Yukihiro Tatemoto

Mechanism of HIF-1α-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells

The transcriptional factor hypoxia‐inducible factor‐1 (HIF‐1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF‐1α has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF‐1α‐induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF‐1α suppressed hypoxia‐induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF‐1α inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca2+ concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase‐9 and caspase‐3. In addition, antiapoptotic Bcl‐2 and Bcl‐XL levels were increased and pro‐apoptotic Bax and Bak levels were decreased in the HIF‐1α‐overexpressing OSCC line. Overexpression of HIF‐1α also increased the levels of phosphorylation of Akt and extracellular signal‐regulated kinases (ERK). These findings indicate that HIF‐1α prevents apoptotic cell death through two mechanisms, including inhibition of cytochrome c release and activation of Akt and ERK. (Cancer Sci 2005; 96: 394–402)

Mn-SOD antisense upregulates in vivo apoptosis of squamous cell carcinoma cells by anticancer drugs and γ-rays regulating expression of the BCL-2 family proteins, COX-2 and p21

ROIs and their scavengers are associated with apoptosis induction by anticancer drugs and γ‐rays, but the details have not been clarified. We examined the effect of transfection of Mn‐SOD antisense on apoptosis by 5‐FU, PLM, CDDP and γ‐rays using nu/nu mice. After inoculation of Mn‐SOD antisense–transfected SCC cells into the subcutis of each mouses back, they slowly multiplied to form tumors sized 1,460 ± 70 mm3 at day 60, while control vector‐transfected SCC cells rapidly multiplied, with a mean tumor size of 2,330 ± 220 mm3. Inversely, mice in the Mn‐SOD antisense group survived longer (mean survival duration 94.4 ± 12.7 days) compared to those in the empty vector group (67.3 ± 6.8 days). After treatment with 5‐FU (5 μg/day), PLM (50 μg/day), CDDP (10 μg/day) and γ‐rays (2 Gy/day), mean survival times were largely prolonged, to 126.3 ± 22.7, 123.0 ± 22.1, 136.3 ± 24.0 and 143.0 ± 20.8 days, respectively, while mean survival times in the empty vector group were 91.7 ± 14.8, 85.7 ± 13.3, 97.5 ± 16.0 and 100.7 ± 17.1 days, respectively. Immunohistologically, tumors in the Mn‐SOD antisense group revealed additional nick end‐labeled cells compared to those in the empty vector group. In comparison, strong expression of Bax, Bak and p21waf1/cip1 and suppressed expression of Bcl‐2, Bcl‐XL and COX‐2 were observed in the Mn‐SOD antisense group and the expression pattern of these proteins was the inverse in the empty vector group. The increased expression of these proapoptotic proteins appeared to be p53‐independent because p53 protein expression was not increased in the antisense group. These immunohistologic results were supported by Western blotting of each protein. In conclusion, Mn‐SOD antisense transfection is advantageous for apoptosis induction of SCC cells by anticancer drugs and γ‐rays through induction of proapoptotic Bcl‐2 family proteins and suppression of antiapoptotic protein expression.

Low malignant intraductal carcinoma on the hard palate: a variant of salivary duct carcinoma?

A rare, minor salivary gland tumour of the hard palate in a middle-aged woman was presented. The small (1.0 X 0.5 cm in diameter) hemispherical tumour was well circumscribed with a fine papillomatous surface. Histopathologically, tumour cells with eosinophilic cytoplasm and a large nucleus were single-strand cuboidal and columnar cells, which showed intraductal growth exhibiting a cribriform pattern. The histological features were distinct from adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma because the tumour lacked the neurotropic infiltration, cord-like proliferation and targetoid arrangement. The tumour could not be identified as a typical salivary-duct carcinoma because Roman bridging, papillary projection, and severe cell atypia were not found. Tumour cells were negative for PAS, Alcian blue, mucicarmine, p53, c-erbB-2, CEA, S-100 protein, alpha-smooth muscle actin, lactoferrin or vimentin. About 5% of the tumour cells were positive for proliferating cell nuclear antigen. Taking these factors into account, together with the clinical features, the name low malignant intraductal carcinoma seems appropriate.

