The Molecular Mechanisms of Oleanane Aldehyde-β-enone Cytotoxicity against Doxorubicin-Resistant Cancer Cells

SIMPLE SUMMARY: Currently, the main reason for the ineffectiveness of systemic chemotherapy is the formation of cancer cells with the phenotype of multiple drug resistance (MDR), which are able to survive at high doses of chemotherapy drugs. One of the MDR mechanisms involves the overproduction of proteins of the ABC transporter family, including P-glycoprotein, which are responsible for the transport of a wide range of drugs from the cell. This paper focuses on a new derivative of the natural triterpenoid betulin, oleanane aldehyde-β-enone (OA), having low toxicity toward normal cells and cytotoxicity toward cancer cells and their doxorubicin-resistant variants. The calculations and experimental methods revealed that this compound is not released from cells like most known drugs. The analysis of molecular cellular targets showed that the mechanism of OA toxic action is associated with the activation of the external and/or internal pathways of the apoptotic death of cancer cells and their drug-resistant variants. Thus, OA, as a highly cytotoxic compound toward doxorubicin-resistant cancer cells, can be a promising candidate for drug development. ABSTRACT: Oleanane aldehyde-β-enone (OA), being the semi-synthetic derivative of the triterpenoid betulin, effectively inhibits the proliferation of HBL-100 and K562 cancer cells (IC(50) 0.47–0.53 µM), as well as the proliferation of their resistant subclones with high P-gp expression HBL-100/Dox, K562/i-S9 and K562/i-S9_Dox (IC(50) 0.45−1.24 µM). A molecular docking study, rhodamine efflux test, synergistic test with Dox, and ABC transporter gene expression were used to investigate the ability of OA to act as a P-gp substrate or inhibitor against Dox-resistant cells. We noted a trend toward a decrease in ABCB1, ABCC1 and ABCG2 expression in HBL-100 cells treated with OA. The in silico and in vitro methods suggested that OA is neither a direct inhibitor nor a competitive substrate of P-gp in overexpressing P-gp cancer cells. Thus, OA is able to overcome cellular resistance and can accumulate in Dox-resistant cells to realize toxic effects. The set of experiments suggested that OA toxic action can be attributed to activating intrinsic/extrinsic or only intrinsic apoptosis pathways in Dox-sensitive and Dox-resistant cancer cells, respectively. The cytotoxicity of OA in resistant cells is likely mediated by a mitochondrial cell death pathway, as demonstrated by positive staining with Annexin V–FITC, an increasing number of cells in the subG0/G1 phase, reactive oxygen species generation, mitochondrial dysfunction, cytochrome c migration and caspases-9,-6 activation.