Induction of invasion in an organotypic oral cancer model by CoCl2, a hypoxia mimetic.

Invasion is a hallmark of malignancy. The aim of this study was to develop an in vitro model that can be used for experimental studies of cancer cell invasion. The organotypic oral cancer model was constructed by growing oral squamous cell carcinoma (OSCC) cells on a collagen matrix in which normal human fibroblasts were incorporated. Immunohistochemical staining of the model showed that the expression of invasion-related molecules such as phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), cyclooxygenase-2 (COX-2), p75(NTR), and hepatocyte growth factor receptor (Met) was similar to that seen in OSCC. Treatment of the model with cobalt chloride (CoCl(2)) to mimic hypoxic conditions increased cancer cell invasion, defined as the appearance of cancer cell islands protruding into the matrix. Models treated with CoCl(2) showed increased expression of p75(NTR) and laminin-5 in the cancer cells, and a more pronounced fragmentation of collagen IV in the basal membrane area, in contrast to models that were left untreated. The results indicate that the present model is well suited for studies on cancer cell invasion in the matrix and that the addition of CoCl(2) on day 3 of the experiment is indicated because it markedly increases the invasion and improves the model.

Epidermal growth factor-induced cyclooxygenase-2 expression in oral squamous cell carcinoma cell lines is mediated through extracellular signal-regulated kinase 1/2 and p38 but is Src and nuclear factor-kappa B independent.

The intracellular signalling cascade(s) mediating epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression is poorly defined in oral carcinomas. Investigation of two different oral squamous cell carcinoma (OSCC) cell lines with high EGF-induced COX-2 expression revealed, however, that this expression was dependent on two mitogen-activated protein kinase (MAPK) pathways [extracellular signal-regulated kinase 1/2 (ERK1/2) and p38] because combined inhibition of these pathways was needed to abolish EGF-induced COX-2 expression. Surprisingly, inhibition of phosphoinositide-3 kinase (PI3K) increased EGF-induced COX-2 expression in the basaloid OSCC cell line (C12), suggesting a PI3K-controlled, inhibitory COX-2-regulating pathway. Neither the transcription factor nuclear factor-kappaB (NF-kappaB), nor Src, was involved in EGF-induced COX-2 expression. The results suggest that EGF-induced COX-2 expression is regulated by several pathways, and emphasizes that individual tumors use different strategies for intracellular signalling.

c-Jun N-terminal kinase negatively regulates epidermal growth factor-induced cyclooxygenase-2 expression in oral squamous cell carcinoma cell lines.

Epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression in squamous cell carcinomas is mediated through the extracellular signal-regulated kinase 1/2 and p38 pathways. Examination of a basaloid and a conventional oral squamous cell carcinoma cell line revealed that inhibition of c-Jun N-terminal kinase (JNK) with SP600125 increased EGF-induced (but not basal) COX-2 transcription 1.5-1.9-fold in extracellular signal-regulated kinase 1/2 and p38 pathway-dependent manners. Although JNK may phosphorylate the cyclosporine A-sensitive transcription factor, nuclear factor of activated T cells c3, it was seemingly not involved because cyclosporine A did not reduce EGF-induced COX-2 expression. Thus, JNK negatively regulated EGF-induced extracellular signal-regulated kinase 1/2 and/or p38-mediated COX-2 transcription, presumably through activating an unidentified phosphatase.

A study of phosphorylated ERK1/2 and COX-2 in early stage (T1-T2) oral squamous cell carcinomas.

BACKGROUND: Histomorphological grading at the invasive front of oral squamous cell carcinomas (OSCCs) may provide useful prognostic information. In the present study, we investigated the presence and prognostic value of activated phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2) and cyclo-oxygenase-2 (COX-2) both at the invasive front and in central/superficial parts of OSCCs. METHODS: Using immunohistochemistry, we assessed the presence of p-ERK1/2 and COX-2 in 53 early stage OSCCs. Clinical data were recorded prospectively. The end point was disease-free survival. RESULTS: p-ERK1/2 staining was present in almost all tumours. The staining was mostly nuclear in the cells of the invasive front and either nuclear or nuclear/cytoplasmic in central/superficial tumour parts. COX-2 was observed in almost all tumours (98%) and the staining was often restricted to focal areas. Most tumours were COX-2 negative at the invasive front. The lowest P-value in survival analyses was P = 0.06 for p-ERK1/2 at the invasive front. COX-2, the histomorphological grading systems and TNM stage were of no prognostic value. CONCLUSION: p-ERK1/2 was present in almost all tumours and p-ERK1/2 may be a prognostic marker at the invasive front of OSCCs. In early stage OSCCs, most tumours did not express COX-2 at the invasive front.

Nerve growth factor beta/pro-nerve growth factor and their receptors in normal human oral mucosa.

Nerve growth factor beta (NGF-beta) and its precursor proNGF are important for the differentiation and survival of neurons and dermal keratinocytes. The aim of this study was to determine the role that NGF might play in the differentiation and wound healing of oral mucosa. Cultured normal human oral mucosal keratinocytes expressed mRNA for NGF-beta/proNGF and for their receptors TrkA and p75(NTR). Lysates from cultured oral mucosal keratinocytes did not contain detectable amounts of mature 14-kDa NGF-beta but did contain several NGF proforms with molecular weights between 32 and 114 kDa. Culture medium from oral mucosal keratinocytes contained 75 kDa proNGF. The addition of NGF-beta significantly enhanced the proliferation of oral mucosal keratinocyte cultures and in vitro scratch closure. Immunostaining of biopsies from normal oral mucosa showed the presence of proNGF in all epithelial layers. NGF staining was observed in the granular and upper spinous cell layers. TrkA immunoreactivity was detected in basal and parabasal cells, with weak to moderate staining in spinous and granular cell layers. p75(NTR) staining was seen in basal cell layers. These findings indicate that NGF-beta/proNGF have mitogenic and motogenic effects on oral mucosal keratinocytes and therefore may aid in the healing of oral wounds. Differential expression of NGF and NGF receptors throughout the epithelium suggests a role in epithelial differentiation.