Bioinformatics and Molecular Evidences

rom & A at al ic in P l ic a n d Huang et al., Med Aromat Plants 2015, 4:5

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 A and acetone (1:1) solution for 10 min. Colonies were stained with 0.1% (5 mg/ml; Qiagen) for 30 min at 37°C to degrade cellular RNA content. crystal violet. Plates were air-dried and scanned for record. Colonies Cells were stained with 250 μl Guava cell cycle reagent (50 μg/ml) were counted and statistical analysis of the colony forming efficiency in for 1 h in dark and analyzed on the Guava cell cycle analyzer (Merk, control and treated cultures was determined as described below. Millipore). The data were analyzed by ModFit software to evaluate the Immuno-blotting: Cell pellets were solubilized in RIPA buffer distribution of cells in different phases of cell cycle. (Thermo Scientific, Waltham, MA). Supernatant containing 15-20 µg Statistical analysis of proteins was resolved in SDS-polyacrylamide gel, and was electro- blotted onto PVDF membranes (Millipore, Billerica, MA) using a All experiments were carried out in triplicate. Data values were semidry transfer unit (ATTO, Tokyo, Japan). Immuno-blotting was expressed as mean ± SEM of three individual experiment sets. Statistical performed with anti-p53, -Bcl-2, -PARP, -NRF-2, -Caspase-9 (Santa analyses were carried out using Student’s t-test or nonparametric Mann- Cruz, CA); p21 (Cell Signaling) and β-actin (Abcam, Cambridge) Whitney U-test; whichever was applicable. Statistical significance was antibodies. Anti-CARF and- mortalin antibodies were generated in defined as p-value ≤ 0.05. our laboratory. The membranes probed with the first antibodies were excessively washed with TBS-T (Tris buffer saline-Tween 20) and Results incubated with secondary horseradish peroxidase (HRP)-conjugated Docking of methoxy-withaferin a with mortalin, p53, goat anti-mouse or anti-rabbit (Santa Cruz) antibodies. Protein p21WAF1 and NRF-2 bands were detected using ECL prime substrate (GE Healthcare, CA). Densito-metric quantitation of three independent immuno-blotting Withanolides, Withaferin A (Wi-A) and Withanone (Wi-N) experiments was performed with the ImageJ software (National from Ashwagandha have been shown to possess anticancer activity Institute of Health). Expression level of each of the proteins in control [12,17-19]. Whereas Wi-N killed cancer cells selectively, Wi-A was and treated cells was calculated with respect to the β-actin (loading found to have cytotoxicity to normal cells also [6,19,20]. Studies on the control). All the experiments were performed in triplicate. molecular mechanism of their action have revealed that these evoke oxidative stress and DNA damage response in cancer cells, and trigger Immuno-staining: Cells (4 × 104/well) were plated on the glass either growth arrest or apoptosis in a dose dependent manner. Loss-of- coverslips placed in a 12-well plate. After the cells had attached well function screenings earlier revealed the involvement of mortalin, p53, to the surface they were treated with indicated doses of either Wi-A or p21, TPX-2, aurora, vimentin and NRF-2 proteins in Wi-A and Wi-N mWi-A for 24 h and then fixed with either pre-chilled methanol: acetone mediated cancer cell killing [6,19,20]. (1:1) or 4% formaldehyde for 10 min on ice or at room temperature, respectively. Fixed cells were permeabilized with PBS-Triton-X-100 Mortalin is a mitochondrial chaperone that has been shown to (0.1%) for 10 min followed by blocking with 2% BSA for 10 min. The be frequently up regulated in many cancers and is known to promote coverslips were incubated with antibodies against p53, NRF-2 (Santa carcinogenesis by inactivation of the tumor suppressor protein p53 Cruz), p21WAF1 (Cell Signaling Technology) CARF, mortalin at and deregulation of apoptosis. Activation of p53 by abrogation of room temperature (1 h) or 4oC (overnight). Cells were then probed its complex with mortalin has been shown to cause growth arrest of with Alexa Fluor-conjugated secondary antibodies (Molecular Probes, cancer cells in several studies [21-23]. We have earlier shown that Invitrogen), and counterstained with Hoechst 33258 (Sigma-Aldrich). Wi-N and Wi-A can interfere with the binding of mortalin to p53. The stained cells were viewed under Zeiss Axioplan 2 microscope and In the present study, the modelled three-dimensional structure of images were captured using a Zeiss AxioCam HRc camera. 3β-methoxy-Withaferin-A (mWi-A) was docked with the substrate-

