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Inhibition of CYP2S1 Mediated Metabolism of Anticancer Prodrug AQ4N by Liarozole

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Inhibition of CYP2S1 Mediated Metabolism of Anticancer Prodrug AQ4N by Liarozole Journal of Applied Pharmaceutical Science Vol. 7 (02), pp. 001-007, February, 2017 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2017.70201 ISSN 2231-3354 Inhibition of CYP2S1 mediated metabolism of anticancer prodrug AQ4N by liarozole Naveen K. M. Singh1, Shawn R. White2, Sudhakar Kalagara3, Samuel Kadavakollu1* 1Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM, USA. 2Department of Chemistry, Western New Mexico University, Silver City, NM, USA. 3Department of Chemistry, University of Texas at El Paso, TX, USA. ABSTRACT ARTICLE INFO Article history: CYP2S1 is an orphan Cytochrome P450 whose expression is elevated in numerous epithelial derived cancers; Received on: 21/04/2016 however, little is known about CYP2S1 inhibitors. Our results indicate that liarozole acts as an effective inhibitor Accepted on: 30/06/2016 of CYP2S1, and decreases the turnover of the prodrugAQ4N to its active metabolites AQ4M and AQ4. Our Available online: 27/02/2017 studies also found that the active drug AQ4 is found only in the perinuclear location, whereas the prodrug AQ4N is located in the cytoplasm. In addition, we showed that AQ4N is non-geno/cytotoxic whereas the bioreduced Key words: metabolite AQ4 is both genotoxic and cytotoxic. Finally, we observed that CYP2S1 metabolizes AQ4N under CYP2S1, Toxicity, Inhibition anoxic conditions and that increasing the CYP2S1 concentration leads to more AQ4N turnover. The important Activity, AQ4N, AQ4, enzymatic role of CYPS21 with regard to the metabolism of the anticancer prodrug AQ4N into AQ4 was Banoxantrone and Liarozole. confirmed. INTRODUCTION identified endogenous and exogenous substrates of CYP2S1. However, very few studies have examined the role of inhibitors on Cytochrome P450 (CYPs) constitute a large CYP2S1 mediated metabolism of substrates like AQ4N. We superfamily of enzymes that are haem-thiolate proteins with therefore examined the role of liarozole, a known inhibitor of CYP ubiquitous expression and distribution in diverse life forms enzymes in CYP2S1 mediated metabolism of AQ4N. including bacteria, fungi, plants and animals (Nelson et al., Banoxantrone (AQ4N) is an anticancer prodrug that requires both 1999). CYPs play an important role in carcinogen and anti- a hypoxic environment and metabolic activation to generate the cancer drug biotransformation (Tang et al., 2015). Within cells, ultimate toxicant, a topoisomerase II inhibitor, AQ4. Heterologous CYPs are localized to the endoplasmic reticulum (ER) and CYP2S1 expression studies have revealed that AQ4N is a substrate mitochondria (Szczesna-Skorupa et al., 2008). In total, 58 human for CYP2S1 (Nishida et al., 2010), and that CYP2S1 metabolizes cytochrome P450s (CYPs) have been identified to date. Most non-cytotoxic AQ4N to AQ4M and cytotoxic AQ4. While there CYPs have been well characterized, and biological substrates have been numerous studies conducted to identify the substrates of have been identified for nearly 75% of all known CYPs. CYP2S1, there is very little information on its inhibitors. Liarozole However there remains 25% of CYPs called “orphan P450s” is an imidazole-containing compound that inhibits the cytochrome which little physiological function has clearly been identified. P-450-dependent metabolism of ATRA in hamster liver CYP2S1 is a gene coding for the Cytochrome P450 2S1 protein, microsomes (Van Wauwe et al., 1992). Previous studies have which has been shown to metabolize AQ4N, an anticancer shown that liarozole inhibits human epidermal retinoic acid 4- prodrug, under anoxic conditions (Xiao et al., 2011, Nishida et hydroxylase activities (Kang et al., 1996). Liarozole acts as a al., 2010). The epoxygenase activity of CYP2S1 is inhibited in blocker of retinoic acid metabolism (retinoic acid metabolism vitro by miconazole and liarozole, an inhibitor of CYP26 blocking agent, RAMBA) and has been shown to inhibit and CYP2S1 (Fromel et al., 2013). Several ongoing studies have CYP26A1 (Nelson et al., 2013). It is a known Cytochrome P450 inhibitor, which inhibits several cytochrome P450 enzymes * Corresponding Author including aromatase (CYP19), a key enzyme in the biosynthesis of Samuel Kadavakollu, Department of Chemistry, Western New Mexico estrogens and retinoic acid 4-hydroxylase (CYP26S), University, Silver City, NM, USA. Email:[email protected] responsible for ATRA metabolism (PavezLorie et al., 2009). © 2017 Naveen K.M. Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial- ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). 