Huang-Hsu Chen

Collagen Biosynthesis in Human Oral Submucous Fibrosis Fibroblast Cultures

To investigate the mechanism of collagen accumulation in oral submucous fibrosis (OSF) tissues, we examined the biosynthesis of collagen in fibroblast cultures established from OSF lesions. Fibroblasts obtained from four of ten OSF specimens showed more than a 1.5-fold increase in the production of collagens compared with fibroblasts from age-, sex-, and passage-matched normal controls (p < 0.05). When the relative amounts of collagen synthesis were estimated by SDS polyacrylamide gel electrophoresis, it was found that both OSF and control cells produced about 85% type I collagen and 15% type III collagen. The ratio of α1(I) to a2(I) chains was about 3:1 in OSF cells instead of the 2:1 expected for type I collagen. The excess al(I) chains could mean that collagen type I trimer was synthesized by the fibroblasts. These findings suggest that collagen overproduction and a reduced degradation of the structure-stable collagen type I trimer synthesized by OSF fibroblasts might contribute to the accumulation of collagen in OSF lesions in vivo. The mechanism(s) of increased procollagen production were analyzed by Northern blot, slot blot, and Southern blot. The OSF fibroblast strains with elevated collagen production also contained higher-than-normal levels of procollagen mRNA, and the ratios of α1(I), a2(I), and α1(III) procollagen mRNAs were compatible with the results of corresponding procollagen a chains. The gene copy number of proa2(I) collagen gene in OSF fibroblasts was about 1.05. No gene amplification was found. These results indicate that expression of these procollagen genes in cultured fibroblasts is regulated at the transcriptional level.

Association between Genetic Polymorphism of Tumor Necrosis Factor-a and Risk of Oral Submucous Fibrosis, a Pre-cancerous Condition of Oral Cancer

Many cytokines have been thought to play important roles in the pathogenesis of oral submucous fibrosis (OSF), an areca nut chewing-specific pre-cancerous condition characterized by the deposition of collagen in oral submucosa. Tumor necrosis factor-a (TNF-a), situated in the class III region of human leukocyte antigen (HLA), is a mediator with multiple functions, including the regulation of inflammatory reaction and transcriptions of collagen and collagenase. In total, 809 male subjects were recruited for assessment of the association of OSF with a bi-allelic promoter-region (-308) polymorphism on the TNFA gene. The high production allele, TNF2, was significantly lower among OSF subjects (n = 166) than in areca-chewing controls (n = 284). This association was independent of oral cancer status. The multivariate-adjusted odds ratio for the TNFA 11 genotype was 2.6 (95% confidence interval = 1.4-4.9 ; p = 0.004). The finding may imply a multifunctional etiological factor of TNF-α in OSF pathogenesis.

Significant reduction of homocysteine level with multiple B vitamins in atrophic glossitis patients

OBJECTIVE This study evaluated whether supplementations of different vitamins and iron could reduce the serum homocysteine levels in 91 atrophic glossitis (AG) patients. MATERIALS AND METHODS Atrophic glossitis (AG) patients with concomitant deficiencies of vitamin B12 only (n = 39, group I), folic acid only (n = 10, group II), iron only (n = 9, group III), or vitamin B12 plus iron (n = 19, group IV) were treated with vitamin BC capsules plus deficient hematinics. AG patients without definite hematinic deficiencies (n = 14, group V) were treated with vitamin BC capsules only. The blood homocysteine and hematinic levels at baseline and after treatment till all oral symptoms had disappeared were measured and compared by paired t-test. RESULTS Supplementations with vitamin BC capsules plus corresponding deficient hematinics for groups I, II, III, IV patients and with vitamin BC capsules only for group V patients could reduce the high serum homocysteine levels to significantly lower levels after a mean treatment period of 8.3-11.6 months (all P-values < 0.05). CONCLUSION Supplementations with vitamin BC capsules plus corresponding deficient hematinics or with vitamin BC capsules only can reduce the high serum homocysteine levels to significantly lower levels in AG patients.

Ultrastructural changes of posterior lingual glands after hypoglossal denervation in hamsters

Posterior lingual glands consist of two sets of minor salivary glands that serve important functions in oral physiology. To investigate the hypothesis that the hypoglossal nerve provides sympathetic innervation to the posterior lingual glands, we examined ultrastructural changes in the glands following hypoglossal denervation. In the posterior deep lingual glands (of von Ebner), the serous acinar cells showed a decrease in the number of secretory granules and an increase in lipofuscin accumulation. The ratios of cells containing lipofuscin granules were 11.39, 36.49 and 50.46%, respectively, of the control, 3‐ and 7‐day post‐axotomy glands (P < 0.001). Intraepithelial phagocytotic activity was increased. The mucous acinar cells in the posterior superficial lingual glands (of Weber) also showed degenerative changes after hypoglossal denervation. One week after nerve transection, marked cytoplasmic vacuolation and fragmentation of organelles were frequently observed. Degenerative changes were also found in unmyelinated axons associated with the glands. We provide the first evidence of the structural and functional connections between the sympathetic component of the hypoglossal nerve and posterior lingual glands.

