Figure 1.
Uptake of erythrosine by pre-malignant (DOK) and malignant (H357) oral epithelial cells was analyzed at 37°C and 4°C. (A) Drug uptake kinetics of erythrosine in DOK cells at 37°C and 4°C. (B) Drug uptake kinetics of erythrosine in H357 cells at 37°C and 4°C. The values are means of four replicate determinations ± S.E.M. Statistical analysis was done via student's t-test with significance set at P<0.05 (* indicates statistical comparison of 37°C data-points vs. the respective data values of 0 µM erythrosine; # indicates comparison of 4°C data-points vs. the respective data values of 0 µM erythrosine and ** indicates comparison between 37°C data-points and the corresponding 4°C data-points).
Figure 2.
The pre-malignant (DOK) and malignant (H357) oral epithelial cells were incubated with different concentrations of erythrosine B (71.03 µM, 142.06 µM, 284.13 µM, 568.27 µM and 1136.50 µM) followed by PDT irradiation with different fluences (0 J/cm2, 40.86 J/cm2, 81.72 J/cm2 and 122.58 J/cm2). Following PDT, the cells were recovered 24 h later and percentage (%) of cell killing caused by PDT was estimated. (A) DOK cell's susceptibility to erythrosine-based PDT and (B) H357 cell's susceptibility to erythrosine-based PDT. The values are means of six replicate determinations ± S.D. Statistical analysis was done via student's t-test with significance set at P<0.05 (* indicates statistical comparison of a data-point within one erythrosine B concentration group vs. the corresponding data values of 0 J/cm2 fluencies for that group).
Figure 3.
Erythrosine co-localization analysis.
Sub-cellular co-localization of erythrosine in both DOK and H357 cells. (A and B) LysoTracker localization (for lysosomal visualization) and MitoTracker localization (for mitochondrial visualization) was compared with 1136.50 µM erythrosine localization in both DOK (A) as well as H357 (B) cells. Also, co-localization frequency scatter plots, Pearson's coefficient (r) and Mander's coefficient (M) were calculated to evaluate the overlap between LysoTracker/MitoTracker localizations and erythrosine localizations. (C) Co-localization frequency colour map indicates the estimation of co-localizations on the basis of ‘heat map schemes’ such that hotter colours tend to indicate high co-localization. Thus, red/maroon indicates 100% co-localization, yellow-green indicates 50% co-localization and purple/dark blue indicates 0% co-localization.
Figure 4.
Mitochondrial trans-membrane potential analysis.
Effects of PDT on mitochondrial trans-membrane potential (ΔΨm) at different recovery time points post-PDT (0, 2 and 24 h) on DOK and H357 cells were analyzed at both low (71.03 µM) and high (1136.50 µM) doses of erythrosine. For both doses, PDT irradiation was done with a fluence of 122.58 J/cm2. A, ΔΨm studies on DOK cells at low (71.03 µM–122.58 J/cm2) and high (1136.50 µM–122.58 J/cm2) doses of erythrosine-PDT. B, ΔΨm studies on H357 cells at low (71.03 µM–122.58 J/cm2) and high (1136.50 µM–122.58 J/cm2) doses of erythrosine-PDT. The presented histograms are representative of three replicate determinations.
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 µM) and high (1136.50 µM) 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.
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 µM) and high (1136.50 µM) 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.
Figure 7.
Confocal Laser Scanning Microscopy (CLSM) Analysis.
CLSM images of DOK (A) and H357 (B) cells at different recovery time points post-PDT (2, 4 and 24 h) were taken for both low (71.03 µM) and high (1136.50 µM) doses of erythrosine. For both doses, PDT irradiation was done with a fluence of 122.58 J/cm2. Green (SYTO9) fluorescence represents both live and dead cells while red (Propidium Iodide) fluorescence stains the dead cells alone. The arrow in the DOK cells (A) indicates the presence of apoptotic bodies whereas the arrow in the H357 cells (B) points to a group of necrotic cells.
Figure 8.
Caspase 3/7 activation in response to PDT was estimated at four different recovery time points post-PDT (0, 2, 4 and 24 hr) in DOK (A) and H357 cells (B) at both low (71.03 µM) and high (1136.50 µM) doses of erythrosine. For both doses, PDT irradiation was done with a fluence of 122.58 J/cm2. In order to bring the data sets of both DOK and H357 cells to the same scale, the luminometer arbitrary values for DOK cells were divided by 10. The values are means of three replicate determinations ± S.D. Statistical analysis was done via student's t-test with significance set at P<0.05 (* indicates statistical comparison as indicated by the lines).