Sittichai Koontongkaew

Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays

Although risk factors for squamous cell carcinomas of the head and neck (HNSCC) are well recognized, very little is known about the molecular mechanisms responsible for this malignancy. Furthermore, the ability to investigate gene expression profiles at different stages of tumor progression is usually limited by the remarkable heterogeneity of these neoplastic lesions. Here, we show the successful use of laser capture microdissection (LCM) to procure specific cell populations. The 5000 cells from representative sets of HNSCC and their matching normal tissues are sufficient to extract RNA of high integrity for the synthesis of labeled amplified cDNA probes which can then be hybridized to these membranes arrayed with known human cancer-related cDNAs. Furthermore, when compared to normal tissues, we demonstrate a consistent decrease in expression of differentiation markers such as cytokeratins, and an increase in the expression of a number of signal transducing and cell cycle regulatory molecules, as well as growth and angiogenic factors and tissue degrading proteases. Unexpectedly, we also found that most HNSCC overexpress members of the wnt and notch growth and differentiation regulatory system, thus suggesting that the wnt and notch pathways may contribute in squamous cell carcinogenesis. This experimental approach may facilitate the identification candidate markers for the early detection of preneoplastic lesions, as well as novel targets for pharmacological intervention in this disease.

A role for p38 MAPK in head and neck cancer cell growth and tumor‐induced angiogenesis and lymphangiogenesis

We have recently gained a remarkable understanding of the mutational landscape of head and neck squamous cell carcinoma (HNSCC). However, the nature of the dysregulated signaling networks contributing to HNSCC progression is still poorly defined. Here, we have focused on the role of the family of mitogen activated kinases (MAPKs), extracellular regulated kinase (ERK), c‐Jun terminal kinase (JNK) and p38 MAPK in HNSCC. Immunohistochemical analysis of a large collection of human HNSCC tissues revealed that the levels of the phosphorylated active form of ERK1/2 and JNK were elevated in less than 33% and 16% of the cases, respectively. Strikingly, however, high levels of active phospho‐p38 were observed in most (79%) of hundreds of tissues analyzed. We explored the biological role of p38 in HNSCC cell lines using three independent approaches: treatment with a specific p38 inhibitor, SB203580; a retro‐inhibition strategy consisting in the use of SB203580 combined with the expression of an inhibitor‐insensitive mutant form of p38α; and short‐hairpin RNAs (shRNAs) targeting p38α. We found that specific blockade of p38 signaling significantly inhibited the proliferation of HNSCC cells both in vitro and in vivo. Indeed, we observed that p38 inhibition in HNSCC cancer cells reduces cancer growth in tumor xenografts and a remarkable decrease in intratumoral blood and lymphatic vessels. We conclude that p38α functions as a positive regulator of HNSCC in the context of the tumor microenvironment, controlling cancer cell growth as well as tumor‐induced angiogenesis and lymphangiogenesis.

Tumor–stroma interactions influence cytokine expression and matrix metalloproteinase activities in paired primary and metastatic head and neck cancer cells

This study evaluates the effects of gingival fibroblasts, type I collagen and autocrine/paracrine elements on cytokine expression in paired primary and metastatic human squamous cell carcinoma (HNSCC) cell lines. Additionally, the effects of IL‐1α, IL‐1β, IL‐6, TNF‐α, TGF‐β and HGF on MMPs and cell invasion were investigated. RT‐PCR results indicated the presence of mRNAs for IL‐1α, IL‐1β, IL‐6, TNF‐α, and TGF‐β in primary and metastatic HNSCC cell lines but high expression of cytokines was not a prerequisite for metastatic cancer cells. HGF mRNA was not detected in the cancer cell lines. Co‐culturing of HNSCC cells with fibroblasts caused increases in cytokine expression. Type I collagen and conditioned media derived from HNSCC cells or fibroblasts enhanced cytokine expression in the cancer cells. Cytokines also enhanced MMP‐2 and MMP‐9 enzymatic activities as well as HNSCC cell invasion. Our findings suggest that the interactions between cancer cells, the extracellular matrix and fibroblasts, as mediated by cytokines, play important roles in the progression of HNSCC.