Arecoline-stimulated placenta growth factor production in gingival epithelial cells: modulation by curcumin

OBJECTIVE Placenta growth factor (PlGF) is associated with the progression and prognosis of oral cancer. MATERIALS AND METHODS This study used ELISA, quantitative polymerase chain reaction, and Western blotting to study the arecoline-stimulated (PlGF) protein or mRNA expression in human gingival epithelial S-G cells. RESULTS Arecoline, a major areca nut alkaloid and an oral carcinogen, could stimulate PlGF protein synthesis in S-G cells in a dose- and time-dependent manner. The levels of PlGF protein secretion increased about 3.1- and 3.8-fold after 24-h exposure to 0.4 and 0.8 mM arecoline, respectively. Pretreatment with antioxidant N-acetyl-l-cysteine (NAC) and ERK inhibitor PD98059, but not NF-κB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580, and PI3-K inhibitor LY294002, significantly reduced arecoline-induced PlGF protein synthesis. ELISA analyses demonstrated that NAC and PD98059 reduced about 43% and 38% of the arecoline-induced PlGF protein secretion, respectively. However, combined treatment with NAC and PD98059 did not show additive effect. Moreover, 10 μM curcumin and 4 mM NAC significantly inhibited arecoline-induced ERK activation. Furthermore, 10 μM curcumin completely blocked arecoline-induced PlGF mRNA expression. CONCLUSION Arecoline-induced PlGF synthesis is probably mediated by reactive oxygen species/ERK pathways, and curcumin may be an useful agent in controlling oral carcinogenesis.

Autofluorescence lifetime measurement on oral carcinogenesis

Normal and cancerous tissues have distinct autofluorescence lifetime because of their biophysical and biochemical differences. Protoporphyrin IX (PplX) is a useful fluorophore, which generally accumulates more in cancerous cells than in normal cells due to heme synthesis pathway, is often employed in photodynamic detection and therapy. Under 410nm excitation, the main emission peak of PplX is at 630nm. Autofluorescence lifetime at 630nm emission would be elongated if PplX gathered more in cells. In this study, we tried to find if there exist significant differences of autofluorescence lifetime at 630nm (under 410nm excitation) between normal and cancerous tissues for in vivo measurement. The result shows that normal tissues in general have shorter lifetime (about 2.8/spl sim/3.5 ns) than that of abnormal tissues. The measured data suggest that lifetime would get longer in accordance with the degree of carcinogenesis. For cancer tissues, the average autofluorescence lifetime was extended to be about 10ns. Furthermore, the efficiency of treatment could also be defined refer to the time-series of lifetime decline.

Nuclear Expression of metastasis-associated Protein 1 (MTA1) is Inversely Related to Progression of Oral Squamous Cell Carcinoma in Taiwan

Overexpression of metastasis-associated protein 1 (MTA1) has been demonstrated in a variety of human cancers and found to be significantly associated with the tumor size, lymph node metastasis, clinical stage, tumor invasion, and prognosis of these cancers. In this study, we examined the expression of MTA1 in 74 specimens of oral squamous cell carcinoma (OSCC), 100 specimens of oral epithelial dysplasia (OED; 33 mild, 44 moderate, and 23 severe OED cases), and 21 specimens of normal oral mucosa (NOM) by immunohistochemistry. The cytoplasmic and nuclear MTA1 labeling indices (LIs) in OSCC, OED, and NOM samples were calculated and compared among these groups. Correlations of the cytoplasmic and nuclear MTA1 LIs in OSCC with clinicopathological parameters and survival of OSCC patients were statistically analyzed. We found that the mean cytoplasmic MTA1 LIs were all over 95% for the NOM, OED, and OSCC samples. There were no significant associations of the cytoplasmic MTA1 LI with any of the clinicopathological parameters of OSCC patients. The mean nuclear MTA1 LIs significantly decreased from NOM (73%±13%) to mild OED (71%±16%), moderate OED (60%±22%), and severe OED (46%±25%) and OSCC samples (30%±30%, p＜0.001). A significant correlation was found between the lower mean nuclear MTA1 LIs and OSCCs located on the buccal mucosa and tongue (p=0.001), with larger tumor sizes (T3 and T4, p=0.020), with regional lymph node metastases (N1, N2, and N3, p=0.024), and with more-advanced clinical stages (stages 3 and 4, p=0.045). Our results suggest that MTA1 is universally expressed in the cytoplasm of normal, dysplastic, and malignant oral epithelial cells. The nuclear expression of MTA1 significantly dropped from NOM through OED to OSCC samples, and was inversely related to the T status, N status, and clinical staging of OSCCs. Therefore, the nuclear MTA1 LI can be used to predict the progression of OSCCs in Taiwan.

