Aim: To study in vitro the molecular mechanism of apoptosis caused by beta-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae).
Materials and methods: The study was carried out on human bladder carcinoma T24 cell line. Determination of cell viability was done using trypan blue exclusion method, apoptosis quantitative estimation - by DAPI staining and agarose gel electrophoresis for DNA fragmentation. Flow cytometry analysis, RT-PCR and Western blot analysis, colorimetric assay of caspase activity were applied as well.
Results: It was found that in micromolar range of concentrations beta-lapachone inhibited the viability of T24 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation. Treatment of T24 cells with beta-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression. beta-lapachone-induced apoptosis was also associated with activation of caspase-3 and caspase-9, inhibition of IAP expression, and degradation of poly (ADP-ribose) polymerase, phospholipase C-gamma1 and beta-catenin proteins. At the same time Fas and FasL levels were inhibited upon treatment with beta-lapachone in a concentration-dependent manner.
Conclusion: beta-lapachone-induced apoptosis in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway.