Analysis of microRNA-203 function in CREB/MITF/RAB27a pathway: comparison between canine and human melanoma cells.

MicroRNA (miR)-203 is downregulated and acts as an anti-oncomir in melanoma cells. Here, using human and canine melanoma cells, we elucidated the effects of miR-203 on cyclic adenosine monophosphate response element binding protein (CREB)/microphthalmia-associated transcription factor (MITF)/RAB27a pathway, which is known to be important for the development and progression of human melanoma. In this study, we showed that miR-203 directly targeted CREB1 and regulated its downstream targets, MITF and RAB27a. miR-203 significantly suppressed the growth of human and canine melanoma cells and inhibited melanosome transport through the suppression of the signalling pathway. In conclusion, miR-203 was shown to be a common tumour-suppressive miRNA in human and canine melanoma and thus to play a crucial role in the biological mechanisms of melanoma development.

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas.

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA-microarray assay and quantitative RT-PCR. Importantly, a decreased expression of miR-203 was significantly associated with a shorter survival time. Also, miR-203 and -205 were markedly down-regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR-205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR-203 is a new prognostic factor in canine oral MMs and that miR-205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.

socs7, a target gene of microRNA-145, regulates interferon-ß induction through STAT3 nuclear translocation in bladder cancer cells.

We recently reported that microRNA (miR)-145 is downregulated and induces apoptosis in human bladder cancer cells. Also, it is suggested that the ectopic expression of miR-145 induces apoptosis with the induction of TRAIL expression in several cancer cells. Here, we demonstrated a novel mechanism of apoptosis induction by miR-145 in bladder cancer cells. Exogenous miR-145 in T24 and NKB1 cells markedly increased the expression levels of interferon (IFN)-ß, 2'-5'-oligoadenylate synthetase 1, which lies upstream of 2'-5' oligoadenylates/RNase L system, and TRAIL, and induced apparent caspase-dependent apoptosis that was suppressed by cotreatment with a pan-caspase inhibitor; moreover, these expression levels were reduced by cotreatment with an miR-145 inhibitor. The apoptosis did not depend on Toll-like receptor 3 (TLR3) expression, because TLR3-silencing failed to inhibit IFN-ß induction by miR-145. Then, we focused on the suppressor of cytokine signaling 7 (socs7), whose expression level was upregulated in bladder cancer cells compared with its level in normal human urothelial cells, as a putative target gene involved in IFN-ß induction by miR-145. Expectedly, exogenous miR-145 decreased the expression level of SOCS7, and socs7-silencing enhanced IFN-ß induction by transfection with a TLR3 ligand, polyinosinic acid-polycytidylic acid (PIC). The results of a luciferase reporter assay revealed that miR-145 targeted socs7. In addition, socs7-silencing significantly decreased the level of p-Akt and suppressed the growth of T24 cells. Furthermore, exogenous miR-145 or socs7-silencing promoted nuclear translocation of STAT3. In conclusion, the machinery of IFN-ß induction through the regulation of SOCS7 by miR-145 was closely associated with the induction of apoptosis. Moreover, exogenous miR-145 promoted IFN-ß induction by targeting socs7, which resulted in the nuclear translocation of STAT3. Additionally, our data indicate that SOCS7 functioned as an oncogene, the finding that revealed a novel mechanism of carcinogenesis in bladder cancer cells.

Fascin-1 expression in canine cutaneous and oral melanocytic tumours.

Fascin-1 expression was examined in 9 cutaneous melanocytomas and 47 oral melanomas. The cases were scored on the basis of extent and intensity of staining, and combined scores were calculated. Fascin-1 expression was observed in 5/9 (56%) melanocytomas and 46/47 (98%) melanomas. The combined score for fascin-1 was significantly greater in stage III/IV melanomas than in stage I/II melanomas (P < 0.05). In addition, strong fascin-1 staining was associated with a significantly shortened survival time (P < 0.05). The results of this study suggest that fascin-1 overexpression correlates with the malignancy of canine melanoma and has the potential to be a new immunohistochemical marker to predict the clinical course of canine melanoma. In addition, targeted therapy for fascin-1 may represent a new strategy for the treatment of canine melanoma.

SIRT1 modulates expression of matrix metalloproteinases in human dermal fibroblasts.