Page 3 of 8 binding domain of mortalin (PDB-id: 3N8E) (Figure 1). As shown in similar to Wi-A, it was predicted to disrupt the mortalin-p53 complex. Table 1, compared to Wi-A, mWi-A was less efficient both in terms of Based on these analyses, it could be perceived that mWi-A may affect binding energy and number of hydrogen bonding interactions. Wi-A mortalin-p53 complex causing its activation and upregulation of and mWi-A exhibited a binding energy of -9.8 and -7.47 Kcal/mol, p21WAF1 protein, a crucial downstream effector of p53 involved in respectively. Of note, both withanolides (Figure 1A) interacted with growth arrest and initiation of S phase of the cell cycle. the residue 513 (Arg) of mortalin (Figure 1B). The latter constitute the “latch region” of mortalin and functions as a “latch” between the We, thus, also investigated the docking efficacy of Wi-A and “lid and cleft regions” that are important for its chaperoning activity mWi-A to p21WAF1. As shown in Figure 2 and Table 1, both Wi-A and (Figure 1C). Thus, it was predicted that mWi-A, although weaker than mWi-A showed interaction to the same sites of p21WAF1 (Figure 2A- Wi-A, might cause an antagonistic effect on the activity of the protein. 2B). However, significant difference in the binding energies of Wi-A We next examined if it would also bind to p53. The modelled structure (7.84 Kcal/mol) and mWi-A (-6.58 Kcal/mol) was observed (Table of mWi-A was docked with the three-dimensional structure of p53 1). Docking analysis of Wi-A and mWi-A to the three-dimensional (PDB-id: 3D09). The results (Figure 1D-E and Table 1) suggested that structures of NRF-2 (PDB-id: 2FLU), an important regulator of mWi-A is less efficient (binding energy: -6.74 Kcal/mol) as compared cellular oxidative stress response revealed that they dock to the same to Wi-A (binding energy: -10.18 Kcal/mol). Similar to Wi-A, it was sites (Figure 2C-2D and Table 1) and similar to the other targets (as predicted to interact with Leu 111 and Arg 282 (Figure 1E), the residues described above) mWi-A showed weaker interaction (binding energy- known to be essential for the stability of the p53 protein. Furthermore, 4.60 Kcal/mol) than Wi-A (binding energy -7.87 Kcal/mol). Taken

Figure 1: Hydrogen bonding interactions between Wi-A and mWi-A with mortalin (PDB-ID: 3N8E) and p53 (PDB-ID: 3D09). (A) Structure of Wi-A (top) and mWi-A (bottom). (B) Hydrogen-bond (H-H) interactions between Wi-A (top) and mWi-A (bottom) with mortalin (PDB-ID: 3N8E). (C) H-H interactions between Wi-A (top) and mWi-A (bottom) with three-dimensional (3D) structure of mortalin (shown in pink). (D-E) Interactions between Wi-A (D) and mWi-A (E) with p53 (PDB-ID: 3D09), wherein H-H (left) and the 3D docked structures (right) are shown.

