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Phytomedicine 19 (2012) 912–923 Contents lists available at SciVerse ScienceDirect Phytomedicine journal homepage: www.elsevier.de/phymed Effect of diallyl disulfide on insulin-like growth factor signaling molecules involved in cell survival and proliferation of human prostate cancer cells in vitro and in silico approach through docking analysis R. Arunkumar a , G. Sharmila a , P. Elumalai a , K. Senthilkumar a , S. Banudevi a , D.N. Gunadharini a , C.S. Benson a , P. Daisy b , J. Arunakaran a,∗ a b Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India PG and Research Department of Biotechnology & Bioinformatics, Holy Cross College, Trichirappalli 620002, India a r t i c l e Keywords: Apoptosis DADS Docking IGF Prostate cancer i n f o a b s t r a c t Purpose: Diallyl Disulfide (DADS) is one of the major components of garlic, which inhibits the proliferation of various cancer cells. Our previous studies showed that DADS inhibits cell growth and induces apoptosis on prostate cancer cells. Insulin like growth factor signaling pathway plays a significant role on prostate cancer cell growth and survival and it’s over expression also identified in human prostate cancers. The molecular mechanism of IGF mediated PI3K/Akt signaling remains to be elucidated. The present study was designed to evaluate the effects of diallyl disulfide on IGF signaling in androgen independent prostate cancer cells (PC-3). Methods: DADS (10–50 M) caused dose-dependent inhibition of PC-3 cells, were analyzed by MTT, IC50 value of PC-3 cells was 40 M for 24 h. Interestingly, DADS also altered the mRNA and protein expressions of IGF signaling and apoptotic molecules which were confirmed by semi quantitative PCR and western blot method. Further the docking study of DADS with IGF receptor was carried out by Ligand Fit of Discovery studio. Accord Excel Package was used for the prediction of ADME properties of the compound. Results: The results suggests that DADS decreases the survival rate of androgen independent prostate cancer cells by modulating the expression of IGF system, which leads to inhibition of phosphorylation of Akt, thereby inhibits cell cycle progression and survival by lowering the expression of cyclin D1, NFkB and anti-apoptotic Bcl-2 molecule and increasing the level of pro-apoptotic (Bad and Bax) signaling molecules which leads to apoptosis. Conclusion: The present investigation showed downregulation of Akt and a concomitant increase in apoptosis in DADS treated prostate cancer cells. Since inhibition of this Akt pathway by DADS leads to inhibition in cancer cell progression, it is highly suggested that DADS has the potential use as a therapy for prostate cancer. © 2012 Elsevier GmbH. All rights reserved. Introduction Dietary intake of allium vegetables including garlic is protective against the risk of various types of malignancies, including prostate cancer (Antosiewicz et al. 2006). Among garlic constituents, diallyl disulfide (DADS) seems to be the most effective in reducing the growth of human tumor cells originating from mammary gland, prostate, colon, liver, lung and skin (Sundaram and Milner 1996a,b). DADS, an organosulfur compound up-regulate p21waf/cip1 protein expression and induce apoptosis which was due to the hyperacetylation of histone H3 and H4 (Arunkumar et al. 2007). Increased ∗ Corresponding author. Tel.: +91 44 24547043; fax: +91 44 24540709. E-mail address: j [email protected] (J. Arunakaran). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.04.009 expression levels and/or enhanced activity of IGF-1Receptor (IGFIR) have been observed in many types of cancer including PCa (Cardillo et al. 2003; Zhang and Yee 2004) and also increased levels of Insulin-like Growth Factor (IGF)-I have been associated with increased risk of PCa (Borugian et al. 2008). IGF-binding protein-3 (IGFBP-3) is a modulator of the IGF-signaling pathway and described as an anti-cancer agent in PCa. IGFBP-3 inhibits IGF-stimulated cell proliferation by blocking IGF-mediated proliferation signals; IGFs stimulate cell proliferation by binding to high-affinity IGF-IR and subsequently activate the receptor tyrosine kinase. The activities of the growth factors IGF-I and IGF-II are modulated by a family of IGF-binding proteins (IGFBPs). The IGFBPs stabilize the IGFs by forming IGF/IGFBP complexes. This leads to the sequestration of IGFs from their cell surface receptors and a resultant inhibition of downstream signaling events (Jones and R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 Clemmons 1995; Hwa et al. 1999). IGF