[show abstract][hide abstract] ABSTRACT: Pim-3, a proto-oncogene with serine/threonine kinase activity, was enhanced in hepatocellular carcinoma (HCC) tissues. To address the roles of Pim-3 in HCC development, we prepared transgenic mice that express human Pim-3 selectively in liver. The mice were born at a Mendelian ratio, were fertile and did not exhibit any apparent pathological changes in the liver until 1 year after birth. Pim-3-transgenic mouse-derived hepatocytes exhibited accelerated cell cycle progression. The administration of a potent hepatocarcinogen, diethylnitrosamine (DEN), induced accelerated proliferation of liver cells in Pim-3 transgenic mice in the early phase, compared with that observed for wild-type mice. Treatment with DEN induced lipid droplet accumulation with increased proliferating cell numbers 6 months after the treatment. Eventually, wild-type mice developed HCC with a frequency of 40% until 10 month after the treatment. Lipid accumulation was accelerated in Pim-3 transgenic mice with higher proliferating cell numbers, compared with that observed for wild-type mice. Pim-3 transgenic mice developed HCC with a higher incidence (80%) and a heavier burden, together with enhanced intratumoral CD31-positive vascular areas, compared with that observed for wild-type mice. These observations indicate that Pim-3 alone cannot cause, but can accelerate HCC development when induced by a hepatocarcinogen, such as DEN.
[show abstract][hide abstract] ABSTRACT: Pim-3, a member of the proto-oncogene Pim family with serine/threonine kinase activity was aberrantly expressed in cancerous lesions of endoderm-derived organs such as liver, pancreas, and colon. The aim of this study was to clarify the role of Pim-3 expression in the tumorigenesis and the development of gastric carcinomas.
Pim-3 expression was immunohistochemically examined on the tissue microarrays containing primary (n = 285) and metastastic (n = 37) sites of gastric carcinomas, in comparison with adenoma (n = 48) and non-cancerous mucosa (n = 84). It was also compared with the clinicopathological parameters of gastric carcinomas.
Pim-3 expression was enhanced in adenoma (64.6%) and metastasis sites of gastric carcinoma (73.0%), to a lesser degree in primary sites of gastric carcinoma (39.3%) when compared to non-cancerous mucosa (13.1%, p < 0.0001). Pim-3 expression levels were higher in intestinal-type than diffuse-type gastric carcinoma (p = 0.018). Pim-3 expression was closely correlated with sex (p = 0.047), lymphatic (p = 0.019) and venous invasion (p = 0.014). Pim-3 expression was correlated significantly with vascular endothelial growth factor (VEGF, p = 0.009) and extracellular matrix metalloproteinase inducer (EMMPRIN, p = 0.032), both of which are presumed to be involved in neovascularization, a crucial step for metastasis. On the contrary, phosphatase and tensin homology deleted from human chromosome 10 (Pten) negative gastric carcinomas exhibited higher Pim-3 expression than Pten positive ones (p = 0.042). There was no relationship between Pim-3 expression and MVD in gastric carcinomas (p = 0.715). Furthermore, patients with Pim-3 positive gastric cancer, showed a lower cumulative survival rate than those with Pim-3 negative gastric cancer (p = 0.014) and Pim-3 positive was also identified as an independent prognostic factor for gastric carcinoma patients (p = 0.006).
Aberrant Pim-3 expression was involved in gastric adenoma-adenocarcinoma sequence and subsequent invasion and metastasis process in gastric cancer. Moreover, Pim-3 may be employed to predict the prognosis of gastric cancer patients. Distinct Pim-3 expression underlies the molecular mechanisms for the differentiation of intestinal-type and diffuse-type carcinomas.
Journal of Cancer Research and Clinical Oncology 04/2008; 134(4):481-8. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.
Journal of Clinical Investigation 03/2008; 118(2):560-70. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously observed that Pim-3 with serine/threonine kinase activity, was aberrantly expressed in malignant lesions of endoderm-derived organs, liver and pancreas. Because Pim-3 protein was not detected in normal colon mucosal tissues, we evaluated Pim-3 expression in malignant lesions of human colon, another endoderm-derived organ. Pim-3 was detected immunohistochemically in well-differentiated (43/68 cases) and moderately differentiated (23/41 cases) but not poorly differentiated colon adenocarcinomas (0/5 cases). Moreover, Pim-3 proteins were detected in adenoma (35/40 cases) and normal mucosa (26/111 cases), which are adjacent to adenocarcinoma. Pim-3 was constitutively expressed in SW480 cells and the transfection with Pim-3 short hairpin RNA promoted apoptosis. In the same cell line, a pro-apoptotic molecule, Bad, was phosphorylated at Ser(112) and Ser(136) sites of phosphorylation that are representative of its inactive form. Ser(112) but not Ser(136) phosphorylation in this cell line was abrogated by Pim-3 knockdown. Furthermore, in human colon cancer tissues, Pim-3 co-localized with Bad in all cases (9/9) and with phospho-Ser(112)Bad in most cases (6/9). These observations suggest that Pim-3 can inactivate Bad by phosphorylating its Ser(112) in human colon cancer cells and thus may prevent apoptosis and promote progression of human colon cancer.