Fibroblasts and extracellular matrix differently modulate MMP activation by primary and metastatic head and neck cancer cells

A genetically related pair of human head and neck cancer (HNSCC) cell lines derived from the same patient at different stages of disease was used to investigate the role of extracellular matrix, integrin, and CXCL12–CXCR4 receptor interactions and their signal pathways in MMP-2 and MMP-9 activation and cell invasion. We found that collagen I enhanced MMP-2 and MMP-9 secretion in both primary and metastatic HNSCC cells. Collagen I acted through α2β1 integrin to activate tyrosine kinases, protein kinase C, ERK1/2, and p38, which in turn activated MMP-2 and MMP-9 production. The signaling function was also involved in the enhancement of cell invasion. Experiments using cocultures between live and fixed cells demonstrated that direct contact between tumor and fibroblast cells was required to activate MMP-2 and MMP-9 secretion in both tumor cells and fibroblasts. The augmentation appears specific for MMP-2. Fibroblasts seem to be responsible for the increased MMP-2 in the coculture. In addition, fibroblast or tumor cell-conditioned media upregulated the secretion of MMP-2 and MMP-9 in HNSCC cells. These findings indicate that autocrine and paracrine factors are involved in the augmented secretion of MMPs in coculture. We also found that CXCL12-enhanced HNSCC cell invasion through paracrine-activated CXCR4, which triggered MMP-dependent cell invasion. Together, our results suggest that cell–matrix and cell–cell interactions including autocrine and paracrine factors play important roles in the invasive behavior of HNSCC via upregulation of MMP-2 and MMP-9.

Inhibition of arachidonic acid metabolism decreases tumor cell invasion and matrix metalloproteinase expression.

Head and neck cancers are known to synthesize arachidonic acid metabolites. Interfering with arachidonic acid metabolism may inhibit growth and invasiveness of cancer cells. In this study we investigate effects of sulindac (the non-selective COX inhibitor), aspirin (the irreversible, preferential COX-1 inhibitor), NS-398 (the selective COX-2 inhibitor), NDGA (nordihydroguaiaretic acid, the selective LOX inhibitor) and ETYA (5,8,11,14-eicosatetraynoic acid, the COX and LOX inhibitor) on cell viability, MMP-2 and MMP-9 activities, and in vitro invasion of cancer cells derived from primary and metastatic head and neck, and colon cancers. The inhibitors of COX and/or LOX could inhibit cell proliferation, MMP activity and invasion in head and neck and colon cancer cells. However, the inhibitory effect was obviously observed in colon cancer cells. Inhibition of arachidonic acid metabolism caused a decrease in cancer cell motility, which partially explained by the inhibition of MMPs. Therefore, COX and LOX pathways play important roles in head and neck cancer cell growth.

Dental caries, cariogenic microorganisms and salivary properties of allergic rhinitis children

OBJECTIVES The aim of this study was to observe the caries activities of allergic rhinitis patients in relation to salivary properties, salivary levels of mutans streptococci (MS) and lactobacillus (LB), oral hygiene and dietary habits. METHODS The study groups composed of 40 allergic rhinitis children and 40 healthy controls aged between 6 and 13 years old. Demographic data, oral hygiene practices and dietary habits were recorded by questionnaire. For permanent teeth, caries experience was expressed as DMFT (D=decayed; M=missing; F=filled; T=teeth) index. The dmft (d=decayed; m=missing; f=filling; t=teeth) index was used for caries prevalence in primary teeth. Unstimulated salivary flow rate, salivary buffering capacity, and salivary MS and LB were also determined in children with allergic rhinitis and controls. RESULTS There were no significant differences in combined DMFT/dmft, salivary flow rate, buffer capacity of saliva, salivary LB levels, and sugary food consumption between cases and controls (p>0.05). However, higher salivary MS levels were observed in allergic rhinitis patients, compared to controls (P<0.05). CONCLUSIONS Our results demonstrated that patients with allergic rhinitis had an increase in the level of salivary MS.

Safety Evaluation of Zingiber cassumunar Roxb. Rhizome Extract: Acute and Chronic Toxicity Studies in Rats

Zingiber cassumunar Roxb. has been used for traditional medicine, but few studies have described its potential toxicity. In this study, the acute and chronic oral toxicity of Z. cassumunar extract granules were evaluated in Sprague-Dawley rats. The extract at a single dose of 5000 mg/kg body weight did not produce treatment related signs of toxicity or mortality in any of the animals tested during the 14-day observation period. However, a decrease in body weights was observed in treated males (P < 0.05). The weights of lung and kidney of treated females were increased (P < 0.05). Treated males were increased in spleen and epididymis weights (P < 0.05). In repeated dose 270-day oral toxicity study, the administration of the extracts at concentrations of 0.3, 3, 30, 11.25, 112.5, and 1,125 mg/kg body weight/day revealed no-treatment toxicity. Although certain endpoints among those monitored (i.e., organ weight, hematological parameters, and clinical chemistry) exhibited statistically significant effects, none was adverse. Gross and histological observations revealed no toxicity. Our findings suggest that the Z. cassumunar extract granules are well tolerated for both single and chronic administration. The oral no-observed-adverse-effect level (NOAEL) for the extract was 1,125 mg/kg body weight/day for males and females.

