Excessive Oxidative Stress in the Synergistic Effects of Shikonin on the Hyperthermia-Induced Apoptosis.

BACKGROUND: Hyperthermia (HT) has been used widely for cancer therapy, and the development of modern devices has made it more efficient. Shikonin (SHK) is a natural naphthoquinone derivative from a Chinese herb. Although the anticancer properties of SHK are evident, the underlying molecular mechanisms are not fully understood. OBJECTIVE: In this study, the effects of combining low doses of SHK with mild HT were investigated in the U937 cell line. METHODS: The cells were subjected to HT at 44°C for 10 min with or without SHK pretreatment, and parameters reflecting apoptosis, ROS generation and intracellular calcium elevation were evaluated by using DNA fragmentation, flow cytometry, and western blot analyses. RESULTS: SHK 0.5 µM significantly enhanced HT-induced apoptosis as indicated by DNA fragmentation and caspase-3 activation with increased generation of ROS and elevation of intracellular calcium. The combined treatment also synergistically activated proapoptotic proteins and inactivated anti-apoptotic proteins. Furthermore, the phosphorylation of JNK and PKC- δ and the dephosphorylation of ERK and AKT were the upstream effects that may have compounded the induction of apoptosis. The modulatory effects of HT and SHK were abrogated with the employment of NAC and JNK-IN-8 by inactivating the MAPK pathway and cleavage of caspase-3. Intracellular calcium was also elevated and was found to be responsible for the induction of cell death evident by the DNA fragmentation with or without the employment of BAPTA-AM. CONCLUSION: Conclusively, this study provides persuasive evidence that SHK in combination with HT is a propitious therapeutic way for augmentation of apoptosis and hence suggest a novel strategy for treating cancers.

Serum n-3 polyunsaturated fatty acids and psychological distress in early pregnancy: Adjunct Study of Japan Environment and Children's Study.

N-3 polyunsaturated fatty acids (PUFAs), especially long-chain types such as docosahexaenoic acid, are important nutrients in pregnancy, but the relationship between n-3 PUFA levels and perinatal and postnatal depression remains controversial. This study examined the possible relationship between serum n-3 PUFA levels and psychological distress among expectant mothers in early pregnancy. Data and specimen samples were obtained in a birth cohort study started at Toyama Regional Center in July 2012 as an adjunct study of the Japan Environment and Children's Study. Blood samples were collected at 9-14 weeks' gestation (75% of samples) or after 15 weeks (25%). Subjects with a Kessler Psychological Distress Scale score (K6) ⩾ 9 were assigned to the psychological distress group (n=283). The control group (n=283) was matched for age, educational level and family income. Fatty acid composition was determined from serum samples by gas chromatography. Associations between fatty acid levels and incident psychological distress were evaluated by logistic regression. After adjusting for possible confounders, eicosapentaenoic acid showed an inverse association with risk of psychological distress, with an odds ratio of 0.47 (95% confidence interval: 0.30, 0.73) for the highest tertile. This inverse association remained even after applying a higher cutoff score (K6 ⩾ 13) indicating severe psychological distress (74 pairs). We believe this is the first study to reveal the associations between serum n-3 PUFAs and risk of psychological distress in early pregnancy. Further research is required to verify the causality of these associations.

Fish consumption and depressive symptoms in undergraduate students: A cross-sectional analysis.

BACKGROUND: Emerging evidence suggests that fish consumption may have beneficial effects on mood disorders. However, no study has been reported on this issue in young adults to date. The aim of this study was to investigate the relationship between fish consumption and depressive symptoms in Japanese undergraduate students. METHODS: The 20-item Center for Epidemiologic Studies Depression Scale was used to measure depressive symptoms with a cut-off score of 16. A total of 4190 completed questionnaires (from 2124 men and 2066 women) were received for analysis. RESULTS: Multivariate logistic analysis showed that fish intake was inversely associated with risk of depressive symptoms in undergraduate students. After adjustment for possible confounders, the odds-ratios (95% confidence intervals) for fish intake 1-2 times/month, 1-2 times/week, 3-4 times/week, and almost every day (compared with "almost never") were 0.78 (0.62-0.99), 0.70 (0.56-0.87), 0.67 (0.53-0.85) and 0.65 (0.46-0.92), respectively. This association tended to be stronger in women than in men. CONCLUSIONS: Frequent fish consumption in undergraduate students seems to moderate depressive symptoms. Further research is warranted to clarify the causality.

