Immunohistochemical expression of mast cell tryptase in giant cell fibroma and inflammatory fibrous hyperplasia of the oral mucosa.

This study analysed the immunohistochemical expression of mast cell tryptase in giant cell fibromas (GCFs). In addition, the possible interaction of mast cells with stellate giant cells, as well as their role in fibrosis and tumour progression, was investigated. For this purpose, the results were compared with cases of inflammatory fibrous hyperplasia (IFH) and normal oral mucosa. Thirty cases of GCF, 30 cases of IFH and 10 normal mucosa specimens used as control were selected. Immunoreactivity of mast cells to the anti-tryptase antibody was analysed quantitatively in the lining epithelium and in connective tissue. In the epithelial component (p=0.250) and connective tissue (p=0.001), the largest mean number of mast cells was observed in IFHs and the smallest mean number in GCFs. In connective tissue, the mean percentage of degranulated mast cells was higher in GCFs than in IFHs and normal mucosa specimens (p<0.001). Analysis of the percentage of degranulated mast cells in areas of fibrosis and at the periphery of blood vessels also showed a larger mean number in GCFs compared to IFHs and normal mucosa specimens (p<0.001). The percent interaction between mast cells and stellate giant cells in GCFs was 59.62%. In conclusion, although mast cells were less numerous in GCFs, the cells exhibited a significant interaction with stellate giant cells present in these tumours. In addition, the results suggest the involvement of mast cells in the induction of fibrosis and modulation of endothelial cell function in GCFs.

Absence of hotspot mutations in exons 9 and 20 of the PIK3CA gene in human oral squamous cell carcinoma in the Greek population.

OBJECTIVE: Phosphatidylinositol-3 kinases (PI3K) are a group of heterodimeric lipid kinases that regulate many cellular processes. Recent studies have reported high frequencies of somatic hotspot mutations in the phosphatidylinositol-3 kinase catalytic alpha (PIK3CA) gene, which encodes for one of these kinases, in several human solid tumors, including oral squamous cell carcinoma (OSCC). The aim of this study was to determine the frequency of hotspot mutations in exons 9 and 20 of the PIK3CA gene in OSCC in the Greek population. STUDY DESIGN: Eighty-six formalin-fixed and paraffin-embedded primary tumor specimens were analyzed by direct genomic DNA sequencing. Chi-square was used for statistical analysis. RESULTS: No hotspot mutations were detected in any of the samples. Two intronic polymorphisms IVS8 and IVS9 were detected, mainly in patients with cancer of the buccal mucosa and lower gingival and alveolus respectively. CONCLUSIONS: PIK3CA hotspot mutations are unlikely to play a major role in the pathogenesis of OSCC in the Greek population.

Cyclic AMP phosphodiesterase activity in human gingival carcinoma.

BACKGROUND: The phosphodiesterases (PDEs) are responsible for the hydrolysis of the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP), to their corresponding monophosphates with a fundamental role in the transduction of the intracellular signals. At least 11 different enzymatic isoforms have been identified, which are listed according to their specificity or affinity for the substratum, identity of the amino acid sequence, cofactor, and inhibitor sensitivity. Variations in PDE activity have been found in different pathologies, and they have also been correlated to different pathological e/o physiological mechanisms, such as cellular differentiation, apoptosis, and tumor invasivity. OBJECTIVES: In this study, we have evaluated cAMP PDE activity in patients with carcinoma of the gingiva, with the purpose of correlating differences in its development and progression. The same enzymatic activity has been used to evaluate differences between patients with lymph node involvement (group N(+)), and patients without lymph node involvement (N(-)). MATERIALS AND METHODS: The analysis of PDE activity and the cAMP assay was performed by reverse-phase HPLC on samples of fresh or frozen gingival tissues. Analysis of cAMP was confirmed with the enzyme-linked immunoabsorption assay (EIA). RESULTS AND CONCLUSIONS: The differences between control and N(-) groups (P = 0.0433), and between control and N(+) groups (P = 0.0156) were statistically significant. PDE3A was also evaluated immunohistochemically in lymph-node negative and lymph-node positive cases. The differences between the two groups were statistically significant (P = 0.0397).

Cyclic guanosine monophosphate phosphodiesterase activity in human gingival carcinoma.

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is an essential second messenger metabolized by phosphodiesterases (PDEs). OBJECTIVES: We looked for a possible correlation of PDE activities in human oral squamous cell carcinoma (OSCC) with and without lymph node metastases. MATERIALS AND METHODS: The analysis of phosphodiesterase activity and the cGMP assay were done by reverse-phase HPLC on samples of fresh or frozen gingival tissues. Analysis of cGMP was confirmed with the enzyme-linked immunoabsorption assay. RESULTS AND CONCLUSIONS: cGMP PDE activity was 34.92 +/- 7.17 SD, 12.89 +/- 4.43 SD, and 35.88 +/- 8.76 SD (nmols/mg of protein), respectively, in controls, samples without lymph node involvement (N-), and specimens with lymph node metastases (N+). cGMP values were 1.97 +/- 0.63 SD, 3.30 +/- 1.47 SD, and 3.49 +/- 1.47 SD (nmols/mg of protein). Our data support the hypothesis of a role for cGMP and PDE in the progression of OSCC.

Cathepsin expression in oral squamous cell carcinoma: relationship with clinicopathologic factors.

OBJECTIVE: Proteases are involved in the invasion and metastasis of carcinoma cells. In vivo, oral carcinoma cells easily invade the bone tissue and metastasize to the submandibular and neck lymph nodes. Cathepsin expression has been shown in some neoplastic tissues and serves as a prognostic indicator. The purpose of this study was to investigate the relationship between clinicopathohistologic grades and cathepsin expressions in oral squamous cell carcinoma and to investigate which cathepsin provides prognostic information for patients with oral carcinoma. STUDY DESIGN: Immunohistochemical studies were performed on 78 carcinoma samples with monoclonal antibodies against cathepsins B, H, and L, and a polyclonal antibody against cathepsin D. Serial sections were stained by hematoxylin-eosin staining and classified by Anneroth's classification. Cathepsin B, H, L and D activities of blood serum were determined. Positive results indicative of the presence of cathepsin were investigated to determine any correlation between a particular cathepsin and histologic malignancy grades, tumor cell growth, serum cathepsin activities, and clinical factors. RESULTS: Cathepsins B, H, L, and D were positive in every case. Although the labeling indices for cathepsins B (CB-LI), H (CH-LI), and D (CD-LI) for the cancer cases showed significant differences from those of controls, cathepsin L (CL-LI) of cancer cases showed no difference from that of controls (P <.05). A close correlation was found between CD-LI and T categories of TNM classification (P <.05), and between CD-LI and PCNA-LI (P <.05). Furthermore, a close correlation was found between CD-LI and N categories in TNM classification (P <.05). Pathologically, a close correlation was found between CB-LI or CD-LI and the pattern and/or stage of invasion (P <.05). CONCLUSION: Cathepsin D and B expression were closely correlated with carcinoma invasion and progression. These proteases may be useful in determining the prognoses of patients with oral carcinoma.

The effect of betel nut extract on cell growth and prostaglandin endoperoxide synthase in human epidermoid carcinoma cells.

The objective of this study was to find out whether prostaglandin endoperoxide synthase (PHS) involves the action of betel nut extract (BNE) on the growth of oral cancers. Therefore, growth and PHS activity were examined in two human oral carcinoma cell lines (OEC-M1 and KB) and one normal fibroblast cell line (NF) in the presence of increasing BNE concentration. BNE at concentrations above 50 microg/ml significantly inhibited the cell growth of OEC-M1 after 72 h in culture, of KB and NF after 48 h in culture. The IC50 of BNE in OEC-M1, KB and NF at 24 h in culture was about 406, 37.5 and 140 microg/ml respectively. PHS activity in OEC-M1 was significantly increased by low BNE concentrations (50 microg/ml, 114%; 100 microg/ml, 33%; 150 microg/ml, 30%) but significantly reduced at higher BNE concentrations (300 microg/ml, 33%; 500 microg/ml, 61%). The PHS activity in KB was significantly inhibited by BNE and this effect was intensified as concentrations increased (50 microg/ml, 31%; 100 microg/ml, 24%; 150 microg/ml, 43%; 300 microg/ml, 60%; 500 microg/ml, 92%). Similar to that in OEC-M1, the PHS activity in NF was significantly increased at low BNE concentrations (50 microg/ml, 139%; 100 microg/ml, 87%;150 microg/ml, 77%) but reduced at higher concentrations (300 microg/ml, 55%; 500 microg/ml, 72%). The PHS activity in all cell lines was almost completely blocked by indomethacin (5 x 10(-6) M). We conclude that these findings suggest that PHS may be an important biochemical mediator of the effect of BNE on the growth of two human oral carcinoma cell lines.

Antisense oligonucleotides against protein kinase CK2-alpha inhibit growth of squamous cell carcinoma of the head and neck in vitro.

BACKGROUND: Human squamous cell carcinomas of the head and neck (SCCHN) overexpress the protein kinase CK2, and elevated CK2 activity correlates with aggressive tumor behavior and poor clinical outcome. We therefore investigated whether interference with CK2 expression would inhibit SCCHN cell growth in vitro. METHODS: We targeted the catalytic (alpha) subunit of CK2 using an antisense oligodeoxynucleotide (ODN) strategy. Human Ca9-22 cells derived from SCCHN were transfected with CK2-alpha sense, nonsense, or antisense ODN; CK2 activity was measured; and the effect on CK2 activity and on cell growth was determined. RESULTS: Transfection of Ca9-22 cells with antisense CK2-alpha ODN resulted in significantly decreased CK2 kinase activity associated with nuclear chromatin and in dose-dependent growth inhibition of Ca9-22 cells in vitro. CONCLUSIONS: Interference with the protein kinase CK2 signal in SCCHN cells may offer a novel anticancer strategy for this malignancy.

Inhibition of Na+,K+-ATPase by cisplatin and its recovery by 2-mercaptoethanol in human squamous cell carcinoma cells.

