Effects of chemotherapeutic drugs on the antioxidant capacity of human erythroleukemia cells with MDR phenotype.

In this work, we identified that different chemotherapeutic drugs may select cells with different antioxidant capacities. For this, we evaluated the sensitivity of two multidrug-resistant (MDR) erythroleukemia cell lines: Lucena (resistant to vincristine, VCR) and FEPS (resistant to daunorubicin, DNR) derived from the same sensitive cell K562 (non-MDR) to hydrogen peroxide. In addition, we evaluated how the cell lines respond to the oxidizing agent in the absence of VCR/DNR. In absence of VCR, Lucena drastically decreases cell viability when exposed to hydrogen peroxide, while FEPS is not affected even without DNR. To analyze whether selection by different chemotherapeutic agents may generate altered energetic demands, we analyzed the production of reactive oxygen species (ROS) and the relative expression of the glucose transporter 1 gene (glut1). We observed that the selection through DNR apparently generates a higher energy demand than VCR. High levels of transcription factors genes expression (nrf2, hif-1α, and oct4) were kept even when the DNR is withdrawn from the FEPS culture for one month. Together, these results indicate that DNR selects cells with greater ability to express the major transcription factors related to the antioxidant defense system and the main extrusion pump (ABCB1) related to the MDR phenotype. Taking into account that the antioxidant capacity of tumor cells is closely related to resistance to multiple drugs, it is evident that endogenous antioxidant molecules may be targets for the development of new anticancer drugs.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations.

BACKGROUND: PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype. OBJECTIVE: In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR). MATERIALS AND METHODS: In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR). RESULTS: Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments. CONCLUSION: Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

Modeling drug-drug interactions of AZD1208 with Vincristine and Daunorubicin on ligand-extrusion binding TMD-domains of multidrug resistance P-glycoprotein (ABCB1).

In the present study, the molecular docking mechanism based on pharmacodynamic interactions between the ligands AZD1208 and recognized chemotherapy agents (Vincristine and Daunorubicin) with human ATP-binding cassette (ABC) transporters (ABCB1) was investigated. For the first time, were combined an in silico approaches like molecular docking and ab initio computational simulation based on Density Functional Theory (DFT) to explain the drug-drug interaction mechanism of aforementioned chemotherapy ligands with the transmembrane ligand extrusion binding domains (TMDs) of ABCB1. In this regard, the theoretical pharmacodynamic interactions were characterized by using the Gibbs free energy (FEB, kcal/mol) from the best ABCB1-ligand docking complexes. The molecular docking results pointing that for the three chemotherapy ABCB1-ligand complexes are mainly based in non-covalent hydrophobic and hydrogen-bond interactions showing a similar toxicodynamic behavior in terms of strength of interaction (FEB, kcal/mol) and very close free binding energies when compared with the FEB-values of the ABCB1 specific-inhibitor (Rhodamine B) = -6.0 kcal/mol used as theoretical docking control to compare with FEB (AZD1208-ABCB1) ∼ FEB (Vincristine-ABCB1) ∼ FEB (Daunorubicin-ABCB1) -6.2 kcal/mol as average. Ramachandran plot suggests that the 3D-crystallographic structure from ABCB1 transporter can be efficiently-modeled with conformationally-favored Psi versus Phi dihedral angles for all key TMDs-residues. Though, the results of DFT-simulation corroborate the existence of drug-drug interaction between (AZD1208/Vincristine) > (AZD1208/Daunorubicin). These theoretical pieces of evidence have preclinical relevance potential in the design of the new drugs to understand the polypharmacology influence in the molecular mechanism of multiple-drugs resistance, contributing with a higher success in chemotherapy and prognosis of cancer patients.