- Lysine-specific histone demethylase 1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch. [Journal Article]
- Sci Rep 2016.:30903.
Lysine-specific histone demethylase 1 (LSD1) regulates histone methylation and influences the epigenetic state of cells during the generation of induced pluripotent stem cells (iPSCs). Here we reported that LSD1 inhibition via shRNA or specific inhibitor, tranylcypromine, promoted reprogramming at early stage via two mechanisms. At early stage of reprogramming, LSD1 inhibition increased the retrovirus-mediated exogenous expression of Oct4, Klf4, and Sox2 by blocking related H3K4 demethylation. Since LSD1 inhibition still promoted reprogramming even when iPSCs were induced with small-molecule compounds in a virus-free system, additional mechanisms should be involved. When RNA-seq was used for analysis, it was found that LSD1 inhibition reversed some gene expression changes induced by OKS, which subsequently promoted reprogramming. For example, by partially rescuing the decreased expression of Hif1α, LSD1 inhibition reversed the up-regulation of genes in oxidative phosphorylation pathway and the down-regulation of genes in glycolysis pathway. Such effects facilitated the metabolic switch from oxidative phosphorylation to glycolysis and subsequently promoted iPSCs induction. In addition, LSD1 inhibition also promoted the conversion from pre-iPSCs to iPSCs by facilitating the similar metabolic switch. Therefore, LSD1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch.
- Antidepressant drugs can modify cytotoxic action of temozolomide. [JOURNAL ARTICLE]
- Eur J Cancer Care (Engl) 2016 Aug 2.
Cancer patients often require antidepressant treatment due to comorbid depressive disorder. However, recent studies have demonstrated that antidepressant drugs affect the efficacy of chemotherapy and promote progression of cancer. Apart from the main mood-improving effect, antidepressant drugs also produce analgesic, anxiolytic, hypnotic and pro-cognitive actions. Patients suffering from brain cancer constitute the greatest percentage of depressive cancer patients. However, vital safety and efficacy issues related to combined therapy with temozolomide, the first-line cytostatic in patients diagnosed with glioblastoma multiforme, and antidepressant drugs have yet to be addressed. The aim of the present studies was to evaluate the effect of three antidepressant drugs (imipramine, fluoxetine and tranylcypromine) on the cytotoxic efficacy of temozolomide on T98G cells, a human glioblastoma cell line. In our experiments, we used a complex experimental in vitro system to mimic the instability of a tumour's oxygen supply, thereby reproducing conditions that occur inside the tumour. The effect of the interaction between temozolomide and antidepressant drugs on viability, apoptosis and intensity of divisions of glioblastoma cells was evaluated under different oxygen conditions. The results of our studies demonstrated that imipramine and tranylcypromine reduced the cytotoxic efficacy of temozolomide under some oxygen conditions while fluoxetine did not demonstrate such effects.
- LSD1: biologic roles and therapeutic targeting. [Journal Article]
- Epigenomics 2016 Aug; 8(8):1103-16.
LSD1 (KDM1A; BHC110; AOF2) was the first protein reported to exhibit histone demethylase activity and has since been shown to have multiple essential roles in mammalian biology. Given its enzymatic activity and its high-level expression in many human malignancies, a significant recent focus has been the development of pharmacologic inhibitors. Here we summarize structural and biochemical knowledge of this important epigenetic regulator, with a particular emphasis on the functional and preclinical studies in oncology that have provided justification for the evaluation of tranylcypromine derivative LSD1 inhibitors in early phase clinical trials.
- Evaluation of herb-drug interaction of a polyherbal Ayurvedic formulation through high throughput cytochrome P450 enzyme inhibition assay. [JOURNAL ARTICLE]
- J Ethnopharmacol 2016 Jul 25.
