A pan-cancer analysis of alternative splicing events reveals novel tumor-associated splice variants of matriptase.

High-throughput transcriptome sequencing allows identification of cancer-related changes that occur at the stages of transcription, pre-messenger RNA (mRNA), and splicing. In the current study, we devised a pipeline to predict novel alternative splicing (AS) variants from high-throughput transcriptome sequencing data and applied it to large sets of tumor transcriptomes from The Cancer Genome Atlas (TCGA). We identified two novel tumor-associated splice variants of matriptase, a known cancer-associated gene, in the transcriptome data from epithelial-derived tumors but not normal tissue. Most notably, these variants were found in 69% of lung squamous cell carcinoma (LUSC) samples studied. We confirmed the expression of matriptase AS transcripts using quantitative reverse transcription PCR (qRT-PCR) in an orthogonal panel of tumor tissues and cell lines. Furthermore, flow cytometric analysis confirmed surface expression of matriptase splice variants in chinese hamster ovary (CHO) cells transiently transfected with cDNA encoding the novel transcripts. Our findings further implicate matriptase in contributing to oncogenic processes and suggest potential novel therapeutic uses for matriptase splice variants.

Ex vivo Akt/HO-1 gene therapy to human endothelial progenitor cells enhances myocardial infarction recovery.

The aim of this study was to evaluate the overexpression of genes central to cell survival and angiogenesis to enhance the function of human late outgrowth endothelial progenitor cells (EPCs) and their utility for infarct recovery. Ischemic myocardial injury creates a hostile microenvironment, which is characterized by hypoxia, oxidative stress, and inflammation. The infarct microenvironment prevents adhesion, survival, and integration of cell transplants that promote neovascularization. EPCs are dysfunctional as a result of risk factors in cardiovascular patients. Protein kinase B (Akt) and heme-oxygenase-1 (HO-1) are intracellular proteins that play an important role in angiogenesis and cell survival. Late outgrowth EPCs transduced ex vivo with Akt and HO-1 demonstrate improved adhesion to extracellular matrix, improved migration toward human cardiomyocytes, and an improved paracrine profile under stress. Enhanced late outgrowth EPCs reduce the tumor necrosis factor-α (TNF-α) burden both in vitro and in vivo, attenuating nuclear factor-κB (NF-κB) activity and promoting cell survival. Akt and HO-1 enhance late outgrowth EPC neovascularization, resulting in improved cardiac performance and reduced negative remodeling after myocardial infarction in nude mice. Alteration of the infarct microenvironment through gene modification of human late outgrowth EPCs enhances the function and integration of transplanted cells for restoration of cardiac function.

Recombinant truncated and microsomal heme oxygenase-1 and -2: differential sensitivity to inhibitors.

Recombinant truncated forms of heme oxygenase-1 and -2 (HO-1 and HO-2) were compared with their crude microsomal counterparts from brain and spleen tissue of adult male rats with respect to their inhibition by azole-based, nonporphyrin HO inhibitors. The drugs tested were an imidazole-alcohol, an imidazole-dioxolane, and a triazole-ketone. Both the recombinant and crude forms of HO-2 were similarly inhibited by the 3 drugs. The crude microsomal spleen form of HO-1 was more susceptible to inhibition than was the truncated recombinant form. This difference is attributed to the extra amino acids in the full-length enzyme. These observations may be relevant in the design of drugs as inhibitors of HO and other membrane proteins.

Induction of prostacyclin by steady laminar shear stress suppresses tumor necrosis factor-alpha biosynthesis via heme oxygenase-1 in human endothelial cells.

Cyclooxygenase (COX)-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas. We have addressed whether the induction of COX-2-dependent prostanoids in endothelial cells by LSS plays a role in restraining endothelial tumor necrosis factor (TNF)-alpha generation, a proatherogenic cytokine, through the induction of heme oxygenase-1 (HO)-1, an antioxidant enzyme. In human umbilical vein endothelial cells (HUVECs) exposed to steady LSS of 10 dyn/cm(2) for 6 hours, COX-2 protein was significantly induced, whereas COX-1 and the downstream synthases were not significantly modulated. This was associated with significant (P<0.05) increase of 6-keto-prostaglandin (PG)F(1alpha) (the hydrolysis product of prostacyclin), PGE(2), and PGD(2). In contrast, TNF-alpha released in the medium in 6 hours (3633+/-882 pg) or detected in cells lysates (1091+/-270 pg) was significantly (P<0.05) reduced versus static condition (9100+/-2158 and 2208+/-300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF-alpha generation and HO-1 induction were abrogated by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific prostacyclin receptor (IP) antagonist RO3244794 illuminates the central role played by LSS-induced COX-2-dependent prostacyclin in restraining endothelial inflammation. Carbacyclin, an agonist of IP, induced HO-1. Similarly to inhibition of prostacyclin biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC15 reversed TNF-alpha reduction by LSS. These findings suggest that inhibition of COX-2-dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking traditional nonsteroidal antiinflammatory drugs (NSAIDs) and NSAIDs selective for COX-2 through downregulation of HO-1, which halts TNF-alpha generation in human endothelial cells.