A case of apolipoprotein C-II deficiency with coronary artery disease.

A 56-year-old male with apolipoprotein C-II deficiency experienced a myocardial infarction without pancreatitis. A coronary angiogram showed complete occlusions of both the right and circumflex coronary arteries. His serum lipid levels were as follows: fasting total cholesterol 3.15 mmol/l; postprandial total cholesterol 3.62 mmol/l; fasting triglycerides 1.46 mmol/A; postprandial triglycerides 6.14 mmol/l; fasting high-density lipoprotein-cholesterol 0.47 mmol/l; and postprandial high-density lipoprotein cholesterol 0.36 mmol/l. His fasting level of plasma apolipoprotein C-II was 0.005 g/l, but his plasma levels of other apolipoproteins were within normal ranges. A DNA sequence analysis of the apolipoprotein C-II gene showed no mutations in exon 1, 2, 3, or 4, where most gene mutations related to apolipoprotein C-II deficiency occur. We report this patient's very rare heterozygous apolipoprotein C-II deficiency with coronary artery disease. Although this patient had some risk factors for coronary artery disease, coronary atherosclerosis in this patient might have occurred as a result of lipoprotein abnormalities caused by at least one mutation in the apolipoprotein C-II gene.

Plasma monocyte chemoattractant protein-1 and pulmonary vascular resistance in chronic thromboembolic pulmonary hypertension.

The pathogenesis of severe pulmonary hypertension seems to be related to inflammatory response in diseased sites. Monocyte chemoattractant protein-1 (MCP-1) has been reported to play a role in the development of congestive heart failure. In this immunological response, activation and migration of leukocytes including macrophages to the inflammatory region are important factors. We hypothesized that the severity of pulmonary hypertension may be related to MCP-1, which is thought to be upregulated by blood pressure or shear stress in pulmonary vasculature as well as by immunological and inflammatory reactions in chronic thromboembolic pulmonary hypertension (CTEPH). Circulating levels of MCP-1, interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) were measured by sandwich ELISA in 14 patients with CTEPH. The plasma level of MCP-1 was significantly correlated with pulmonary vascular resistance. In IL-1beta and TNF-alpha, on the other hand, there was no correlation between cytokines and pulmonary hemodynamics. Pathological specimens obtained from the patients with CTEPH undergoing thromboendarterectomy demonstrated immunoreactivity of MCP-1 in endothelium, smooth muscle cells, and macrophages within neointima in the hypertensive large elastic pulmonary artery. We conclude that MCP-1 is upregulated in the remodeling of pulmonary arteries in close association with increased pulmonary vascular resistance in CTEPH.

[How do universities confront and manage environmental problems?].

Occupational medicine is concerned with the recognition and prevention of diseases related to the work environment. The special tools, namely, epidemiology, toxicology, and public health and clinical expertise, are joined by another specialty, environmental medicine. The Environmental Science Center (ESC) of the University of Tokyo was established in April 1975 for the purpose of treating chemically-hazardous wastes deriving from the university. The ESC houses various sections including research, education, operations, management, and also a waste-collection and-treatment division. In this review, the author intends to summarize the activity of the ESC, and then review the approaches we have applied in order to deal with the environmental problems we have faced.

WISP-2 as a novel estrogen-responsive gene in human breast cancer cells.