BACKGROUND: SIRT1, an NAD(+) -dependent histone/protein deacetylase, controls a broad range of cellular functions. OBJECTIVES: We examined if SIRT1 is involved in the regulation of matrix metalloproteinase (MMP) expression in human dermal fibroblasts. METHODS: We studied the effect of inhibition of SIRT1 by specific inhibitor and small interfering RNA (siRNA) on MMP-1 and MMP-3 expression in human dermal fibroblasts. RESULTS: Treatment with a potent and selective inhibitor of SIRT1, EX-527, increased the basal expression levels of MMP-1 and MMP-3 proteins. Knockdown of endogenous SIRT1 by siRNA led to increased expression of MMP-1 and MMP-3 at both mRNA and protein levels. SIRT1 knockdown also upregulated MMP protein induction caused by an inflammatory cytokine, interleukin (IL)-1ß. Moreover, treatment with a SIRT1 activator, resveratrol, significantly suppressed IL-1ß-mediated induction of MMP-1, which was attenuated by pretreatment with EX-527. Finally, MMP-1 promoter activity was increased by EX-527 in cells treated with or without IL-1ß. CONCLUSIONS: Our findings suggest that SIRT1 exerts a negative regulatory role in the production of MMP-1 and MMP-3 in human dermal fibroblasts.

Role of anti-oncomirs miR-143 and -145 in human colorectal tumors.

We examined the expression levels of microRNAs (miRNAs (miRs)) in colorectal tumors (63 cancer specimens and 65 adenoma specimens) and paired non-tumorous tissues. Decreased expression of miR-143 and -145 was frequently observed in the adenomas and cancers tested, compared with miR-34a downregulation and miR-21 upregulation. Expression profiles of miR-143 and -145 were not associated with any clinical features. As the downregulation of miR-143 and -145 was observed even in the early phase of adenoma formation, the decreased expression of both miRs would appear to contribute mainly to the initiation step of tumorigenesis, not to the progression stage, and not to clinical prognostic factors. For clinical application, we changed the sequences of the passenger strand in the miR-143 duplex and performed chemical modification at the 3'-overhang portion of miR-143, leading to greater activity and stability to nuclease. The cell growth inhibitory effect of the chemically modified synthetic miR-143 in vitro was greater than that of endogenous miR-143. The miR-143 showed a significant tumor-suppressive effect on xenografted tumors of DLD-1 human colorectal cancer cells. These findings suggest that miR-143 and -145 are important onco-related genes for the initiation step of colorectal tumor development and that the chemically modified synthetic miR-143 may be a hopeful candidate as an RNA medicine for the treatment of colorectal tumors.

Neuroprotection by propargylamines in Parkinson's disease: intracellular mechanism underlying the anti-apoptotic function and search for clinical markers.

In Parkinson's and other neurodegenerative diseases, a therapeutic strategy has been proposed to halt progressive cell death. Propargylamine derivatives, rasagiline and (-)deprenyl (selegiline), have been confirmed to protect neurons against cell death induced by various insults in cellular and animal models of neurodegenerative disorders. In this paper, the mechanism and the markers of the neuroprotection are reviewed. Propargylamines prevent the mitochondrial permeabilization, membrane potential decline, cytochrome c release, caspase activation and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase. At the same time, rasagiline induces anti-apoptotic pro-survival proteins, Bcl-2 and glial cell-line derived neurotrophic factor, which is mediated by activated ERK-NF-kappaB signal pathway. DNA array studies indicate that rasagiline increases the expression of the genes coding mitochondrial energy synthesis, inhibitors of apoptosis, transcription factors, kinases and ubiquitin-proteasome system, sequentially in a time-dependent way. Products of cell survival-related gene induced by propargylamines may be applied as markers of neuroprotection in clinical samples.

The effect of neuromelanin on the proteasome activity in human dopaminergic SH-SY5Y cells.