mediated Akt trigger a cascade of response, from cell growth and proliferation to survival and motility that drive tumor progression (Vivanco and Sawyers 2002). The Akt family of serine/threonine kinase composed of Akt1, Akt2, and Akt3. Upon activation of growth factor receptors, Akt family members become phosphorylated on two residues (Thr308 and Ser473) by the phosphoinositide-dependent kinase 1 (PDK1) and the mammalian target of rapamycin-rictor complex, respectively (Stambolic and Woodgett 2006; Engelman 2009). Disruption of the expression of Akt isoforms revealed that they have overlapping but not identical functions. Thus, Akt kinases have isoform-specific functions in the regulation of cell migration, invasion, and metastasis (Stambolic and Woodgett 2006). Akt regulates at least four different but interacting pathways: cell survival, growth, metabolism and progression through the cell cycle (Engelman 2009). Glycogen synthase kinase-3 (GSK-3), which phosphorylates and inactivates crucial cell cycle regulators and transcription factors, including -catenin, cyclin D1, and cMyc, is a negatively regulated key target of Akt (Majumder and Sellers 2005; Liu et al. 2009). The inhibition of GSK-3 by Akt prevents the phosphorylation of the cytoplasmic signaling molecule -catenin, which impedes its degradation; hence it is translocated to the nucleus. Once in the nucleus, -catenin combines with different transcription factors, like TCF/LEF-1, to induce the expression of several genes, one such is Cyclin D1, which induces cell cycle progression via regulation of Retinoblastoma (Rb) hyperphosphorylation and inactivation. Akt also phosphorylates P21/Waf1/Cip1 and P27/Kip2, and inhibits their anti-proliferative effects by retaining them in the cytoplasm (Shin et al. 2002). Another target of Akt is the human double minute-2 (HDM-2) E3 ubiquitin ligase which is responsible for ubiquitination of the p53 tumor suppressor. Activation of HDM-2 represses p53 activity and targets it for degradation (Engelman 2009). In addition, activated Akt targets several proteins that regulate the process of apoptosis, such as BAD, caspase 9, and Mcl-1 (Amaravadi and Thompson 2005; Li et al. 2006). NFkB is a key regulator of genes involved in cell activation and proliferation (Grilli et al. 1993). NFkB activity is normally regulated through its cytoplasmic sequestration by specific inhibitors including IkB and related proteins. Upon stimulation by a variety of stimuli including cytokines, radiation, and oxidative stress etc., Ik is phosphorylated by IkB kinase (IKK), allowing the release of NFkB dimers to enter the nucleus and subsequent activation of target genes. Akt has been shown to activate NFkB by phosphorylation of IKK at a critical regulatory site Thr23 and subsequent degradation of IkB (Ozes et al. 1999; Romashkova and Makarov 1999). Additionally, Akt may also contribute to NFkB regulation through p65/RelA phosphorylation, as observed in HepG3 cells during IL-1 stimulation, where it appears to be independent of IKK activation and IkB degradation (Sizemore et al. 1999). Moreover, Akt has been found to enhance the degradation of IkB, which led to activation of NFkB in the Jurkat T-cell line (Kane et al. 1999). Akt is a downstream target of NFkB, because over expression of p65 led to higher Akt phosphorylation (Meng et al. 2002). The phosphatidylinositol 3-kinase/Akt signaling pathway plays a key role in the regulation of cell division and survival in cancer cells (Engelman 2009; Liu et al. 2009). Loss of phosphatase and tensin homolog deleted on chromosome ten (PTEN), a negative regulator of Akt activation, results in constitutive activation of Akt, which is frequent in prostate cancer (Stambolic and Woodgett 2006) and correlates with poor prognosis (Armstrong and Carducci 2006). This renders the Akt pathway a promising target for the development of novel therapeutic approaches (Liu et al. 2009). The effect of DADS on IGF mediated cell proliferation, survival and apoptosis pathway in prostate cancer has not been extensively studied. So, the present study is aimed to investigate the effect of DADS on IGF signaling pathway and its downstream targets. 