Expression of p53 and p21 Proteins in Oral Squamous Cell Carcinoma : Correlation with Lymph Node Metastasis and Response to Chemoradiotherapy

p53 protein, a product of the p53 cancer suppressor gene, and p21 protein, a cyclin-dependent kinase inhibitor, were immunohistochemically investigated in 150 oral squamous cell carcinomas (SCCs) and the relationship between their expression and clinicopathological findings were evaluated. The positivity for p53 and p21 proteins was not correlated with the T-stage, mode of tumor cell invasion or tumor cell differentiation. However, the expression of p53 and p21 proteins was correlated with lymph node metastasis. Of 62 SCCs with regional lymph node metastasis, 45 SCCs (72.6%) were positive for p53 while 45 (52.9%) of 88 SCCs without metastasis expressed p53 protein (p < 0.02). In addition, p21 protein was observed in 25 (38.5%) and 18 (21.2%) SCCs with and without metastasis, respectively (p < 0.05). Furthermore, p53 protein was inversely correlated with the histopathological effect of inductive chemoradiotherapy; the rate of chemoradiotherapy-induced lethal degeneration (56.7%) in p53-negative SCCs was significantly higher than that (28.9%) in p53-positive SCCs (p < 0.005). However, no clear difference in the effect was observed between p21-positive and p21-negative SCCs. Finally, the 5-year-survival rate was highest in p53(-)-p21(+) (80.0%) followed by 76.3% in p53(-)-p21(-), 65.9% in p53(+)-p21(+) and 65.4% in p53(+)-21p(-) SCCs. These results indicate that although the expression of p21 protein is only weakly correlated with the clinico-histopathological findings, p53 protein is a useful prognostic marker and that inductive chemoradiotherapy can be successfully planned by immunohistochemical examination of p53 protein.

Tumorigenicity of cell lines established from oral squamous cell carcinoma and its metastatic lymph nodes

We investigated the biological and histopathological characteristics of seven human tumour cell lines established from primary tongue squamous cell carcinoma (OSC-1), from metastasised lymph nodes of the gingival carcinoma (OSC-2 and OSC-3) and from tongue carcinoma (remaining four lines). The doubling time ranged from 22 h (OSC-2 and OSC-4) to 55 h (OSC-7), and did not correlate with tumour cell stratification in a collagen gel matrix. An invasive tendency was most prominent in OSC-2 and OSC-4; with the other cell lines, except OSC-6 and OSC-7, only a few sporadic invading cells were found in the tissue culture. In the cell lines established from the metastatised tumours, originally exhibiting grade 3 invasion, the invasion became more sporadic when the tumour cells were xenografted into the tongues of nude mice, while an invasion similar to the original was observed in the cell lines obtained from the original site (OSC-1) and from tumours of Grade 4C invasion. These findings suggest that the biological behaviour of the established tumour cells is markedly different even in tumours of the same tissue origin, and strongly invasive cells may selectively invade, and metastatise to the lymph nodes.

Enhanced expression of Toll-like receptor 2 in lesional tissues and peripheral blood monocytes of patients with oral lichen planus

Some members of the Toll‐like receptor (TLR) family, which plays key roles in both innate and adaptive immune responses, are involved in the pathogenesis of autoimmune, chronic inflammatory and infectious diseases. However, the role of TLR in the pathogenesis of oral lichen planus (OLP) has not been investigated. The aim of this study was to understand the roles of TLR in OLP. The expression of TLR genes in OLP tissues was analyzed by cDNA microarray and reverse transcription polymerase chain reaction, and TLR protein expression in OLP tissues and peripheral blood monocytes was examined by immunohistochemical analysis and flow cytometry, respectively. Furthermore, TLR ligand‐induced cytokine production from peripheral blood monocytes was measured by enzyme‐linked immunosorbent assay. Among 10 TLR genes, the average expression ratio of the genes for TLR1, 2, 3, 5, 6 and 10 in OLP tissues compared to that in the normal buccal mucosae was more than 1.0. In contrast, the average ratio of the genes for TLR7, 8 and 9 was less than 1.0. TLR2 but not TLR4 was highly expressed in the cells of the spinous layer and infiltrating monocytes in OLP tissues, and the mean fluorescence intensity of TLR2 on peripheral blood monocytes was significantly higher in OLP patients than in healthy controls. Furthermore, the peripheral blood monocytes from OLP patients produced considerably higher amounts of interleukin (IL)‐12 and lower amounts of IL‐10 than those from healthy controls. In OLP, the T‐helper cell (Th)1/Th2 balance appears to shift toward Th1 dominance, probably depending on the upregulation of TLR2 expression and these alterations in TLR2‐mediated immunity may be involved in the pathogenesis and maintenance of OLP.