Inhibitors of the heme oxygenase - carbon monoxide system: on the doorstep of the clinic?

The past decade has seen substantial developments in our understanding of the physiology, pathology, and pharmacology of heme oxygenases (HO), to the point that investigators in the field are beginning to contemplate therapies based on administration of HO agonists or HO inhibitors. A significant amount of our current knowledge is based on the judicious application of metalloporphyrin inhibitors of HO, despite their limitations of selectivity. Recently, imidazole-based compounds have been identified as potent and more selective HO inhibitors. This 'next generation' of HO inhibitors offers a number of desirable characteristics, including isozyme selectivity, negligible effects on HO protein expression, and physicochemical properties favourable for in vivo distribution. Some of the applications of HO inhibitors that have been suggested are treatment of hyperbilirubinemia, neurodegenerative disorders, certain types of cancer, and bacterial and fungal infections. In this review, we address various approaches to altering HO activity with a focus on the potential applications of second-generation inhibitors of HO.

Inhibition of the enzymatic activity of heme oxygenases by azole-based antifungal drugs.

Ketoconazole (KTZ) and other azole antifungal agents are known to have a variety of actions beyond the inhibition of sterol synthesis in fungi. These drugs share structural features with a series of novel heme oxygenase (HO) inhibitors designed in our laboratory. Accordingly, we hypothesized that therapeutically used azole-based antifungal drugs are effective HO inhibitors. Using gas chromatography to quantify carbon monoxide formation in vitro and in vivo, we have shown that azole-containing antifungal drugs are potent HO inhibitors. Terconazole, sulconazole, and KTZ were the most potent drugs with IC(50) values of 0.41 +/- 0.01, 1.1 +/- 0.4, and 0.3 +/- 0.1 microM for rat spleen microsomal HO activity, respectively. Kinetic characterization revealed that KTZ was a noncompetitive HO inhibitor. In the presence of KTZ (2.5 and 10 microM), K(m) values for both rat spleen and brain microsomal HO were not altered; however, a significant decrease in the catalytic capacity (V(max)) was observed (P < 0.005). KTZ was also found to weakly inhibit nitric-oxide synthase with an IC(50) of 177 +/- 2 microM but had no effect on the enzymatic activity of NADPH cytochrome P450 reductase. Because these drugs were effective within the concentration range observed in humans, it is possible that inhibition of HO may play a role in some of the pharmacological actions of these antimycotic drugs.

Determination of in vivo carbon monoxide production in laboratory animals via exhaled air.

INTRODUCTION: In vitro assays play an important role in the understanding of the heme oxygenase (HO)/carbon monoxide (CO) pathway. However, because physiological roles for the products of this pathway are hypothesized, it is becoming increasingly important to perform in vivo studies. Since CO production is primarily mediated by HO and is excreted mainly by the lungs, measurements of total body CO excretion (VeCO) via the breath allow continuous, noninvasive monitoring of heme degradation and CO and bilirubin production. Here, we describe a modified flow-through method for the collection and quantitation of CO from small laboratory animals. METHODS: Mice and rats were studied in gas-tight chambers supplied with a continuous flow of CO-free air. CO in the exhaust air was measured by gas chromatography with a reduction gas analyzer. After establishing baseline VeCO levels, animals were administered various xenobiotics known to alter HO activity and further monitored for changes in CO production for up to 12 h without observable distress. RESULTS: Administration of heme (substrate for HO) resulted in reproducible increases in CO production; whereas, prior administration of zinc protoporphyrin (ZnPP, HO inhibitor) or cobalt protoporphyrin (CoPP, HO inducer) resulted in respective dose-dependent decreases and increases in the heme-induced CO production. DISCUSSION: We have demonstrated that this noninvasive method of CO quantitation reliably estimates heme degradation with sensitivity to distinguish between different types of HO-manipulating xenobiotics in a dose-dependant manner in both mouse and rat models. Furthermore, VeCO measurements allow nearly real-time determinations of CO and bilirubin formation, which helps to illustrate the time course of drug action.