Na+,K+-ATPase (EC 3.6.1.37) is assumed to be involved in the transport of cisplatin [cis-diamminedichloroplatinum(II)] into cells and to act as a modulator of 5-fluorouracil (5-FU) in combination therapy of cisplatin and 5-FU. Whereas inhibition of Na+,K+-ATPase activity by cisplatin is expected to have effects on both anti-cancer therapy and nephrotoxicity, the inhibition mechanism remains to be elucidated. We studied the inhibition of Na+,K+-ATPase activity by cisplatin using an enzyme partially purified from Ca9-22 cells derived from a human squamous cell carcinoma of the gingiva. Cisplatin inhibited the Na+,K+-dependent ATP hydrolysis activity, and this inhibition depended on both the concentration of cisplatin and the preincubation time with cisplatin. The time-dependent inhibition was thought to be caused by a slow change of cisplatin from the inactive to the active form. We further tested the effect of cisplatin on the partial reactions of the enzyme, Na+-dependent ATP hydrolysis and K+-dependent pnitrophenylphosphate hydrolysis activities to determine which step in the reaction sequence of Na+,K+-ATPase was inhibited. Cisplatin inhibited both activities depending on its concentration and the preincubation time, whereas the Na+-dependent ATP hydrolysis activity was inhibited even at lower concentrations. Formation of a phosphointermediate of Na+,K+-ATPase was also inhibited by cisplatin depending on the concentration and preincubation time. Cisplatin (500 microM) and 8-fold higher concentration of 2-mercaptoethanol (2-ME; 4 mM) prevented inactivation of the enzyme by cisplatin, and the Na+,K+-ATPase activity inhibited by pretreatment with cisplatin was also recovered almost completely by 2-ME. These results suggest that the active form of cisplatin inhibits the Na+,K+-ATPase activity by inhibiting the formation of a phosphointermediate of the enzyme and that the inhibition by cisplatin is arrested by an addition of thiol group.

Apoptosis and growth inhibition of head and neck tumor cell line induced by epidermal growth factor receptor tyrosine kinase inhibitor.

Overexpression of the epidermal growth factor (EGF) receptor, a hallmark of aerodigestive squamous cell carcinoma of the head and neck (SCCHN), correlates with aggressive tumor behavior. There is evidence that SCCHN cells auto-activate their EGF receptors. The receptor has therefore attracted interest as a potential therapeutic target. We tested the in vitro therapeutic efficacy of PD153035--a potent, specific inhibitor of the tyrosine kinase intrinsic to the EGF receptor--by employing a well-characterized cell line derived from human gingival SCCHN. DNA-synthesis and cell number were assayed for growth-inhibitory effects, phosphorylation of the EGF receptor was quantitated by immunoblot, and cell apoptosis was detected by terminal deoxytransferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) in situ assay. PD153035, at nanomolar concentrations, inhibited autophosphorylation of the EGF receptor induced by EGF stimulation and the inhibition occurred in a dose-dependent manner. Under the same conditions, PD153035 inhibited cell growth, and induced apoptosis of SCCHN cells in vitro. We conclude that selective inhibition of the EGF receptor tyrosine kinase completely abolishes EGF receptor phosphorylation resulting from receptor stimulation, and results in growth inhibition and apoptosis of SCCHN cells in vitro. By inducing cytostasis and apoptosis, this new class of inhibitors may be of therapeutic value against SCCHN.

Regulation of PG synthase by EGF and PDGF in human oral, breast, stomach, and fibrosarcoma cancer cell lines.

Prostaglandins may inhibit or promote tumor cell replication, depending on the cell system that is investigated. In our laboratory, we have established and characterized four different specific human cancer cell lines. The objectives of this study were to examine and compare the prostaglandin endoperoxide synthase (PG synthase, EC 1.14.99.1) activity of these cell lines by measuring the conversion of arachidonate to 3H-PGE2 and 3H-PGF2 alpha. We found that the oral epidermal carcinoma cell line (OEC-M1) had a moderate degree of PG synthase activity. Enzyme activity could be partially blocked (statistically significant) by the addition of epidermal growth factor (EGF) at 20 ng/mL and almost completely inhibited by platelet-derived growth factor at (PDGF) 20 mU/mL. By contrast, we discovered that the human breast adenocarcinoma cell line (BC-M1) did not contain significant PG synthase, and enzyme activity could be significantly activated by the addition of epidermal growth factor at 20 ng/mL and platelet-derived growth factor at 20 mU/mL. We also found that the human stomach adenocarcinoma cell line (SCM-1) had a significant amount of PG synthase activity, and these PG synthase activities were not activated or inhibited by EGF at 20 ng/mL or PDGF at 20 mU/mL. Furthermore, the human fibrosarcoma (FS-M1) cell line also contained a moderate degree of PG synthase activity, which could be significantly inhibited by PDGF at 20 mU/mL but was not inhibited by EGF at 20 ng/mL. The results suggest that EGF and PDGF may be involved in the regulation of the PG synthase activities of human oral, breast, stomach, and fibrosarcoma cancer cells.