Masafumi Ito

Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells

Tumor suppressor p53 transcriptionally regulates expression of microRNA-34a, which confers translational inhibition and mRNA degradation of genes involved in cell cycle control and apoptosis. In various cancers, miR-34a expression is lost or reduced. Here, we investigated the role of miR-34a in prostate cancer cell lines. MiR-34a expression was markedly reduced in p53-null PC3 cells and p53-mutated DU145 cells compared with LNCaP cells expressing wild-type p53. In PC3 cell, ectopic expression of miR-34a decreased the SIRT1 mRNA and protein levels as well as protein levels of known direct target genes. Reporter assays revealed that miR-34a-induced SIRT1 inhibition occurred at the transcriptional but not post-transcriptional level despite the presence of a potential miR-34a binding site within its 3-UTR. Ectopic miR-34a expression resulted in cell cycle arrest and growth inhibition and attenuated chemoresistance to anticancer drug camptothecin by inducing apoptosis, suggesting a potential role of miR-34a for the treatment of p53-defective prostate cancer.

MiR-148a Attenuates Paclitaxel Resistance of Hormone-refractory, Drug-resistant Prostate Cancer PC3 Cells by Regulating MSK1 Expression

MicroRNAs are involved in cancer pathogenesis and act as tumor suppressors or oncogenes. It has been recently reported that miR-148a expression is down-regulated in several types of cancer. The functional roles and target genes of miR-148a in prostate cancer, however, remain unknown. In this report, we showed that miR-148a expression levels were lower in PC3 and DU145 hormone-refractory prostate cancer cells in comparison to PrEC normal human prostate epithelial cells and LNCaP hormone-sensitive prostate cancer cells. Transfection with miR-148a precursor inhibited cell growth, and cell migration and invasion, and increased the sensitivity to anti-cancer drug paclitaxel in PC3 cells. Computer-aided algorithms predicted mitogen- and stress-activated protein kinase, MSK1, as a potential target of miR-148a. Indeed, miR-148a overexpression decreased expression of MSK1. Using luciferase reporter assays, we identified MSK1 as a direct target of miR-148a. Suppression of MSK1 expression by siRNA, however, showed little or no effects on malignant phenotypes of PC3 cells. In PC3PR cells, a paclitaxel-resistant cell line established from PC3 cells, miR-148a inhibited cell growth, and cell migration and invasion, and also attenuated the resistance to paclitaxel. MiR-148a reduced MSK1 expression by directly targeting its 3′-UTR in PC3PR cells. Furthermore, MSK1 knockdown reduced paclitaxel-resistance of PC3PR cells, indicating that miR-148a attenuates paclitaxel-resistance of hormone-refractory, drug-resistant PC3PR cells in part by regulating MSK1 expression. Our findings suggest that miR-148a plays multiple roles as a tumor suppressor and can be a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.

MiR-34a attenuates paclitaxel-resistance of hormone-refractory prostate cancer PC3 cells through direct and indirect mechanisms

Patients with hormone‐refractory prostate cancer are treated with taxane drugs, but eventually become drug resistant. We aimed to elucidate the molecular mechanisms underlying paclitaxel resistance of hormone‐refractory prostate cancer with a special focus on the roles of miR‐34a and SIRT1.

A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells

SIRT1, which belongs to the family of type III histone deacetylase, is implicated in diverse cellular processes. We have determined the expression levels of SIRT1 in human prostate cancer cell lines and have examined the roles of SIRT1 in cell growth and chemoresistance. SIRT1 expression was markedly up-regulated in androgen-refractory PC3 and DU145 cells compared with androgen-sensitive LNCaP cells and its expression level was correlated with cell growth in PC3 cells. Treatment with a SIRT1 inhibitor, sirtinol, inhibited cell growth and increased sensitivity to camptothecin and cisplatin. Silencing of SIRT1 expression by siRNA also suppressed cell proliferation and reduced camptothecin resistance in PC3 cells, mimicking the chemosensitizing effect caused by sirtinol. Also in DU145 cells, sirtinol treatment enhanced sensitivity to camptothecin and cisplatin. These results suggest that up-regulation of SIRT1 expression may play an important role in promoting cell growth and chemoresistance in androgen-refractory PC3 and DU145 cells.

Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease

Molecular hydrogen serves as an antioxidant that reduces hydroxyl radicals, but not the other reactive oxygen and nitrogen species. In the past year, molecular hydrogen has been reported to prevent or ameliorate eight diseases in rodents and one in human associated with oxidative stress. In Parkinsons disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra. We examined effects of approximately 50%-saturated molecular hydrogen in drinking water before or after the stereotactic surgery on 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinsons disease. Methamphetamine-induced behavioral analysis showed that molecular hydrogen prevented both the development and progression of the nigrostrital degeneration. Tyrosine hydroxylase staining of the substantia nigra and striatum also demonstrated that pre- and post-treatment with hydrogen prevented the dopaminergic cell loss. Our studies suggest that hydrogen water is likely able to retard the development and progression of Parkinsons disease.

Molecular hydrogen suppresses FcεRI-mediated signal transduction and prevents degranulation of mast cells

Molecular hydrogen ameliorates oxidative stress-associated diseases in animal models. We found that oral intake of hydrogen-rich water abolishes an immediate-type allergic reaction in mice. Using rat RBL-2H3 mast cells, we demonstrated that hydrogen attenuates phosphorylation of the FcepsilonRI-associated Lyn and its downstream signal transduction, which subsequently inhibits the NADPH oxidase activity and reduces the generation of hydrogen peroxide. We also found that inhibition of NADPH oxidase attenuates phosphorylation of Lyn in mast cells, indicating the presence of a feed-forward loop that potentiates the allergic responses. Hydrogen accordingly inhibits all tested signaling molecule(s) in the loop. Hydrogen effects have been solely ascribed to exclusive removal of hydroxyl radical. In the immediate-type allergic reaction, hydrogen exerts its beneficial effect not by its radical scavenging activity but by modulating a specific signaling pathway. Effects of hydrogen in other diseases are possibly mediated by modulation of yet unidentified signaling pathways. Our studies also suggest that hydrogen is a gaseous signaling molecule like nitric oxide.

