Reena V. Kartha

Competing endogenous RNAs (ceRNAs): new entrants to the intricacies of gene regulation

The discovery of microRNAs (miRNAs) has led to a paradigm shift in our basic understanding of gene regulation. Competing endogenous RNAs (ceRNAs) are the recent entrants adding to the complexities of miRNA mediated gene regulation. ceRNAs are RNAs that share miRNA recognition elements (MREs) thereby regulating each other. It is apparent that miRNAs act as rheostats that fine-tune gene expression and maintain the functional balance of various gene networks. Thus MREs in coding and non-coding transcripts have evolved to become the crosstalk hubs of gene interactions, affecting the expression levels and activities of different ceRNAs. Decoding the crosstalk between MREs mediated by ceRNAs is critical to delineate the intricacies in gene regulation, and we have just begun to unravel this complexity.

Enhanced detection of hydrogen sulfide generated in cell culture using an agar trap method

Lack of reliable methods to accurately measure hydrogen sulfide (H(2)S) produced in vitro has impeded research on the physiology of this gaseous mediator. Current in vitro methods involve measurement of H(2)S in cell culture media following incubation with H(2)S-releasing compounds. However, this method is inaccurate because H(2)S gas has a short life and thus evades detection. To overcome this, we have adapted a method that employs a modified agar layer to instantly trap H(2)S, allowing measurement of H(2)S accumulated with time. The amount of H(2)S trapped in the agar is quantified using an in situ methylene blue assay. We were able to detect H(2)S produced from sodium hydrogen sulfide (NaHS) added at concentrations as low as 10 μM. Following a 24-h incubation of endothelial-like or vascular smooth muscle cells with 50 μM NaHS, we were able to recover twice more H(2)S than conventional methods. When H(2)S-releasing compounds L-cysteine and N-acetylcysteine were added to the cell culture, the amount of H(2)S increased in a concentration-, time-, and cell line-dependent manner. In conclusion, we have developed an improved method to quantify H(2)S generated in vitro. This method could be used to screen compounds to identify potential H(2)S donors and inhibitors for therapeutic use.

Intravenous Administration of Stable-Labeled N-Acetylcysteine Demonstrates an Indirect Mechanism for Boosting Glutathione and Improving Redox Status

There is an increasing interest in using N-acetylcysteine (NAC) as a treatment for neurodegenerative disorders to increase glutathione (GSH) levels and its redox status. The purpose of this study was to characterize the biosynthesis of NAC to GSH using a novel stable isotope-labeled technique, and investigate the pharmacodynamics of NAC in vivo. Female wild-type mice were given a single intravenous bolus dose of 150 mg kg(-1) stable-labeled NAC. Plasma, red blood cells (RBC), and brain tissues were collected at predesignated time points. Stable-labeled NAC and its metabolite GSH (both labeled and unlabeled forms) were quantified in blood and brain samples. Molar ratios of the reduced and oxidized forms of GSH (GSH divided by glutathione disulfide, redox ratio) were also determined. The elimination phase half-life of NAC was approximately 34 min. Both labeled and unlabeled GSH in RBC were found to increase; however, the area under the curve above baseline (AUCb0-280 ) of labeled GSH was only 1% of the unlabeled form. These data indicate that NAC is not a direct precursor of GSH. In addition, NAC has prolonged effects in brain even when the drug has been eliminated from systemic circulation.

Repeated‐Dose Oral N‐Acetylcysteine in Parkinson's Disease: Pharmacokinetics and Effect on Brain Glutathione and Oxidative Stress

