A Testosterone Metabolite 19-Hydroxyandrostenedione Induces Neuroendocrine Trans-Differentiation of Prostate Cancer Cells via an Ectopic Olfactory Receptor.

Olfactory receptor OR51E2, also known as a Prostate Specific G-Protein Receptor, is highly expressed in prostate cancer but its function is not well understood. Through in silico and in vitro analyses, we identified 24 agonists and 1 antagonist for this receptor. We detected that agonist 19-hydroxyandrostenedione, a product of the aromatase reaction, is endogenously produced upon receptor activation. We characterized the effects of receptor activation on metabolism using a prostate cancer cell line and demonstrated decreased intracellular anabolic signals and cell viability, induction of cell cycle arrest, and increased expression of neuronal markers. Furthermore, upregulation of neuron-specific enolase by agonist treatment was abolished in OR51E2-KO cells. The results of our study suggest that OR51E2 activation results in neuroendocrine trans-differentiation. These findings reveal a new role for OR51E2 and establish this G-protein coupled receptor as a novel therapeutic target in the treatment of prostate cancer.

Exercise-Induced Alterations in Skeletal Muscle, Heart, Liver, and Serum Metabolome Identified by Non-Targeted Metabolomics Analysis.

BACKGROUND: The metabolic and physiologic responses to exercise are increasingly interesting, given that regular physical activity enhances antioxidant capacity, improves cardiac function, and protects against type 2 diabetes. The metabolic interactions between tissues and the heart illustrate a critical cross-talk we know little about. METHODS: To better understand the metabolic changes induced by exercise, we investigated skeletal muscle (plantaris, soleus), liver, serum, and heart from exercise trained (or sedentary control) animals in an established rat model of exercise-induced aerobic training via non-targeted GC-MS metabolomics. RESULTS: Exercise-induced alterations in metabolites varied across tissues, with the soleus and serum affected the least. The alterations in the plantaris muscle and liver were most alike, with two metabolites increased in each (citric acid/isocitric acid and linoleic acid). Exercise training additionally altered nine other metabolites in the plantaris (C13 hydrocarbon, inosine/adenosine, fructose-6-phosphate, glucose-6-phosphate, 2-aminoadipic acid, heptadecanoic acid, stearic acid, alpha-tocopherol, and oleic acid). In the serum, we identified significantly decreased alpha-tocopherol levels, paralleling the increases identified in plantaris muscle. Eleven unique metabolites were increased in the heart, which were not affected in the other compartments (malic acid, serine, aspartic acid, myoinositol, glutamine, gluconic acid-6-phosphate, glutamic acid, pyrophosphate, campesterol, phosphoric acid, creatinine). These findings complement prior studies using targeted metabolomics approaches to determine the metabolic changes in exercise-trained human skeletal muscle. Specifically, exercise trained vastus lateralus biopsies had significantly increased linoleic acid, oleic acid, and stearic acid compared to the inactive groups, which were significantly increased in plantaris muscle in the present study. CONCLUSIONS: While increases in alpha-tocopherol have not been identified in muscle after exercise to our knowledge, the benefits of vitamin E (alpha-tocopherol) supplementation in attenuating exercise-induced muscle damage has been studied extensively. Skeletal muscle, liver, and the heart have primarily different metabolic changes, with few similar alterations and rare complementary alterations (alpha-tocopherol), which may illustrate the complexity of understanding exercise at the organismal level.

Non-Targeted Metabolomics Analysis of the Effects of Tyrosine Kinase Inhibitors Sunitinib and Erlotinib on Heart, Muscle, Liver and Serum Metabolism In Vivo.