Mitochondrial DNA Haplogroup D4a Is a Marker for Extreme Longevity in Japan

We report results from the analysis of complete mitochondrial DNA (mtDNA) sequences from 112 Japanese semi-supercentenarians (aged above 105 years) combined with previously published data from 96 patients in each of three non-disease phenotypes: centenarians (99–105 years of age), healthy non-obese males, obese young males and four disease phenotypes, diabetics with and without angiopathy, and Alzheimers and Parkinsons disease patients. We analyze the correlation between mitochondrial polymorphisms and the longevity phenotype using two different methods. We first use an exhaustive algorithm to identify all maximal patterns of polymorphisms shared by at least five individuals and define a significance score for enrichment of the patterns in each phenotype relative to healthy normals. Our study confirms the correlations observed in a previous study showing enrichment of a hierarchy of haplogroups in the D clade for longevity. For the extreme longevity phenotype we see a single statistically significant signal: a progressive enrichment of certain “beneficial” patterns in centenarians and semi-supercentenarians in the D4a haplogroup. We then use Principal Component Spectral Analysis of the SNP-SNP Covariance Matrix to compare the measured eigenvalues to a Null distribution of eigenvalues on Gaussian datasets to determine whether the correlations in the data (due to longevity) arises from some property of the mutations themselves or whether they are due to population structure. The conclusion is that the correlations are entirely due to population structure (phylogenetic tree). We find no signal for a functional mtDNA SNP correlated with longevity. The fact that the correlations are from the population structure suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations.

Women With Mitochondrial Haplogroup N9a Are Protected Against Metabolic Syndrome

To identify mitochondrial haplogroups that confer resistance against or susceptibility to metabolic syndrome, we performed a large-scale association study on 1,337 unrelated Japanese individuals, including 871 subjects with metabolic syndrome and 466 control subjects. Metabolic syndrome was diagnosed according to modified National Cholesterol Education Program Adult Treatment Panel III guidelines, using the cutoff point for obesity as a BMI of ≥25 kg/m2 instead of waist circumference. The genotypes for 25 polymorphisms in the coding region of the mitochondrial genome were determined, and the haplotypes were classified into 10 major haplogroups, i.e., F, B, A, N9a, M7a, M7b, G1, G2, D5, and D4. Multivariate logistic regression analysis revealed that the haplogroup N9a was significantly associated with resistance against metabolic syndrome in women with an odds ratio (OR) of 0.21 (95% CI 0.07–0.58, P = 0.0042). Women with haplogroups G1 and D5 tended to be resistant against metabolic syndrome with an OR of 0.22 (0.06–0.68, P = 0.0129) for G1 and with an OR of 0.32 (0.10–0.96, P = 0.0469) for D5, respectively. These results indicate that mitochondrial haplogroup N9a may be a protective factor against metabolic syndrome in Japanese women.

Sirtuin 1 overexpression mice show a reference memory deficit, but not neuroprotection.

Sirtuin 1 (SIRT1) is the closest mammalian homologue of yeast silent information regulator 2 (Sir2) and has a role in lifespan modulation. Reportedly, SIRT1 is also linked to neurodegenerative diseases. However, there are limited studies that report the relation between SIRT1 and neurodegenerative diseases using in vivo transgenic (Tg) methods. In the present study, we generated neuron-specific enolase (NSE) SIRT1 Tg mice that overexpress human SIRT1 in neurons. We examined possible neuroprotective effects of SIRT1 overexpression and compared their higher brain functions with those of wild-type (WT) mice. Overexpression of SIRT1 did not have any neuroprotective effects against the neuronal damage induced by ischemia or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, SIRT1 Tg mice exhibited a reference memory deficit. These findings suggest that an excessive expression of SIRT1 might induce the memory deficit in mice, but not neuroprotective effects.

Enrichment of longevity phenotype in mtDNA haplogroups D4b2b, D4a, and D5 in the Japanese population

We report new results from the re-analysis of 672 complete mitochondrial (mtDNA) genomes of unrelated Japanese individuals stratified into seven equal sized groups by the phenotypes: diabetic patients, diabetic patients with severe angiopathy, healthy non-obese young males, obese young males, patients with Alzheimer’s disease, patients with Parkinson’s disease and centenarians. Each phenotype had 96 samples over 27 known haplogroups: A, B4a, B4b, B4c, B*, B5, D*, F1, F2, M*, M7a, M7b, M8, M9, D4a, D4b1, D4b2, D4d, D4e, D4g, D4h, D5, G, Z, M*, N9a, and N9b. A t-test comparing the fraction of samples in a haplogroup to healthy young males showed a significant enrichment of haplogroups D4a, D5, and D4b2 in centenarians. The D4b2 enrichment was limited to a subgroup of 40 of 61 samples which had the synonymous mutation 9296Cxa0>xa0T. We identified this cluster as a distinct haplogroup and labeled it as D4b2b. Using an exhaustive procedure, we constructed the complete list of “mutation patterns” for centenarians and showed that the most significant patterns were in D4a, D5, and D4b2b. We argue that if a selection for longevity appeared only once, it was probably an autosomal event which could be dated to after the appearance of the D mega-group but before the coalescent time of D4a, D5, and D4b2b. Using a simple procedure, we estimated that this event occurred 24.4xa0±xa00.9 kYBP.