The expression and action mechanisms of Akt, Hsp90, mTOR and cyclin D1 proteins in cultured cells of squamous cell carcinoma and dysplastic cells after being irradiated with low level laser therapy

Author(s)

Felipe Fornias Sperandio

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia (FO/SDO)
• Defense date: 2012-12-06

Examining board members

Suzana Cantanhede Orsini Machado de Sousa; Luciana Corrêa; Daiane Thaís Meneguzzo; Décio dos Santos Pinto Júnior; Renata Tucci

• Advisor: Suzana Cantanhede Orsini Machado de Sousa

Abstract

Head and neck squamous cell carcinoma (HNSCC) is an epithelial malignant neoplasm that accounts for approximately 500.000 new cases yearly around the world. Several studies have been conducted to elucidate the mechanisms of proliferation and invasion of this lesion, whereas the Akt/mTOR signaling pathway with its related proteins is being pointed out as one of the main pathways involved in HNSCC`s progression. Neoplastic cells, as well as cells that originate from different tissues may have their behavior modified by low level laser therapy (LLLT); however, the mechanisms through which the low level laser light interacts with these cells remain poorly understood. Thus, this study sought to evaluate the cell viability and the expression levels of Akt, pAkt, Hsp90, S6, pS6 and Cyclin D1 proteins in two oral squamous cell carcinoma cell lineages (SCC9 and SCC25) and in one oral dysplastic human keratinocyte cell line (DOK) after they had been treated with LLLT. The laser device was a semiconductor diode of Gallium and Aluminum Arsenate (GaAlAs), operating with wavelengths of 660nm (red) and 780nm (infrared), with a fixed power of 40mW and giving three different energy densities: 2.05J/cm², 3.07J/cm² and 6.15J/cm². Apoptosis was analyzed through TUNEL test and the protein expression was accessed with Immunofluorescence and Western blotting. After statistical analysis through two-way ANOVA and Tukey or Student`s T test, all of them with a level of significance of 5%, it was concluded that LLLT induced distinct behaviors to each of the studied cell lines. Increases and inhibitions in cell viabilities were detected depending on the wavelength and also on the irradiated cell line. The energy density of 2.05J/cm² produced the most significant findings over SCC9. On the other hand, in SCC25 the most relevant results were detected with 3.07J/cm², while the most prominent findings were seen with 6.15J/cm² when the cell line DOK was evaluated. In that way, these respective doses were chosen for each cell line to continue with Western blotting and Immunofluorescence. Among the most relevant findings, the variation of pS6 and Cyclin D1 levels can be cited for DOK in some evaluated periods. SCC9 presented both pAkt and Cyclin D1 variations in the studied periods. Besides that, SCC25 also had pAkt, pS6 and Cyclin D1 levels modified by LLLT. Interestingly, the appearance and maintenance of an Hsp90 isoform was found in SCC9 and SCC25 after laser irradiation. Moreover, the induction of apoptosis was detected for the SCC25 cell line. Finally, the LLLT employed herein was able to enhance the expression of proteins related to progression and invasion in all of the studied cell lines. In addition, there was a single laser irradiation, although it caused prolonged effects, sometimes through the latest evaluated period. (AU)

• FAPESP's process: 09/08452-2 - Action mechanisms and expression analysis of cyclin D1, akt, mTOR and hsp90 proteins in squamous cell carcinoma in vitro after low intensity laser irradiation
• Grantee: Felipe Fornias Sperandio
• Support Opportunities: Scholarships in Brazil - Doctorate