002 Singh et al. / Journal of Applied Pharmaceutical Science 7 (02); 2017: 001-007 Liarozole also displays antitumor activity against electrophoresis solution, and washed by immersing twice in androgen-dependent and independent rat prostate carcinomas neutralization buffer for 5 min each and in 70% ethanol for 5 min (Smets et al., 1995). A hypoxic environment is exhibited by solid followed by air-drying at room temperature for 10-15 min. 100 µl tumors due to poor vascularization resulting from the rapid growth of diluted SYBR® Gold was placed onto each circle of dried of tumors. Tirapazamine (Brown et al., 1999), mitomycin (Tomasz agarose and stained for 30 min at room temperature in the dark. 1995), NLCQ-1 (Papadopoulou and Bloomer 2003), KS119 (Seow The slides were allowed to dry completely at 37°C and analyzed et al., 2005) and AQ4N (Patterson et al., 2002, Lalani et al., 2007) using View slides by epifluorescence microscopy using Nikon are a few anticancer agents that are selectively activated in an e600 microscope using fluorescein filter with excitation/emission hypoxic environment (Nishida et al., 2010). AQ4 itself is not at 496 nm/522 nm. The relative DNA damage (Genotoxicity) was suitable as an anticancer drug because it is highly toxic to normal analyzed using olive tail moment, a product of tail length and the cells (Nishida et al., 2010) whereas AQ4N has negligible fraction of total DNA in the tail. Prism statistical software topoisomerase inhibitory activity (Patterson et al., 1994, Smith et (Graphpad Prism 5.0) was used to graph the results obtained. al., 1997). When AQ4Nreaches hypoxic tumor cells the drug is selectively converted into its active cytotoxic form AQ4. The Cell Cytotoxicity Assays active form of the drug AQ4 has been shown to be a potent The cell cytotoxicity assays were performed using ® topoisomerase II inhibitor and DNA intercalator (Patterson et al., AlamarBlue cell viability reagent (Life Technologies, Cat. No.: 2000). This work focuses on the role of the liarozole inhibitor on DAL1100) according to the manufacturer’s protocol. BEAS-2B CYP2S1 and its subsequent effect on metabolism of AQ4N. cells were plated at 2,000 to 4,000 cells per well, in triplicates, of at least four per treatment or genotype, in a 96 well plate. The cell MATERIALS AND METHODS numbers for plating were calculated using the hemocytometer after trypsinizing BEAS2B cells in a T-75 flask grown to 90% Cell Lines and Reagents confluency. The cells were allowed to grow overnight at 5% CO2 A549 and BEAS2B were obtained from ATCC. A549s at 37°C. were grown in DMEM (Invitrogen, Cat. No. 31053-036) with 10% The following day media with three different FBS media (Invitrogen, Cat. No. 16000-044) and BEAS2B were concentrations (1, 10 or 100 μM) of the drug (AQ4N, AQ4 or grown in LHC9 media (Invitrogen, Cat. No.12680-013). The cell Mitoxantrone) was added to three different plates each lines were kept in 37° C incubator with 5% CO2 conditions. The representing one of the drug treatments. Drug preparations were media was changed every other day until the cells were 80 – 90% done in DMSO as described elsewhere in experimental confluent, and then the cells were split using Trypsin 0.25% procedures. For negative controls, DMSO and untreated media EDTA (Invitrogen, Cat. No. 25200-056) for A549s and TrpLE for were used.For the positive control, fully reduced alamarBlue® Beas2Bs (Invitrogen, Cat. No. 12604-021). Phosphate buffer prepared by autoclaving media with 10% alamarBlue® was used. Saline (PBS) was used to wash the cells (Invitrogen, Cat. No. Finally, media without cells was used as a blank to establish the 10010-023). background. The 96 well plates were now placed in a hypoxic chamber for 24 hrs at 1% O2, 5% CO2 / 37°C, and control samples Cell Genotoxicity Assays were placed in a 5% CO2 incubator at 37°C. After 24 hr, Genotoxicity (DNA damage) in BEAS2B was evaluated alamarBlue® was added to a final concentration of 10%, after using Trevigen Comet AssayTMkit (Trevigen Inc., Gaithersburg, which the cells were incubated for four more hours at previously MD, Catalog No. # 4250-050-K) according to the manufacturers described conditions. protocol. BEAS2B was plated in six well plates at a density of The fluorescence measurements of the plates were ~1.2 X 106 cells/mlfor treatment with AQ4N, AQ4 or recorded with the following parameters: excitation wavelength at Mitoxantrone. Three different concentrations of drug 1, 10 and 530-560nm and emission wavelength at 590 nm. Percentage 100 μM, were used. BEAS2B adherent cells were trypsinized and reduction of alamarBlue® was calculated and these values were cells were resuspended in ice cold PBS to a concentration of 1 X used to compare cells. All data points were normalized to blank 5 10 cells/ml. The cells were combined with low melting agarose (at media wells (alamarBlue® background fluorescence levels). The 42°C) at a ratio of 1:10 (v/v) and immediately pipetted (50 µl) experiments were conducted at least three times under identical onto CometSlide™. The slides were incubated in the dark at 4 °C conditions. for 10 min and them immersed in 4°C Lysis Solution for 60 min. Fluorescence measurements were analyzed using Prism The slides were then transferred to a beaker with Alkaline statistical software (Graphpad Prism 5.0), analyzing linear Unwinding Solution for 20 min at room temperature or 1 hr at cytotoxicity levels at different concentrations of the drug.
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