In order to search for novel estrogen-responsive genes, we performed serial analysis of gene expression (SAGE) for estrogen-treated MCF-7 human breast cancer cells. SAGE analysis of 31,000 and 30,856 tags from non-treated and 17 beta-estradiol (E2)-treated cells for 24 h, respectively, facilitated the identification of 15,037 different transcripts. Comparison of these two SAGE libraries indicated a remarkable similarity in expression profiles. Among the identified transcripts, four genes were found to be markedly increased for E2-treated cells compared with control cells. Three of the transcripts were cathepsin D, pS2 and high mobility group 1 protein, which have been described as estrogen-inducible genes. The fourth gene was WISP-2 (Wnt-1 inducible signaling pathway protein 2) which has recently been reported as an up-regulated gene in the mammary epithelial cell line C57 MG transformed by the Wnt-1 oncogene. The increase in WISP-2 mRNA was completely prevented by co-incubation with a pure anti-estrogen ICI 182,780, but not by coincubation with cycloheximide, indicating that WISP-2 is directly regulated by the estrogen receptor. The WISP-2 gene was also induced by treating with environmental estrogens, such as bisphenol-A or nonylphenol. This study represents the first comprehensive gene expression analysis of estrogen-treated human breast cancer cells.

Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer.

Tumor cells stimulate the formation of stroma that secretes various mediators pivotal for tumor growth, including growth factors, cytokines, and proteases. However, little is known about the local regulation of these soluble mediators in the human tumor microenvironment. In this study, the local expression of cytokines, chemokines, and angiogenic factors was investigated in primary breast cancer tissue. The concentrations of interleukin (IL)-1, IL-4, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-8, macrophage chemoattractant protein (MCP)-1, epithelial-neutrophil activating peptide-78, vascular endothelial growth factor, and thymidine phosphorylase (TP) were measured in 151 primary breast cancer extracts by ELISA. Tumor-associated macrophages (TAMs) were also examined by immunohistochemistry with anti-CD68 antibodies. The correlation between soluble mediators and the relationship between TAM count and soluble mediators were evaluated. MCP-1 concentration was correlated significantly with the level of vascular endothelial growth factor, TP, TNF-alpha, and IL-8, which are potent angiogenic factors. IL-4 concentration was correlated significantly with IL-8 and IL-10. On the other hand, an inverse association was observed between TP and IL-12. The level of MCP-1 was associated significantly with TAM accumulation. In the immunohistochemical analysis, MCP-1 expression was observed in both infiltrating macrophages and tumor cells. Prognostic analysis revealed that high expression of MCP-1, as well as of VEGF, was a significant indicator of early relapse. These findings indicate that interaction between the immune network system and angiogenesis is important for progression of human breast cancer, and that MCP-1 may play an important role in the regulation of angiogenesis and the immune system.

TNF-alpha is a potent inducer for IFN-inducible protein-10 in hepatocytes and unaffected by GM-CSF in vivo, in contrast to IL-1beta and IFN-gamma.

We have recently shown that IFN-inducible protein 10 (IP-10), a member of the CXC chemokine family, is induced in hepatocytes surrounded by infiltrative mononuclear cells in human livers with chronic hepatitis. Hence, we examined the kinds of stimuli that can induce IP-10 expression in hepatocytes in vivo. While the liver expressed three chemokine genes (IP-10, JE/MCP-1, KC/GRO) in a tissue-specific fashion following systemic treatment with pro-inflammatory cytokines, IP-10 mRNA expression showed the most marked liver-specificity. Pretreatment with GM-CSF selectively inhibited IL-1beta, but not TNF-alpha-induced IP-10 mRNA expression. In situ hybridization analysis in the liver and Northern hybridization analysis in isolated liver cell fractions from rodents treated with pro-inflammatory cytokines revealed cellular sources of chemokine expression. IP-10 mRNA expression in hepatocytes was induced by i.v. administration of TNF-alpha, and to a much lesser extent in response to IL-1beta and IFN-gamma, whereas Kupffer cells and endothelial cells expressed IP-10 mRNA equivalently in response to these three stimuli. On the other hand, JE/MCP-1 mRNA expression was detected only in non-parenchymal cells in response to TNF-alpha and IL-1beta, but not in response to IFN-gamma. KC/GRO mRNA expression was also induced mainly in sinusoidal cells by treatment with TNF-alpha or IL-1beta, although it was detected to a lesser extent in hepatocytes. Our results demonstrated that chemokine induction is stimulus-, tissue- and cell type-specific and that IP-10 (but not MCP-1) is inducible in hepatocytes by TNF-alpha most potently, even in the presence of GM-CSF, suggesting the specific role of TNF-alpha-induced IP-10 on intralobular mononuclear infiltration in chronic hepatitis.