Background: More than 90 tyrosine kinases have been implicated in the pathogenesis of malignant transformation and tumor angiogenesis. Tyrosine kinase inhibitors (TKIs) have emerged as effective therapies in treating cancer by exploiting this kinase dependency. The TKI erlotinib targets the epidermal growth factor receptor (EGFR), whereas sunitinib targets primarily vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR).TKIs that impact the function of non-malignant cells and have on- and off-target toxicities, including cardiotoxicities. Cardiotoxicity is very rare in patients treated with erlotinib, but considerably more common after sunitinib treatment. We hypothesized that the deleterious effects of TKIs on the heart were related to their impact on cardiac metabolism. Methods: Female FVB/N mice (10/group) were treated with therapeutic doses of sunitinib (40 mg/kg), erlotinib (50 mg/kg), or vehicle daily for two weeks. Echocardiographic assessment of the heart in vivo was performed at baseline and on Day 14. Heart, skeletal muscle, liver and serum were flash frozen and prepped for non-targeted GC-MS metabolomics analysis. Results: Compared to vehicle-treated controls, sunitinib-treated mice had significant decreases in systolic function, whereas erlotinib-treated mice did not. Non-targeted metabolomics analysis of heart identified significant decreases in docosahexaenoic acid (DHA), arachidonic acid (AA)/ eicosapentaenoic acid (EPA), O-phosphocolamine, and 6-hydroxynicotinic acid after sunitinib treatment. DHA was significantly decreased in skeletal muscle (quadriceps femoris), while elevated cholesterol was identified in liver and elevated ethanolamine identified in serum. In contrast, erlotinib affected only one metabolite (spermidine significantly increased). Conclusions: Mice treated with sunitinib exhibited systolic dysfunction within two weeks, with significantly lower heart and skeletal muscle levels of long chain omega-3 fatty acids docosahexaenoic acid (DHA), arachidonic acid (AA)/eicosapentaenoic acid (EPA) and increased serum O-phosphocholine phospholipid. This is the first link between sunitinib-induced cardiotoxicity and depletion of the polyunsaturated fatty acids (PUFAs) and inflammatory mediators DHA and AA/EPA in the heart. These compounds have important roles in maintaining mitochondrial function, and their loss may contribute to cardiac dysfunction.

A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.

Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2µm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

Structure and Inhibition of Microbiome ß-Glucuronidases Essential to the Alleviation of Cancer Drug Toxicity.

The selective inhibition of bacterial ß-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative ß-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a ß-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the ß-glucuronidase active site. Finally, we establish that ß-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial ß-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.

Evaluation of drug loading, pharmacokinetic behavior, and toxicity of a cisplatin-containing hydrogel nanoparticle.

Cisplatin is a cytotoxic drug used as a first-line therapy for a wide variety of cancers. However, significant renal and neurological toxicities limit its clinical use. It has been documented that drug toxicities can be mitigated through nanoparticle formulation, while simultaneously increasing tumor accumulation through the enhanced permeation and retention effect. Circulation persistence is a key characteristic for exploiting this effect, and to that end we have developed long-circulating, PEGylated, polymeric hydrogels using the Particle Replication In Non-wetting Templates (PRINT®) platform and complexed cisplatin into the particles (PRINT-Platin). Sustained release was demonstrated, and drug loading correlated to surface PEG density. A PEG Mushroom conformation showed the best compromise between particle pharmacokinetic (PK) parameters and drug loading (16wt.%). While the PK profile of PEG Brush was superior, the loading was poor (2wt.%). Conversely, the drug loading in non-PEGylated particles was better (20wt.%), but the PK was not desirable. We also showed comparable cytotoxicity to cisplatin in several cancer cell lines (non-small cell lung, A549; ovarian, SKOV-3; breast, MDA-MB-468) and a higher MTD in mice (10mg/kg versus 5mg/kg). The pharmacokinetic profiles of drug in plasma, tumor, and kidney indicate improved exposure in the blood and tumor accumulation, with concurrent renal protection, when cisplatin was formulated in a nanoparticle. PK parameters were markedly improved: a 16.4-times higher area-under-the-curve (AUC), a reduction in clearance (CL) by a factor of 11.2, and a 4.20-times increase in the volume of distribution (Vd). Additionally, non-small cell lung and ovarian tumor AUC was at least twice that of cisplatin in both models. These findings suggest the potential for PRINT-Platin to improve efficacy and reduce toxicity compared to current cisplatin therapies.