Erythrosine-Based PDT of Oral Epithelial Cells Figure 5. Scanning Electron Microscopy (SEM) Analysis on DOK cells. SEM images showing DOK cell morphologies at different recovery time points post-PDT (0, 2 and 24 h) were taken for 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. These images demonstrate the occurrence of apoptosis (and secondary necrosis) following PDT with low dose erythrosine and necrosis in the high dose condition. doi:10.1371/journal.pone.0034475.g005 (pre-malignant) cells showed more susceptibility to erythrosine- based PDT than H357 (malignant) cells. This difference in susceptibility to PDT could be occurring for various reasons. One possible reason is the expression of telomerase which promotes cell survival by protecting cells from apoptosis [36,37,38]. Other protective mechanisms that might play a role in the resistance of H357 cells to erythrosine-based PDT include; (1) induction of drug-detoxifying mechanisms, (2) resistance to drug-induced apoptosis [39], (3) Mutational difference between DOK and H357 cells (p53 and Ha-ras gene is mutated in H357 cells) and (4) increased expression of anti-oxidants. Further, it was noted that for low PDT-doses, the standard deviation was large whereas for high PDT-doses the data spread was much lower. This variability in data might be explained by the fact that low PDT-stress resulting from low dosage has the ability of inducing several different responses in the treated cells e.g. apoptosis, repair, survival tactics and autophagy, while high PDT-stress results in induction of quick responses such as necrosis [21]. Erythrosine was found to be predominantly localizing in the mitochondria, for DOK as well as H357 cell types. We therefore attempted to find out whether erythrosine-based PDT causes any change in the mitochondrial trans-membrane potential (Dym). Our results showed that the H357 cells are far more resistant to changes in Dym when compared to the DOK cells. Dissipation of Dym in DOK cells was observed with both high and low doses of erythrosine which suggests it as a primary result of PDT-induced damage. However, there was less variation in the Dym of H357 cells where both the high and low doses produced a shift in potential only at the 24 hr recovery time point. This suggests that in the H357 cells there is a mechanism occurring at the cellular level which is capable of rescuing the cells from the effect of PDT. The precise mechanism by which depletion of Dym is causing cell death is unclear, but it has been reported that the dissipation of Dym will actually cause the release of cytochrome c and activate apoptosis [40,41]. Other reports conclude that there is no relationship between Dym and release of cytochrome c along with apoptosis [42]. Nevertheless, our data suggests that the dissipation of Dym following PDT has significantly deleterious effects on cell survival. Interestingly, it has also been reported previously that the dissipation in Dym actually enhances the permeability of lysosomal membranes, resulting in the release of lysosomal proteins and subsequent activation of cell death pathways accompanied by autophagy [43]. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34475