Cancer Science 04/2007; 98(3):321-8. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pancreatic cancer still remains a serious health problem with <5% 5-year survival rate for all stages. To develop an effective treatment, it is necessary to identify a target molecule that is crucially involved in pancreatic tumor growth. We previously observed that Pim-3, a member of the proto-oncogene Pim family that expresses serine/threonine kinase activity, was aberrantly expressed in human and mouse hepatomas but not in normal liver. Here, we show that Pim-3 is also expressed in malignant lesions of the pancreas but not in normal pancreatic tissue. Moreover, Pim-3 mRNA and protein were constitutively expressed in all human pancreatic cancer cell lines that we examined and colocalized with the proapoptotic protein Bad. The ablation of endogenous Pim-3 by small hairpin RNA transfection promoted apoptosis, as evidenced by increases in a proportion of cells in the sub-G(1) fraction of the cell cycle and in phosphatidyl serine externalization. A proapoptotic molecule, Bad, was phosphorylated constitutively at Ser(112) but not Ser(136) in human pancreatic cancer cell lines and this phosphorylation is presumed to represent its inactive form. Phosphorylation of Bad and the expression of an antiapoptotic molecule, Bcl-X(L), were reduced by the ablation of endogenous Pim-3. Thus, we provide the first evidence that Pim-3 can inactivate Bad and maintain the expression of Bcl-X(L) and thus prevent apoptosis of human pancreatic cancer cells. This may contribute to the net increase in tumor volume or tumor growth in pancreatic cancer.
Cancer Research 07/2006; 66(13):6741-7. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously observed that a chemokine, macrophage inflammatory protein-1 alpha/CCL3, and its receptor, CCR1, were aberrantly expressed in human hepatocellular carcinoma (HCC) tissues. Here, we show that CCL3 and CCR1 are also expressed in 2 different models of this cancer; N-nitrosodiethylamine (DEN)-induced HCC and HCC induced by hepatitis B virus surface (HBs) antigen-primed splenocyte transfer to myelo-ablated syngeneic HBs antigen transgenic mice. At 10 months after DEN treatment, foci number and sizes were remarkably reduced in CCR1- and CCL3-deficient mice, compared with those of wild-type (WT) mice, although tumor incidence were marginally, but significantly, higher in CCR1- and CCL3-deficient mice than in WT mice. Of note is that tumor angiogenesis was also markedly diminished in CCL3- and CCR1-deficient mice, with a concomitant reduction in the number of intratumoral Kupffer cells, a rich source of growth factors and matrix metalloproteinases (MMPs). Among growth factors and MMPs that we examined, only MMP9 and MMP13 gene expression was augmented progressively in liver of WT mice after DEN treatment. Moreover, MMP9, but not MMP13, gene expression was attenuated in CCR1- and CCL3-deficient mice, compared with that of WT mice. Furthermore, MMP9 was expressed mainly by mononuclear cells but not hepatoma cells, and MMP9-expressing cell numbers were decreased in CCR1- or CCL3-deficient mice, compared with WT mice. These observations suggest the contribution of the CCR1-CCL3 axis to HCC progression.
International Journal of Cancer 05/2006; 118(8):1869-76. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The liver parenchyma is populated by hepatocytes and several nonparenchymal cell types, including Kupffer cells and hepatic stellate cells. Both Kupffer cells and hepatic stellate cells are responsive to the chemokine CCL2, but the precise roles of CCL2 and these cells in liver tumor formation remain undefined. Hence, we investigated the effects of the lack of the major CCL2 receptor, CCR2, on liver tumor formation induced by intraportal injection of the murine colon adenocarcinoma cell line, colon 26. Wild-type mice showed macroscopic tumor foci in the liver 10 days after injection of colon 26 cells. After 10 days, CCL2 proteins were detected predominantly in tumor cells, coincident with increased intratumoral macrophage and hepatic stellate cell numbers. Although tumor formation occurred at similar rates in wild-type and CCR2-deficient mice up to 10 days after tumor cell injection, the number and size of tumor foci were significantly attenuated in CCR2-deficient mice relative to wild-type mice thereafter. Moreover, neovascularization and matrix metalloproteinase 2 expression were diminished in CCR2-deficient mice with a concomitant reduction in the accumulation of macrophages and hepatic stellate cells. Furthermore, matrix metalloproteinase 2 was detected predominantly in hepatic stellate cells but not in macrophages. We provided the first definitive evidence that the absence of CCR2-mediated signals can reduce the trafficking of hepatic stellate cells, a main source of matrix metalloproteinase 2, and consequently can diminish neovascularization during liver tumor formation.
International Journal of Cancer 02/2006; 118(2):335-45. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Molecular signals regulating adult neurogenesis in primates are largely unknown. Here the authors used differential display to analyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and characterized its expression pattern at the protein level. In contrast with approximately threefold upregulation of Dscam gene on days 5 and 7, immunoblotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or betaIII-tubulin, while another is astrocytes positive for S100beta. Young astrocytes were in intimate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of primate hippocampus upregulated after ischemia.
