Mitsunobu Sato

Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma.

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.

Possible contribution of active MMP2 to lymph‐node metastasis and secreted cathepsin L to bone invasion of newly established human oral‐squamous‐cancer cell lines

We have established human oral‐squamous‐cancer cell lines, BHY and HN, derived from non‐metastatic cancer and metastatic cancer respectively. We examined the expression of matrix‐degrading enzymes and their inhibitors in these cell lines. Both cell lines expressed pro‐matrix metalloproteinase (MMP)1, proMMP2, proMMP9, membrane‐type MMP and urokinase‐type plasminogen activator. In addition to these enzymes, BHY cells secreted proMMP7 and procathepsin L, while HN cells secreted a large amount of active MMP2. BHY cells secreted a tissue inhibitor of matrix metalloproteinase, TIMP2, but only a trace level of TIMP1. Contrary to BHY cells, HN cells secreted TIMP1, but only a trace level of TIMP2. When we inoculated these cells into the masseter muscle of nude mice, both types of cell formed solid tumors, whose microscopic appearance was identical to that of the original tumors. BHY tumors were highly differentiated squamous‐cell carcinomas, and invasive to the masseter muscle and the mandibular bone. Despite their local aggressiveness, BHY tumors did not metastasize to any distant organs. HN tumors were poorly differentiated squamous‐cell carcinomas, weakly invasive to the muscle, but not to the mandibular bone. However, HN tumors frequently metastasized to cervical lymph nodes. These results suggest that the net activity of MMP2 (active MMP2/TIMP2) and cathepsin L secreted from cancer cells may contribute respectively to lymph‐node metastasis and to bone invasion by oral cancer cells.

Expression of Toll-Like Receptor 4 on Dendritic Cells Is Significant for Anticancer Effect of Dendritic Cell-Based Immunotherapy in Combination with an Active Component of OK-432, a Streptococcal Preparation

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432, a Streptococcus-derived anticancer immunotherapeutic agent. In the present study, we first examined the effect of OK-PSA on the maturation of dendritic cells (DCs) in vitro by using the DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without expression of toll-like receptor 4 (TLR4) or MD-2 mRNA. OK-PSA treatment effectively increased the surface expression of MHC class II, CD80, CD83, and CD86. OK-PSA-stimulated DCs secreted the cytokines that can induce helper T-cell 1 (Th1)-type T-cell response, and stimulated allogeneic T cells to produce IFN-γ and to elicit an allogeneic antigen-specific cytotoxicity. These activities almost depended on expression of TLR4 and MD-2 genes. We next investigated the in vivo anticancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4−/−) mice were used. Although OK-PSA accelerated the antitumor effect of intratumoral DC administration in wild-type mice bearing syngeneic tumors, the antitumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4−/− mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked antitumor effect even in the TLR4−/− mice. These findings suggest that OK-PSA may be a potent adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anticancer activity even in a TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.

Role of HGF/c-met system in invasion and metastasis of oral squamous cell carcinoma cells in vitro and its clinical significance

We examined the role of the hepatocyte growth factor (HGF)/c‐met system on invasion and metastasis of oral squamous cell carcinoma (SCC) cells. In monolayer culture, exogenous HGF marginally affected the growth of oral SCC cells (BHY, HN, IH) and human gingival epithelial cells (GE). In type I collagen matrix, however, HGF significantly enhanced the invasive growth of the cancer cells (p < 0.05). We detected the expression of c‐met (HGF receptor) mRNA in all of the cancer cells, but not in human gingival fibroblasts (GF). Oral SCC cells did not secret HGF protein into the medium, but GF secreted a large amount of HGF protein (15 ng/ml). Furthermore, HGF markedly enhanced the migration of cancer cells in a Transwell invasion chamber. Then, we examined the serum levels of HGF in oral SCC patients, or HGF concentrations in oral cancer tissues. Serum levels of HGF in the patients were significantly higher than those in healthy volunteers (p < 0.05). After initial treatment, all of the tumor‐free survivors showed a marked decline in the serum HGF levels. Furthermore, HGF concentrations in metastatic cancer tissues were significantly higher than those of non‐metastatic cancer tissues and normal gingiva (p < 0.01). These results suggest that HGF plays an important role in invasion and metastasis of oral SCC cells as a paracrine factor, and an elevated HGF level in the cancer tissue can be a predictive marker for metastasis formation in patients with oral SCC.