Molecular analysis of the inhibition of monocyte chemoattractant protein-1 gene expression by estrogens and xenoestrogens in MCF-7 cells.

Xenoestrogens (XEs) are a diverse group of chemicals that mimic estrogenic actions and may have adverse effects on human health. The influence of these compounds on cytokine production or immune system function remains unclear. In this study we have examined the effects of 17beta-estradiol (E2) and XEs on chemoattractant cytokine (chemokine) production and analyzed the molecular mechanism. Monocyte chemoattractant protein-1 (MCP-1), also termed monocyte chemotactic and activating factor, is a member of the chemokine family and attracts mainly blood monocytes. Human mammary tumor cell line MCF-7 cells produce a large quantity of MCP-1 in response to interleukin-1alpha (IL-1alpha). Addition of E2 to MCF-7 cells inhibited MCP-1 production in a dose-dependent manner. XEs, bisphenol A, and NP also inhibited MCP-1 production, although the potency was 3-4 orders of magnitude lower than that of E2. E2, bisphenol A, and NP inhibited MCP-1 messenger RNA expression in MCF-7 cells. Two closely located nuclear factor-kappaB sites, A1 and A2, have been identified in the promoter of the human MCP-1 gene. A luciferase construct containing this enhancer region (pGLM-ENH) was activated by IL-1alpha, and a mutation at either the A1 or A2 site resulted in a loss of IL-1alpha responsiveness. Treatment with E2 or XEs decreased the IL-1alpha-inducible pGLM-ENH luciferase activity significantly. In an electrophoretic mobility shift assay and supershift analysis, we found that treatment with E2 or XEs diminished the IL-1alpha-induced complex formation with both A1 and A2 probes, which was identified immunochemically to consist of nuclear factor-kappaB, p50, and p65. The IL-1alpha-induced p50/c-Rel complex to the A2 probe was also, to a lesser extent, decreased by E2 or XE treatment. The effects of E2 and XEs on the expression of MCP-1 seem to be much more dramatic than the effects of these agents on the promoters used in the luciferase assay, suggesting the involvement of an additional site(s) of the promoter region of the MCP-1 gene or posttranscriptional regulation of MCP-1 gene expression by E2 and XEs. This work represents the first report describing possible regulation of immune system function by XEs through inhibiting chemokine production.

Increase in circulating levels of monocyte chemoattractant protein-1 with aging.

Monocyte chemoattractant protein-1 (MCP-1) is a member of chemokines with chemoattractant activity for monocytes, T cells, mast cells, and basophils. Precursor mRNA or protein was detected at high levels in the lesions of several diseases, such as pulmonary fibrosis, rheumatoid arthritis, atherosclerosis, and some types of tumors. The regulation of MCP-1 production and the role of this chemokine in pathophysiologic states, however, remain largely unknown. In this study, using an enzyme-linked immunosorbent assay (ELISA), we measured the circulating MCP-1 levels in 405 healthy Japanese subjects of various ages, eliciting a profound age-dependent MCP-1 increase. Multivariate regression analysis revealed that significant predictors of MCP-1 value for males were age (p = 0.033) and serum triglyceride (p = 0.039). For females, age was also a significant predictor (p = 0.00002). One possible explanation is that the plasma MCP-1 concentration might reflect the existence of atherosclerosis, although the plasma MCP-1 concentration from patients with coronary artery disease or cerebrovascular accidents appears not to differ from age-matched, disease-free controls. This is the first report linking an increase in a particular chemokine level with aging.

Administration of a small amount of lard enhances intimal thickening in the balloon catheter injury model without affecting serum lipids.

Effects of fatty acids on intimal thickening induced by a balloon catheter injury model were investigated by feeding rabbits a small amount of either lard [L] or fish oil [F]. Serum lipids of these groups were not different from those of basal diet-fed rabbits [controls] after 4 weeks of feeding. Serum saturated fatty acids such as 14:0, 16:0, and 18.0 were significantly greater in the L-fed rabbits compared with controls, but those of the aorta were not significantly different. Fatty acid composition of the F-fed rabbits was only different from that of the controls in that n-3 fatty acids slightly increased. The mean and maximum intimal thickening 2 weeks after ballooning, carried out 2 weeks after feeding, were significantly higher in the carotid arteries of the L-fed rabbits than in the controls. The intimal thickening of the F-fed rabbits did not significantly differ from that of the controls. These results suggest that lard promotes the formation of the smooth muscle cell dominant type of arteriosclerosis without affecting serum lipid levels.