In Parkinson's disease (PD), the selective depletion of dopamine neurons in the substantia nigra, particular those containing neuromelanin (NM), is the characteristic pathological feature. The role of NM in the cell death of dopamine neurons has been considered either to be neurotoxic or neuroprotective, but the precise mechanism has never been elucidated. In human brain, NM is synthesized by polymerization of dopamine and relating quinones, to which bind heavy metals including iron. The effects of NM prepared from human brain were examined using human dopaminergic SH-SY5Y cells. It was found that NM inhibits 26S proteasome activity through generation of reactive oxygen and nitrogen species from mitochondria. The mitochondrial dysfunction was also induced by oxidative stress mediated by iron released from NM. NM accumulated in dopamine neurons in ageing may determine the selective vulnerability of dopamine neurons in PD.

Overexpression of amyloid precursor protein induces susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells.

In Alzheimer's disease amyloid beta peptide (Abeta) produced from amyloid precursor protein (APP) is considered to induce cell death. To clarify the molecular mechanism underlying Abeta neurotoxicity, we established the cell line overexpressing wild or mutant (His684Arg) APP in human SH-SY5Y cells. This paper presents that overexpression of wild-APP in the cells (SH/w-APP) increased the levels of APP and Abeta(1-40) but not Abeta(1-42), and reduced Bcl-2 level and proteasome activity with increased susceptibility to oxidative stress. The intracellular levels of reactive oxygen species in SH/w-APP increased significantly by H(2)O(2) treatment. The level of Bcl-2 protein, but not mRNA, was markedly decreased in SH/w-APP cells, which was inversely correlated with APP expression among subcloned SH/w-APP cells. These results indicate that increased expression of wild type APP renders neuronal cells more vulnerable to oxidative stress leading to cell death.

Neuromelanin induces oxidative stress in mitochondria through release of iron: mechanism behind the inhibition of 26S proteasome.

Parkinson's disease is characterized by the selective depletion of dopamine neurons in the substantia nigra, particular those containing neuromelanin. Involvement of neuromelanin in the pathogenesis may be either cytotoxic or protective. Recently we found that neuromelanin reduces the activity of 26S proteasome. In this paper, the detailed mechanisms behind the reduced activity were studied using neuromelanin isolated from the human brain. Neuromelanin increased the oxidative stress, but synthetic melanin did not. Superoxide dismutase and deferoxamine completely suppressed the increase, indicating that superoxide produced by an iron-mediated reaction plays a central role. Iron was shown to reduce in situ 26S proteasome activity in SH-SY5Y cells and the reduction was protected by antioxidants. These results suggest that iron released from neuromelanin increases oxidative stress in mitochondria, and then causes mitochondrial dysfunction and reduces proteasome function. The role of neuromelanin is discussed in relation to the selective vulnerability of dopamine neurons in Parkinson's disease.

N-Propargylamine protects SH-SY5Y cells from apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol, through stabilization of mitochondrial membrane and induction of anti-apoptotic Bcl-2.

Propargylamine derivatives, rasagiline and (-)deprenyl, are anti-Parkinson agents and protect neurons from cell death as shown by in vivo and in vitro experiments. The studies on the chemical structure-activity relationship proved that the propargyl moiety is essentially required for the neuroprotective function. In this paper, neuroprotective activity of free N-propargylamine was studied using SH-SY5Y cells expressing only type A monoamine oxidase (MAO) against apoptosis induced by an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol. N-Propargylamine prevented apoptosis, whereas N-methylpropargylamine and propiolaldehyde did not. N-Propargylamine stabilized mitochondrial membrane potential and induced anti-apoptotic Bcl-2 at 1 microM-10 nM. N-Propargylamine inhibited MAO-A in competition to substrate with the apparent K(i) value of 28 microM, which was significantly higher than the concentration required for neuroprotection. It indicates that MAO inhibition is not prerequisite for the protective function of N-propargylamine. The anti-apoptotic function of N-propargylamine is discussed in terms of neuroprotection by propargylamines in neurodegenerative diseases, including Parkinson's disease.

Involvement of type A monoamine oxidase in neurodegeneration: regulation of mitochondrial signaling leading to cell death or neuroprotection.

In neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, apoptosis is a common type of cell death, and mitochondria emerge as the major organelle to initiate death cascade. Monoamine oxidase (MAO) in the mitochondrial outer membrane produces hydrogen peroxide by oxidation of monoamine substrates, and induces oxidative stress resulting in neuronal degeneration. On the other hand, a series of inhibitors of type B MAO (MAO-B) protect neurons from cell death. These results suggest that MAO may be involved in the cell death process initiated in mitochondria. However, the direct involvement of MAO in the apoptotic signaling has been scarcely reported. In this paper, we present our recent results on the role of MAO in activating and regulating cell death processing in mitochondria. Type A MAO (MAO-A) was found to bind an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, and induce apoptosis in dopaminergic SH-SY5Y cells containing only MAO-A. To examine the intervention of MAO-B in apoptotic process, human MAO-B cDNA was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even though the activity and protein of MAO-B were expressed markedly. MAO-B oxidized dopamine with production of hydrogen peroxide, whereas in control cells expressing only MAO-A, dopamine autoxidation produced superoxide and dopamine-quinone, and induced mitochondrial permeability transition and apoptosis. Rasagiline and other MAO-B inhibitors prevent the activation of apoptotic cascade and induce prosurvival genes, such as bcl-2 and glial cell line-derived neurotrophic factor, in MAO-A-containing cells. These results demonstrate a novel function of MAO-A in the induction and regulation of apoptosis. Future studies will clarify more detailed mechanism behind regulation of mitochondrial death signaling by MAO-A, and bring out new strategies to cure or ameliorate the decline of neurons in neurodegenerative disorders.

Neuromelanin inhibits enzymatic activity of 26S proteasome in human dopaminergic SH-SY5Y cells.

Recently, impairment of the ubiquitin-proteasome system is suggested to be responsible for the neuronal death in ageing and Parkinson's disease. The specific degeneration of dopamine neurons containing neuromelanin (NM) suggests that NM itself may be involved in the cellular dysfunction and death, even though the direct link has never been reported. We examined the effects of NM isolated from the human substantia nigra on the proteasome activity in human dopaminergic SH-SY5Y cells. NM reduced the activities of 26S proteasome, as shown in situ using a green fluorescent protein homologue targeted to 26S proteasome and also in vitro using ubiquitinated lysozyme as a substrate. However, NM did not affect 20S proteasome activity in vitro. NM reduced the amount of PA700 regulatory subunit of 26S proteasome, but did not affect that of alpha- and beta-subunits of 20S proteasome. These results suggest that NM may inhibit the ubiquitin-26S proteasome system, and determine the selective vulnerability of dopamine neurons in ageing and related disorders.

Overexpression of a DEAD box/RNA helicase protein, rck/p54, in human hepatocytes from patients with hepatitis C virus-related chronic hepatitis and its implication in hepatocellular carcinogenesis.

Hepatitis C virus (HCV) infection is the most common cause of chronic hepatitis, which frequently progresses to hepatocellular carcinoma. The pathogenesis of its persistent infection and tumour progression has not been fully characterized yet. The RCK gene was previously cloned at the breakpoint of the t(11;14)(q23;q32) chromosome translocation observed in human B-cell lymphoma cell line RC-K8. The RCK protein, rck/p54, which is a 54-kDa cytoplasmic protein belonging to the DEAD box/RNA helicase family, is considered to facilitate the translation of mRNA(s) of genes for cell proliferation and malignant transformation not only in B-cell lymphomas having the t(11;14) translocation but also in other solid tumours. The aim of this work was to examine the involvement of rck/p54 in carcinogenesis of hepatocellular carcinoma from HCV-related chronic hepatitis. We examined the expression of rck/p54 in 29 cases of HCV-related chronic hepatitis and eight cases of hepatocellular carcinoma by immunohistochemistry and Western blot analysis. Twenty-six of 29 cases with HCV-related chronic hepatitis and all cases with hepatocellular carcinoma tested overexpressed rck/p54 protein. The expression of rck/p54 was lowered by treatment with IFN-alpha in two cases who showed the decrease in HCV RNA levels. These findings suggest that rck/p54 protein is possibly involved in the replication of HCV genomes in hepatocytes and in tumourigenesis of hepatocellular carcinomas.

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5'-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.

Mitochondria determine the survival and death in apoptosis by an endogenous neurotoxin, N-methyl(R)salsolinol, and neuroprotection by propargylamines.

