Masahiko Shibazaki

Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines

Alterations of several microRNA (miRNA) have been linked to cancer development and its biology. To search for unique miRNA that might play a role in the development of anaplastic thyroid carcinoma (ATC), we examined the expression of multiple miRNA and their functional effects on target genes in human thyroid carcinoma cell lines. We quantitatively evaluated the expression of multiple miRNA in 10 ATC and five papillary thyroid carcinoma (PTC) cell lines, as well as primary tumors from 11 thyroid carcinoma patients (three ATC and eight PTC), using the stem‐loop‐mediated reverse transcription real‐time polymerase chain reaction method. We also examined the target gene specificity of unique miRNA that showed differences in expression between ATC and PTC cell lines. One miRNA, miR‐138, was significantly downregulated in ATC cell lines in comparison with PTC (P < 0.01). Eleven miRNA (including miR‐138) potentially targeting the human telomerase reverse transcriptase (hTERT) gene were totally downregulated in both ATC and PTC cell lines in comparison with normal thyroid tissues. A tendency for an inverse correlation between miR‐138 and hTERT protein expression was observed in the thyroid cancer cell lines, although this failed to reach significance (r = –0.392, P = 0.148). We demonstrated that overexpression of miR‐138 induced a reduction in hTERT protein expression, and confirmed target specificity between miR‐138 and the hTERT 3′‐untranslated region by luciferase reporter assay. These results suggest that loss of miR‐138 expression may partially contribute to the gain of hTERT protein expression in ATC, and that further multiple miRNA targeting hTERT mRNA might be involved in the development of thyroid carcinoma. (Cancer Sci 2008; 99: 280–286)

Translocation of platelets into Disse spaces and their entry into hepatocytes in response to lipopolysaccharides, interleukin-1 and tumour necrosis factor: the role of Kupffer cells

BACKGROUND/AIMS Injection into mice of a small dose of either a lipopolysaccharide or interleukin-1 induces a slowly developing accumulation of 5-hydroxytryptamine, predominantly in the liver. We have established that this 5-hydroxytryptamine accumulation is the result of the translocation of platelets to hepatic sinusoidal spaces and, further, into Disse spaces, and that the platelets make direct contact with hepatocytes. In the present study, we report our recent findings on this phenomenon. METHODS Platelets contain a large amount of 5-hydroxytryptamine, but the 5-hydroxytryptamine content of the liver is normally very small. Therefore, the translocation of platelets to the liver was assessed by measuring 5-hydroxytryptamine as in previous studies, and it was also analysed by electron microscopy. RESULTS Anti-platelet agents, such as heparin and inhibitors of prostaglandin synthesis, were ineffective in preventing the lipopolysaccharide-induced accumulation of 5-hydroxytryptamine in the liver. Of the various cytokines tested, only interleukin-1 and tumour necrosis factor induced such an accumulation of 5-hydroxytryptamine. Intravenous injection of liposomes encapsulating dichloromethylene bisphosphonate resulted in an almost complete depletion of macrophages from the liver. The lipopolysaccharide- and cytokine-induced hepatic accumulations of 5-hydroxytryptamine were abolished almost completely in such macrophage-depleted mice. Electron microscopy revealed no accumulation of platelets in the liver after injection of lipopolysaccharide into the macrophage-depleted mice. Surprisingly, in normal mice injected with lipopolysaccharide, several platelets were found inside some hepatocytes, even though there was no visible damage to these hepatocytes. In fact, there were many polysomes around the degranulated platelets within the hepatocytes, suggesting an enhanced protein synthesis. CONCLUSION These results suggest that, in response to lipopolysaccharide, interleukin-1 or tumour necrosis factor, platelets translocate into the liver in a way that is different from aggregation, and that some, at least, enter hepatocytes. During these processes, hepatic macrophages play an essential role.

Enhancement by galactosamine of lipopolysaccharide(LPS)-induced tumour necrosis factor production and lethality: its suppression by LPS pretreatment

D‐Galactosamine (GalN) depletes UTP primarily in the liver, resulting in decreased RNA synthesis in hepatocytes. Co‐injection of GalN and lipopolysaccharide (LPS) into mice produces fulminant hepatitis with severe hepatic congestion, resulting in rapid death. Although the underlying mechanism is uncertain, GalN enhances the sensitivity to tumour necrosis factor (TNF). Administration of uridine (a precursor of UTP) prior injection of either LPS itself or interleukin‐1 (IL‐1) reduces the lethality of GalN+LPS. The present study focused on the effects of these agents on TNF production. Intraperitoneal injection of GalN+LPS into mice greatly elevated serum TNF. Although large doses of LPS alone also greatly elevated serum TNF, LPS itself induced neither hepatic congestion nor rapid death. Administration of a macrophage depletor, liposomes encapsulated with dichloromethylene bisphosphonate, reduced both the TNF production and mortality induced by GalN+LPS. Uridine, when injected 0.5 h after the injection of GalN+LPS, reduced the production of TNF. Prior injection of LPS, but not of IL‐1, also reduced this TNF production. Serum from LPS‐injected mice reduced the TNF production induced by GalN+LPS, but it was less effective at reducing the lethality. Its ability to reduce TNF production was abolished by heat‐treatment. We hypothesize that a factor inhibiting TNF production by macrophages is produced by hepatocytes in response to LPS. Possibly, production of this hepatocyte‐derived TNF‐down‐regulator (TNF‐DRh) may be: (i) inhibited by GalN, causing over‐production of TNF by macrophages and (ii) stimulated by LPS‐pretreatment (and restored by uridine), causing reduced TNF production.

Contrasting Effects of Lipopolysaccharides (Endotoxins) from Oral Black-Pigmented Bacteria and Enterobacteriaceae on Platelets, a Major Source of Serotonin, and on Histamine-Forming Enzyme in Mice

By measurement of serotonin levels, the translocation of platelets to various tissues was examined following intravenous injection of a lipopolysaccharide (LPS) into C3H/HeN mice. There was a rapid platelet accumulation (within 5 min and particularly in the lung), followed by a slower accumulation in the liver, which reached its plateau 3-5 h later. The severity of the anaphylactoid shock corresponded well with the magnitude of the rapid response. LPSs from the oral black-pigmented bacteria, Porphyromonas gingivalis and Prevotella intermedia, were much more potent in inducing the rapid platelet response than were those from the Enterobacteriaceae Escherichia coli and Salmonella typhimurium. However, LPSs from these Enterobacteriaceae were significantly more potent than those from black-pigmented bacteria in inducing the slow platelet response. There was also a contrast between their abilities to induce histidine decarboxylase, which forms histamine from histidine: LPSs from the Enterobacteriaceae were much more potent than those from black-pigmented bacteria.

Loss of HOXD10 expression induced by upregulation of miR-10b accelerates the migration and invasion activities of ovarian cancer cells

Small and large non-coding RNAs (ncRNAs) contribute to the acquisition of aggressive tumor behavior in diverse human malignancies. Two types of ncRNAs, miRNA‑10b (miR-10b) and homemobox (HOX) transcript antisense RNA (HOTAIR), can suppress the translation of the HOXD10 gene, an mRNA encoding a transcriptional repressor that inhibits the expression of cell migration/invasion-associated genes. Using epithelial ovarian cancer cell lines and primary tumors, we investigated whether miR‑10b and/or HOTAIR can regulate the expression of HOXD10, and whether it permits gain of pro‑metastatic gene products, matrix metallopeptidase 14 (MMP14) and ras homolog family member C (RHOC). Overexpression of miR-10b induced a decrease in HOXD10 protein expression, and upregulated the migration and invasion abilities in ovarian cancer cell lines (P<0.05). In these cells, a significant increase of MMP14 and RHOC protein was observed. No significant upregulation of the HOXD10 protein was observed in cells with the treatment of HOTAIR-siRNA. Positive signals for HOXD10 and MMP14 proteins were observed in 47 (69%) and 25 (37%) of 68 patients with epithelial ovarian cancers. An inverse correlation between HOXD10 and MMP14 immunoreactivities was observed (P<0.05), and miR-10b expression was also inversely correlated with HOXD10 protein expression (P<0.05). These results suggested that downregulation of HOXD10 expression by miR-10b overexpression may induce an increase of pro-metastatic gene products, such as MMP14 and RHOC, and contribute to the acquisition of metastatic phenotypes in epithelial ovarian cancer cells.