913 Materials and methods Chemicals Diallyl disulfide, DMEM medium, MTT, -actin (mouse monoclonal), Acrylamide, bis-acrylamide, Ammonium persulfate, Bovine Serum Albumin (BSA), N,N,N ,N Tetramethylethylene diamine (TEMED), and Sodium bicarbonate were purchased from Sigma–Aldrich Chemicals Pvt Ltd (USA). Primary antibodies caspase-9 and -10 (Rabbit monoclonal) were purchased from Merck Biosciences (USA) antibodies for AKT, Phospho AKT, Bad, PI3K, IGF1R (Rabbit polyclonal) and cyclin D (mouse monoclonal) were purchased from Cell Signaling Technology. Polyvinilidine difluoride (PVDF) membrane was purchased from Millipore, Bangalore, India. Fetal bovine serum (FBS), penicillin/streptomycin solution and Trypsin EDTA were purchased from Gibco. The secondary antibodies, Horseradish peroxidase (HRP)-Goat-Anti Rabbit IgG were obtained from GENEI, Bangalore. Other chemicals were obtained from Sisco Research Laboratories (SRL Pvt Ltd), India. All the chemicals used were extra pure and were of culture grade. Cell culture Human prostate cancer cell line PC-3 was procured from National Centre for Cell Science, Pune, India and were cultured in DMEM culture medium containing 10% FBS and 5% CO2 at 37 ◦ C. Cells were passaged at 70–80% confluence using Trypsin EDTA. DADS preparation Diallyldisulfide was dissolved in dimethylsulfoxide (DMSO) to prepare 1 mM stock solution. From the stock DADS was prepared at different micromolar concentrations with serum free medium. In all the preparation, the concentration of DMSO never exceeded 0.01%. Cell viability assay Viable cells were measured by a colorimetric assay composed of solutions of a tetrazolium compound MTT (dimethyl thiazolyl tetrazolium bromide). MTT is bioreacted by cells into a formazan product that is soluble and the absorbance of the formazan at 570 nm is measured directly. Cells were seeded at a density of 5 × 103 cells/well, in a 96-well plate and incubated for 24 h at 37 ◦ C in 5% CO2 incubator. After attachment, the cells were washed with PBS and then serum-free medium (SFM) was added for 6–12 h. The cells were added into fresh medium containing different concentrations of DADS and allowed to grow for an additional 24 and 48 h after DADS treatment. The medium was removed and washed twice with PBS and 100 l of 0.5 mg/1 ml MTT solution was added to each well and incubated for 2–3 h. After incubation 100 l of DMSO was added for solubilization of cells and then kept in dark for 1 h. The intensity of the color developed was read at 570 nm in an ELISA reader. The cell viability was calculated as follows. = absorbance of treated cells × 100%. absorbance of control cells Triplicate measurements with different DADS concentrations were performed, and the concentration that gave a 50% reduction in the number of living cells (IC50) was estimated. RNA isolation and RT-PCR The total RNA was isolated by using Tri Reagent (Sigma). Total RNA (2 g) from each sample was subjected to reverse transcription using a Superscript first strand cDNA synthesis kit (Invitrogen) 914 R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 according to the manufacturer’s protocol. PCR reactions were then carried out by mixing 1 l of cDNA, 10 l of KAPA Fast PCR Master mix, 1 l of specific gene primer pair, and glyceraldehyde-3phosphate dehydrogenase primer pair (Internal control) and made up to 20 l with sterile water and then amplified for 35 cycles. Each cycle consisted of denaturation for 5 min at 94 ◦ C, annealing for 30 sec at appropriate annealing temperature and polymerization for 30 sec at 72 ◦ C. The PCR products were resolved by electrophoresis through a 2% agarose gel and stained with ethidium bromide. The densities of PCR products in the agarose gel were scanned with a Gel Doc image scanner (Bio-Rad), and quantified by Quantity One Software (Bio-Rad). Preparation of ligand PC3 cells were plated at the concentration of 1 × 106 cells/plate in medium containing 10% FBS. Once the cells attain 70–80% confluence, the medium was removed and replaced with serum-free medium and treated with DADS at 20 and 40 M concentration for PC-3 cells. At the end of DADS treatment, cells were washed once with ice-cold PBS, and 600 l of ice-cold RIPA buffer containing 40 l of protease inhibitor cocktail was added. Samples were collected into a 1.5 ml tube, and centrifuged for 10 min at 12,000 rpm at 4 ◦ C. The supernatants were collected in new tubes and protein concentrations were determined by Lowry’s method. The structure of DADS was drawn using ChemSketch. ACD/ChemSketchTM software is an integrated software package from Advanced Chemistry Development, Inc., Toronto, Canada, (http://www.acdlabs.com/com), for generating chemical structures of bioactive compounds, 2D structure cleaning, 3D optimization etc. After ligand preparation, hydrogen bonds were added and energy minimization was done using CHARMM force field. TSAR, an Accelrys software package mainly describes Quantitative Structure Activity Relationship (QSAR) and correlates the variations in biological activity