Apoptosis induction by 13-acetoxyrolandrolide through the mitochondrial intrinsic pathway

Abstract

The aim of this study was to evaluate the mechanisms of cytotoxicity of the sesquiterpene lactone 13-acetoxyrolandrolide, a nuclear factor kappa B (NF-κB) inhibitor that was previously isolated from Rolandra fruticosa. The effects associated with the inhibition of the NF-κB pathway included dose-dependent inhibition of the NF-κB subunit p65 (RelA) and inhibition of upstream mediators IKKβ and oncogenic Kirsten rat sarcoma (K-Ras). The inhibitory concentration of 13-acetoxyrolandrolide on K-Ras was 7.7 µM. The downstream effects of the inhibition of NF-κB activation were also investigated in vitro. After 24 h of treatment with 13-acetoxyrolandrolide, the mitochondrial transmembrane potential was depolarized in human colon cancer (HT-29) cells. The mitochondrial oxidative phosphorylation was also negatively affected, and reduced levels of nicotinamine adenine dinucleotide phosphate (NAD(P)H) were detected after 2 h of 13-acetoxyrolandrolide exposure. Furthermore, the expression of the pro-apoptotic protein caspase-3 increased in a concentration-dependent manner. Cell flow cytometry showed that 13-acetoxyrolandrolide induced cell cycle arrest at G1 , indicating that the treated cells had undergone caspase-3-mediated apoptosis, indicating negative effects on cancer cell proliferation. These results suggest that 13-acetoxyrolandrolide inhibits NF-κB and K-Ras and promotes cell death mediated through the mitochondrial apoptotic pathway.

Keywords: HT-29; K-Ras; NF-κB; apoptosis; colon cancer; mitochondria; reactive oxygen species; sesquiterpene.