Specific MRP4 Inhibitor Ceefourin-1 Enhances Apoptosis Induced by 6-Mercaptopurine in Jurkat Leukemic Cells, but Not in Normal Lymphoblast Cell Line CRL-1991.

Background and objectives: The multidrug resistance protein 4 (MRP4) is a member of the ABC transporter, which has been extensively related to many types of cancer including leukemia. MRP4 overexpression and activity over the efflux of some chemotherapeutic drugs are the main causes of chemoresistance. 6-mercaptopurine (6-MP) is a chemotherapeutic drug widely used in the consolidation and maintenance phases of leukemia treatment. However, 6-MP is a substrate of MRP4, which decreases its chemotherapeutic efficacy. Current research is focused on the development of MRP4 inhibitors to combat chemoresistance by allowing the accumulation of the drug substrates inside the cells. To date, the only specific MRP4 inhibitor that has been developed is ceefourin-1, which has been reported to inhibit MRP4 in many cancer cells and which makes it an excellent candidate to enhance the activity of 6-MP in a combined treatment in vitro of leukemic cells. Materials and methods: in the present work, we determined the enhancing activity of ceefourin-1 on the antiproliferative and apoptotic effect of 6-MP in leukemic Jurkat cells by trypan blue assay and flow cytometry. Besides, we determined the 6-MP and ceefourin-1 binding sites into MRP4 by molecular docking and molecular dynamics. Results: ceefourin-1 enhanced the apoptotic activity of 6-MP in Jurkat cells, while in CRL-1991 cells both antiproliferative and apoptotic effect were significantly lower. Ceefourin-1 additively cooperates with 6-MP to induce apoptosis in leukemic cells, but normal lymphoblast CRl-1991 showed resistance to both drugs. Conclusion: ceefourin-1 and 6-MP cooperates to trigger apoptosis in leukemic Jurkat cells, but the full mechanism needs to be elucidated in further works. In addition, our perspective is to test the cooperation between ceefourin-1 and 6-MP in samples from patients and healthy donnors.

Study of Endogen Substrates, Drug Substrates and Inhibitors Binding Conformations on MRP4 and Its Variants by Molecular Docking and Molecular Dynamics.

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.

Glutamic Acid Increased Methotrexate Polyglutamation and Cytotoxicity in a CCRF-SB Acute Lymphoblastic Leukemia Cell Line.

Background and Objectives: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in childhood. The majority of patients respond to treatment, but those with resistant phenotypes suffer relapse or death. The antifolate methotrexate (MTX) is the most commonly used drug against ALL due to its efficacy. Once inside leukemic cells, MTX is metabolized into methotrexate polyglutamates (MTX-PG) by action of the enzyme folylpolyglutamate synthetase (FPGS), leading to a longer action compared to that of MTX alone. Materials and Methods: In this work, we demonstrated that the combination treatment of methotrexate and 5 and 10 mM glutamic acid could enhance methotrexate cytotoxicity in CCRF-SB (B-ALL) cells. In addition, MTX plus 20 mM glutamic acid was able to improve the synthesis of MTX-PG5. Results: All treatments induced an increase in FPGS expression compared to that of the control group. Furthermore, we detected different cellular expression patterns of FPGS in the different treatments. Conclusion: Based on these findings, we demonstrated that levels of methotrexate polyglutamates (MTX-PGs) could be a key determinant of methotrexate-induced cytotoxicity in CCRF-SB acute lymphoblastic leukemia cells.

Green Synthesis and Antiproliferative Activity of Gold Nanoparticles of a Controlled Size and Shape Obtained Using Shock Wave Extracts from Amphipterygium adstringens.

In this study, green chemistry was used as a tool to obtain gold nanoparticles using Amphipterygium adstringens extracts as a synthesis medium. Green ethanolic and aqueous extracts were obtained using ultrasound and shock wave-assisted extraction. Gold nanoparticles with sizes ranging between 100 and 150 nm were obtained with ultrasound aqueous extract. Interestingly, homogeneous quasi-spherical gold nanoparticles with sizes between 50 and 100 nm were achieved with shock wave aqueous-ethanolic extracts. Furthermore, 10 nm gold nanoparticles were obtained by the traditional methanolic macerate extraction method. The physicochemical characteristics, morphology, size, stability, and Z potential of the nanoparticles were determined using microscopic and spectroscopic techniques. The viability assay in leukemia cells (Jurkat) was performed using two different sets of gold nanoparticles, with final IC50 values of 87 µM and 94.7 µM, reaching a maximum cell viability decrease of 80% The results do not indicate a significant difference between the cytotoxic effects produced by the gold nanoparticles synthesized in this study and vincristine on normal lymphoblasts (CRL-1991).