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Binding in both experiments (Figure 3A and 3B), in Wi-A treated cells only. In Hydrogen bond Distance Receptor Ligand energy interactions (Å) order to delineate the mechanism of upregulation, we also performed Kcal/mol RT-PCR analysis and found a low, but significant, upregulation of both Glu (483) O2-H...O 3.20 p53 and p21WAF1 levels in only Wi-A treated cells (Supp. Figure 1). Arg (513) NH1-H…O4 2.97 Arg (513) NH1-H…O6 3.13 Wi-A -9.80 Based on the above data, we investigated if the nuclear enrichment Arg (513) NH2-H…O6 2.88 Mortalin Gly (587) N-H…O6 2.84 and activation of p53 was a result of disruption of mortalin-p53 Asn (583) O6-H…O 2.61 complexes in cell cytoplasm due to strong docking of Wi-A in these (PDB-ID: 3N8E) Arg(513) NH1-H...O11 1.9 proteins as predicted by bioinformatics analysis described above. Arg(513) NH2-H...O11 1.9 mWi-A -7.47 Immuno-blotting and immuno-staining of mortalin in Wi-A treated Leu (450) N-H...O4 2.6 Gln(479) NE2-H...O6 2.2 and control cells showed decrease in mortalin and shift in its staining Arg (282) NH1-H…O6 pattern from the perinuclear to pancytoplasmic region (Figure 4A 3.23 Tyr (126) O6-H…O and 4B; data not shown). Of note, mWi-A cells showed no difference. 2.77 Tyr (126) N-H…O6 2.92 Consistent with this, increase in nuclear p53 was observed in Wi-A, Leu (111) O-H…O1 Wi-A 3.13 -10.18 but not in mWi-A, treated cells (Supp. Figure 2). These data endorsed Leu (111) N-H…O1 3.29 p53 Asn (268) ND2 -H… the above findings that docking of Wi-A to mortalin and p53 proteins (A) 3.08 O2 disrupted their interactions, causing nuclear translocation and 2.98 (PDB-ID: 3D09) Asn (268) O2-H…OD1 (A) activation of p53 as determined by upregulation of p21WAF1 protein. Leu(111) O8-H...O 2.2 Importantly, mWi-A is found incapable to induce such activation of His(115) ND1-H...O50 2.1 mWi-A -6.74 Arg(282) NH1-H...O5 2.1 p53-p21 pathway. Arg(282) NH2-H...O6 1.7 Induction of oxidative stress and DNA damage by Wi-A and Met (147) O2-H…O 2.44 Tyr (151) N-H…O2 3.37 mWi-A His (152) O-H…O1 2.67 Ile (158) N-H…O6 3.40 NRF-2 is an important regulator of cellular oxidative stress Wi-A -7.84 Arg (156) O6-H…O 2.81 p21WAF1 response and shown to induce antioxidant response in cells. NRF-2 Arg (156) O-H…O4 2.79 is upregulated in many cancers and provides an advantage of survival Arg (156) N-H…O5 3.07 (PDB-ID: 1AXC) Arg (155) NH1-H…O4 2.71 against oxidative stress. We earlier showed that NRF-2 is an important Arg (155) N-H...O74 2.00 target of Wi-A [25]. The level of NRF-2 was examined in mWi-A Arg (156) NE-H...O4 2.10 mWi-A -6.58 treated and control cells. Whereas no change, either in expression Leu (157) O8-H...O 1.80 level (Figure 4A) or staining intensity (Supp. Figure 3) was observed Phe (159) N-H...O8 2.30 in mWi-A treated cells, high doses of Wi-A (0.6 to 3.0 µg/ml) lead to Ala (69) N-H…O2 2.74 Phe (71) N-H…O2 3.44 decrease in NRF-2 expression, also observed at the transcript level Wi-A Ala (72) N-H…O3 2.52 -7.87 (Supp. Figure 4). NRF-2 Gln (75) OE1-H…O4 3.51 Leu (76) O6-H…O 2.87 Consistent with these data, we found that the cells treated with (PDB-ID: 2FLU) Ala (69) N-H...O11 1.8 Wi-A showed increase in ROS level; mWi-A treated cells remained Ala (69) N-H...O8 2.3 mWi-A -4.60 unaffected (Figure 4C). DNA damage response has been implicated as a Phe (70) N-H...O8 1.9 Phe (71) N-H...O11 1.8 predominant mechanism of action of several anticancer drugs. We have earlier shown that CARF (Collaborator of ARF) is involved in DNA Table 1: Details of molecular interaction of Wi-A and mWi-A with their targets proteins. damage response and checkpoint signaling pathways [26]. Increase in CARF expression was seen to correlate with the increased expression together, these predictions envisaged that in contrast to Wi-A, mWi-A of DNA damage response and checkpoint proteins. We examined the exhibit weaker interactions with the target proteins and hence may level of CARF in control, Wi-A and mWi-A treated cells. Consistent possess weak potency, and low efficacy as an anticancer drug. with the anticancer potency of Wi-A, we found that it resulted in Effect of Wi-A and mWi-A on p53-p21WAF1 pathway moderate, but significant, induction of CARF both at the protein and mRNA levels (Figure 5A and 5B). Immunostaining of CARF revealed In order to validate the above bioinformatics predictions, we its upregulation in Wi-A treated cells, while its expression remain examined the activation of p53 in control and treated human bone unchanged in mWi-A treated cells (Figure 5C). Therefore, mWi-A cancer (U2OS; possess wild type p53 protein) cells. Induction of p53 treated cells did not show such changes correlating with its low efficacy was observed in cells treated with Wi-A (Figure 3A), at a dose as or lack of anticancer activity. Consistent with the above finding, levels low as 0.6 µg/ml. Whereas, mWi-A treated cells showed no change of Gadd45a and γH2AX were also found upregulated in Wi-A treated in the expression level of p53 even with five fold higher doses (3 µg/ cells only (data not shown). ml) (Figure 3A). Immuno-cytochemical analysis of p53 in control and Wi-A treated cells revealed strong nuclear p53 staining in majority of Anticancer efficacy of Wi-A and mWi-A the cells with doses 0.6 µg/ml and above (Figure 3B). In view of several We further examined the cytotoxicity of Wi-A and mWi-A by cell other studies [24,25], the present data suggested the (i) transcriptional viability and colony forming assays. Wi-A exhibited strong cytotoxicity activation function of p53 and (ii) abrogation of its complex with (Figure 6A and Supp. Figure 5). In above observation, it was found mortalin in the cell cytoplasm. Of note, mWi-A treated cells showed no that the cells undergo growth arrest at low dose, but apoptosis at high change in the staining intensity or localization of p53 (Figure 3B). We dose of Wi-A, respectively. By contract, equivalent doses of mWi-A next examined the activation of p53 function by analysing the level of remained ineffective both in short-term viability and long-term p21WAF1. As anticipated, p21WAF1 exhibited upregulation, affirmed clonogenic assays (Figure 6B, Supp. Figures 6 and 7). mWi-A treated

