Abstract
Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-κB and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed. Key words:Carcinoma, squamous cell, head and neck neoplasms, extracellular matrix, cell culture techniques, signal transduction.
Highlights
As one of the most common cancers, Head and Neck Squamous Cell Carcinoma (HNSCC) is an aggressive malignancy that arises in the oral cavity, larynx and pharynx, resulting in 500.000 new cases per year in the world [1] and nearly 11.000 deaths in the United States alone [2]
A’-Focal cytoplasmatic staining of pAkt in the HN6 cells; B -Nuclear translocation of pAkt in the HN6 cells when cultured three-dimensionally; C-HN30 cell line showing cytoplasmatic expression of NF-κB; D NF-κB expression remains in the cytoplasm of HN30 cells cultured with the matrix system; E – HN31 cell line showing nuclear expression of Cyclin D1; F – Cyclin D1 expression remains in the nuclei of the HN31 cells when cultured with the three-dimensional mode
Three-dimensional culture models are currently being used to understand the signals that regulate the normal function of tissues and how they may change during pathological processes, such as cancer [18]
Summary
As one of the most common cancers, Head and Neck Squamous Cell Carcinoma (HNSCC) is an aggressive malignancy that arises in the oral cavity, larynx and pharynx, resulting in 500.000 new cases per year in the world [1] and nearly 11.000 deaths in the United States alone [2]. Among the most studied proteins related to HNSCC invasion, cell proliferation and metastasis, pAkt, NF-κB and Cyclin D1 can be cited [5]. The PI3K-Akt signaling pathway, when activated, may prevent the cell death by inactivating pro-apoptotic factors and by activating transcription factors that upregulate anti-apoptotic genes, including the transcription factor NF-κB, which is an essential component of intracellular regulatory circuitries of cell proliferation and survival [8]. In HNSCCs, the expression and activity of NF-κB is often up-regulated, and its levels increase gradually from pre-malignant lesions to invasive cancer [9,10], which suggests that NF-κB signaling pathway may play an important role at the early stages of carcinogenesis. This work sought to evaluate the expression of pAkt, NF-κB and Cyclin D1 in HNSCC cell lines cultured with the traditional bidimensional method (monolayer) and with the threedimensional model. Recombinant extracellular matrix composed by some components of human extracellular matrix, such as laminin, type IV collagen, nidogen, entactin and heparan sulfate [13] was used to mimic threedimensional microenvironment
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