with the properties or molecular structures, was used for the calculation of Lipinski’s Rule of Five. Accord for excel ADME data, whether experimentally measured or computationally predicted, provide key insights into how a drug will ultimately be treated or accepted by the body. The ADMET properties for DADS which satisfies the Lipinski properties were calculated preliminarily for its toxicity parameters by using Accord Excel 6.1. Accord for excel use the Accord Chemistry engine to handle chemistry structures and incorporates a number of add-ins to perform chemical calculations. The Accord Chemistry tool bar provides an alternative method of accessing Accord commands and also provides access to additional display operations and functions short-cuts. The molecular descriptors widely adopted in ADME/T predictions were predicted for DADS. Western blot analysis Docking analysis Cell lysates (20–50 g) were electrophoresed in 12% SDS polyacrylamide gel and then transferred into PVDF membranes. The membranes were incubated with primary antibodies against Bax, Bad, Bcl-2, IGF1R, IGFBP3, PI3K, XIAP, caspases, Cyclin D (1: 2000) and GSK-3, p-GSK-3, IKK␣, IKK, NFkB, p-Akt, Akt (1:1000) in Tris-buffered saline. After washing, the membranes were incubated with HRP conjugated anti-mouse IgG (1:5000) or HRP conjugated Goat-anti rabbit IgG (1:5000). Protein bands were detected using chemiluminescence system (ECL Kit) and quantified in Chemi Doc XRS Imaging System, Bio-Rad (USA). To explore the interaction and accurate binding model for the active site of Insulin like growth factor with DADS, molecular docking analysis was carried out by using ligand fit of Discovery Studio (http://www. accelrys.com/product/dstudio/) Accelrys® software corporation, San Diego, USA. The mechanism of ligand placement is based on fitting points. Fitting points are added to hydrogen bonding groups on the protein and ligand. Scoring functions implemented in docking programs make various assumptions and simplifications in the evaluation of modeled complexes, which includes terms of hydrogen bonds employed by Discovery Studio to rank the docked bases and to assess the binding site and the number of rotatable bonds present. Preparation of cell lysate In silico analysis: docking – Discovery studio Docking study is performed for the active organosulfur compound DADS with Insulin like growth factor 1 beta receptor (IGF-IR) by Discovery Studio version 2.1. Accelrys Discovery studio (2.1) is a licensed life science modeling and simulation suite of application focused on optimizing the drug discovery process. Preparation of protein structure The 3D co-ordinates of the crystal structure of Insulin like growth factor 1  (PDB ID: 1K3A) was downloaded from the protein data bank (http://www.rcsb.org/pdb/) established by Brookhaven National Laboratories (BNL) in 1971. It contains the structural information of the macromolecules determined by X-Ray crystallographic and NMR methods. Before docking, water molecules were removed from protein file 1K3A. Crystallographic disorders and unfilled valence atoms were corrected using alternate conformations and valence monitor options and were subjected to energy minimization by applying CHARMM (Chemistry at HARvard Macromolecular Mechanics) force fields. CHARMM is program for macromolecular dynamics; it can be used for energy minimization, normal modes and crystal optimizations and also incorporates free energy methods for chemical and conformational free energy calculations. The druggability site of the protein (1K3A) was defined where the ligand can bind and interact after energy minimization. Statistical analysis The data were analyzed using the SPSS Windows Students version software. For all the measurement, one-way ANOVA followed by Student–Newman–Keul’s (SNK) test was used to assess the statistical significance of difference between control and DADS treated. A statistically significant difference was considered at the level of p < 0.05. Results Drug (DADS) ADME/T property So while a drug lead may exhibit phenomenal efficacy in vitro, poor ADME results will almost invariably terminate its development. Table 1 shows the ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicology) properties of the DADS that have already satisfied Lipinski’s properties. The ADMET properties include Fast Polar Surface Area (FPSA) Aqueous solubility, Blood Brain penetration level, Cytochrome 450 (CYP450 2D6), Hepatotoxicity, Human Intestinal Absorption and Plasma protein binding level. DADS have satisfied all the ADMET properties by being within the defined specified ranges (Table 1). 