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Figure 2: Hydrogen bond interactions between Wi-A and mWi-A with p21WAF1 (PDB-ID: 1AXC) and NRF2 (PDB-ID: 2FLU). (A-B) H-H interactions between Wi-A (A) and mWi-A (B) with p21WAF1 (PDB-ID: 1AXC). (C-D) Interactions between Wi-A (A) and mWi-A (B) with Nrf-2 (PDB-ID: 2FLU). Images showing H-H (left) and the 3D docked structures (right), whereas Wi-A (strong binding; pink) and mWi-A (weak binding; black) are marked in 3D structures.

Figure 3: Methoxy-withaferin (mWi-A) and activation of p53/p21 pathway. (A) Immunoblotting showing p53 and p21 protein levels in Wi-A and mWi-A treated cells. Quantitation of their expressions is shown below. (B) Immunostaining showing p53 and p21WAF1 expression in the above treated cells.

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Figure 4: Methoxy-withaferin (mWi-A) and activation of p53 and oxidative stress. (A) Immunoblotting with mortalin and NRF-2 proteins in control, Wi-A and mWi-A treated cells; quantitation of their expression level is shown below. (B-C) Immunostaining with mortalin in these cells and elevated ROS levels in Wi-A treated cells, but not in mWi-A.

Figure 5: Methoxy-withaferin (mWi-A) and DNA damage response. (A) Protein expressions of CARF (above) and its quantitation (below), (B) Reverse- transcriptase PCR affirmed CARF transcript levels (above) and its quantitation (below), while (C) showing CARF immunostaining in Wi-A and mWi-A cells.