Arishtas are Ayurvedic formulation made with decoction of herbs. Arjunarishta formulation is being used in Ayurveda for cardio-protective activity. Ashwagandharishta formulation possesses antioxidant, anti-atherosclerotic and anti-stress properties. Ridayarishta, a novel empirical formulation was prepared using combination of selected ingredients from these two formulations to support healthy heart functions and to reduce stress.Aim of the Study was to investigate herb-drug interaction (HDI) of Ridayarishta formulation through human hepatic cytochrome P450 (CYP450) enzyme inhibition assay.Ridayarishta formulation was phyto-chemically standardized against arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A using high performance thin layer chromatography (HPTLC) analysis. The formulation was standardized with respect to ethanol by gas chromatographic (GC) analysis. HDI was evaluated with Ridayarishta formulation and amlodipine besilate, atenolol, atorvastatin, metformin, glipizide glimepiride cocktail using high throughput CYP450 enzyme inhibition assay; against CYP1A2, 2C19, 2D6 and 3A4 isozymes.Contents of arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A in Ridayarishta formulation were found to be 1.76±0.12, 1.51±0.09, 1.85±0.05, 3.2±0.12, 1.21±0.08, and 2.16±0.09ppm, respectively. Quantity of ethanol in Ridayarishta was found to be 7.95±0.023% (V/V). Ridayarishta showed significantly higher (P<0.001) IC50 value against CYP1A2 (IC50-13.80±1.96µg/mL), 2C19 (IC50-14.343±2.28µg/mL), 2D6 (IC50-0.897±0.28µg/mL) and 3A4 (IC50-32.057±2.51µg/mL) compared to positive controls such as furafylline, tranylcypromine, quinidine and ketoconazole respectively. Cocktail of herbal formulation and cardio protective, antihypertensive, anti-diabetic drugs showed significantly (P<0.001and P<0.01) less or negligible HDI.Ridayarishta formulation alone and cocktail with amlodipine besilate, atenolol, atorvastatin, metformin, glipizide, glimepiride had negligible or insignificant effect on CYP450 inhibition. It may be concluded that consumption of Ridayarishta along with selective cardio protective, antihypertensive and anti-diabetic conventional medicine is safe with negligible or without any significant CYP450 (CYP1A2, 2C19, 2D6 and 3A4) inhibition mediated HDI.
- Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers. [JOURNAL ARTICLE]
- Blood 2016 Jul 12.
Overcoming the silencing of the fetal γ-globin gene has been a long standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called LCR, dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells the LCR can be re-directed from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom designed zinc finger proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacological reactivation of fetal hemoglobin (HbF), hematopoietic cells from SCD patients were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double of that observed with decitabine and pomalidomide; butyrate had an intermediate effect while tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle Hb (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared to the drug regimens support its therapeutic potential for the treatment of SCD.
- Rebalancing gene haploinsufficiency in vivo by targeting chromatin. [Journal Article]
- Nat Commun 2016.:11688.
Congenital heart disease (CHD) affects eight out of 1,000 live births and is a major social and health-care burden. A common genetic cause of CHD is the 22q11.2 deletion, which is the basis of the homonymous deletion syndrome (22q11.2DS), also known as DiGeorge syndrome. Most of its clinical spectrum is caused by haploinsufficiency of Tbx1, a gene encoding a T-box transcription factor. Here we show that Tbx1 positively regulates monomethylation of histone 3 lysine 4 (H3K4me1) through interaction with and recruitment of histone methyltransferases. Treatment of cells with tranylcypromine (TCP), an inhibitor of histone demethylases, rebalances the loss of H3K4me1 and rescues the expression of approximately one-third of the genes dysregulated by Tbx1 suppression. In Tbx1 mouse mutants, TCP treatment ameliorates substantially the cardiovascular phenotype. These data suggest that epigenetic drugs may represent a potential therapeutic strategy for rescue of gene haploinsufficiency phenotypes, including structural defects.
- Significant inhibitory impact of dibenzyl trisulfide and extracts of Petiveria alliacea on the activities of major drug-metabolizing enzymes in vitro: An assessment of the potential for medicinal plant-drug interactions. [Journal Article]
- Fitoterapia 2016 Jun.:138-46.
Dibenzyl trisulfide (DTS) is the major active ingredient expressed in Petiveria alliacea L., a shrub widely used for a range of conditions, such as, arthritis, asthma and cancer. Given its use alone and concomitantly with prescription medicines, we undertook to investigate its impact on the activities of important drug metabolizing enzymes, the cytochromes P450 (CYP), a key family of enzymes involved in many adverse drug reactions. DTS and seven standardized extracts from the plant were assessed for their impact on the activities of CYPs 1A2, 2C19, 2C9, 2D6 and 3A4 on a fluorometric assay. DTS revealed significant impact against the activities of CYPs 1A2, 2C19 and 3A4 with IC50 values of 1.9, 4.0 and 3.2μM, respectively, which are equivalent to known standard inhibitors of these enzymes (furafylline, and tranylcypromine), and the most potent interaction with CYP1A2 displayed irreversible enzyme kinetics. The root extract, drawn with 96% ethanol (containing 2.4% DTS), displayed IC50 values of 5.6, 3.9 and 4.2μg/mL respectively, against the same isoforms, CYPs 1A2, 2C19 and 3A4. These investigations identify DTS as a valuable CYP inhibitor and P. alliacea as a candidate plant worthy of clinical trials to confirm the conclusions that extracts yielding high DTS may lead to clinically relevant drug interactions, whilst extracts yielding low levels of DTS, such as aqueous extracts, are unlikely to cause adverse herb-drug interactions.
