Hiroki Kuniyasu

Frequent amplification of the c-met gene in scirrhous type stomach cancer.

Amplification of the c-met gene, that encodes hepatocyte growth factor receptor, was examined on human esophageal, gastric and colorectal carcinomas. Six (55%) of the 11 gastric carcinoma cell lines and 15 (23%) of the 64 advanced gastric carcinomas showed the c-met gene amplification. Among them, c-met amplification was detected in 5 gastric cancer cell lines, derived from scirrhous gastric carcinoma and in 5 (38%) of 13 scirrhous gastric carcinoma tissues. Furthermore, patients of gastric carcinoma with c-met amplification showed significantly advanced tumor stage and poorer prognosis than those without the amplification. Conversely, no amplification was detected in any of the esophageal and colorectal carcinoma cell lines as well as carcinoma tissues except one colonic carcinoma. These results overall suggest that amplification of the c-met gene might participate in carcinogenesis and progression of stomach cancer, especially scirrhous type stomach carcinoma.

Expression of receptors for advanced glycation end‐products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer

The receptor for advanced glycation end‐products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S‐oligodeoxynucleotide was nine times less than that of sense S‐oligodeoxynucleotide‐treated cells. Growth of cells under RAGE antisense S‐oligodeoxynucleotide treatment was not different from that seen under sense S‐oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non‐RAGE‐expressing cell line). RAGE antisense S‐oligodeoxynucleotide treatment suppressed the invasive activity of RAGE‐positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen‐coated membrane under RAGE antisense S‐oligodeoxynucleotide treatment was significantly lower than under RAGE sense S‐oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S‐oligodeoxynucleotide‐treated RAGE‐negative MKN45 cells showed no difference. A wound‐healing assay showed that no RAGE antisense S‐oligodeoxynucleotide‐treated MKN28 cells migrated into the scraped area, whereas sense S‐oligodeoxynucleotide‐treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE‐positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE‐positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer. Copyright

Frequent amplification of the cyclin E gene in human gastric carcinomas

We searched for genetic alterations of the cyclin D1 and cyclin E genes in 45 human gastric carcinoma tissues. Expression of cyclin E mRNA and protein was also analyzed in eight of them by Northern and Western blots and immunohistochemical staining. The cyclin E gene was amplified 3–10 fold in seven gastric cancer tissues (15.6%), of which six were advanced gastric cancers. All of the cases with the cyclin E gene amplification displayed lymph node metastasis. Moreover, the case with the gene amplification overexpressed the cyclin E mRNA and protein. One of eight gastric cancer cell lines, MKN‐7, shared the cyclin E gene amplification, and all of the gastric cancer cell lines expressed high levels of the cyclin E mRNA and protein even without gene amplification. Amplification of the cyclin D1 gene was not observed in any of the gastric carcinoma tissues or gastric carcinoma cell lines. These results suggest that the gene amplification and overexpression of cyclin E play an important role in the abnormal growth and progression of gastric carcinoma.

Production of Experimental Malignant Pleural Effusions Is Dependent on Invasion of the Pleura and Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor by Human Lung Cancer Cells

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.

Accumulation of DNA Methylation Is Associated with Tumor Stage in Gastric Cancer

The authors purpose in this study was to clarify the difference in terms of clinicopathologic features between gastric cancer (GC) with high numbers of DNA methylated genes and CpG island methylator phenotype (CIMP)‐positive GC as originally defined.

Differential effects between amphoterin and advanced glycation end products on colon cancer cells

Amphoterin is 1 ligand of the receptor for advanced glycation end products (RAGE). We studied expression of amphoterin and RAGE mRNA and proteins in colorectal carcinoma cells and investigated their associations with the invasive activities of cells exposed to advanced glycation end products (AGE). Expression of RAGE and amphoterin was examined in 4 colorectal carcinoma cell lines. All cell lines expressed both RAGE and amphoterin. The effects of RAGE and amphoterin on cell growth (MTT assay), migration (wound healing assay) and invasion (in vitro invasion assay) were tested by treatment of cells with RAGE and amphoterin antisense S‐oligodeoxynucleotides (ODNs). Cell growth, migration and invasion were inhibited significantly in Colo320 and WiDr carcinoma cells treated with RAGE and amphoterin antisense S‐ODNs compared with sense‐treated cells. Differences in ligand activity between amphoterin and AGE were examined with AGE‐bovine serum albumin (BSA). AGE‐BSA decreased cell growth, migration and invasion of amphoterin antisense S‐ODN‐treated Colo320 and WiDr cells compared with those of cells treated with Colo320 conditioned medium. Phosphorylation of extracellular signal‐regulated kinase‐1/2, Rac1 and AKT and production of matrix metalloproteinase 9 were increased to a greater degree by amphoterin than by AGE‐BSA. In contrast, production of inducible nitric oxide synthase and nuclear factor‐κBp65 were increased to a greater degree by AGE‐BSA than by amphoterin.

Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9‐cis‐retinoic acid resistant (MKN‐7 and Ho‐1‐N‐1) and IFN‐β resistant cell lines (MKN‐7, ‐28 and ‐45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP‐ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21WAF1, CREB‐binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F‐1, E2F‐4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell‐to‐cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis‐regulating proteins. Int. J. Cancer 88:992–997, 2000.

Inhibition of heme oxygenase-1 by zinc protoporphyrin IX reduces tumor growth of LL/2 lung cancer in C57BL mice

Heme oxygenase (HO)‐1 is a key player reducing cytotoxicity and enhancing protumoral effects of nitric oxide (NO). We examined zinc protoporphyrin (ZnPP) IX, an HO‐1 inhibitor, to affect tumor growth of LL/2 mouse lung cancer cells. ZnPPIX reduced HO‐1 expression and HO activity in LL/2 cells, whereas cobalt PPIX (CoPPIX), an HO‐1 activator, increased both. LL/2 cells treated with sodium nitropurusside, an NO donor, showed growth inhibition dose‐dependently, which was enhanced by ZnPPIX cotreatment, but was reduced by CoPPIX. In mice tumors, ZnPPIX decreased HO‐1 expression. LL/2‐tumors were found in 88% (7/8) vehicle‐treated mice, whereas tumors were found in 38% (3/8) and 25% (2/8) mice treated with 5 and 20 μg/mouse ZnPPIX, respectively (p = 0.0302). Tumor growth was inhibited dose‐dependently by ZnPPIX. Vascular endothealial growth factor concentration in tumors was reduced by ZnPPIX (p = 0.0341). Microvessel density (MVD) in ZnPPIX‐treated tumors was lower than that in vehicle‐treated tumors (p = 0.0362). Apoptotic cell count in ZnPPIX‐treated tumors was higher than that in vehicle‐treated tumors (p = 0.0003). In contrast, CoPPIX treatment increased HO‐1 expression, enhanced tumorigenicity and MVD and reduced apoptosis. From these findings, inhibition of HO‐1 by ZnPPIX provides relevant antitumoral effects.

Downregulation of miR-126 induces angiogenesis and lymphangiogenesis by activation of VEGF-A in oral cancer

Background:MicroRNA (miRNA)-126 (miR-126) is an endothelial-specific miRNA located within intron 7 of epidermal growth factor-like domain 7 (EGFL7). However, the role of miR-126 in cancer is controversial.Methods:We examined the function of miR-126 in oral squamous cell carcinoma (OSCC) cells. Furthermore, a series of 118 cases with OSCC were evaluated for the expression levels of miR-126.Results:MicroRNA-126 (miR-126) was associated with cell growth and regulation of vascular endothelial growth factor-A activity, and demethylation treatment increased expression levels of miR-126 and EGFL7 in OSCC cells. A significant association was found between miR-126 expression and tumour progression, nodal metastasis, vessel density, or poor prognosis in OSCC cases. In the multivariate analysis, decreased miR-126 expression was strongly correlated with disease-free survival.Conclusion:The present results suggest that miR-126 might be a useful diagnostic and therapeutic target in OSCC.

Molecular diagnosis of gastric cancer: present and future.

Although histopathological diagnosis is extremely useful for the definitive as well as the supportive diagnosis of gastric cancer in clinical practice, it is limited in certain respects. Over the past 15 years, integrated research in molecular pathology has clarified the details of genetic and epigenetic abnormalities of cancer-related genes in the course of the development and progression of gastric cancer. These abnormalities, which include telomerase activation, genetic instability, and abnormalities in oncogenes, tumor suppressor genes, cell-cycle regulators, cell adhesion molecules, and DNA repair genes, could be effective markers in the molecular diagnosis of gastric cancer. It is possible that the molecular analysis of these alterations in histopathology specimens may overcome deficiencies in diagnoses that depend only on histomorphology, and, consequently, we may be able to improve the differential diagnosis of cancer, obtain information on the grade of malignancy, and identify patients at high risk of developing multiple primary cancers. In Hiroshima, we have established a system of molecular-pathological diagnosis as a routine service; about 5000 lesions of the stomach have been subjected to this diagnosis, and much useful information has been obtained. In the near future, genetic analysis by means of DNA microarray may become routine in the diagnosis of gastric cancer. Genetic analysis of histopathology specimens may make clear the characteristics of individual cancers; indicating the common and specific features of molecular pathogenesis that may be directly connected with gene therapy or molecular-targeted therapy. By analyzing the relationship between single-nucleotide polymorphisms and cancer susceptibility, we will be able to obtain information on cancer prevention from histopathology samples.