In neurodegenerative disorders, such as Parkinson's disease, selective neuronal death characterizes clinical signs and symptoms. Recently apoptosis was reported to be a common type of cell death in some disorders, and well-controlled apoptotic cascade is proposed to be a target of neuroprotective therapy. In our studies to find endogenous neurotoxins as a pathogenic factor in Parkinson's disease, dopamine-derived N-methyl(R)salsolinol was found to induce apoptosis in dopamine neurons of rat models of Parkinson's disease. In human dopaminergic SH-SY5Y cells, apoptosis was initiated by decline in mitochondrial membrane potential, and anti-apoptotic Bcl-2 family protein regulated apoptotic signal transduction. In addition, a series of propargylamines were found to prevent apoptosis through stabilization of mitochondrial membrane potential, which also involved Bcl-2. The role of mitochondria and the involvement of Bcl-2 in apoptosis and neuroprotection were clearly demonstrated using isolated mitochondria. These results indicate that mitochondria are the site to determine the cell death induced by neurotoxins and also the neuroprotection by anti-apoptotic propargylamines.

Co-overexpression of DEAD box protein rck/p54 and c-myc protein in human colorectal adenomas and the relevance of their expression in cultured cell lines.

The RCK gene was cloned through a study of the breakpoint of the t(11;14)(q23;q32) chromosomal translocation observed in a human B-cell lymphoma and overexpression of the protein (rck/p54) due to the translocation was shown to be associated with malignant transformation. The rck/p54 protein belongs to the DEAD box protein/RNA helicase family, which has a variety of functions such as translation initiation, pre-mRNA splicing and ribosome assembly. It is considered that rck/p54 protein may have significant effects on the mRNA structure of genes associated with cell proliferation, facilitating protein synthesis. Expression of rck/p54 in colorectal adenomas, which are a premalignant lesion of colorectal cancer, was examined by Western blot analysis and immunohistochemistry. The rck/p54 protein was found to be overexpressed in tumor tissues resected from 17 of 26 cases (65.4%) of colorectal adenomas and 13 of 14 c-myc-positive cases (92.8%) also co-overexpressed rck/p54 protein. Thus, a significant correlation between rck/p54 and c-myc co-overexpression was found (Spearman's rank correlation, P = 0.0018). We demonstrate that overexpression of rck/p54 in two different cell lines, COS 7 and human colorectal cancer cell line SW480, caused an increase in c-myc protein levels by enhancement of its translation efficiency and/or stabilization of its mRNA. These results suggest that rck/p54 of the DEAD box protein/RNA helicase family may contribute to cell proliferation and carcinogenesis in the development of human colorectal tumors at the translational level by increasing synthesis of c-myc protein.

Transfection-enforced Bcl-2 overexpression and an anti-Parkinson drug, rasagiline, prevent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase induced by an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol.

An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, was found to induce apoptosis in human dopaminergic SH-SY5Y cells by step-wise activation of apoptotic cascade; collapse in mitochondrial membrane potential, DeltaPsim, activation of caspases, and fragmentation of DNA. Recently, accumulation of gylceraldehyde-3-phosphate dehydrogenase (GAPDH) in nuclei was proposed to play an important role in apoptosis. In this paper, involvement of GAPDH in apoptosis induced by N-methyl(R)salsolinol was studied. The isoquinoline reduced DeltaPsim within 3 h, as detected by a fluorescence indicator, JC-1, then after 16 h incubation, GAPDH accumulated in nuclei by detection with immunostaining. To clarify the role of GAPDH in apoptotic process, a stable cell line of Bcl-2 overexpressed SH-SY5Y cells was established. Overexpression of Bcl-2 prevented the decline in DeltaPsim and also apoptotic DNA damage induced by N-methyl(R)salsolinol. In Bcl-2 transfected cells, nuclear translocation of GAPDH was also completely suppressed. In addition, a novel antiparkinsonian drug, rasagiline, prevented nuclear accumulation of GAPDH induced by N-methyl(R)salsolinol in control cells. These results suggest that GAPDH may accumulate in nuclei as a consequence of signal transduction, which is antagonized by anti-apoptotic Bcl-2 protein family and rasagiline. The results are discussed in concern to intracellular mechanism underlying anti-apoptotic function of rasagiline analogues.

Involvement of phospholipase D in sphingosine 1-phosphate-induced activation of phosphatidylinositol 3-kinase and Akt in Chinese hamster ovary cells overexpressing EDG3.

Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.