PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences PUFAs enhance oxidative stress and apoptosis in tumour cells exposed to hypericin-mediated PDT Martin Kello, Jarom´ ır Mikeˇ s, Rastislav Jendˇ zelovsk´ y, J ´ an Kova ˇ l and Peter Fedoroˇ cko* Received 7th April 2010, Accepted 20th July 2010 First published as an Advance Article on the web 12th August 2010 DOI: 10.1039/c0pp00085j Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin. Introduction Polyunsaturated fatty acids (PUFAs) and their metabolites play a variety of physiological roles encompassing energy provision, membrane structure, cell signalling and regulation of gene expression. 1,2 However, alongside these essential functions, certain particular dietary fatty acids have been identified as having effects, both positive and negative, on cancer progression. This has led to individual PUFAs being proposed as possible chemotherapeutic agents, or potential adjuvants to radio- or chemotherapy/cancer therapies. Although the cellular and molecular mechanisms of tumour growth inhibition by n-3 fatty acids such as docosahexaenoic or eicosapentaenoic acid (DHA, EPA) are not precisely known, it has been suggested that they might alter membrane phospholipid turnover, 2 release of membrane arachidonic acid (AA) from phospholipids and prostaglandin synthesis via cyclooxygenase enzymes, reactive oxygen species (ROS) production, 2,3 membrane lipid oxidation, 4 reactive nitrogen species (RNS) accumulation, 5 transcriptional or translational regulation, 6,7 caspase activation 8 and intracellular signal transduction. 9 On the other hand, arachidonic acid is a well-known biologically-active n-6 PUFA. It has been reported to act as a potent inflammatory mediator 10 and stimulator of human breast 11 and prostate cancer cell growth. 12 But there are some indications that arachidonic acid (AA) exerts antitumour activity too. For example, an addition of exogenous unes- terified AA to different cancer cell lines inhibits cell growth Institute of Biology and Ecology, Faculty of Science, P. J. ˇ Saf´ arik University in Koˇ sice, Moyzesova 11, 040 01, Koˇ sice, Slovakia. E-mail: peter.fedorocko@upjs.sk; Fax: +421-55-6222124; Tel: +421-55-2341182 and induces apoptosis. 13 In addition, oxidized AA breakdown products such as 4-hydroxynonenal induce apoptosis. 14 More- over, AA also stimulates the hydrolysis of sphingomyelin, releasing ceramide which is implicated in the final phase of apoptosis. 15 Photodynamic therapy (PDT) is an approach well-suited for the treatment of superficial skin and mucosal cancers. Selective activation of photosensitizer by light induces local ROS produc- tion leading to destruction and cell death, by both apoptosis and necrosis, in vitro as well as in vivo. 16 Naturally occurring hydroxylated phenanthroperylenequinone hypericin, 15 a promis- ing highly-effective second-generation photosensitizer, is being studied mostly for applications in photodynamic diagnosis and therapy. 17 Due to its amphiphilic nature, the membranes have been postulated as the principal target of hypericin, 18,19 though the mitochondria have proved to be the most effective one, 20 probably for their central role in cell death signaling. On the other hand, activation of the stress kinases such as JNK1 and p38 MAPK has been identified as an important signal of cellular resistance to hypericin-induced apoptosis. 21 Various studies have demonstrated that some tumour cell lines are more sensitive to the cytotoxic and antiproliferative effects of PUFAs or PDT than normal cells. 22–25 Elevated PUFA concentrations affect cell membrane fluidity and possibly the biochemical properties of transporter proteins, 26,27 so they might interfere with trans-membrane transport. Since PUFAs are easily oxidized, increased lipid peroxidation induced by PDT might possibly lead to higher therapeutic efficiency. For these reasons, we focused on the characterization of cytokinetical and cyto- physiological parameters in two human tumour cell lines of different histological origin (HeLa and HT-29) pre-treated with n-3 and n-6 PUFAs and subsequently exposed to PDT with hypericin. 1244 | Photochem. Photobiol. Sci., 2010, 9, 1244–1251 This journal is © The Royal Society of Chemistry and Owner Societies 2010