[show abstract][hide abstract] ABSTRACT: Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre-malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre-malignant lesions, we focused on Pim-3, a member of a proto-oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full-length human Pim-3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, HepG2. The obtained 2,392 bp human Pim-3 cDNA encodes a predicted open reading frame consisting of 326 amino acids. Pim-3 mRNA was selectively expressed in human hepatoma cell lines, but not in normal liver tissues. Moreover, Pim-3 protein was detected in human hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human hepatoma cell lines by the ablation of Pim-3 gene with RNA interference. These observations suggest that aberrantly expressed Pim-3 can cause autonomous cell proliferation or prevent apoptosis in hepatoma cell lines.
International Journal of Cancer 03/2005; 114(2):209-18. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to clarify the roles of tumor necrosis factor (TNF)-alpha in lung metastasis, we injected Renca cells intravenously into TNF receptor p55-deficient (TNF-Rp55 KO) and wild-type (WT) mice. Microscopic and macroscopic metastasis foci appeared in lungs at 7 and 14 days after the tumor injection, respectively. Moreover, metastasis foci expanded at similar rates in both WT and TNF-Rp55 KO mice until 21 days, and lungs were occupied with metastasis foci. However, later than 21 days after the injection, metastasis foci spontaneously regressed in TNF-Rp55 KO mice, whereas WT mice exhibited a progressive growth of metastasis foci. Moreover, metastasis foci remained reduced sizes in TNF-Rp55 KO mice even at 26 days, when all WT mice died with lungs filled with metastasis foci. Later than 21 days after the tumor injection, the number of apoptotic tumor cells was increased in TNF-Rp55 KO mice. In contrast, neovascularization was less evident in TNF-Rp55 KO than WT mice, with depressed hepatocyte growth factor (HGF) gene in TNF-Rp55 KO mice at 21 days after the tumor injection. Thus, TNF-Rp55-mediated signals can maintain tumor neovascularization at least partly by inducing HGF expression, and eventually support lung metastasis process.
International Journal of Cancer 01/2005; 112(6):927-33. · 6.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cervical cancers at advanced stages or with recurrent status are mainly treated by platinum-based chemotherapy, such as cisplatin. However, a novel strategy to reduce the minimally effective dose is required to prevent severe adverse effects that limit the effectiveness of the treatment. Monocyte chemoattractant protein-1 (MCP-1) is a subtype of chemokines that can promote monocyte/macrophage infiltration and enhance their phagocytosis at not only sites of inflammatory lesions but also of tumors. The present study applies MCP-1-based gene therapy to treat cervical cancers. To achieve tumor-specific expression of MCP-1, retroviral expression vector was constructed using the human telomerase reverse transcriptase gene (hTERT) promoter. Retroviral expression of MCP-1 into cervical cancer ME180 cells did not affect their proliferation either in vitro or in vivo. However, when combined with a suboptimal low dose of cisplatin, tumor formation was obviously reduced in clones transduced with MCP-1, but not in control clones. Histological examination revealed that a substantial number of macrophages infiltrated the tumor sites of MCP-1-transduced cells, but not of controls. These findings suggest that MCP-1 expression sensitizes cervical cancer cells to an otherwise ineffective low dose of cisplatin, possibly by inducing the migration of macrophages to eradicate tumor cells. This system may be a novel strategy for chemotherapy combined with immunogene therapy against otherwise intractable cervical cancers.
Cancer Gene Therapy 02/2004; 11(1):1-7. · 2.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1 alpha and IL-1 beta. Finally, IL-1 alpha and IL-1 beta were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner.
American Journal Of Pathology 05/2003; 162(4):1249-58. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: TNF-alpha has pleiotropic functions, but its role in liver fibrosis has not yet been clarified. To understand the pathophysiologic role of the TNF-alpha/TNF receptor (TNFR) p55 signals in liver fibrosis, 10 mg/kg of dimethylnitrosamine, a specific hepatotoxicant, was administered twice a week into the peritoneal cavity of both TNFRp55 knock-out (KO) and wild-type mice, and the severity of fibrosis was monitored histologically and biochemically. In wild-type mice, histologic analysis demonstrated evident fibrotic changes 1 week after the initiation of dimethylnitrosamine administration, consistent with increased liver collagen contents. Concomitantly, the numbers of Kupffer cells and activated hepatic stellate cells (HSCs) were increased in liver tissue. On the contrary, fibrotic changes were attenuated and the numbers of Kupffer cells and HSCs were decreased in TNFRp55-KO mice. Moreover, gene expression of TNF-alpha and monocyte chemoattractant protein-1, which are involved in Kupffer cell activation or migration, was decreased in the liver of TNFRp55-KO mice. Collectively, TNFRp55-mediated signals may regulate activation of Kupffer cells and HSCs and eventually enhance fibrotic process.