0 Binding is <90% = 0 Binding is ≥90% = 1 Binding is ≥95% = 2 1 Good absorption = 0 Moderate absorption = 1 Low absorption = = 2 Very low absorption = 3 Plasma protein binding Human intestinal absorption Fig. 1. Effects of DADS on PC-3 cell viability. The cells were grown and treated with serum-free DMEM medium containing indicated doses of DADS. The cell viability of PC-3 cells was measured by MTT assay for 24 and 48 h. Each bar represents the mean ± SEM of six independent observations. ‘a’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. Low = 2 Undefined = 4 Hepatotoxic = 1 Inhibitors of CYP40 2D6 = 1 High = 1 Medium = 3 0 Non-hepatotoxic = 0 0 Non-inhibitors of CYP450 2D6 = 0 Poor at permeating cell membranes 3 Extremely low soluble = Less than −8.0 to −6.0 Low soluble = −8.0 to −6.0 Good = −4.0 to −2.0 Optimal = −2.0 to 0 Highly soluble = greater than 0 1 Very high = 0 Effect of DADS on mRNA and protein expression of IGF1, IGF1R and IGFBP3 0 FPSA ≥ 150.0 Hepatotoxicity CYP2D Blood–brain barrier penetration level The effect of DADS on cell viability was determined using MTT assay (Fig. 1). Percentage of cell viability was significantly decreased with increase in concentration of DADS. In PC-3 cells 50% inhibition was found at 40 M and hence for further studies we selected 20 and 40 M to identify the effect of DADS in other signaling molecules. Aqueous solubility level DADS Reference range 915 Diallyl disulfide (DADS) inhibits cell growth in human prostate cancer cells (PC-3) Fast Polar Surface Area (FPSA) Table 1 Assessment of ADME/T for DADS. R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 IGF system molecule and its downstream molecules play a vital role in cell survival of prostate cancer cell progression by inducing cell cycle progression and inhibiting apoptosis. To see the effect of DADS on IGF system molecule the mRNA and protein expressions were analyzed. mRNA and protein expressions of IGF-I and its receptor was significantly decreased with 20 and 40 M concentration of DADS treatment. IGFBP-3 significantly increased at 20 and 40 M concentration of DADS (Figs. 2 and 7). Regulation of PI3K/AKT pathway by diallyl disulfide In most cancer cells, AKT is constitutively active and enhances cell proliferation. In the present study mRNA expression of PI3K showed significant decrease with 20 and 40 M DADS concentration. But there is no significant effect on Akt mRNA expression. Protein expression of PI3K, P-Akt and cyclin D1 were significantly decreased with 20 and 40 m concentration of DADS but the total Akt was not altered. DADS decrease only the phosphorylation of Akt (Figs. 2 and 8) thereby it is suggested that DADS inhibits cell proliferation by regulating PI3K/AKT pathway. Effect of DADS on GSK-3ˇ, pGSK-3ˇ and cyclin D1 expression Due to inhibition of Akt phosphorylation further studies were proceeded with the protein expression of downstream molecules of Akt. Studies showed that DADS inhibits phosphorylation of GSK3 and increases total GSK3 leads to inhibition of cyclin D1 which is also shown in the Fig. 3. Cyclin D1 which is the key regulator of cell cycle which cause cell cycle arrest. Thus DADS alters the cell cycle progression of PC-3 by decreasing the level of p-Akt and alters the downstream signaling molecules. 916 R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 Fig. 2. Effect of DADS on IGF1R, p-IGF1R, IGFBP3, PI3K, p-Akt and Total Akt protein expression in PC-3 cells. Protein expressions were analyzed by western blotting. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. Fig. 3. Effect of DADS on GSK, p-GSK and cyclin D1 protein expression in PC-3 cells. Protein expressions were analyzed by western blotting. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. Effect of DADS on IKK˛, IKKˇ and NfkB expression Apoptosis-induction by DADS in prostate cancer cell lines Recent reports showed that the NFkB activation is regulated by Akt signaling pathway. The present study shows that DADS significantly decreased the IKK␣, IKK and NfkB expression thereby inhibited the Akt and NFkB mediated cell survival (Fig. 4). In DADS exposed PC-3 cells, the m-RNA and protein expressions of Bad, Bax, caspase 8 and 9 were significantly increased with DADS 20 and 40 M concentration but decreases the protein expression of anti-apoptotic molecule Bcl2. XIAP is an important regulator R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 917 Fig. 4. Effect of DADS on IKK␣, IKK and NfkB protein expression in PC-3 cells. Protein expressions were analyzed by western blotting. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. Fig. 5. Effect of DADS on BAD, BAX and Bcl-2 protein expression in PC-3 cells. Protein expressions were analyzed by western blotting. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. 918 R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 Fig. 6. Effect of DADS on Caspase 8, 9 and XIAP protein expression protein expression in PC-3 cells. Protein expressions were analyzed by western blotting. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. Fig. 7. Effects of DADS on mRNA expressions of IGF1, IGF1R and IGFBP3. The cells were cultured and treated with indicated concentrations of DADS for 24 h. RT-PCR was performed for IGF1, IGF1R and IGFBP3 mRNA expression. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. Abbreviations: M – 100 bp DNA ladder; L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 919 Fig. 8. Effects of DADS on mRNA expressions of PI3K and Akt. The cells were cultured and treated with indicated concentrations of DADS for 24 h. RT-PCR was performed for PI3K and Akt mRNA expression. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. Abbreviations: M – 100 bp DNA ladder; L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. Fig. 9. Effects of DADS on mRNA expressions of BAD, BAX and FAS. The cells were cultured and treated with indicated concentrations of DADS for 24 h. RT-PCR was performed for BAD, BAX and FAS mRNA expression. Each bar represents the mean ± SEM of three independent observations. ‘*’ represents statistical significance between control versus DADS treatment groups at p < 0.05 level using Student–Newman–Keul’s test. Abbreviations: M – 100 bp DNA ladder; L1 – control; L2 – 20 M DADS; L3 – 40 M DADS. of apoptosis which inhibits caspase activity, DADS inhibits XIAP protein expression thereby promoting downstream effector caspases activity. Further, DADS increased the mRNA expression of FAS which mediates the extrinsic mediated apoptosis. Increased level of caspases and FAS and inhibition of XIAP leads to apoptosis (Figs. 5, 6 and 9). Detection of apoptosis by DAPI staining DAPI nuclear staining was performed to confirm apoptotic changes. Nuclear chromosome condensation, shrunken nuclei, and eventually the formation of apoptotic bodies were characteristics of apoptosis in DAPI staining. Apoptotic changes were significantly 920 R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 Fig. 10. DAPI staining. Photomicrograph of DAPI staining of the PC-3 cells incubated for 24 h and visualized and photographed at 40× magnification under fluorescence microscope (Nikon microscope, Japan). (A) Control cells treated with serum-free DMEM medium, viable cells shows blue stained nuclei. (B) 20 M and (C) 40 M DADS treated cells showed condensed chromatin with high fluorescence staining. Results were confirmed by three independent observations. Fig. 13. Docked complex of DADS (yellow color) with IGF-beta receptor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Fig. 11. Structure of DADS, drawn using Chemsketch, (the yellow color indicates oxygen, white color indicates hydrogen, ash color indicates carbon). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) 1 hydrogen bond at Gln182, S5 of DADS and the HE21 of Gln182 of receptor involved in bond formation with a bond distance of 2.171 Å. The libdock score was 65.456 and the energy value was 4.931 (Fig. 14). The energy required for the drug-receptor binding directly correlates to the effective binding of the drug to the target receptor. Discussion Fig. 12. Structure of IGF beta receptor retrieved from PDB database. induced at the 20 and 40 M concentration of DADS when compared to control (Fig. 10). Docking analysis The structure of DADS was generated using Chemsketch (Fig. 11a and b) and was docked with IGFIR (Fig. 12), which was carried out by Ligand Fit of Discovery studio (Version 2.1, Accelry’s Software Inc.). DADS and IGFIR produced a total number of 7 interactions while docking (Fig. 13). DADS interacts with IGFIR with IGF-IR is composed of two covalently linked polypeptide chains, each with an extracellular ␣-subunit and a transmembrane subunit, which possesses tyrosine kinase activity (Grzmil et al. 2004). Activated IGF-IR recruits and phosphorylates adaptor proteins belonging to the insulin receptor substrate (IRS) family or Shc. The phosphorylated adaptor proteins then serve as docking sites for other signaling molecules, resulting in the activation of the downstream pathways such as PI3K and extracellular signal-regulated kinase 1/2 of the mitogen-activated protein kinase (MAPK) pathways that lead to cell survival and proliferation (Chen et al. 2009). Downregulation