Abstract B152: The selective effect of omega-3 polyunsaturated fatty acids on normal and neoplastic oral keratinocyte apoptosis
Authors: Nikolakopoulou Zacharoula , Michael-Titus Adina T. , Parkinson Eric K. 
Published: June 2012

Abstract
Background: Head and neck cancer (HNSCC) is the sixth most common malignancy worldwide. Squamous cell carcinomas (SCCs) of the aerodigestive tract often recur following therapy because of incomplete excision or the appearance of second primary or second field cancers. This makes the cost-effective prevention of second tumors an important priority. Recent evidence suggests that natural dietary products, such as omega-3 polyunsaturated fatty acids (PUFAs), contained in oily fish, have antitumorigenic activities, Therefore, PUFAs might be successful chemopreventive compounds and adjuvant agents for effective treatment when combined with chemotherapeutic drugs. In the present study, we tested the potential of omega-3 PUFAs to act as selective chemopreventive and therapeutic agents against oral and epidermal SCCs.
Materials and Methods: We investigated the effect of the long chain omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the malignant keratinocyte cell lines SCC-13 (epidermal), SCC-25 (oral) and the pre-malignant immortal line SVFHK and also on the normal keratinocyte lines, NHEK-131 and HEK-127. Cell viability, upon treatment with PUFAs, was determined by the MTT assay. The Annexin-V/ DAPI flow cytometric assay was carried out to assess apoptosis. Western blot analysis was performed to investigate the cleavage of caspases and the cell signaling pathways involved.
Results: Treatment with increasing concentrations of omega-3 PUFAs (0.5–5 µM) caused a dose-dependent inhibition of growth after only 4 days, as determined by the MTT assay. Under the same conditions, these natural products seemed to have little or no effect on the normal epidermal keratinocytes, at doses that killed the malignant cells. The cell death was confirmed by the Annexin-V/DAPI apoptosis assay. The assay determined that the malignant cell lines were undergoing an apoptotic death after treatment with PUFAs for 48 h, showing an increase in the Annexin-V positive /DAPI negative cell population. Western blot analysis confirmed that the PUFAs cause apoptosis via cleavage of caspase 3. The involvement of the intrinsic and extrinsic pathways of apoptosis was also demonstrated through cleavage of caspase 9 and 8, respectively. Moreover, Western blot analysis indicated that the treatment of oral SCC-25 with PUFAs resulted in the upregulation of phospho-ERK1/2. The increase in ERK1/2 phosphorylation is noticed already during the first few hours of treatment and continues to increase for 24 hours. The role of ERK1/2 activation in the apoptosis process and the involvement of other signaling pathways are under investigation.
Conclusions: The study demonstrates that the omega-3 PUFAs DHA and EPA display a significant antitumor effect against SCC keratinocytes at concentrations that do not eliminate normal cells, giving them a great potential as future therapeutic and prophylactic tools against HNSCC.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B152.