Mitochondrial-Mediated Apoptosis by the Sesquiterpene Oil α-Bisabolol and Its Synergism with Imatinib and Nilotinib In BCR-ABL+ Cells

Abstract

AbstractAbstract 4456 Background.The plant-derived agent α-bisabolol is a small oily sesquiterpene alcohol that has been demonstrated to be cytotoxic against human malignant non-hematological and leukemic cells (Bonifacio M et al, Blood, 2009 ASH annual meeting abstracts;114:4800). Here we tested its activity against BCR-ABL+ cell lines and primary cells from patients, alone or in combination with the Tyrosine-Kinase Inhibitors (TKIs) Imatinib and Nilotinib. Also, the mechanism of α-bisabolol cytotoxicity in BCR-ABL+ cells was assessed. Methods.We used the BCR-ABL+ K562, LAMA-84 and CML-T1 cell lines and primary leukemic cells from 14 patients with BCR-ABL+ Acute Lymphoblastic Leukemia at diagnosis. First, the citotoxicity of single-agent α-bisabolol was determined by MTT. Then, mitochondrial membrane potential of treated cells was evaluated by the JC-1 dye in flow cytometry and fluorescence microscopy. Permeabilized leukemic cells were assayed for oxygen consumption by measuring mitochondrial state 3 and uncoupled respiration. Reactive oxygen species (ROS) production in α-bisabolol treated cells were quantified in flow cytometry by oxidation of CM-H2DCFDA, measuring the fluorescence intensity of the DCF products. Apoptosis was studied by the poly(ADP-ribose) polymerase (PARP) cleavage and internucleosomal DNA laddering analysis. Finally, the combination effects between α-bisabolol and Imatinib or Nilotinib (kindly provided by Novartis) were analyzed according to the median-effect method of Chou and Talalay using the CalcuSyn software. Results.α-bisabolol reduced the viability of BCR-ABL+ cells in a dose-dependent manner. The mean IC50 values of α-bisabolol were 46±11 μ M for primary leukemic cells and ranged from 62 to 115 μ M in the cell lines. JC-1 staining of BCR-ABL+ primary leukemic cells treated with 40 μ M α-bisabolol for 3 to 5 hours demonstrated a dissipation of the mitochondrial transmembrane potential (ΔΨm), thus indicating the start of the apoptotic process. Moreover, NADH-supported state 3 respiration in α-bisabolol treated leukemic cells was significantly decreased in comparison with untreated leukemic controls (140.0±70.5 vs 280.7±11.9 pmol O2/min/106 cells; p<.05). Finally, PARP cleavage and DNA laddering followed α-bisabolol exposure of leukemic BCR-ABL+ blasts. The apoptosis induction was accompanied by ROS production. When tested in combination at constant ratio with Imatinib or Nilotinib, α-bisabolol showed overall slight to strong synergistic effects, without evidence for antagonism across a range of doses (Table 1). In 3 patients with mutation of BCR-ABL (T315I, E255V and Y253H, respectively) we observed full activity of α-bisabolol as single agent and confirmed the synergism between α-bisabolol and Imatinib.Table 1Combination indices (CI) for synergysm experiments using drugs in constant ratioCell Lineα-bisabolol plus Imatinibα-bisabolol plus NilotinibCI at IC50CI at IC75CI at IC90CI at IC95CI at IC50CI at IC75CI at IC90CI at IC95K5620.8500.5630.3770.2890.6860.5760.4840.431LAMA-840.7410.6860.6350.6030.8990.7770.6350.552CML-T10.6830.6130.5530.5170.8580.7970.7420.707 Conclusion.This study indicates that α-bisabolol is an effective pro-apoptotic agent for human acute BCR-ABL+ leukemia cells via induction of mitochondrial membrane damage. The combination of α-bisabolol with Imatinib or Nilotinib allows a dose reduction up to 90% of each drug to obtain the same cytotoxic effect, so indicating a clear synergism. α-bisabolol may be a potential candidate for the treatment of BCR-ABL+ leukemias and the effective dose of TKIs could be reduced in a combined treatment with α-bisabolol. Disclosures:No relevant conflicts of interest to declare.