Trans-differentiation of myoblasts to adipoblasts: triggering effects of fatty acids and thiazolidinediones.

Long-chain fatty acids (LCFA) and thiazolidinediones are potent activators of differentiation of preadipose cells. These adipogenic effects are, at least in part, mediated by nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) subfamily. This report describes the effects of these agents on the differentiation pathway of myoblasts. Exposure of C2C12 myoblasts to LCFA or thiazolidinediones prevents the formation of multinucleated myotubes and the expression of specific muscle markers, leading in parallel to the expression of a typical adipose differentiation program. Similar transdifferentiation also occurs in mouse muscle satellite cells maintained in primary cell culture. These observations indicate that PPAR activators, such as LCFA or thiazolidinediones, convert the differentiation pathway of myoblasts into that of adipoblasts. This phenomenon could explain the appearance of adipocytes into muscle which occurs in some pathological states characterized by an increase of fatty acid disposal, such as obesity or mitochondrial myopathy.

Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells.

Fatty acids and thiazolidinediones act as potent activators of the adipose differentiation program in established preadipose cell lines. In this report, the effects of these agents on the differentiation pathway of myoblasts have been investigated. Exposure of C2C12N myoblasts (a subclone of the C2C12 cell line) to thiazolidinediones or fatty acids prevents the expression of myogenin, alpha-actin, and creatine kinase, thus abolishing the formation of multinucleated myotubes. These treatments lead in parallel to the expression of a typical adipose differentiation program including acquisition of adipocyte morphology and activation of adipose-related genes. A similar transition toward the adipose differentiation pathway also occurs in mouse muscle satellite cells maintained in primary culture. Thiazolidinediones exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes are insensitive to the compounds. Continuous exposure to inducers after growth arrest is not required to maintain the adipose phenotype, but proliferation of adipose-like C2C12N cells leads to a complete reversion toward undifferentiated cells able to undergo either myogenic or adipogenic differentiation depending on the composition of culture medium. These results indicate that adipogenic inducers, such as thiazolidinediones or fatty acids, specifically convert the differentiation pathway of myoblasts into that of adipoblasts.

Treatment of obesity by personality classification-oriented program.

It is apparent that personality is related to the pathogenesis of obesity, and that understanding the personality of the patient may be a key to successful treatment of the disease. Using the Rorschach test and interviews by a psychiatrist, the types of personality were classified into four groups according to the healthiness of personalities. The judgment of healthiness was based mainly on the scores obtained from the Rorschach test. This classification revealed that the occurrence of mental and physical symptoms during therapy with a very low calorie diet (VLCD) and subsequent rebound of bodyweight were more frequently observed in patients with relatively less healthy personalities. We used this classification to adapt our program to treat obese patients. In this program, severe diet restrictions were applied to patients with relatively healthy personalities. These restrictions were applied with modifications to patients with less healthy personality, because severe restrictions would be possibly very stressful for them and would bring about an undesirable reaction. For strengthening the patients' motivation for therapy, the significance of body weight reduction was explained in different ways to patients with different types of personality. The target of bodyweight reduction, reward for patients with successful weight reduction, and the duration of therapy were set up differently for patients with different personalities types. The results showed that bodyweight rebound one or two years after treatment was reduced with the personality-oriented therapy program compared to that observed with the previous conventional therapies. Also, the incidence of psychological problems was remarkably decreased.

[Apolipoprotein C-II deficiency].

Apo C-II has a central role in triglyceride metabolism as a cofactor for lipoprotein lipase (LPL), the enzyme that catalyzes the hydrolysis of triglycerides on plasma lipoproteins. Apo C-II deficiency is a rare genetic disorder that is inherited as an autosomal recessive trait. Patients with this syndrome have marked alterations of triglyceride metabolism which include elevated fasting triglycerides, chylomicrons, and VLDL. Clinical features also include lipemia retinalis, eruptive xanthomas, and an increased incidence of pancreatitis. The initial description of the first patient with apo C-II deficiency by Breckenridge et al. established the important role of apo C-II as a cofactor for LPL. Since then, many kindreds with apo C-II deficiency have been described and the underlying molecular defect characterized.