Loss of Class III β-Tubulin Induced by Histone Deacetylation Is Associated with Chemosensitivity to Paclitaxel in Malignant Melanoma Cells

Overexpression of class III beta-tubulin (TUBB3) is an important mechanism of taxane resistance. Using 7 melanoma cell lines, 2 normal neonatal human epidermal melanocyte (NHEM) cultures, and 49 primary melanomas, we investigated TUBB3 expression, its relationship to chemosensitivity to taxane derivatives, and the epigenetic mechanism controlling TUBB3 gene expression. Normal melanocytes in vitro and in vivo strongly expressed TUBB3 protein. NHEMs exhibited marked chemoresistance to paclitaxel-induced apoptosis. A subset (10 of 49, 20%) of primary malignant melanomas was TUBB3 negative. The incidence of TUBB3-negative melanomas increased with stage of progression. TUBB3 protein expression varied among cell lines; one (HMV-I) of the seven cell lines exhibited an extremely low endogenous level. TUBB3 protein expression correlated well with chemosensitivity to paclitaxel-induced apoptosis (P<0.05). Treatment with a histone deacetylase (HDAC) inhibitor restored TUBB3 expression in HMV-I. Chromatin immunoprecipitation assays revealed that histones H3 and H4 were hypoacetylated at the TUBB3 gene in HMV-I as compared with a TUBB3-overexpressing cell type (HMV-II). Treatment with the HDAC inhibitor induced gain of histone acetylation only in HMV-I. These results suggest that loss of TUBB3 protein may be induced by histone deacetylation in a subset of malignant melanomas, and may be associated with chemosensitivity to taxane.

Inhibition of inflammatory actions of aminobisphosphonates by dichloromethylene bisphosphonate, a non‐aminobisphosphonate

When injected intraperitoneally into mice in doses larger than those used clinically, all the amino derivatives of bisphosphonates (aminoBPs) tested induce a variety of inflammatory reactions such as induction of histidine decarboxylase (HDC, the histamine‐forming enzyme), hypertrophy of the spleen, atrophy of the thymus, hypoglycaemia, ascites and accumulation of exudate in the thorax, and an increase in the number of macrophages and/or granulocytes in the peritoneal cavity of blood. On the other hand, dichloromethylene bisphosphonate (Cl2MBP) a typical non‐aminoBP, has no such inflammatory actions. In the present study, we found that this agent can suppress the inflammatory actions of aminoBPs. Cl2MBP, when injected into mice before or after injection of 4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonic acid (AHBuBP; a typical aminoBP), inhibited the induction of HDC activity by AHBuBP in a dose‐ and time‐dependent manner. The increase in HDC activity induced by AHBuBP was largely suppressed by the injection of an equimolar dose of Cl2MBP. Cl2MBP also inhibited other AHBuBP‐induced inflammatory reactions, as well as the inflammatory actions of two other aminoBPs. However, Cl2MBP did not inhibit the increase in HDC activity induced by lipopolysaccharide (LPS). We have previously reported that AHBuBP augments the elevation of HDC activity and the production of interleukin‐1β (IL‐1β) that are induced by LPS. These actions of AHBuBP were also inhibited by Cl2MBP. Based on these results and reported actions of bisphosphonates, the mechanisms underlying the contrasting effects of aminoBPs and Cl2MBP, a non‐aminoBP are discussed. The results suggest that combined administration of Cl2MBP and an aminoBP in patients might be a useful way of suppressing the inflammatory side effects of aminoBPs.

Contrasting effects of an aminobisphosphonate, a potent inhibitor of bone resorption, on lipopolysaccharide‐induced production of interleukin‐1 and tumour necrosis factor α in mice

