The Sesquiterpene Oil α-Bisabolol Induces Apoptosis of B-Chronic Lymphocytic Leukemia Primary Cells
Authors: Bonifacio Massimiliano , Rigo Antonella , Bonalumi Angela , Guardalben Emanuele , Nichele Ilaria , Sissa Cinzia , Cavalieri Elisabetta , Bergamini Christian , Pizzolo Giovanni , Suzuki Hisanori , Vinante Fabrizio 
Published: October 2019

Abstract
Abstract 1319
We have recently demonstrated that the sesquiterpene oil α-bisabolol is cytotoxic against primary acute leukemia cells ex vivo and in chronic myeloid leukemia cell lines. It enters cells via lipid rafts and activates the mitochondrial-dependent intrinsic pathway of apoptosis, exerting a preferential toxicity against malignant vs normal cells probably due to their higher content in lipid rafts. Here we investigated the in vitro activity of α-bisabolol in primary cells from patients with B-Chronic Lymphocytic Leukemia (B-CLL).
Twenty-six patients with newly diagnosed B-CLL gave their informed consent to the study. Cells were collected before any treatment, purified and cultured for 24 hours with serial dilutions of α-bisabolol. Citotoxicity was quantified in flow cytometry by the BD Trucount™ technology to allow comparison between neoplastic and normal residual lymphocytes. B-CLL cells (IC50 42±15 μM) were significantly more sensitive towards α-bisabolol than normal B- (IC50 82±34 μM, p=.005) and T-cells (IC50 120±35 μM, p<.001). Citotoxicity was similar between the IgVH mutated (n=11) and the IgVH unmutated samples (n=7), as well as between the Binet stage A (n=20) and B-C (n=6) patients.
To investigate the mechanisms of α-bisabolol-induced toxicity we treated B-CLL cells with 40 μM α-bisabolol for up to 3 hours. We observed a time-dependent increase in fluorescence of cells treated with the membrane-impermeant nucleic acid stain TO-PRO-3, already detactable after 30 minutes. When cells were loaded with the Ca2+ indicator Fluo-4 AM, an increase of Ca2+ influx was revealed already after 15 minutes. These early events indicate that α-bisabolol induces the loss of cellular membrane integrity, so triggering the apoptotic cascade.
Then we assessed the mitochondrial transmembrane potential (ΔΨm) with the fluorochrome JC-1 to confirm that a mitochondrial damage is a concurrent mechanism in the apoptotic process induced by α-bisabolol. By flow cytometry we demonstrated that, after 3-hour incubation with 40 μM α-bisabolol, ΔΨm dissipation was already detectable in leukemic cells, while T-lymphocytes, evaluated as internal control in the same samples, stayed vital. To investigate the mitochondrial target of α-bisabolol we examined the function of the mitochondrial permeability transition pore (mPTP). After 5-hour incubation with 40 μM α-bisabolol we loaded cells with the calcein AM dye and added CoCl2 to distinguish between intact and damaged mitochondria, confirming that the function of mPTP was compromised in B-CLL cells but not in normal controls. Finally, to determine whether α-bisabolol affects the oxydative state of treated cells, we evaluated the intracellular concentration of reactive oxygen species (ROS) by measuring the fluorescent signal of CM-H2DCFDA loaded cells. When B-CLL cells were exposed to 40 μM a-bisabolol for 3 hours, they exhibited a clear fluorescence increase, indicating the striking generation of ROS: this was completely abrogated by the addition of N-acetylcysteine, a scavenger of intracellular ROS. Clues about the molecular mechanistics of α-bisabolol have also emerged from in vitro models based on treating cells previously transfected with BH3-only molecules. In this setting, α-bisabolol exposed cells seem to undergo detrimental, non-selective autophagy-like phenomena.
Our data indicate that α-bisabolol exerts a level of cytotoxicity against B-CLL cells at concentrations that only partially affect normal B- and T-cells. Moreover, a brief exposure (3–5 hours) to α-bisabolol is sufficient to elicit multiple pro-apoptotic signals independently of the patients' mutational status.

Disclosures:
No relevant conflicts of interest to declare.