Tomonori Sasahira

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.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.

The expression of receptor for advanced glycation end products is associated with angiogenesis in human oral squamous cell carcinoma

The receptor for advanced glycation end products (RAGE) is associated with cancer progression in several human cancers. In this study, we examined the roles of RAGE in the angiogenesis of oral squamous cell carcinoma (OSCC). RAGE concentration was examined in 20 OSCC tumors by enzyme-linked immunosorbent assay (ELISA). The microvessel density (MVD) and lymph vessel density (LVD) were examined by immunostaining. Concentrations of vascular endothelial growth factor (VEGF) and VEGF-C were examined in tumor tissues by ELISA. Tumoral RAGE concentration was associated with higher tumor MVD (P = 0.0123) and tumor VEGF concentration (P = 0.0344), but not with LVD and VEGF-C concentration. Treatment with RAGE ligand, high-mobility group box (HMGB)-1 increased the secretion of VEGF but not that of VEGF-C in human OSCC cell lines, HSC-3 and HSC-4. The effect of HMGB-1 was abrogated by RAGE down-regulation by antisense S-oligodeoxynucleic acid. These results suggest that RAGE expression is closely associated to angiogenesis in OSCC.

High mobility group box‐1‐inducible melanoma inhibitory activity is associated with nodal metastasis and lymphangiogenesis in oral squamous cell carcinoma

Melanoma inhibitory activity (MIA) is an 11‐kDa secretory protein isolated from malignant melanoma cells that is correlated with invasion and metastasis in various human malignancies. We examined MIA expression in 62 oral squamous cell carcinomas (OSCC) by immunohistochemistry. MIA expression was significantly associated with nodal metastasis (P = 0.00018). MIA expression was also associated with expression of high mobility group box‐1 (HMGB1) (P < 0.0001) and lymph vessel density (P < 0.0001). Expression levels of MIA, HMGB1, nuclear factor kB (NFkB) p65 and HMGB1–NFkB p65 binding were significantly higher in a metastatic human OSCC cell line (HSC3) than those in a non‐metastatic OSCC cell line (HSC4). Treatment with receptor for advanced glycation end products (RAGE) antisense or small interfering RNA and human recombinant HMGB1 (hrHMGB1) did not affect MIA expression, whereas HMGB1 antisense or siRNA treatment decreased MIA expression in HSC3 cells. Then HMGB1 enhanced MIA expression as an NFkB cofactor but not as a RAGE ligand. MIA neutralization by MIA antibodies increased extracellular signal‐related kinase 1/2 phosphorylation, but decreased p38 phosphorylation and the expression of vascular epithelial growth factor (VEGF)‐C and ‐D. Treatment with p38 inihibitor decreased VEGF‐C and ‐D expression in HSC3 cells. These results suggest that MIA expression is enhanced by the interaction of intracellular HMGB1 and NFkBp65 and MIA is closely involved in tumor progression and nodal metastasis by the increments of VEGF‐C and VEGF‐D in OSCC. (Cancer Sci 2008; 99: 1806– 1812)

High mobility group box 1 released from necrotic cells enhances regrowth and metastasis of cancer cells that have survived chemotherapy

The role of the high mobility group box 1 (HMGB1) protein in chemotherapy-induced cell death was examined. CT26 mouse colon cancer cells were treated with trichostatin A (TSA; apoptosis inducer) or doxorubicin (DXR; necrosis inducer). DXR increased HMGB1 concentration in CT26 cell culture medium, whereas TSA did not. In a CT26 bilateral subcutaneous tumour model, DXR or TSA was injected in a single tumour. After injection, serum HMGB1 concentration in DXR-treated mice was 10 times higher than that in TSA-treated mice. After DXR treatment, the contralateral and remnant tumours showed more pronounced growth than did those treated with TSA. In mouse models, lung and liver metastasis was enhanced by DXR but not by TSA. DXR-enhanced metastasis was abrogated by anti-HMGB1 antibody treatment. In a cancer dormancy model, DXR induced regrowth of quiescent CT26 cells. HMGB1 induced tumour necrosis factor-α secretion via Toll-like receptor (TLR)4 in U937 monocytes; however, HMGB1 decreased the number of U937 cells, resulting in restriction of immune activation via receptor for advanced glycation endproducts (RAGE). RAGE showed a more pronounced effect on nuclear factor kappa B activation than did TLR4 in CT26 cells. These findings suggest that HMGB1 released from necrotic cancer cells treated with a necrosis inducer enhances regrowth and metastasis of remnant cancer cells via RAGE activation.