Oral Nevi-A Clinicopathological Study of 29 Cases

Oral nevi are uncommon oral mucosal lesions. Twenty-nine cases of oral nevi including 21 intramucosal, 2 compound, and 6 common blue nevi were analyzed in this study. These 29 nevi were excised from 8 male and 19 female patients. Two patients each had 2 nevi. The mean age of patients at the time of diagnosis was 33 (range, 7~56) years. The more-common sites for oral nevi were the hard palate (12 cases) and vermilion border (9 cases). All 6 common blue nevi were located on the hard palate. The greatest dimension of the lesion was ≧1 cm in 3 cases and ＜1 cm in 24 cases. Excisional biopsy was the treatment of choice in 27 cases and wide excision in 2 cases. No recurrence of these lesions was found after surgical excision. Type A, type B, type C, and multinucleated giant nevus cells were found in 23 (100%), 19 (83%), 14 (61%), and 12 (52%) of 23 cases of intramucosal and compound nevi. The mean percentages of type A, type B, and type C nevus cells in total nevus cells were 73%±25%, 17%±15%, and 11%±18% in 23 cases of intramucosal and compound nevi, respectively. All 23 cases of intramucosal and compound nevi more or less contained nevus cells with pigmentation which varied from 1% to 91% (mean, 20%±26%). We concluded that the most-common type of oral nevi is an intramucosal nevus. Intramucosal and compound nevi were composed of approximately 3/4 of type A and 1/4 of type B and type C nevus cells. Oral nevi in this study occurred more often in female patients and in the third and fourth decades of life. The more-commonly affected sites were the hard palate and vermilion border. About 90% of these oral nevi were smaller than 1 cm.

Oral Neurilemmomas-A Clinicopathological Study of 21 Cases

Oral neurilemmomas (ON) are rare oral lesions. Twenty-one cases of ONs diagnosed and treated in our institute during the period from January 1991 to June 2004 were analyzed in this study. There were 14 male and 7 female patients. The mean age of the 21 patients at the time of diagnosis was 30 (range, 14-67) years with a peak incidence of tumor occurrence in the third decade. The more-common sites for an ON were the tongue (8 cases) and buccal mucosa (5 cases). The clinical appearance of ONs was frequently a pink, movable mass. In only 1 case was palpation tenderness noted. The greatest dimension of the lesion was 2 cm with a mean of 1.0±0.5 cm. The duration of the lesion ranged from 10 days to 10 years with a median of 6 months. Of the 21 cases, the clinical diagnosis was a fibroma in 6, a mucocele in 4, a tongue or buccal tumor in 5, and others in 6. Surgical excision was the treatment of choice for all 21 lesions. None of the lesions showed recurrence after surgical excision. On the average, our 21 ONs contained 77%±16% Antoni A tissue and 23%±16% Antoni B tissue. We conclude that ONs occur most often in the third decade. The more-commonly affected sites are the tongue and buccal mucosa. ONs often present as a painless and movable mass which is very difficult to diagnose just based on their clinical features. Our ONs were mostly composed of Antoni A tissue, with much-smaller content of Antoni B tissue.

A Clinicopathological Study of 22 Cases of Oral Melanotic Macule

Oral melanotic macules, or melanosis, are uncommon oral mucosal lesions. Twenty-two cases of oral melanotic macule diagnosed and treated in our institute during the period from January 1989 to June 2004 were analyzed in this study. There were 8 male and 14 female patients. The mean age of patients at the time of diagnosis was 41.2 (range, 5~80) years. The most-common sites for melanotic macule were the buccal mucosa (7 cases) and lower lip (6 cases). Multiple lesions were noted in 5 cases. The clinical appearance of the lesion was frequently a black macule (19 cases). The greatest dimension of the lesion was 1.5cm with a mean of 0.5cm. Of the 22 cases, the clinical diagnosis was oral melanosis in 12, oral melanotic macule in 6, intramucosal nevus in 2, and others in 2. Excisional biopsy was the treatment of choice in 21 cases and incisional biopsy in 1 case. Recurrence of the lesion was found in 3 cases. We found that buccal mucosal lesions had a significantly lower mean density of pigmented basal epithelial cells/mm of the basement membrane (14±26 cells/mm) than of the lower lip (89±62 cells/mm, p=0.014) or gingival lesions (76±62 cells/mm, p=0.041). In contrast, buccal mucosal lesions had a significantly higher mean density of melanophages/mm of basement membrane(52±29 cells/mm) than gingival lesions (14±2 cells/mm, p=0.031). However, there was no significant difference ii the mean density of melanophages/mm of the basement membrane between buccal mucosal lesions and lower lip lesions (28±16 cells/mm, p=0.1). We concluded that oral melanotic macules in this study occurred most often in the fifth decade. The most-commonly affected sites were the buccal mucosa and lower lip. The black pigmentation of these buccal mucosal melanotic macules might have been due to an increase in the number of melanophages in the subepithelial connective tissue, and the black pigmentation of lower lip or gingival melanotic macule might have been due to an increase in the number of melanin-pigmented basal epithelial cells.