A naturally occurring mutation at the second base of codon asparagine 43 in the proposed N-linked glycosylation site of human lipoprotein lipase: in vivo evidence that asparagine 43 is essential for catalysis and secretion.

The patient was a 20-year-old male. His fasting plasma triglyceride and cholesterol levels were 1258 mg/dl and 138 mg/dl, respectively. The lipoprotein lipase (LPL) activity and mass from postheparin plasma of the patient were 0.00 mumol/ml/h (normal range: 5.51 +/- 1.12) and 23 ng/ml (normal range: 220 +/- 42), respectively. DNA sequence analysis of the LPL gene from the patient revealed a homozygous nucleotide change: a A-->G transition at nucleotide position 383, resulting in an amino acid substitution of Ser for Asn43, which is believed to be an N-linked glycosylation site of the LPL mature protein. Expression studies of this mutant LPL cDNA produced an inactive LPL protein which was not secreted into the media.

Lipoprotein(a) is a risk factor for diabetic retinopathy in the elderly.

OBJECTIVE: To assess whether serum lipoprotein(a) is a risk factor for diabetic retinopathy in the elderly. DESIGN: A cross-sectional study. SETTING: Outpatient diabetic clinic. PATIENTS: One hundred four noninsulin-dependent diabetic patients (35 males, 69 females). Twenty-three were less than 60 years of age (middle-aged), and 81 were 60 years or older (elderly). MEASUREMENT: Levels of lipoprotein(a) (Lp(a)) and lipids were measured in fasting serum. HbA1c was also measured as an indicator of diabetic control. Other indicators possibly related to retinopathy were also checked. Retinopathy was estimated by photographs of fundi. RESULTS: Significantly higher indicators in the group with retinopathy than in the group without were: HbA1c, Lp(a), duration of diabetes, and systolic blood pressure (BP) in the total cases; HbA1c, duration of diabetes, and Lp(a) in the middle-aged; HbA1c, systolic BP, and Lp(a) in the elderly. Multiple logistic regression analysis showed that only HbA1c and Lp(a) were independent risk factors for retinopathy in all cases and in the elderly. The incidence of retinopathy was positively correlated to serum Lp(a) levels. CONCLUSION: Lp(a) is an independent risk factor for diabetic retinopathy.

Diabetic control and progression of retinopathy in elderly patients: five-year follow-up study.

OBJECTIVE: To assess whether control of diabetes mellitus is as important in the elderly as in young and middle-aged diabetic patients in terms of progression of retinopathy. DESIGN: A 5-year longitudinal cohort study. SETTING: Outpatient diabetic clinic. PATIENTS: One hundred fourteen non-insulin-dependent diabetic patients (30 males, 84 females) > or = 60 years of age. MEASUREMENTS: Retinopathy was checked at the beginning and end of the follow-up period. During the 5-year follow-up period, demographic variables, body mass index, HbA1c, blood pressure, and plasma lipids were monitored. Retinopathy was classified as follows: grade 0, no lesion; grade 1, non-proliferative retinopathy; grade 2, pre-proliferative retinopathy; grade 3, proliferative retinopathy. Progression of retinopathy during the 5-year follow-up was defined as an increase in its grade. RESULTS: At the start of the study, 13% of the patients already had retinopathy, all of grade 1. The 5-year follow-up study showed that progression of retinopathy was 23.6% in all cases, 22.2% in those with grade 0 initially, and 33.3% in those with grade 1 initially. The progression rates of retinopathy as a function of the mean HbA1c during the follow-up were as follows: lower than 7%, 2%; 7-8%, 20%; 8-9%, 40%; more than 9%, 61%. Multiple logistic regression analysis showed that, of the parameters examined, only HbA1c was a significant risk factor for progression of retinopathy. CONCLUSIONS: Control of diabetes mellitus is the most important factor associated with prevention of progression of retinopathy in elderly patients.