Kazunori Yoneda

Serum cytokines, interleukin-2 receptor, and soluble intercellular adhesion molecule-1 in oral disorders

Serum levels of soluble intercellular adhesion molecule-1, soluble interleukin-2 receptor, and cytokines such as interleukin-3, interleukin-4, interleukin-6, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor were examined in patients with oral disorders with 20 healthy persons used as control subjects. Patients studied included 30 with squamous cell carcinoma, 26 with oral lichen planus, 20 with recurrent aphthous ulcer, 19 with acute odontogenic bacterial infection, 16 with pseudomembranous candidiasis, and 16 with herpetic gingivostomatitis. Compared with levels in control subjects, detectable serum levels of interleukin-3 (> or = 10 pg/ml) existed more frequently in pseudomembranous candidiasis (13/16), acute odontogenic bacterial infection (14/19), and squamous cell carcinoma (24/30) and of granulocyte-macrophage colony-stimulating factor (> or = 4 pg/ml) more frequently in recurrent aphthous ulcer (15/20) and squamous cell carcinoma (21/30). These cytokine levels were increased with T stage of squamous cell carcinoma. About 20 pg/ml of interleukin-4 was detected in serum from one third to one fourth of patients with oral lichen planus, recurrent aphthous ulcer, and squamous cell carcinoma. Tumor necrosis factor-alpha was hardly detected in most patients except those with oral lichen planus and squamous cell carcinoma in which about one third of the patients had more than 40 pg/ml of tumor necrosis factor-alpha in serum. More than 10 pg/ml of interleukin-6 was frequently detected in all disorders, especially recurrent aphthous ulcer (18/20), pseudomembranous candidiasis (12/16), and acute odontogenic bacterial infection (17/19).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Manganese Superoxide Dismutase Negatively Regulates the Induction of Apoptosis by 5-Fluorouracil, Peplomycin and γ-Rays in Squamous Cell Carcinoma Cells

We investigated the relationship between manganese superoxide dismutase (Mn‐SOD) activity and apoptosis induced by anticancer drugs and radiation. Although the activity of copper, zinc‐SOD did not differ greatly among 9 squamous cell carcinoma (SCC) cell lines (OSC‐1 to OSC‐9), the Mn‐SOD activity did differ among the cell lines. The Mn‐SOD activity was increased by treatments with 5‐fluorouracil (5‐FU), peplomycin and 137Cs, reaching plateau levels at 12 h after treatment and then decreasing gradually. When OSC‐1 and OSC‐3, and OSC‐2 and OSC‐4 were examined as representative cell lines with low and high Mn‐SOD activity, respectively, the decrease was more prominent in OSC‐1 and OSC‐3 than in OSC‐2 and OSC‐4. The intracellular levels of superoxide and hydrogen peroxide (H2O2) were increased after treatment with the anti‐cancer agents, and the increases were larger in OSC‐1 and OSC‐3 than in OSC‐2 and OSC‐4. The decrease of mitochondrial membrane potential (Δψm) by the anticancer agents was marked in OSC‐1 and OSC‐3. Correspondingly, the release of cytochrome c, the activation of caspase‐3 and the cleavage of poly(ADP‐ribose)polymerase were stronger in OSC‐3 than in OSC‐4. In addition, apoptosis induced by the anticancer agents was prominent in OSC‐3, exhibiting a close relationship with the Δ7psi;m and the H2O2 level. These results indicate that Mn‐SOD in SCC cells modulates apoptosis induction and the inactivation of Mn‐SOD might be a promising strategy for SCC treatment.

Treatment of Xerostomia with the Bile Secretion-Stimulating Drug Anethole Trithione: A Clinical Trial

BACKGROUND Saliva protects the oral mucosa, inhibiting microbial overgrowth. Hyposalivation, therefore, induces multiple oral disorders, although treatment of hyposalivation is very difficult. METHODS A cholagogue, anethole trithione (AT) was administered to patients with symptomatic hyposalivation (xerostomia) caused by senile hypofunction (4 men and 17 women; senile group), medications (6 men and 17 women; drug group), and oral cancer therapy (two men and three women; cancer group). For control groups, an artificial saliva was administered to 45 patients consisting of senile hypofunction (10 men and 16 women), drug-induced xerostomia (3 men and 10 women) and oral cancer therapy-induced xerostomia (four men and two women). RESULTS Two weeks after administration of AT (6 tablets per day), both nonstimulated salivary flow rate (SFR) and stimulated SFR increased in a statistically significantly manner from 0.76 +/- 0.41 and 5.18 +/- 3.02 to 1.54 +/- 1.33 (P<0.05) and 9.07 +/- 4.10 mL/10 min (P<0.05), respectively. Of the three groups, the drug group showed the largest increases in both SFRs, from 0.90 +/- 0.54 and 6.29 +/- 4.12 to 1.69 +/- 1.65 and 12.09 +/- 5.10 mL/10 min (P<0.05 and P<0.02, respectively). Patients in the control group had almost constant SFRs. After AT administration, the salivary viscosity was, however, mildly decreased and concentrations of secretory-immunoglobulin A, lactoferrin, potassium, and chloride in nonstimulated saliva were almost constant. Corresponding with the increase of salivation, oral discomfort and inflammation were improved or resolved in 41 patients of the AT group within about 4 weeks, whereas improvement was observed in only nine patients of the control group. CONCLUSIONS These results indicate that AT sufficiently stimulates salivation and improves xerostomia.