Macrolides Attenuate Phorbol Ester-Induced Tumor Necrosis Factor-α and Mucin Production from Human Airway Epithelial Cells

Background/Aims: Macrolide antibiotics are effective drugs in chronic bronchiolitis and chronic rhinosinusitis with mucus hypersecretion. However, the mechanism of action is unclear. This study was designed to investigate the effect of azithromycin (AZM; 15-membered) and midecamycin acetate (MDM; 16-membered) on MUC5AC and MUC2 gene expression and secretion from human airway epithelial cells. The effects of the two macrolides on tumor necrosis factor-α (TNF-α) release were also examined. Methods: Confluent NCI-H292 human mucoepidermoid airways epithelial cells were pretreated with AZM or MDM for 2 h and then stimulated with 200 nmol/l phorbol 12-myristate 13-acetate (PMA) for 8 h. The MUC5AC and MUC2 gene expression was measured by real-time quantitative RT-PCR. Total mucin in culture supernatants was measured using enzyme-linked lectin assay. Enzyme-linked immunosorbent assay was used to determine MUC5AC, MUC2 and TNF-α released by the cells. Results: AZM and MDM attenuated PMA-induced MUC5AC and MUC2 gene and protein expression in NCI-H292 cells. They also suppressed PMA-mediated TNF-α in the cells. Conclusion: The present study demonstrates that AZM and MDM suppress the synthesis of mucin and TNF-α from human airway epithelial cells.

Effects of ameloblastoma-associated fibroblasts on the proliferation and invasion of tumor cells

CONTEXT Ameloblastoma is the most common odontogenic tumor, however, the molecular pathology, especially the role of the tumor stroma, is still unclear. AIMS To investigate the effects of the ameloblatoma-associated fibroblast (AAFs) on the proliferation and invasion of tumor cells. SETTINGS AND DESIGN Cell culture, ELISA, proliferation and invasion assays. METHODS AND MATERIAL The cocultivations and three-dimensional organotypic cultures of the AAFs and the tumor cells were performed. The gingival fibroblasts (GFs) were used as the control. The levels of TGF-β and HGF in the conditioned media were analyzed using ELISA technique. The MTT proliferation assays and Boyden chamber chemoinvasion assays were also performed. STATISTICAL ANALYSIS USED ANOVA. RESULTS Both AAFs and GFs stimulated tumor cell growth. The TGF-β level in the AAF group was more than those of GF group, whereas the HGF levels were not different. The AAF conditioned media also stimulated tumor cell proliferation and invasion more than the GF conditioned media. However, no difference in the thickness and morphology between the AAF and GF groups could be demonstrated in the organotypic models. CONCLUSIONS Both AAFs and GFs support the proliferation of the tumor cells in cocultivation experiment and three-dimensional organotypic cultures. However, the AAFs have a tendency to stimulate the proliferation and induce the invasion more than GFs. Increased TGF-β levels in the AAF condition media suggested the possible role of this growth factor in the ameloblastoma biology.

Sandblasting and fibronectin-derived peptide immobilization on titanium surface increase adhesion and differentiation of osteoblast-like cells (MC3T3-E1)

Background/purpose Various chemical titanium (Ti) surface modifications have been reported for enhancing cellular activities that promote early osseointegration. The purpose of this study was to determine if sandblasted Ti coated with or without fibronectin (FN) or FN-derived peptides stimulated osteoblast-like cell adhesion, spreading, proliferation, and differentiation. Materials and methods Osteoblast-like cells (MC3T3-E1) were cultured on sandblasted Ti disks immobilized with FN or FN-derived peptides [GRGDSP (Gly-Arg-Gly-Asp-Ser), PHSRN (Pro-His-Ser-Arg-Asn), or GRGDSP/PHSRN]. Surface topography, cell morphology, cell adhesion, cell proliferation, analysis of osteogenesis-related genes and protein expression, alkaline phosphatase, and alizarin red staining of mineralization were evaluated. Results The sandblasted Ti coated with FN or FN-derived peptides enhanced cell adhesion and cell proliferation. However, the Ti coated with FN or FN-derived peptides groups were similar in cell spreading. Osteogenic differentiation was observed in the peptide-modified Ti surface groups, compared with that of the noncoated Ti group. FN and GRGDSP/PHSRN coating enhanced the gene and protein expression of Runx2, osteocalcin, and bone sialoprotein. Alkaline phosphatase activity and matrix mineralization were also markedly enhanced in the Ti coated groups. Conclusion The sandblasted Ti coated with FN or FN-derived peptides (GRGDSP/PHSRN) markedly enhance adhesion, proliferation, and differentiation of osteoblast-like cells compared with uncoated sandblasted Ti.