Page 7 of 8 cells exhibited normal morphology (Figure 6A) and showed negligible cell cycle profiles showed increased G0/G1 and arrested S phase in Wi-A effect on the colony forming efficacy at doses as high as 3 µg/ml (Figure treated cells affirmed efficacy of Wi-A, and not of mWi-A. Activation 6B). Cell cycle analysis also revealed G1 and G2 arrest in Wi-A, and not of ROS and CARF expression further demonstrated induced oxidative mWi-A, treated cells at dose of 0.3 µg/ml (Figure 6C). Whereas high stress and DNA damage response pathway in Wi-A treated cells, doses (1.5 and 3.0 µg/ml) of Wi-A, and not mWi-A, caused apoptosis, though mWi-A failed in activating above response. In conjunction as also supported by expression analysis of control and treated cells. with the bioinformatics analyses, our study showed that small chemical Consistent with their phenotypes, down-regulation of anti-apoptotic proteins, Bcl-2, total PARP and Caspase 9 was detected in Wi-A treated modifications i.e., methylation of withaferin A exerts major impact cells indicating activation of apoptosis pathway in these cells, while mWi-A on its binding efficacy to the target proteins and its cellular outcomes. failed to modulate expression of apoptosis markers (Figure 6D). Moreover, alkyl modification, besides improving drug potency by stability and activity of organic/steroidal compounds, may also cause Discussion attenuation of their chemotherapeutic potency. Altogether, present Advancement of computational biology has provided a investigation implies that (i) the bioinformatics predictions are reliable comprehensive platform to model, predict and validate putative and good predictive parameters and (ii) sufficient care is warranted in molecular interaction. It has provided an insight into detailed the use of these phytochemicals as anticancer reagents. structural annotation that may predict atomics bonding, localization, and its strength to further affirm it in wet lab. Prediction of binding Disclosure Statement of mortalin, p53, p21WAF1 and NRF-2 with Wi-A and its methoxy The authors have no conflict of interest derivative (mWi-A) at the first step demonstrated strength of their interactions that was further affirmed by molecular validations. Author’s Contributions Strength of mWi-A interaction with their target proteins was lower in comparison to Wi-A, and thus it reflected in further expression KS, ZZ, SCK and RW designed the study and drafted the validations. The strong interaction of Wi-A with target proteins led to manuscript. CC, KV and RSK have carried out all the experiments. All activation of p53/p21WAF1 pathway, while it modulated mortalin and the authors read and approved the final version of the manuscript. NRF-2 levels. On the other hand, mWi-A remained ineffective in doing Acknowledgements so, reflecting its weaker interaction with target proteins predicted with bioinformatics analysis. Consistent with initial predictions, Wi-A The study was supported by grants from the AIST. RSK is a recipient of Japan Society for Promotion of Science (JSPS) Post-doctoral Fellowship. Authors thank mediated activation of p53/p21 pathway further found to lead growth Navjot Shah, Anupama Chaudhary and Jay Prakash for their contributions to this arrest followed by apoptosis, validated by associated markers. Altered study.

Figure 6: Anticancer efficacies of mWi-A and Wi-A. (A) Phase contrast representative images of mWi-A and Wi-A treated cells. (B) Colony forming efficacy of Wi-A and mWi-A treated cells. (C) Cell cycle analysis showing percent of Wi-A and mWi-A (Conc. - 0.3 µg/ml) treated cells in G1, S, G2 and M phases of cell cycle. (D) Immunoblotting of Pro-PARP, Pro-Caspase-3 and Bcl-2 proteins and their quantitation (above) in control and treated cell.

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References A and X-ray irradiation enhances apoptosis in U937 cells. Toxicol In Vitro 25: 1803-1810. 1. Mishra LC, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern Med Rev 5: 334-346. 14. Yang ES, Choi MJ, Kim JH, Choi KS, Kwon TK (2011) Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen 2. Siddique AA, Joshi P, Misra L, Sangwan NS, Darokar MP (2014) 5,6-de-epoxy- species, Bcl-2 downregulation and Akt inhibition. Chem Biol Interact 190: 9-15. 5-en-7-one-17-hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal leaves. Nat Prod Res 28: 392-398. 15. Sy-Cordero AA, Graf TN, Runyon SP, Wani MC, Kroll DJ, et al. (2013) Enhanced bioactivity of silybin B methylation products. Bioorg Med Chem 21: 3. Joshi P, Misra L, Siddique AA, Srivastava M, Kumar S, et al. (2014) Epoxide 742-747. group relationship with cytotoxicity in withanolide derivatives from Withania somnifera. Steroids 79: 19-27. 16. Walle T (2009) Methylation of dietary flavones increases their metabolic stability and chemopreventive effects. Int J Mol Sci 10: 5002-5019. 4. Wijeratne EM, Xu YM, Scherz-Shouval R, Marron MT, Rocha DD, et al. (2014) Structure-activity relationships for withanolides as inducers of the cellular heat- 17. Gao R, Shah N, Lee JS, Katiyar SP, Li L, et al. (2014) Withanone-rich combination shock response. J Med Chem 57: 2851-2863. of Ashwagandha withanolides restricts metastasis and angiogenesis through hnRNP-K. Mol Cancer Ther 13: 2930-2940. 5. Deocaris CC, Widodo N, Wadhwa R, Kaul SC (2008) Merger of ayurveda and tissue culture-based functional genomics: inspirations from systems biology. J 18. Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, et al. (2004) Transl Med 6: 14. Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis 7: 115-122.