1 Aminobisphosphonates (aminoBPs), potent inhibitors of bone resorption, have been reported to induce inflammatory reactions such as fever and an increase in acute phase proteins in human patients, and to induce the histamine‐forming enzyme, histidine decarboxylase, in mice. In the present study, we examined the effect of aminoBP, 4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonic acid (AHBuBP), on the production of the pro‐inflammatory cytokines, IL‐1 and TNFα, in mice. 2 Intraperitoneal injection of AHBuBP did not itself produce detectable levels of IL‐1 (α and β) and TNFα in the serum. However, the elevation of serum IL‐1 induced by lipopolysaccharide (LPS) was greatly augmented in mice injected with AHBuBP 3 days before the LPS injection, whereas the LPS‐induced elevation of serum TNFα was almost completely abolished. 3 Spleen and bone marrow cells taken from mice injected with AHBuBP produced IL‐1β in vitro spontaneously, and the production was augmented following the addition of LPS. Cells that accumulated in the peritoneal cavity in response to AHBuBP produced a particularly large amount of IL‐1β. However, AHBuBP treatment of mice did not lead to an impairment of the in vitro production of TNFα by these three types of cells. 4 Liposomes encapsulating dichloromethylene bisphosphonate (a non‐amino BP) selectively deplete phagocytic macrophages. When an intraperitoneal injection of these liposomes was given 2 days after an injection of AHBuBP, there was a marked decrease in the LPS‐induced elevation of serum IL‐1 (α and β) (LPS being injected 3 days after the injection of AHBuBP). 5 These results indicate that AHBuBP has contrasting effects on the in vivo LPS‐induced production of IL‐1 and TNFα in mice, enhancing the production of IL‐1 by phagocytic macrophages and suppressing the production of TNFα, although underling mechanisms remain to be clarified.

Protein kinase Cθ activity is involved in the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced signal transduction pathway leading to apoptosis in L-MAT, a human lymphoblastic T-cell line

The aromatic hydrocarbon receptor (AhR)‐dependent pathway involved in 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD)‐induced immunotoxicity has been studied extensively, but the AhR‐independent molecular mechanism has not. In previous studies we found that the AhR is not expressed in L‐MAT, a human lymphoblastic T‐cell line. In this report, we provide the following evidence that the protein kinase C (PKC)θ activity is functionally involved in the AhR‐independent signal transduction mechanism that participates in the TCDD‐induced L‐MAT cell apoptosis. First, only rottlerin, a novel PKC (nPKC)‐selective inhibitor, blocked the apoptosis completely, in a dose‐dependent manner. Second, PKCθ was the major nPKC isoform (compared to PKCδ) expressed in the L‐MAT cell line. Third, a cell‐permeable myristoylated PKCθ pseudosubstrate peptide inhibitor also blocked the apoptosis completely, in a dose‐dependent manner. Fourth, both rottlerin and myristoylated PKCθ pseudosubstrate peptide inhibitor completely inhibited PKCθ kinase activity in vitro at doses that effectively blocked TCDD‐induced L‐MAT cell apoptosis. TCDD treatment induced a time‐dependent activation of nPKC kinase activity in L‐MAT cells, and moreover, TCDD induced a translocation of PKCθ from the cytosolic fraction to the particulate fraction in L‐MAT cells. Finally, transient over‐expression of a dominant negative PKCθ (a kinase‐dead mutant, K/R 409) in L‐MAT cells conferred significant protection against TCDD‐induced apoptosis.

Downregulation of microRNA-211 is involved in expression of preferentially expressed antigen of melanoma in melanoma cells

MicroRNAs (miRNAs) are small non-coding RNAs whose aberrations are involved in the initiation and progression of human cancers. To seek unique miRNAs contributing to melanoma tumorigenesis, we investigated the global miRNA expression profile of 7 melanoma cell lines and 3 primary cultures of neonatal human epidermal melanocytes (NHEMs) using the stem-loop real-time PCR method. We found 7 miRNAs that were commonly downregulated and 18 that were upregulated in all of the melanoma cell lines in comparison with the 3 primary cultures of NHEMs. We focused on one commonly downregulated miRNA (miR-211), and analyzed its relationship to the expression of preferentially expressed antigen of melanoma (PRAME) protein, which is a potential target of miR-211. We found that all melanoma cell lines exhibited marked down--regulation of miR-211 and upregulation of PRAME mRNA/protein expression in comparison with NHEMs (P<0.05). A significant inverse correlation between miR-211 and PRAME protein expression was found in melanoma cell lines and primary cultures of NHEMs (correlation coefficient of -0.733, P<0.05). We demonstrated that overexpression of miR-211 induced a reduction of PRAME protein levels, and confirmed the target specificity between miR-211 and PRAME by luciferase reporter assay. These results suggest that downregulation of miR-211 may be partly involved in aberrant expression of the PRAME protein in melanoma cells.