phenylphosphonothioate (CYP), on the fatty acid composition and the subsequent effects on the oxidative phosphorylation system in the brain of rats were studied. After 6 days exposure in pesticides, polyunsaturated fatty acids in brain free fatty acid fractions of CYP treated rats decreased, and the unsaturation index in them were lower than those in the control rats. The polyunsaturated/saturated ratio (P/S ratio) in brain total lipids of CYP treated rats was lower than that in the control. The fatty acid composition in the brain of EPN treated rats had the same inclination as that of CYP treated rats. In the P/S ratio and unsaturation index in serum no difference was observed between CYP treated rats and the control, therefore CYP could affect the fatty acid composition in the rat brain directly. Free fatty acid contents in the brain of EPN and CYP treated rats decreased after 6 days exposure. Activities of complex I of brains were significantly higher in the EPN and CYP exposed rats than in the control rats in spite of the fact that no difference of ATP productivity was observed between them. These results suggest that EPN and CYP may affect the free fatty acid content, especially the polyunsaturated fatty acid content and consequently the enzyme activities in the oxidative phosphorylation system in the brain. Those phenomena, however, were not observed after 28 days exposure in pesticide, therefore those effects may be a passing phenomenon in an acute stage.

Effects of paraquat on mitochondrial electron transport system and catecholamine contents in rat brain.

The effects of paraquat on rat brain were studied. Activities of complex I (NADH: ubiquinone oxidoreductase) in mitochondrial electron transport system, lipid peroxidation and the amount of catecholamines in rat brain were measured after acute paraquat exposure. Complex I activities were significantly lower and lipid peroxides were higher in the brains of a paraquat-treated group than in those of a control group. Lipid peroxide in rat serum, however, did not increase after paraquat exposure. A study of the time dependency of paraquat effects disclosed that mitochondrial complex I activities in rat brain as well as those in rat lung and liver gradually decreased prior to the appearance of respiratory dysfunction. As compared to controls, the dopamine in rat striatum was significantly lower in the paraquat-treated group. These results suggest that paraquat after crossing the blood-brain barrier might be reduced to the radical in rat brain, which may damage the brain tissue, especially dopaminergic neurons in striatum. We therefore propose that cerebral damage should be taken into consideration on paraquat exposure. Patients may therefore need to be followed up after exposure to high doses of paraquat.

Radical formation site of cerebral complex I and Parkinson's disease.

Paraquat was reduced to the paraquat radical via complex I in bovine cerebral mitochondria and accelerated lipid peroxidation. Thirty-kilodalton subunit of complex I was considered to be the radical formation site, because of its marked destruction by the paraquat radical. The lipid peroxidation by the paraquat radical was suppressed not only by superoxide dismutase (SOD) but also by mannitol. The destruction of complex I subunits via lipid peroxidation must have been caused by the hydroxyl radical which was formed from the superoxide radical. The same phenomenon was observed by using 1-methylnicotinamide (MNA), which contains the same partial structure as paraquat in itself and is metabolized from nicotinamide in a living body. We observed NADH oxidation by MNA via cerebral complex I (Km = 26.3 mM), and MNA destroyed some complex I subunits, especially 30-kilodalton protein. Paraquat might be useful for studying the pathogenesis of Parkinson's disease (PD) in vitro, and MNA is expected to be one of the causal substances of PD from the viewpoint of the oxidative stress theory.

[Development of quality of life (QOL) questionnaire for use of lung cancer patients in palliative therapy--study of validity and reliability. No.1].

We investigated the validity and reliability of QOL questionnaire for lung cancer patients in palliative therapy. The questionnaire covered twelve items: appetite, feelings, sleep, mental and physical fatigue, pain, anxiety, daily activity, abdominal and respiratory conditions, linear and face scales. The data were collected from 65 patients and analyzed with principal component analysis and correlation analysis. 1) The percentage of complete answers was 81.5%. 2) Appetite, feelings, sleep, mental fatigue, anxiety and mental scale, pain, respiratory condition, abdominal condition, physical fatigue and physical scale, and satisfied internal consistency. 3) The test-retest reliability was satisfied 4) The inquiry items were grouped into physical, mental and activity scales, and these scales belonged to different dimension. 5) There were correlations between a linear scale, face scale, total score and items. 6) In concurrent validity, there were correlations between performance status and the activity scale, SDS, STAI and the mental scale. 7) In sensitivity, the total score and face score were worst within one week after chemotherapy, and then recovered. This questionnaire was indeed valid and reliable for use as a QOL questionnaire for lung cancer patients in palliative therapy.

Mechanism of cytotoxicity of paraquat. III. The effects of acute paraquat exposure on the electron transport system in rat mitochondria.

The effects of acute paraquat exposure on mitochondrial function in rat lung were studied. The paraquat dose-response study and time-effective study were performed to prove our hypothesis, enzyme toxicity especially in electron transport system following lipid peroxidation of mitochondrial inner membrane. In dose-response study, lipid peroxidation was increased by high dose paraquat exposure (40 mg/kg body weight) in rat lung, but not by low dose exposure (10 mg/kg body weight). But paraquat inhibited NADH:ubiquinone oxidoreductase (complex I) activities, especially NADH:ubiquinone reaction (NQR), even in low dose exposure. The lipid peroxide concentration did not correspond to the damage of complex I activity. In paraquat time-effective study, both lung and blood lipid peroxides increased after 6 h of paraquat exposure, decreased after 12 and 24 h and increased again after 48 h. After first peak of lipid peroxidation, NQR velocity decreased earlier than NADH:ferricyanide reaction (NFR) velocity. From these results, the cytotoxicity via mitochondrial dysfunction by acute paraquat exposure might be caused by complex I toxicity following lipid peroxidation of mitochondrial inner membrane.

[Changes in ischemic findings in ECG, obesity and serum lipids in the residents of an agricultural area in Shimane Prefecture].

Changes in ischemic findings in ECG, obesity and serum lipids and the relations among them were investigated in Sada Town, a typical agricultural and mountaineous area in Shimane Prefecture, where the mortality rate from ischemic heart disease had increased. Compared with 1983, the percentage of ischemic findings in ECG in 1990 increased, and a marked elevation of total serum cholesterol level, and relative and absolute lowering of HDL cholesterol level were observed in 1990. By observing the relations of total serum cholesterol, HDL cholesterol and the atherosclerotic index to the ischemic findings in electrocardiograms (ECG), it was found that males showed a significantly lower HDL cholesterol level and higher atherosclerotic index than the control group. In males, a relation between obesity and ischemic findings in ECG was observed. In females, a relation between obesity and the atherosclerotic index was observed; however, no relations were observed between ischemic findings and obesity. These results support the hypothesis that a marked elevation of the total serum cholesterol level, relative and absolute lowering of the HDL cholesterol level and obesity have an effect on the recent increase of male deaths due to ischemic heart disease in Sada Town, Shimane Prefecture.