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
Induction of Apoptosis by Rhinacanthone in HeLaCells In order to examine whether the inhibitory effect ofrhinacanthone on the growth of HeLa cells is due to apoptosis,we confirmed the apoptotic characterizations in HeLacells by several approaches, e.g., morphological changes,DNA fragmentation, cell cycle progression, TUNEL stainingassay detection, agarose gel electrophoresis, and FACScanflow cytometry.In the morphological change analysis, the cells treatedwith 3, 6, and 10 mM rhinacanthone for 4 and 8 h were examinedby using a confocal laser scanning microscope afterstaining the cells with in situ TUNEL assay. As shown in Fig.3, marked morphological changes were observed such as becominground in shape, membrane blebbing and apoptoticbodies; these were the characteristics of apoptotic cells.20—25)The levels of apoptosis gradually increased in a dose-dependentmanner. Similar results were obtained with b-lapachoneunder the same treatment conditions (data not shown).Subsequently, we analyzed whether DNA fragmentationwas induced by rhinacanthone in HeLa cells using an agarosegel electrophoresis. After the treatment of HeLa cells with6 mM rhinacanthone for 4 and 8 h, a typical ladder pattern ofDNA internucleosomal fragmentation was apparently observed,that is a hallmark of apoptotic cell death18) (Fig. 4).b-Lapachone and adriamycin (6 mM) treatments also affected
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