Slocation was detected in mitochondrial fraction at different times and solubilized doses of abisabolol. a-tubulin and Hsp60 were used as markers for the cytosol and mitochondria fractions, respectively. A representative case is shown. (C) Permeabilized leukemic cells and healthy lymphocytes were incubated for 10 minutes in respiration buffer at 30 in the presence or in the absence of 3 M a-bisabolol. In treated leukemic cells, the G/M oxygen consumption was clearly lower than in untreated leukemic controls (p < 0.05). The S/G3P oxygen consumption was not modified by treatment, and the mitochondrial respiration was not stimulated by FCCP addition. This is in line with a direct effect of a-bisabolol on mitochondrial integrity. Healthy lymphocyte respiration PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28380356 was not affected by treatment. G/M: glutamate plus malate; S/G3P: succinate plus glycerol-3-phosphate; FCCP: carbonylcyanide-4-(trifluoromethoxy)-phenyl-hydrazone. Means ?SD of 6 leukemias and 6 normal donors are depicted.exists as a green-fluorescent monomer. Accordingly, the ratio red/green JC-1 fluorescence can be used as a sensitive measure of m [23]. Disruption of m (a hallmark of cytochrome c translocation and the start of the apoptotic process) is indicated by a loss of red fluorescence and an increase in green fluorescence. Figure 7A shows the representative case Ph-B-ALL #01 out of the 6 tested. Microscopy revealed that in untreated leukemic cells well-polarized mitochondria were marked by punctate red fluorescent staining (Figure 7A, left side). After a 3-hour incubation with 40 M a-bisabolol, this pattern was replaced by diffuse green fluorescence in leukemic cells (Figure 7A, center and right side). Flow cytometry showed that untreated blasts with well-polarized, red-emitting mitochondria localized in the upper region of the plot (Figure 7A, left plot: high m ). Blasts exposed to 40 M a-bisabolol underwent aprogressive loss of red fluorescence, indicated by a shift right and downward over 3 (Figure 7A, central plot: intermediate m) and 5 hours (Figure 7A, right plot: low m ). In contrast, normal lymphocytes used as a negative control did not suffer any changes in their microscopy or cytofluorimetric pattern when exposed to a similar a-bisabolol concentration, indicating that there was no mitochondrial damage (Figure 7B, images and plots), and that the cells remained vital. Finally, the same blasts depicted in Figure 7A underwent PARP cleavage and DNA laddering following a-bisabolol exposure (Figure 7C).Discussion Forecasting the fraction of the lipophilic compound a-bisabolol that was dissolved in water at given times was a basic preliminary step to standardize the drug useCavalieri et al. Journal of Translational Medicine 2011, 9:45 http://www.translational-medicine.com/content/9/1/Page 11 ofAuntreated3 hours5 hourshighmintmlowmJC-1 monomersBhighmJC-1 monomersC116 KDa 85 KDaC 3h 2h 1h C 1h 3h 5hPARP cleavage DNA ladderFigure 7 a-bisabolol-induced mitochondrial damage in primary leukemic blasts. Cells were stained with JC-1. In non-damaged cells, JC-1 forms red-emitting aggregates in the mitochondrial matrix. A loss of red fluorescence and an increase in cytoplasmic green-emitting monomers Procyanidin B1 site signal the disruption of the mitochondrial transmembrane potential (m). (A) The representative case Ph-B-ALL #01 is shown out of the 6 leukemias tested. Microscopy (magnification, ?400). Whereas untreated leukemic blasts showed well-polarized mitochondria marked by punctated red fluore.