Induction of Apoptosis by Rhinacanthone in HeLa Cells In order to examine whether the inhibitory effect of rhinacanthone on the growth of HeLa cells is due to apoptosis,we confirmed the apoptotic characterizations in HeLa cells by several approaches, e.g., morphological changes,
DNA fragmentation, cell cycle progression, TUNEL staining assay detection, agarose gel electrophoresis, and FACScan flow cytometry.In the morphological change analysis, the cells treated with 3, 6, and 10 mM rhinacanthone for 4 and 8 h were examined by using a confocal laser scanning microscope after staining the cells with in situ TUNEL assay. As shown in Fig.
3, marked morphological changes were observed such as becoming round in shape, membrane blebbing and apoptotic bodies; these were the characteristics of apoptotic cells.20—25)The levels of apoptosis gradually increased in a dose-dependent manner. Similar results were obtained with b-lapachone under the same treatment conditions (data not shown).Subsequently, we analyzed whether DNA fragmentation was induced by rhinacanthone in HeLa cells using an agarose gel electrophoresis. After the treatment of HeLa cells with 6 mM rhinacanthone for 4 and 8 h, a typical ladder pattern of DNA internucleosomal fragmentation was apparently observed,
that is a hallmark of apoptotic cell death18) (Fig. 4).b-Lapachone and adriamycin (6 mM) treatments also affected