Risk Factors of Metastasis in Oral Squamous Cell Carcinomas

Lymph node and distant metastasis were comparatively studied in 225 oral carcinomas, and factors predisposing toward metastasis were investigated using clinical and immunohistopathological approaches. Neither the sites of tumors nor T-stage was correlated with either type of metastasis. Tumor cell differentiation was weakly correlated with lymph node metastasis, and stromal reaction (the degree of cell infiltration) did not differ greatly between metastasis-positive and negative tumors, although natural killer (NK) activities were correlated with lymph node metastasis. However, the mode of tumor cell invasion was closely associated with both lymphnode and distant metastases. In grade 4C and 4D tumors, distant and lymph node metastases were observed in 8 (16%) and 31 (62%) cases, respectively, while of 68 grade 1 and 2 tumors, distant metastasis was not observed in any, and lymph node metastasis occurred in only 15 (22.1%). In addition, the expression of p53 protein was correlated with lymph node metastasis; of 70 tumors without p53 protein expression, 23 (32.9%) revealed lymph node metastasis, while it occurred in 54 out of 96 tumors positive for p53 protein. However, p53 protein expression was not associated with distant metastasis, and p24 protein, a cyclin-dependent kinase inhibitor, did not show any relationship with either type of metastasis. These results indicate that lymph node metastasis is correlated with multiple factors in the host and tumor cells, but distant metastasis is only correlated with the mode of tumor cell invasion, suggesting that the former can be highly accurately predicted by invasion mode, p53 protein expression and NK activity.

Characteristic cytokine generation patterns in cancer cells and infiltrating lymphocytes in oral squamous cell carcinomas and the influence of chemoradiation combined with immunotherapy on these patterns

Objectives: Cytokines produced by tumor cells and tumor-infiltrating lymphocytes (TIL) appear to regulate tumor cell growth and the cytotoxic activity of TIL. The objectives of the present study were to investigate cytokine generation patterns in tumor cells and TIL and to examine the influence of cancer therapy on this cytokine production and the cytotoxic activity of TIL. Methods: We determined the levels of cytokines produced by tumor cells and TIL in vitro and measured the cytotoxic activity of TIL against Daudi cells in patients with oral squamous cell carcinoma (OSC) before and 1 week after the start of concomitant chemo-radio-immunotherapy. Results: Before the therapy, OSC cells generated higher levels of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) than did oral keratinocytes isolated from the noninflamed gingivae of healthy individuals, but both kinds of cells generated similar levels of interleukin (IL)-1β and IL-6. Compared with peripheral blood mononuclear cells (PBMC) of the patients, TIL produced higher levels of IL-1β, IL-6, IL-10, TNF-α and TGF-β, whereas their production of IL-12 and interferon-γ (IFN-γ) was only slightly higher than that in PBMC. After 1 week of therapy, the cytokine production by OSC cells had largely decreased, while the production of TNF-α, IFN-γ, TGF-β and IL-12 by TIL had increased greatly, although other cytokine levels were almost constant during the investigations. The cytotoxic activity of TIL was higher than that of PBMC before the therapy, and this activity was strongly increased by 1 week of therapy. Conclusions: These results suggest that the cytokine productivities of TIL and tumor cells differ from those of PBMC and normal keratinocytes, respectively, and that chemo-radio-immunotherapy modulates in situ cytokine generation, which is advantageous for inhibition of tumor cell growth and activation of TIL.

Mucinous adenocarcinoma of the submandibular gland

A rare tumor not easily classifiable among published histologic categories for salivary gland tumors is reported. The neoplasm developed within the submandibular gland of a 78‐year‐old woman with invasion of the mandible and metastasis to regional lymph nodes. Histopathologically, cuboidal cells possessing clear cytoplasm and displaced round nuclei proliferated and exhibited an adenomatous pattern. Many cystic spaces surrounded by tumor cell strands were seen, mucus substance filled in the cystic spaces, and the tumor cells seemed mucus‐secreting, but neither epidermoid cells nor papillary appearance could be observed. Electromicroscopically, numerous mucous droplets of low electron density were prominent in the cytoplasm, and the tumor cells had sparce irregular microvilli on the luminal surface. Mucin histochemistry, including paradoxical concanavalin A staining, revealed that the tumor cells contained neutral and acid mucins, and these were identified as class II and III mucosubstances. No other neoplastic lesion, except recurrent metastatic neck nodes, has been detected 6 years after the first examination, and it seems that the tumor is a rare primary mucinous adenocarcinoma of the submandibular gland.

Isolated metastasis from hepatocellular carcinoma to the mandibular condyle with no evidence of any other metastases: a case report

Regional metastasis of hepatocellular carcinoma (HCC), the most common primary tumour of the liver to the mandible is rare. We present a case of a HCC metastasis to the mandibular condyle with no other extrahepatic metastases sites in a 59-year-old man. Incidental extrahepatic lesions in more uncommon sites should perhaps be investigated as potential areas for metastases, even if metastatic disease has not been found in the more common areas.