In Vitro Studies on Erythrosine-Based Photodynamic Therapy of Malignant and Pre-Malignant Oral Epithelial Cells Abhishek D. Garg1,2, Muthiah Bose1,2, Mohammed I. Ahmed1,2, William A. Bonass1, Simon R. Wood 1 * 1 Department of Oral Biology, Leeds Dental Institute, Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom, 2 Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom Abstract Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37uC the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ,80% of DOK cell killing was achieved as compared to ,60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans- membrane potential (DYm) studies showed that the H357 cells were far more resistant to the changes in DYm when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT’s usage for cancer cell killing. Citation: Garg AD, Bose M, Ahmed MI, Bonass WA, Wood SR (2012) In Vitro Studies on Erythrosine-Based Photodynamic Therapy of Malignant and Pre-Malignant Oral Epithelial Cells. PLoS ONE 7(4): e34475. doi:10.1371/journal.pone.0034475 Editor: Rossella Rota, Ospedale Pediatrico Bambino Gesu` , Italy Received November 28, 2011; Accepted March 5, 2012; Published April 2, 2012 Copyright: 2012 Garg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MSc projects carried out at University of Leeds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: s.r.wood@leeds.ac.uk Introduction Oral cancer is one of the most disfiguring and debilitating malignancies [1] and an important problem especially in those countries where tobacco chewing or smoking is prevalent [2]. In 2002, oral cancers accounted for approximately 274,000 cases world-wide [3]. Surgery, radiation therapy and chemotherapy are the typical traditional methods used for oral cancer treatment. Although these traditional oral cancer treatment strategies have advanced several fold over last few decades (e.g. discovery of new chemotherapeutics, powerful imaging tools allowing for better diagnostics-driven surgery and fluorescence/bioluminescence- guided surgery), the 5-year survival rate for oral cancer victims hasn’t improved significantly over the past 50 years [4]. In addition to this, these treatment strategies have several mild to severe side-effects e.g. salivary gland dysfunction, mucositis/ stomatitis, dental caries/demineralization, neurotoxicity and osteoradionecrosis [1,4]. Thus, an alternative treatment-strategy is desirable which could be utilized for treating tumours in the oral cavity in a simple, efficient, cost-effective and patient-friendly manner [4]. One such emerging treatment strategy is Photody- namic Therapy (PDT) [5,6,7]. The principle of PDT is that an administered photosensitizer (PS) is selectively retained by cancer cells [7] and when the tumour is irradiated with light of the appropriate wavelength, the PS is activated and causes tumour cell death by the production of reactive oxygen species (ROS) and free radicals [7,8,9]. The main cancer-types for which PDT has been widely applied include malignancies of head and neck, brain, lung, prostate, ovaries, skin and oesophagus [6,10,11,12]. In the past, PDT has been applied to oral cancer using various photosensitizers, e.g., dihaematoporphyrin, meta tetrahydroxy phenyl chlorine (mTHPC) or Foscan and 5-aminolaevulinic acid [13]. Though PDT treatments for oral cancer using the above photosensitizers are effective there are some limitations [14,15] including the lengthy clinical and legislative assessments that significantly delay translation of many such photosensitizers into the clinic. For instance, while EU has approved usage of Foscan-based PDT for treatment of early head and neck cancers; FDA has not yet approved PDT treatment for head and neck squamous cell carcinoma [4]. Such scenarios, have encouraged the search for other photosensitizers with more immediate benefits and possibil- ities of fast clinical translation [15]. To this end, our research has PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34475