Erythrosine-Based PDT of Oral Epithelial Cells Figure 4. Mitochondrial trans-membrane potential analysis. Effects of PDT on mitochondrial trans-membrane potential (DYm) at different recovery time points post-PDT (0, 2 and 24 h) on DOK and H357 cells were analyzed at both low (71.03 mM) and high (1136.50 mM) doses of erythrosine. For both doses, PDT irradiation was done with a fluence of 122.58 J/cm2. A, DYm studies on DOK cells at low (71.03 mM–122.58 J/cm 2 ) and high (1136.50 mM–122.58 J/cm2) doses of erythrosine-PDT. B, DYm studies on H357 cells at low (71.03 mM–122.58 J/cm2) and high (1136.50 mM– 2 122.58 J/cm ) doses of erythrosine-PDT. The presented histograms are representative of three replicate determinations. doi:10.1371/journal.pone.0034475.g004 increased in the early recovery time points (Figure 8) and returned back to the control levels by 24 hr after treatment suggesting the occurrence of caspase-dependent apoptosis at early recovery time point culminating into secondary necrosis following PDT. This confirms the results seen with CLSM and SEM studies. Following 2 high dose erythrosine-PDT (1136.5 mM–122.58 J/cm ), DOK cells showed caspase 3/7 activity below the control levels suggesting the occurrence of necrosis (Figure 8A). Again this confirms the results obtained with the CLSM and SEM studies. Contrary to this, the H357 cells showed an increase in caspase 3/7 activity following high PDT dose treatment suggesting the occurrence of apoptosis and a more limited necrotic response (Figure 8B). This limited necrotic response of H357 cells might go some way toward explaining the apparent resistance of these cells to erythrosine-PDT. Discussion In common with other therapeutic strategies, PDT is constantly evolving and continues to be improved further [7,30]. A defining feature of the development of PDT is the discovery of newer and more powerful photosensitizers [5,14,30]. To this end we have been investigating Erythrosine B as a potential photosensitizer for PDT-based treatment of oral malignancies. The concentration of photosensitizer (PS) taken up by the cells is a vital parameter in cell killing efficacy [30]. Erythrosine uptake in DOK and H357 cells increased in an erythrosine concentration- dependent manner. In addition, the overall uptake was higher at 37uC than at 4uC suggesting that both active and passive uptake pathways are operating. Cells usually take up chemicals/drugs through either passive diffusion or through active transport [31] usually in association with various serum proteins such as lipoproteins, albumin and globulins [32] or by pinocytosis especially via bovine/calf serum albumin [33]. Lower temperatures affect lipid membrane structure [34] thereby preventing active transport of drugs into the cell [35]. Thus at 4uC, passive transport is usually predominant. DOK and H357 cells showed a dose-dependent response to the PDT treatment. Maximum cell killing was observed at 1136.5 mM erythrosine and 122.58 J/cm2 irradiation fluence in DOK (,80%) as well as H357 (,60%) cells. It is apparent that DOK PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34475