- Inhibitors of LSD1 as a potential therapy for acute myeloid leukemia. [Journal Article]
- Expert Opin Investig Drugs 2016 Jul; 25(7):771-80.
Epigenetic dysregulation plays a critical role in the pathogenesis of acute myeloid leukemia (AML). Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor suppression and cell cycling, resulting in myeloid maturation arrest and proliferation of early myeloid progenitors. One promising approach targeting chromatin regulatory proteins is inhibition of lysine specific demethylase-1 (LSD1), an enzyme responsible for demethylation of histone H3 as well as other functionsAvailable literature on LSD1 in normal and malignant hematopoiesis was identified in PubMed and reviewed. Areas addressed here include the biology of LSD1, pharmacologic inhibitors, and preclinical data supporting the rationale for LSD1 inhibition in AML therapy.LSD1 inhibitors represent a promising novel epigenetic approach for AML therapy. Preclinical studies have revealed that pharmacologic LD1 inhibitors function primarily by altering stem cell programs and restoring myeloid differentiation to AML cells. These effects are markedly enhanced in combination with trans-retinoic acid or histone deacetylase inhibitors with little toxicity. Currently, multiple oral LSD1 inhibitors are undergoing phase 1 investigation in patients with AML. The results of these clinical trials are eagerly awaited.
- An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s. [Journal Article]
- Acta Pharmacol Sin 2016 May; 37(5):708-18.
The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay.In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT).The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay.The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates.
- Tranylcypromine, a lysine-specific demethylase 1 (LSD1) inhibitor, suppresses lesion growth and improves generalized hyperalgesia in mouse with induced endometriosis. [Journal Article, Research Support, Non-U.S. Gov't]
- Reprod Biol Endocrinol 2016.:17.
Growing evidence indicates that endometriosis is an epigenetic disease. Encouragingly, histone deacetylases (HDACs) and DNA methyltransferases have been shown to be promising targets by numerous in vitro studies. However, only a few studies have shown promising effects of HDAC inhibition in preclinical studies in endometriosis. While lysine-specific demethylase 1 (LSD1) is recently found to be aberrantly expressed in endometriosis, and that the treatment of endometriotic stromal cells with tranylcypromine (TC), an LSD1 inhibitor, significantly reduced cellular proliferation, cell cycle progression, and invasiveness, the in vivo effect of TC treatment is currently lacking. This study sought to evaluate the effect of TC in a mouse model of endometriosis.Forty-seven female C57BL/6 mice were used in this experimentation. All mice, except those randomly selected to form Sham surgery (M) and specificity control (S) groups, received an endometriosis-inducing surgery. Group S was set up mainly to ensure that the reduced generalized hyperalgesia in mice treated with TC is not due to any possible analgesic effect of TC, but rather resulting from the treatment effect specific to endometriosis. Two weeks after the surgery, mice that received surgery were further divided randomly into 3 groups: 1) untreated group (U); 2) low-dose TC group (L); 3) high-dose TC group (H). Group S received the same treatment as in group H. Two weeks after treatment, all mice were sacrificed and their ectopic endometrial tissues were harvested and analyzed by immunohistochemistry analysis. Hotplate test was administrated to all mice before the induction, treatment and sacrifice. Lesion size, hotplate latency, immunoreactivity against markers of proliferation, angiogenesis, H3K4 methylation, and of epithelial-mesenchymal transition (EMT).TC treatment significantly and substantially reduced the lesion size and improved generalized hyperalgesia in a dose-dependent fashion in mice with induced endometriosis. In addition, TC treatment resulted in reduced immunoreactivity to biomarkers of proliferation, angiogenesis, and H3K4 methylation, leading to arrested EMT and lesion growth.In light of our previously reported reduced cellular proliferation, cell cycle progression and invasiveness resulting from the LSD1 inhibition in in vitro studies, our data strongly suggest that LSD1 is a promising therapeutic target for endometriosis.Not applicable.