6. Widodo N, Priyandoko D, Shah N, Wadhwa R, Kaul SC (2010) Selective killing 19. Widodo N, Kaur K, Shrestha BG, Takagi Y, Ishii T, et al. (2007) Selective of cancer cells by Ashwagandha leaf extract and its component Withanone killing of cancer cells by leaf extract of Ashwagandha: identification of a tumor- involves ROS signaling. PLoS One 5: e13536. inhibitory factor and the first molecular insights to its effect. Clin Cancer Res 13: 2298-2306. 7. Lee J, Sehrawat A, Singh SV (2012) Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells. Breast Cancer Res Treat 136: 45-56. 20. Wadhwa R, Sugihara T, Yoshida A, Nomura H, Reddel RR, et al. (2000) Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the 8. Das T, Roy KS, Chakrabarti T, Mukhopadhyay S, Roychoudhury S (2014) hsp70 family protein mot-2 and reactivation of p53 function. Cancer Res 60: Withaferin A modulates the Spindle assembly checkpoint by degradation of 6818-6821. Mad2-Cdc20 complex in colorectal cancer cell lines. Biochem Pharmacol 91: 31-39. 21. Deocaris CC, Lu WJ, Kaul SC, Wadhwa R (2013) Druggability of mortalin for cancer and neuro-degenerative disorders. Curr Pharm Des 19: 418-429. 9. Hahm ER, Moura MB, Kelley EE, Van Houten B, Shiva S, et al. (2011) Withaferin A-induced apoptosis in human breast cancer cells is mediated by 22. Sane S, Abdullah A, Boudreau DA, Autenried RK, Gupta BK, et al. (2014) reactive oxygen species. PLoS One 6: e23354. Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells. Cell 10. Yu SM, Kim SJ (2013) Production of reactive oxygen species by withaferin A Death Dis 5: e1118. causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes. Exptl Cell 23. Wadhwa R, Ando H, Kawasaki H, Taira K, Kaul SC (2003) Targeting mortalin Res 319: 2822-2834. using conventional and RNA-helicase-coupled hammerhead ribozymes. EMBO Rep 4: 595-601. 11. Thaiparambil JT, Bender L, Ganesh T, Kline E, Patel P, et al. (2011) Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by 24. Hahm ER, Lee J, Huang Y, Singh SV (2011) Withaferin a suppresses estrogen inducing vimentin disassembly and serine 56 phosphorylation. Int J Cancer receptor-Î± expression in human breast cancer cells. Mol Carcinog 50: 614-624. 129: 2744-2755. 25. Vaishnavi K, Saxena N, Shah N, Singh R, Manjunath K, et al. (2012) Differential 12. Antony ML, Lee J, Hahm ER, Kim SH, Marcus AI, et al. (2014) Growth arrest activities of the two closely related withanolides, Withaferin A and Withanone: by the antitumor steroidal lactone withaferin A in human breast cancer cells is bioinformatics and experimental evidences. PLoS One 7: e44419. associated with down-regulation and covalent binding at cysteine 303 of beta- tubulin. J Biol Chem 289: 1852-1865. 26. Singh R, Kalra RS, Hasan K, Kaul Z, Cheung CT, et al. (2014) Molecular characterization of collaborator of ARF CARF as a DNA damage response and 13. Yang ES, Choi MJ, Kim JH, Choi KS, Kwon TK (2011) Combination of withaferin cell cycle checkpoint regulatory protein. Exptl Cell Res 322: 324-324.

Med Aromat Plants ISSN: 2167-0412 MAP, an open access journal Volume 4 • Issue 5 • 1000219