Acquisition of lymph node, but not distant metastatic potentials, by the overexpression of CXCR4 in human oral squamous cell carcinoma

Recently, it has been suggested that chemokine/receptor interactions determine the destination of the invasive tumor cells in several types of cancer. It has also been proposed that the stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system might be involved lymph node metastasis in oral squamous cell carcinoma (SCC). In order to further clarify the role of the SDF-1/CXCR4 system in oral SCC, we generated CXCR4 stable transfectants (IH-CXCR4) using oral SCC cells, and compared them to IH, which did not express CXCR4 and which did not have lymph node metastatic potentials in vivo. We introduced enhanced green fluorescent protein (GFP) fused-CXCR4 into IH cells, and detected the GFP fluorescence in the cytoplasm and cell membrane in approximately 60% of the G418-resistant cells. This bulk-transfectant expressed a high level of CXCR4 mRNA and protein, and exhibited the characteristic calcium fluxes and chemotactic activity observed in treatment with SDF-1. SDF-1 biphasically activated extracellular signal-regulated kinase (ERK)1/2, but continuously activated Akt/protein kinase B (PKB) in IH-CXCR4 cells. Most importantly, IH-CXCR4 cells frequently metastasized to the cervical lymph node, but not to the distant organs in the orthotopic inoculation of nude mice. Furthermore, these lymph node metastases were inhibited by the treatment of a mitogen-activated protein kinase/ERK kinase inhibitor, U0126, or a phosphatidylinositol 3 kinase inhibitor, wortmannin. These results indicate that SDF-1/CXCR4 signaling mediates the establishment of lymph node metastasis in oral SCC via ERK1/2 or Akt/PKB pathway.

Search for specific markers of neoplastic epithelial duct and myoepithelial cell lines established from human salivary gland and characterization of their growth in vitro

The neoplastic epithelial duct cells human salivary gland (HSG) and myoepithelial cells human pleomorphic adenoma (HPA) established from human salivary gland were examined by the immunoperoxidase method for the presence of specific antigens such as carcioembryonic antigen (CEA), S‐100 protein, secretory component (SC), lactoferrin (LF), and myosin. Isolation of the cells and their morphologic features were reported previously. Consequently, the presence of CEA, SC, and LF in the HSG cells was demonstrated. The HPA cells were identified to express the specific antigens reactive to anti‐S‐100 protein, anti‐myosin and anti‐CEA sera in addition to the presence of oxytocin receptor. When the two cell lines were co‐cultured in monolayer culture or within the sponge matrix, a large number of ductlike or tubular structures were formed in an optimal ratio of 1:2 in HSG and HPA cells, whereas the cultures of HSG cells only grew with occasional formation of ductlike structure. In addition, in HSG and HPA cells in an area with their contact in the mixed cultures, CEA staining was intensified as compared with the culture of HSG or HPA cells only and further S‐100 protein was detected in HSG cells, whereas S‐100 protein was not detected in the culture of HSG cells only. These findings strongly suggest that the intercalated duct and myoepithelial cells from human salivary gland propagate with their interaction together in the expression of specific antigens such as CEA and S‐100 protein or in the morphogenesis of salivary gland epithelial cells.

Enhanced radiosensitization and chemosensitization in NF-κB-suppressed human oral cancer cells via the inhibition of γ-irradiation- and 5-FU-induced production of IL-6 and IL-8

