Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. Withaferin A (WA) is a plant derived steroidal lactone which holds promise as a therapeutic agent for treatment of breast cancer (BC). We determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines, following 72h treatment to a sub-cytotoxic concentration of WA. In contrast to the DNA demethylating agent 5-aza-2’-deoxycytidine (DAC), WA treatment of MDA-MB-231 cells rather tackles an epigenetic cancer network through gene-specific DNA hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Gene expression and pathway enrichment analysis further reveals epigenetic suppression of multiple cancer hallmarks associated with cell cycle regulation, cell death, cancer cell metabolism, cell motility and metastasis. Remarkably, DNA hypermethylation of corresponding CpG sites in PLAU, ADAM8, TNSF12, GSTM1 and ME3 genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ESR)/progesterone receptor (PR) status in primary BC tumors. Moreover, upon comparing differentially methylated WA responsive target genes with DNA methylation changes in different clinical subtypes of breast cancer patients in the cancer genome atlas (TCGA), we found that WA silences HER2/PR/ESR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks, with an improved therapeutic sensitivity. In this respect, WA may represent a novel and attractive phyto-pharmaceutical for TNBC treatment.
In this study, we designed and synthesized heterobivalent ligands targeting heteromers consisting of the metabotropic glutamate 5 receptor (mGluR5) and the dopamine D receptor (DR). Bivalent ligand 22a with a linker consisting of 20 atoms showed 4-fold increase in affinity for cells coexpressing DR and mGluR5 compared to cells solely expressing DR. Likewise, the affinity of 22a for mGluR5 increased 2-fold in the coexpressing cells. Additionally, 22a exhibited a 5-fold higher mGluR5 affinity than its monovalent precursor 21a in cells coexpressing DR and mGluR5. These results indicate that 22a is able to bridge binding sites on both receptors constituting the heterodimer. Likewise, cAMP assays revealed that 22a had a 4-fold higher potency in stable DR and mGluR5 coexpressing cell lines than 1. Furthermore, molecular modeling reveals that 22a is able to simultaneously bind both receptors by passing between the TM5-TM6 interface and establishing six protein-ligand H-bonds.
BackgroundIn gene expression studies via RT-qPCR many conclusions are inferred by using reference genes. However, it is generally known that also reference genes could be differentially expressed between various tissue types, experimental conditions and animal models. An increasing amount of studies have been performed to validate the stability of reference genes. In this study, two rodent-specific Short Interspersed Nuclear Elements (SINEs), which are located throughout the transcriptome, were validated and assessed against nine reference genes in a model of Temporal Lobe Epilepsy (TLE). Two different brain regions (i.e. hippocampus and cortex) and two different disease stages (i.e. acute phase and chronic phase) of the systemic kainic acid rat model for TLE were analyzed by performing expression analyses with the geNorm and NormFinder algorithms. Finally, we performed a rank aggregation analysis and validated the reference genes and the rodent-specific SINEs (i.e. B elements) individually via Gfap gene expression.ResultsGeNorm ranked Hprt1, Pgk1 and Ywhaz as the most stable genes in the acute phase, while Gusb and B2m were ranked as the most unstable, being significantly upregulated. The two B elements were ranked as most stable for both brain regions in the chronic phase by geNorm. In contrast, NormFinder ranked the B1 element only once as second best in cortical tissue for the chronic phase. Interestingly, using only one of the two algorithms would have led to skewed conclusions. Finally, the rank aggregation method indicated the use of the B1 element as the best option to normalize target genes, independent of the disease progression and brain region. This result was supported by the expression profile of Gfap.ConclusionIn this study, we demonstrate the potential of implementing SINEs -notably the B1 element- as a stable normalization factor in a rodent model of TLE, independent of brain region or disease progression.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor control deficits, which is associated with the loss of striatal dopaminergic neurons from the substantia nigra. In parallel to dopaminergic denervation, there is an increase of acetylcholine within the striatum, resulting in a striatal dopaminergiccholinergic neurotransmission imbalance. Currently, available PD pharmacotherapy (e.g., prodopaminergic drugs) does not reinstate the altered dopaminergic-cholinergic balance. In addition, it can eventually elicit cholinergic-related adverse effects. Here, we investigated the interplay between dopaminergic and cholinergic systems by assessing the physical and functional interaction of dopamine D 2 and muscarinic acetylcholine M 1 receptors (D 2 R and M 1 R, respectively), both expressed at striatopallidal medium spiny neurons. First, we provided evidence for the existence of D 2 R-M 1 R complexes via biochemical (i.e., co-immunoprecipitation) and biophysical (i.e., BRET 1 and NanoBiT R ) assays, performed in transiently transfected HEK293T cells. Subsequently, a D 2 R-M 1 R co-distribution in the mouse striatum was observed through doubleimmunofluorescence staining and AlphaLISA R immunoassay. Finally, we evaluated the functional interplay between both receptors via behavioral studies, by implementing the classical acute reserpine pharmacological animal model of experimental parkinsonism. Reserpinized mice were administered with a D 2 R-selective agonist (sumanirole) and/or an M 1 R-selective antagonist (VU0255035), and alterations in PD-related behavioral tasks (i.e., locomotor activity) were evaluated. Importantly, VU0255035 (10 mg/kg) potentiated the antiparkinsonian-like effects (i.e., increased locomotor activity and decreased catalepsy) of an ineffective sumanirole dose (3 mg/kg). Altogether, our data suggest the existence of putative striatal D 2 R/M 1 R heteromers, which might be a relevant target to manage PD motor impairments with fewer adverse effects.
The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.
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