Antitumor Effect of Brusatol in Acute Lymphoblastic Leukemia Models Is Triggered by Reactive Oxygen Species Accumulation.

Acute lymphoblastic leukemia (ALL) is one of the most common hematological malignancies at pediatric ages and is characterized by different chromosomal rearrangements and genetic abnormalities involved in the differentiation and proliferation of lymphoid precursor cells. Brusatol is a quassinoid plant extract extensively studied due to its antineoplastic effect through global protein synthesis and nuclear factor erythroid 2-related factor-2 (NRF2) signaling inhibition. NRF2 is the main regulator of cellular antioxidant response and reactive oxygen species (ROS), which plays an important role in oxidative stress regulation. This study aimed to evaluate the effect of brusatol in in vitro models of ALL. KOPN-8 (B-ALL), CEM (T-ALL), and MOLT-4 (T-ALL) cell lines were incubated with increasing concentrations of brusatol, and the metabolic activity was evaluated using the resazurin assay. Flow cytometry was used to evaluate cell death, cell cycle, mitochondrial membrane potential (Δψmit), and to measure ROS and reduced glutathione (GSH) levels. Our results show that brusatol promoted a decrease in metabolic activity in ALL cell lines in a time-, dose-, and cell-line-dependent manner. Brusatol induced a cytostatic effect by cell cycle arrest in G0/G1 in all cell lines; however, cell death mediated by apoptosis was only observed in T-ALL cells. Brusatol leads to an oxidative stress imbalance by the increase in ROS levels, namely, superoxide anion. Redox imbalance and cellular apoptosis induced by brusatol are highly modulated by mitochondria disruption as a decrease in mitochondrial membrane potential is detected. These data suggest that brusatol might represent a new therapeutic approach for acute lymphoblastic leukemia, particularly for ALL T-cell lineage.

Interactions between Rhodamine Dyes and Model Membrane Systems-Insights from Molecular Dynamics Simulations.

BACKGROUND: rhodamines are dyes widely used as fluorescent tags in cell imaging, probing of mitochondrial membrane potential, and as P-glycoprotein model substrates. In all these applications, detailed understanding of the interaction between rhodamines and biomembranes is fundamental. METHODS: we combined atomistic molecular dynamics (MD) simulations and fluorescence spectroscopy to characterize the interaction between rhodamines 123 and B (Rh123 and RhB, respectively) and POPC bilayers. RESULTS: while the xanthene moiety orients roughly parallel to the membrane plane in unrestrained MD simulations, variations on the relative position of the benzoic ring (below the xanthene for Rh123, above it for RhB) were observed, and related to the structure of the two dyes and their interactions with water and lipids. Subtle distinctions were found among different ionization forms of the probes. Experimentally, RhB displayed a lipid/water partition coefficient more than two orders of magnitude higher than Rh123, in agreement with free energy profiles obtained from umbrella sampling MD. CONCLUSIONS: this work provided detailed insights on the similarities and differences in the behavior of bilayer-inserted Rh123 and RhB, related to the structure of the probes. The much higher affinity of RhB for the membranes increases the local concentration and explains its higher apparent affinity for P-glycoprotein reconstituted in model membranes.