Dopamine Receptor D1 Contributes to Cocaine Epigenetic Reprogramming of Histone Modifications in Male Germ Cells.

Paternal environmental perturbations, including cocaine intake, can affect the development and behavior of the offspring through epigenetic inheritance. However, the mechanism by which cocaine alters the male germ cells epigenome is almost unexplored. Here, we report that cocaine-treated male mice showed alterations on specific histone post-translational modifications (PTMs) including increased silent chromatin marks H3K9me3 and H3K27me3 and decreased active enhancer and promoter marks H3K27ac and H3K4me3 in isolated germ cells. Also, cocaine increased H3K9ac and H4K16ac levels, involved in the replacement of histones by protamines that take place at round spermatid stage. Cocaine also altered histones H3/H4 epigenetic enzymes by increasing acetyltransferase KAT8/MOF, deacetylase SIRT1 and methyltransferase KMT1C/G9A, and decreasing deacetylases HDAC1/2 and demethylase KDM1A/LSD1 protein levels. Moreover, a pre-treatment with dopamine receptor 1 (DRD1) antagonist SCH23390 (SCH) blocked cocaine effects on H3K4me3, H3K27me3, and H4K16ac epigenetic marks. Interestingly, treatment with SCH-only was able to modify most of the histone marks tested here, pointing to a dopamine role in controlling histone PTMs in germ cells. Taken together, our data suggest a key role for DRD1 in mediating cocaine-triggered epigenetic modifications related to the silencing of gene transcription and the histone-to-protamine replacement that controls chromatin architecture of maturing sperm cells, and pinpoints a novel role of the dopaminergic system in the regulation of male germ cells reprogramming.

Analogues of the Lignan Pinoresinol as Novel Lead Compounds for P-glycoprotein (P-gp) Inhibitors.

To find novel P-gp-inhibitors, a library of pregnane X receptor (PXR) ligands and the ZINC DrugsNow library were superimposed on the P-gp inhibitor (+)-pinoresinol (1) used as a query for a three-dimensional similarity search. After determining the TanimotoCombo index of similarity with 1, eight compounds from the PXR library and two ZINC compounds were selected for biological evaluation. The P-gp inhibition study showed that compounds 7, 8, and 9 successfully increased intracellular doxorubicin (DOX) accumulation in the P-gp overexpressed Lucena 1 cells from 25, 12.5, and 6.25 µM, respectively. Among a series of analogues of 9, compounds 26-30 were shown to be active, with 26 and 27 causing a significant increase in DOX accumulation from 1.56 µM and rendering Lucena 1 sensitive to DOX from 1.56 and 0.78 µM, respectively. Molecular modeling studies showed that both compounds bind to the P-gp at transmembrane helices (TMH) 4, 5, and 6, with 27 also showing contacts with TMH 3.

Mechanism Underlying the Reversal of Drug Resistance in P-Glycoprotein-Expressing Leukemia Cells by Pinoresinol and the Study of a Derivative.

P-glycoprotein (P-gp) is a membrane protein associated with multidrug resistance (MDR) due to its key role in mediating the traffic of chemotherapeutic drugs outside cancer cells, leading to a cellular response that hinders efforts toward successful therapy. With the aim of finding agents that circumvent the MDR phenotype mediated by P-gp, 15 compounds isolated from native and naturalized plants of Argentina were screened. Among these, the non-cytotoxic lignan (±) pinoresinol successfully restored sensitivity to doxorubicin from 7 µM in the P-gp overexpressed human myelogenous leukemia cells, Lucena 1. This resistance-reversing effect was confirmed by competitively increasing the intracellular doxorubicin accumulation and by significantly inhibiting the efflux of doxorubicin and, to a lesser extent, that of rhodamine 123. The activity obtained was similar to that observed with verapamil. No such results were observed in the sensitive parental K562 cell line. To gain deeper insight into the mode of action of pinoresinol, its effect on P-gp function and expression was examined. The docking simulations indicated that the lignan bound to P-gp at the apex of the V-shaped transmembrane cavity, involving transmembrane helices 4, 5, and 6, and partially overlapped the binding region of tariquidar, which was used as a positive control. These results would shed some light on the nature of its interaction with P-gp at molecular level and merit further mechanistic and kinetic studies. In addition, it showed a maximum 29% activation of ATP hydrolysis and antagonized verapamil-stimulated ATPase activity with an IC50 of 20.9 µM. On the other hand, pinoresinol decreased the presence of P-gp in the cell surface. Derivatives of pinoresinol with improved activity were identified by docking studies. The most promising one, the non-cytotoxic 1-acetoxypinoresinol, caused a reversion of doxorubicin resistance from 0.11 µM and thus higher activity than the lead compound. It also caused a significant increase in doxorubicin accumulation. Results were similar to those observed with verapamil. The results obtained positioned these compounds as potential candidates for effective agents to overcome P-gp-mediated MDR, leading to better outcomes for leukemia chemotherapy.