Parkinsons disease (PD) is associated with oxidative stress and decreased nigral glutathione (GSH), suggesting that therapies that boost GSH may have a disease‐modifying effect. Intravenous administration of a high dose of N‐acetylcysteine (NAC), a well‐known antioxidant and GSH precursor, increases blood and brain GSH in individuals with PD and with Gaucher disease and in healthy controls. To characterize the pharmacokinetics of repeated high oral doses of NAC and their effect on brain and blood oxidative stress measures, we conducted a 4‐week open‐label prospective study of oral NAC in individuals with PD (n = 5) and in healthy controls (n = 3). Brain GSH was measured in the occipital cortex using 1H‐MRS at 3 and 7 tesla before and after 28 days of 6000 mg NAC/day. Blood was collected prior to dosing and at predetermined collection times before and after the last dose to assess NAC, cysteine, GSH, catalase, malondialdehyde (MDA) and 4‐hydroxynonenal (4‐HNE) concentrations and the reduced‐to‐oxidized GSH ratio (GSH/ glutathione disulfide [GSSG]). Symptomatic adverse events were reported by 3 of the 5 subjects with PD. NAC plasma concentration–time profiles were described by a first‐order absorption, 1‐compartment pharmacokinetic model. Although peripheral antioxidant measures (catalase and GSH/GSSG) increased significantly relative to baseline, indicators of oxidative damage, that is, measures of lipid peroxidation (4‐HNE and MDA) were unchanged. There were no significant increases in brain GSH, which may be related to low oral NAC bioavailability and small fractional GSH/GSSG blood responses. Additional studies are needed to further characterize side effects and explore the differential effects of NAC on measures of antioxidant defense and oxidative damage.

A hierarchical Bayesian approach for combining pharmacokinetic/pharmacodynamic modeling and Phase IIa trial design in orphan drugs: Treating adrenoleukodystrophy with Lorenzo’s oil

ABSTRACT X-linked adrenoleukodystrophy (X-ALD) is a rare, progressive, and typically fatal neurodegenerative disease. Lorenzo’s oil (LO) is one of the few X-ALD treatments available, but little has been done to establish its clinical efficacy or indications for its use. In this article, we analyze data on 116 male asymptomatic pediatric patients who were administered LO. We offer a hierarchical Bayesian statistical approach to understand LO pharmacokinetics (PK) and pharmacodynamics (PD) resulting from an accumulation of very long-chain fatty acids. We experiment with individual- and observational-level errors and various choices of prior distributions and deal with the limitation of having just one observation per administration of the drug, as opposed to the more usual multiple observations per administration. We link LO dose to the plasma erucic acid concentrations by PK modeling, and then link this concentration to a biomarker (C26, a very long-chain fatty acid) by PD modeling. Next, we design a Bayesian Phase IIa study to estimate precisely what improvements in the biomarker can arise from various LO doses while simultaneously modeling a binary toxicity endpoint. Our Bayesian adaptive algorithm emerges as reasonably robust and efficient while still retaining good classical (frequentist) operating characteristics. Future work looks toward using the results of this trial to design a Phase III study linking LO dose to actual improvements in health status, as measured by the appearance of brain lesions observed via magnetic resonance imaging.

microRNAs in the Malignant Transformation Process

Many cancers originate as benign neoplasms that transform into malignant cancerous tumors in a multistep progression that is regulated, in part, by microRNAs. Benign neoplasms, by definition, lack the ability to invade adjacent tissues or spread to distant sites through metastasis. The benign to malignant transition is a critical intervention stage as tumors diagnosed in subsequent nonlocalized and malignant stages are exponentially more difficult to treat successfully. This chapter explores the critical roles that microRNAs play in the transformation from benign to malignant in four representative cancers: colorectal cancer, pancreatic cancer, malignant peripheral nerve sheath tumor, and prostate cancer. Understanding how these microRNAs control this progression and transformation will lead to new therapeutic targets and diagnostic biomarkers, resulting in improved treatments and patient outcomes.

MicroRNA Control of Apoptotic Programs in Cancer

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that are involved in diverse cellular functions, including control of cell growth, differentiation, and apoptosis. Expression of miRNAs is deregulated in most cancers and plays a significant role in all aspects of cancer development. Evasion of apoptosis, or programmed cell death, is one of the hallmarks of cancer. Apoptosis is a well-orchestrated, conserved, irreversible cellular mechanism that balances the effects of cell proliferation and cell death. Although the complex role of miRNAs in the regulation of cellular apoptosis is not fully understood, recent data strongly suggest that miRNAs play a key role in regulating apoptosis and downstream signaling pathways associated with it. Many of the apoptotic genes, irrespective of their proapoptotic or antiapoptotic functions, are regulated by miRNAs. Thus, the miRNAs involved in apoptotic pathways can be broadly classified as proapoptotic and antiapoptotic miRNAs. In this chapter, we discuss the emerging role of miRNAs in regulating cellular apoptosis in cancer.