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Sulfabenzamide promotes autophagic cell death 45 Figure 1. A) Chemical structure of sulfabenzamide and doxorubicin. B) Viability curve of sodium sulfabenzamide and doxorubicin treated T-47D cells. Percent viability of cells incubated with sodium sulfabenzamide and doxorubicin was calculated relative to the related untreated controls after 24, 48 and 72 h. Each point relates to the mean value of at least three independent experiments. The related correlation coefficient (r 2 ) was adjusted until the best fit for the selected mathematical function was used to interpolate the experimental points. T-47D cells do not exhibit DNA fragmentation and apoptotic morphology in the presence of sulfabenzamide or doxorubicin Unlike DNA fragmentation patterns observed in DNA extracted from U937 cells incubated with camptothecin (as a positive control of DNA laddering kit), the gel electrophoresis of DNA prepared from cells incubated with sulfabenzamide (<strong>10</strong>.8 mM) or doxorubicin (0.33 µM) showed no DNA ladder or smear pattern confirming lack of apoptosis or necrosis in these cells (Figure 2A). Morphological analysis of sulfabenzamide and doxorubicin treated cells, double stained with Annexin-FITC and PI, evaluated by fluorescent microscopy and confirmed the results of DNA laddering analysis. There were few cells having morphological characteristics of apoptotic and necrotic cells (Figures 2B, 2C). Early (young) apoptotic cells have rounded shape and shiny green membrane because PI cannot penetrate into the cells and Annexin-FITC binds to the externally membrane-exposed phosphatidylserines (Figures 2B, 2C). Late apoptotic and necrotic cells have membrane permeability for PI so their nuclei are stained red (Figure 2C). The main difference between necrotic and late apoptotic cells is the potency of late apoptotic cells for simultaneous staining of nuclei and membrane-exposed phosphatidylserines with PI and Annexin-FITC, respectively. Membrane blebbing, which is a common feature of apoptotic cells was seen in Figure 2B. Evidently, healthy cells cannot be seen under fluorescent microscope since they were not stained with either of the fluorescent dyes (Figure 2E).
46 Raziye MOHAMMADPOUR et al. Figure 2. A) DNA laddering analysis. 1-3 µg DNA prepared from 2×<strong>10</strong> 6 cells was resolved by electrophoresis in a 1% agarose gel. DNA fragmentation was observed only in positive control (camptothecin treated) cells, but it was not detected in control or cells incubated with doxorubicin or sodium sulfabenzamide. B) Observation of the morphology of early apoptotic cells using fluorescent microscopy following double staining with Annexin V-FITC and PI. Morphological characteristics of early apoptotic cells (rounded green shiny cells showing membrane blebbing) in sulfabenzamide treated cells. C) Observation of the morphology of late apoptotic and necrotic cells using fluorescent microscopy following double staining with Annexin V-FITC and PI. Morphological characteristics of late apoptotic (flattened green shiny cells showing red dense nuclei) and necrotic cells (red dense spheres lacking green shiny membrane) in sulfabenzamide treated cells. Similar results were observed for doxorubicin (not shown). Living cells due to lack of staining with dyes are not detectable in fluorescent visual field (E) but are visible using phase contrast microscopy (D).
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