We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to γ‐irradiation (IR) and 5‐Fluorouracil (5‐FU) in human oral carcinoma cells (B88) in which NF‐κB activity was constitutively suppressed. Three super‐repressor form of IκBα cDNA‐transfected cell (B88mI) clones and 1 empty vector‐transfected cell clone (B88neo) have been established. We found that the tumor‐forming ability in nude mice of B88mI clones was significantly lower than that of B88 or B88neo. This suppressed ability in tumorigenicity was attributed to the down‐regulation of the expression of interleukin (IL)‐1α, IL‐6, IL‐8, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)‐9 in B88mI cell clones as compared to that in B88 or B88neo. IR and 5‐FU induced a much greater degree of apoptosis, as evidenced by flow cytometry analysis and annexin V staining, in B88mI cell clones than in B88 or B88neo. When tumor‐bearing nude mice were treated with IR or 5‐FU, the suppression of tumor growth was significantly augmented in B88mI cell clones as compared to that in B88 or B88neo. ELISA analysis indicated that although a remarkable increase in production of IL‐6 and IL‐8 was observed in B88 and B88neo after in vitro exposure to IR or treatment with 5‐FU, radiotherapy and chemotherapy‐induced production of these cytokines was significantly suppressed in B88mI cell clones. These findings suggest that production of angiogenic factors and growth factors in response to radiotherapy and chemotherapy is a principal mechanism of inducible radioresistance and chemoresistance in human oral cancers, and establish the inhibition of NF‐κB as a rational approach to improve conventional radiotherapy and chemotherapy outcomes.

Involvement of an Autocrine Stromal Cell–Derived Factor-1/CXCR4 System on the Distant Metastasis of Human Oral Squamous Cell Carcinoma

We have previously shown that a stromal cell–derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis, but not in that of distant metastasis, in oral squamous cell carcinoma (SCC). In this study, we investigated the role of the autocrine SDF-1/CXCR4 system, with a focus on distant metastasis in oral SCC cells. The immunohistochemical staining of SDF-1 and CXCR4 using primary oral SCCs and metastatic lymph nodes showed a significantly higher number of SDF-1–positive cases among the metastatic lymph nodes than among the primary oral SCCs, which was associated with a poor survival rate among those of the former group. The forced expression of SDF-1 in B88 cells, which exhibit functional CXCR4 and lymph node metastatic potential (i.e., the autocrine SDF-1/CXCR4 system), conferred enhanced cell motility and anchorage-independent growth potential onto the cells. Orthotopic inoculation of the transfectant into nude mice was associated with an increase in the number of metastatic lymph nodes and more aggressive metastatic foci in the lymph nodes. Furthermore, the SDF-1 transfectant (i.e., the autocrine SDF-1/CXCR4 system) exhibited dramatic metastasis to the lung after i.v. inoculation, whereas the mock transfectant (i.e., the paracrine SDF-1/CXCR4 system) did not. Under the present conditions, AMD3100, a CXCR4 antagonist, significantly inhibited the lung metastasis of the SDF-1 transfectant, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that, in cases of oral SCC, the paracrine SDF-1/CXCR4 system potentiates lymph node metastasis, but distant metastasis might require the autocrine SDF-1/CXCR4 system. (Mol Cancer Res 2007;5(7):685–94)

Enhanced IκB kinase activity is responsible for the augmented activity of NF-κB in human head and neck carcinoma cells

Abstract The nuclear transcription factor κB (NF-κB) plays an important role in the development and progression of cancers. However, the mechanism by which cancer cells in the head and neck region acquire high NF-κB activity has not yet been clarified. In this study, we examined the NF-κB binding activity and the expression of the signal-transduction-related proteins of NF-κB in head and neck carcinoma cell lines. These cancer cells showed significantly higher NF-κB binding activity than normal oral epithelial and salivary gland cells. We also demonstrated the increased phosphorylation and degradation of IκB-α protein in cancer cells. Thus, enhanced NF-κB activity in cancer cells is attributable to the rapid phosphorylation and degradation of IκB-α protein. To further elucidate the mechanism involved in this phenomenon, we analyzed both the expression levels of upstream kinases (IκB kinase- (IKK-) α, IKK-β, IKK-γ, and NF-κB-inducing kinase (NIK)) and the IKK activity in cells. Although there was no significant difference in the expression levels of NIK, IKK-β, or IKK-γ in cancer cell lines compared to those in normal cells, increased expression of IKK-α protein was observed in cancer cells. In addition, IKK activity was significantly augmented in cancer cells as compared to normal cells. Thus, our results suggest that enhanced NF-κB activity in head and neck cancer cells may be due to the augmentation of IKK activity.

Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-κB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-κB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.