Bioinformatics and Molecular Evidences

Arom & at al ic in P l ic a n d Huang et al., Med Aromat Plants 2015, 4:5 t e s M Medicinal & Aromatic Plants DOI: 10.4172/2167-0412.1000219 ISSN: 2167-0412 ResearchResearch Article Article OpenOpen Access Access 3β-Methoxy Derivation of Withaferin-a Attenuates its Anticancer Potency: Bioinformatics and Molecular Evidences Huang C1,2, Vaishnavi K3, Kalra RS1, Zhang Z2, Sekar K3*, Kaul SC1* and Wadhwa R1* 1Drug Discovery and Assets Innovation Lab, DBT-AIST International Laboratory for Advanced Biomedicine (DAILAB), National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan 2Graduate School of Life & Environmental Sciences, University of Tsukuba, Japan 3Indian Institute of Science (IISc), Bangalore, India Abstract Withaferin A (Wi-A), a potent anticancer withanolide extracted from Withania somnifera- a tropical medicinal plant, was earlier shown to activate p53 tumor suppressor and oxidative stress pathways in cancer cells causing either growth arrest or apoptosis. In view of the information that alkyl derivatives of several bioactive steroidal compounds exhibit improved stability and activity through mechanisms that have not been completely understood, we investigated the effect of Withaferin A and its methoxy-derivative (2,3-dihydro-3- methoxy-Withaferin A) on human cancer cells to analyze the molecular interactions and efficacy. Systematic bioinformatics and molecular experimental approaches comprising expression analysis and imaging techniques were employed to demonstrate the interactions of Withaferin A and 3β-methoxy Withaferin A with their molecular targets. We demonstrate that 3β-methoxy derivation of Withaferin A possess weaker docking potential to its molecular targets and therefore possess attenuated anticancer activity as compared to Withaferin A, and was affirmed by a number of biochemical and in vitro experiments. Based on the Bioinformatics and experimental data we report that the 3β-methoxy derivation of Withaferin A attenuates its anticancer activities, and hence sufficient care is warranted in the use of these phytochemicals as anticancer reagents. Keywords: Withania somnifera; Withaferin A; Methoxy-Withaferin Materials and Methods A; p53; Apoptosis Molecular docking studies Abbreviations Protein preparation: The three-dimensional atomic coordinates of Wi-A: Withaferin A; mWi-A: 3β-methoxy Withaferin A; DMSO: mortalin, p53, NRF-2 and p21WAF1 proteins (PDB-ID: 3N8E, 3D09, Dimethyl sulfoxide 2FLU and 1AXC) were downloaded from its archives the Protein Data Bank (PDB). All the crystallographic water molecules were removed Introduction from the structures, and hydrogen atoms were added using the Withaferin A (Wi-A) was the first member of the withanolide family “Hydrogen” module and correct ionization and tautomeric states were obtained. Further, Kollman united atom partial charges and salvation to be isolated from the roots of Ashwagandha (Withania somnifera). parameters were assigned. At the end of the protein preparation Several others including Withanolide A, Withanolide B, Withanolide process, a PDBQT file was obtained (for each protein molecule) which D, Withanone and their derivatives [1-4] have subsequently been was essential for the execution of AutoGrid and AutoDock. identified and shown to possess bioactivities in animal as well as cell culture experimental models [5]. The systemic clinical use of this herb Ligand preparation: The three-dimensional coordinates of the molecule Withaferin A (Wi-A) was used as a template to model has not yet been possible due to the lack of identification of (i) active methoxy-Withaferin A (mWi-A). The energy was minimized using constituents and their biological activities, (ii) cellular targets, (iii) GROMOS 96.1 force field through PRODRG server with default bioavailability and efficacy profiles of its constituent phytochemicals parameters. and (iv) pharmokinetics. Amongst various studies describing activities of Wi-A using animal and cell culture models, the anticancer activity Ligand docking: AutoDock 4.2 suite (an automated tool for the prediction of protein-ligand interactions) was used. The Lamarckian is most supported, involving a number of pathways, identified over the Genetic Algorithm was used with a population of 250 dockings. As years [6-12]. Furthermore, treatment with Wi-A has been suggested as a radiosensitizer in cancer treatment [13,14]. Comparison of the cytotoxicity and cytoprotective heat shock-inducing activity of Wi-A *Corresponding author: Kaul SC, Wadhwa R, National Institute of Advanced and its 36 derivatives revealed that the enone ring is an essential Industrial Science & Technology, Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki component for this activity. Chemical modifications, such as, 305-8562, Japan, Tel: 81-29-861-6713; Fax: 81-29-861-2900; E-mail: [email protected]; [email protected] acetylation or hydroxylation affected both activities suggesting that the understanding of the structure-function association may inspire K. Sekar, Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560012, India, Tel: 91-80-22933059; Fax: 91-80-23600085; development of new drugs [4]. Specifically, alkylated (e.g., methyl E-mail: [email protected] or ethyl) secondary metabolites have been shown to acquire greater Received November 17, 2015; Accepted November 23, 2015; Published bioactivity, metabolic stability and chemopreventive potential [15,16]. November 27, 2015 Taking this into account, we investigated the molecular interactions Citation: Huang C, Vaishnavi K, Kalra RS, Zhang Z, Sekar K, et al. (2015) 3β-Methoxy and potency of Withaferin A and its methoxy-derivative (2,3-dihydro- Derivation of Withaferin-a Attenuates its Anticancer Potency: Bioinformatics and Molecular Evidences. Med Aromat Plants 4: 219. doi:10.4172/2167-0412.1000219 3-methoxy-Withaferin A). In line with reported anticancer potential of Withaferin A identified in several studies [11,17,18], we investigated Copyright: © 2015 Huang C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted the efficacy of Wi-A and mWi-A as anticancer drugs by bioinformatics use, distribution, and reproduction in any medium, provided the original author and and biochemical experimental approaches. source are credited. Med Aromat Plants ISSN: 2167-0412 MAP, an open access journal Volume 4 • Issue 5 • 1000219 Citation: Huang C, Vaishnavi K, Kalra RS, Zhang Z, Sekar K, et al. (2015) 3β-Methoxy Derivation of Withaferin-a Attenuates its Anticancer Potency: Bioinformatics and Molecular Evidences. Med Aromat Plants 4: 219. doi:10.4172/2167-0412.1000219 Page 2 of 8 the three-dimensional structures were native (not complexed with Reverse-transcription PCR (RT-PCR): Total RNA was extracted any small molecule) an appropriate grid box was generated to cover with QIAGEN RNAeasy kit. Complementary DNA (cDNA) was the entire protein molecule and docking was performed. Based on the synthesized from 2 µg of RNA using the Thermoscript Reverse results, another grid box was generated in order to cover the possible Transcriptase (QIAGEN, Germany) following the manufacturer’s ligand-binding site and the final docking calculations were performed. protocol. The synthesized cDNA was subjected to PCR amplification Similar conformations were clustered based on the root mean square consisting of an initial 10 min denaturation step at 95°C followed by 30 deviations (RMSD’s) and orientations. cycles at 95°C for 45 s, 56°C for 1 min and 72°C for 45 s, with final annealing step at 72°C for 10 min. PCR amplifications were performed using specific Biochemical and visual assays primers for (i) p53-Sense: 5′-CTGCCCTCAACAAGATGTTTTG-3′& Cell culture and cytotoxicity assays: Human bone cancer (U2OS) Antisense: 5′-CTATCTGAGCAGCGCTCATGG-3′, (ii) cells were maintained in Dulbecco’s Modified Eagle’s Medium p21WAF1: Sense: 5′-ATGAAATTCACCCCCTTTCC-3′& (DMEM; Invitrogen, Carlsbad, CA)-supplemented with 10% fetal Antisense: 5′-CCCTAGGCTGTGCTCACTTC-3′, (iii) bovine serum in a humidified incubator (37°C and 5% CO2). For CARF: Sense: 5′-CAGCCAAAGCAGCAGCAGCG-3′ & cytotoxicity assays, cells (5000/well in three independent replicates) Antisense: 5′-AGCCCAACAAGGGCACCTCG-3′, (iv) were plated in 96-well plate and were treated with indicated doses NRF2: Sense: TACTCCCAGGTTGCCCACA-3′ & Antisense: of either Withaferin A (Wi-A) or methoxy-Withaferin A (mWi-A). 5′-CATCTACAAACGGGAATGTCTGC-3′, (v) GAPDH: Viability of control and treated cells was determined using tetrazolium Sense: 5’-ACCTGACCTGCCGTCTAGAA-3’ & Antisense: dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 5’-TCCACCACCCTGTTGCTGTA-3’. The PCR products were bromide (MTT reagent, Roche), incubated for 4 h. In order to compare then run on a 1% agarose gel and stained with ethidium bromide for the long term cell proliferation and colony-forming efficiency of visualization. control and treated cells, cells (500/well in triplicates) were plated in Cell cycle analysis: Assessment of different phases of cell cycle was 6-well plates and allowed to grow for 10-15 days under conditions of performed through PI cell cycle staining. Cells were harvested (Trypsin- control or treated (as indicated). Medium was replaced every 3-4 days. EDTA) and fixed in 70% chilled ethanol for 15-30 min at 4°C. Cell Experiment was terminated by fixation of the cells in ice-cold methanol pellets was suspended in PBS and incubated with 50 μl Ribonuclease

Load more