p53- and p21-independent apoptosis of squamous cell carcinoma cells induced by 5-fluorouracil and radiation

Apoptosis-inducing therapy is becoming a new strategy in cancer therapy. We investigated the influence of 5-fluorouracil (5-FU) and radiation (gamma-ray) on the cell cycle of tumor cells, and their apoptosis-inducing activity using four oral squamous cell carcinoma lines (OSC-1 and OSC-4 with wild type p53; OSC-2 and OSC-3 with mutant type p53). The expression of p53 and cyclin-dependent kinase 2 (Cdk2) proteins was not increased even after cell treatment with 5-FU and gamma-rays in any cell lines. Although the promoter of p21 gene was not activated, p21-mRNA expression was increased by 5-FU and gamma-rays. p21 protein was expressed by irradiation in parallel with the increase in the messages but not by 5-FU in any OSC lines. Despite the increased p21 protein expression, cyclin E/Cdk2 kinase activity was not suppressed in irradiated cells. With the increased expression of cyclin E protein, 5-FU augmented the kinase activity in OSC-1, OSC-2 and OSC-3 cells. However, with a constant cyclin E level the kinase activity in OSC-4 was not increased by 5-FU. Without correlation to the kinase activity, 5-FU strongly induced apoptosis in OSC-2, OSC-3 and OSC-4 accumulating cells in the S phase, but 5-FU only very weakly induced apoptosis in OSC-1. While irradiated cells were in the G2/M phase, they exhibited apoptosis, to the same degree, in all OSC lines. Furthermore, the expression of Bax protein was not increased by 5-FU or gamma-rays, although apoptosis was induced by both treatments. These findings indicate that 5-FU and gamma-rays induce apoptosis of squamous cell carcinoma cells in p53- and p21-independent manners, in the S and G2/M phases, respectively.

Candidiasis May Induce Glossodynia without Objective Manifestation

BACKGROUND The causes of glossodynia in the absence of objective abnormalities range widely and differential diagnosis of glossodynia is very difficult. METHODS Based on the examination results of peripheral blood, stimulated and nonstimulated salivary flow rate (SFR), glossal pain threshold, and C. albicans cell culture and the response to treatment, we identified the cause of vague pain of the tongue in 98 patients who lacked objective findings and identified candidiasis as the cause of glossodynia in 26 patients. RESULTS These patients revealed hyposalivation and decreased glossal pain thresholds and C. albicans cell overgrowth. Pain thresholds in the painful portion (54.6+/-2.9 degrees C) were significantly decreased compared with those in the painless portion (57.7+/-3.4 degrees C) (P < 0.05) and the pain thresholds were largely increased after treatment (57.2+/-1.6 degrees C). Nonstimulated SFR before treatment was lower than that of age- and gender-matched healthy people, although stimulated SFR was decreased only slightly. C. albicans cell overgrowth was detected by the number of C. albicans colonies that formed in Sabourauds agar plate (539.3+/-198.4/dish). After the subsidence of glossal pain by mouth washing with a 3% amphotericin B solution, the C. albicans colonies were decreased to 31.5+/-19.3/dish, which was almost same as the control level, 14.1+/-8.4/dish. CONCLUSION These results indicate that candidiasis in conjunction with hyposalivation may induce pain in the tongue without manifestation of objective abnormalities.

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.

An immunohistochemical study of odontogenic mixed tumours

Five cases of odontogenic mixed tumour comprising of an ameloblastic fibroma, an adenomatoid odontogenic tumour, an odonto-ameloblastoma and two ameloblastic fibro-odontomas were immunohistochemically investigated. Odontogenic epithelial cells were fully positive for cytokeratin detected by antibody KL-1, although there were some differences in its intensity. In contrast, for tenascin, only immature dental papilla-like mesenchymal tissue, especially around the dental lamina-like odontogenic epithelium, was positive, while the myxomatous area and connective tissue were negative. Positive vimentin staining was observed in some areas of immature dental papilla-like cells as well as the basement membrane of odontogenic epithelium in the ameloblastic fibroma, suggesting that this tumour had developed at the early stage of tooth formation. Proliferating nuclear cell antigen-positive cells were generally rarely seen, but were frequently observed in epithelial cells of the ameloblastic fibroma and odonto-ameloblastoma. These observations suggest that tumour cells in each odontogenic mixed tumour possess characteristic proteins associated with proliferation potential and that ameloblastic fibroma and odonto-ameloblastoma have higher proliferation potential among the tumours examined.

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.