Association of Expression of Receptor for Advanced Glycation End Products and Invasive Activity of Oral Squamous Cell Carcinoma

Objectives: The receptor for advanced glycation end products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. Nevertheless, the involvement of RAGE in the development and progression of oral squamous cell carcinomas has not been elucidated. This study investigated the expression of RAGE in ten oral squamous cell carcinoma cell lines including primary and metastatic cell lines and its association with invasion and metastasis. Methods: Reverse transcriptase-polymerase chain reaction, antisense phosphorothioate (S)-oligodeoxynucleotide assay, preparation of antibody, immunohistochemical staining, immunoblot analysis, migration assay, in vitro invasion assay, and wound-healing assay were used. Results: RAGE protein expression of metastatic cancer cells treated with RAGE antisense S-oligodeoxynucleotide was significantly reduced compared to that of sense S-oligodeoxynucleotide-treated cells. The migration assay showed that invasive activity was significantly reduced in metastatic cancer cells treated with RAGE antisense S-oligodeoxynucleotide. Similarly, during invasion assays, numbers of invading cells were also reduced with the addition of RAGE antisense S-oligodeoxynucleotide-treated cells. A wound-healing assay showed that only a few RAGE antisense S-oligodeoxynucleotide-treated cancer cells migrated into the scraped area, whereas sense S-oligodeoxynucleotide-treated cells showed many budding nests in the scraped area of the metastatic cell lines. Immunohistochemically, oral squamous cell carcinoma cells in the tumour mesenchymal border were often immunopositive, whereas basal cells in the normal mucosa were scarcely positive. Conclusions: These results suggest that RAGE expression appears to be closely associated with the invasiveness of oral squamous cell carcinoma and represents a promising candidate for assessing the future therapeutic potential in treating patients with oral carcinoma.

Receptor for advanced glycation end products (RAGE) is important in the prediction of recurrence in human oral squamous cell carcinoma.

Aims:  Receptor for advanced glycation end products (RAGE) has recently been recognized as a cancer‐associated protein responsible for cancer progression and metastasis in gastrointestinal cancers. The aim was to examine the role of RAGE in oral squamous cell carcinoma (OSCC).

Suppression of dendritic cells by HMGB1 is associated with lymph node metastasis of human colon cancer.

High mobility groupbox-1 (HMGB1) is a multifunctional cytokine secreted by cancer cells, which accelerates cell growth, invasion and angiogenesis in cancer, and induces apoptosis in macrophages. Thioglycolate-stimulated mouse peritoneal macrophages were induced to differentiate into dendritic cells by co-treatment with IL-4 and GM-CSF. The number of mouse peritoneal macrophage-derived dendritic cells (PMDDCs) showed a dose-dependent decrease in hrHMGB1 treatment. HMGB1-treated PMDDCs showed obvious apoptosis and increased the level of phosphorylated JNK. Intraperitoneal administration of HMGB1 decreased CD205-positive splenic dendritic cells in C57BL mice. To confirm the HMGB1-induced inhibitory effect on dendritic cells, 16 cases of human colon cancer invaded into the subserosal layer were examined. The 8 nodal metastasis-positive cases showed higher nodal HMGB1 concentrations (74 ± 23 vs. 41 ± 15 μg/ml, p = 0.0116) in lymph node tissues and lower CD205-positive nodal dendritic cell numbers (86 ± 22 vs. 137 ± 43/mm2, p = 0.0224) than those in the 8 metastasis-negative cases. Primary tumor tissues of metastasis-positive cases showed higher tumor HMGB1 levels (116 ± 33 vs. 37 ± 18 μg/ml, p = 0.0007) and lower CD205-positive intratumoral dendritic cell numbers (21 ± 13 vs. 62 ± 23 /mm2, p = 0.0068) than those in metastasis-negative cases. These findings suggest that HMGB1 produced by colon cancer cells suppressed nodal dendritic cells to disturb host anti-cancer immunity.

Conjugated linoleic acid inhibits peritoneal metastasis in human gastrointestinal cancer cells

The effect of conjugated linoleic acid (CLA) on peritoneal metastasis was examined by in vitro treatment of cancer cells and mouse peritoneal metastasis models. First, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by CLA in a dose‐dependent manner with an increment in apoptosis. CLA significantly inhibited invasion into type IV collagen‐coated membrane of MKN28 and Colo320 cells (p < 0.05). CLA‐induced growth inhibition was recovered by the exposure to antisense S‐oligodeoxynucleotide for peroxisome proliferator‐activated receptor (PPAR)‐γ in both cell lines. BALB/c nu‐nu mice were inoculated with MKN28 and Colo320 cells into their peritoneal cavity, and administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did not affect food intake or weight gain of mice. CLA treatment significantly decreased metastatic foci of both cells in the peritoneal cavity (p < 0.005). Survival rate in mice inoculated with MKN28 or Colo320 cells was significantly recovered by CLA treatment (p = 0.0025 and 0.0052, respectively). Protein production in MKN28 and Colo320 cells treated with CLA showed a decrease in epidermal growth factor receptor and transforming growth factor‐α and an increase in Bax. These findings suggest that CLA inhibits metastasis of human gastric and colon cancer cells.