Erythrosine-Based PDT of Oral Epithelial Cells Figure 6. Scanning Electron Microscopy (SEM) Analysis on H357 cells. SEM images showing H357 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.g006 Cell death morphology analysis using CLSM and SEM, demonstrated the occurrence of apoptosis following low dose PDT and necrosis with high dose in both the cell lines. In H357 cells high dose PDT induced apoptosis in addition to necrosis. It has been reported previously by Castano et al that higher doses of PDT result predominantly in necrosis depending upon the cell line and the low dose in PDT results in apoptosis [44]. Kessel et al have also reported the higher incidence of apoptosis during low dose PDT and inhibition of apoptosis at high dose with aluminum phthalo- cyanine chloride and two other porphycene sensitizers [45]. This is confirmed by the results of the caspase 3/7 activity analysis presented in this study which suggested the occurrence of apoptosis following low dose PDT in both cell lines during the initial recovery time points. An increase in caspase 3/7 activity was also seen using high dose PDT in H357 cells suggesting the occurrence of apoptosis alongside necrosis in this cell line. As expected, high dose PDT of DOK cells resulted in caspase 3/7 activity below control levels which is supported by the SEM/ CLSM data and suggests that necrosis is the main mechanism of cell death at this dose. This occurrence of extensive necrosis in the DOK cells is probably the main reason for the higher death percentage in DOK cells when compared to the H357 cells. Kessel et al (1997) and Shah et al (1996) have reported that extensive necrosis is promoted when there is an increased photo-inactivation of caspases and inhibition of apoptosis [45,46]. The result of this study has shown us that the H357 are far more resistant than the DOK cells. To conclude, we have found that erythrosine is an effective photosensitizer for PDT treatment of both malignant and pre- malignant oral cell lines. In fact, to the best of our knowledge, this is the first instance of erythrosine-based PDT being applied to kill cancer cells. However, the malignant H357 cell line was more resistant to PDT which may be related to a lower necrotic response following PDT. Further studies are needed to elucidate the precise mechanisms of cell killing at a molecular level. Moreover it would be also vital in the near future, to analyze if erythrosine offers better pharmacokinetics and physio-chemical properties than existing photosensitizers. Materials and Methods Cell Culture Conditions The two oral epithelial cell lines (DOK and H357) were purchased from the European Collection of Cell Cultures PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34475