in the IGF-IR mRNA expression, suggesting that it regulates IGF-IR expression at the level of transcription thereby it decreases cell survival in PC-3 cell line (Senthilkumar et al. 2010). Similarly, in the present study also there is a significant decrease in the mRNA and protein expressions of IGF1, IGF-IR and increase in IGFBP-3 in DADS at 20 M and 40 M treated PC-3 cell line suggesting that it may inhibit the cell survival by downregulating the IGF signaling molecules. PCa specifically shows biochemical abnormalities related to Akt that may be important in sustaining tumor growth by preventing apoptosis and promoting proliferation and angiogenesis (Nelson et al. 2007). Akt over expression has been demonstrated in prostate R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 921 Fig. 14. Interaction between residues and drug (green dotted line H bond and magenta line bump interactions). Libdock score = 65.456, Energy value = 4.931, No. of H bonds = 1, Gln182 (HE21) – S5 = 2.171 Å, no. of bump interactions = 7. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) cancer (Edwards et al. 2003). Complete activation of the catalytic activity of Akt requires phosphorylation of a threonine residue at 308 and a serine residue at 473. It is possible that Akt shows partial activation with phosphorylation at the threonine 308 position (Nicholson and Anderson 2002). Hence, phosphorylation at ser 473 is the key for the complete activation of Akt. In the present study, DADS significantly decreased the expression of p-Akt in PC-3 cells at 40 M concentration and also caused a significant decrease in the expression of ser 473 phosphorylated Akt. This is supported by the study conducted by Xiao et al. (2006), in which treatment with Diallyl trisulfide, a constituent of processed garlic, mediated Akt inactivation and dephosphorylation of Bad and its mitochondrial translocation resulting in apoptosis. In the present study, since there is a decrease expression of AKT phosphorylation due to DADS treatment there is also activation of pro-apoptotic proteins Bad and Bax which will finally lead to apoptosis in prostate cancer cells. Thus apoptotic effects of DADS are mediated through inhibition of Akt phosphorylation. Previous studies showed that DADS suppressed the proliferation of prostate cancer cell (Arunkumar et al. 2005; Gayathri et al., 2009) through cell cycle arrest and apoptosis. Cancer cells escape normal biochemical systems regulating the balance between apoptosis and survival. Akt or protein kinase B generally acts to promote survival through inhibition of proapoptotic (BAD, BAX, and BID) factors and activation of anti-apoptotic (Bcl-XL) factors. Akt inhibits the activity of pro-apototic members such as BAD, BAX and BID while activating anti-apoptotic members such as Bcl-XL (Datta et al., 1997; Majewski et al., 2004). In our study also DADS alters the expression of apoptotic molecule by decreasing the expression of Bcl-2 a key regulator of apoptosis and also increase the mRNA and protein expressions of apoptotic molecule such as Bad and Bax. So, on the whole it is suggested that DADS induces apoptosis through inhibition of Akt phosphorylation. Three central regulators of the cell cycle affected by Akt are cyclin D, p21 and p27. Cyclin D is necessary for cyclin-dependent kinase (CDK) activity regulating entry into the cell cycle. p21 and p27 are CDK inhibitors, which oppose cell-cycle progression. Akt phosphorylates and inactivates p21 and p27 thereby eliminating a critical negative regulator of CDK activity and promoting progression through the cell cycle. Cyclin D1 is important for the development and progression of several cancers including those of the breast, oesophagus, bladder and lung (Vermeulen et al., 2003; Knudsen et al., 2006; Musgrove, 2006). Over expression of cyclin D1 has been reported in several cancers, and also been linked to the development of endocrine resistance in breast cancer cells (Hui et al., 2002; Hodges et al., 2003). GSK-3 was eventually identified as being capable of phosphorylating cyclin D1 on T286 and inducing its rapid turnover (Diehl et al., 1998). Since GSK3 is negatively regulated by the Ras-phosphatidylinositol 3 kinase-Akt pathway, these findings also linked cyclin D1 stability to mitogenic stimulation. In the present study, cyclinD1 expression was significantly decreased at 20 and 40 M concentration of DADS in PC-3 cells which is indirectly related to inhibition of cell cycle progression. Akt involved in growth factor-stimulated cell cycle progression from G0/G1 to S phase. Akt phosphorylates and inhibits GSK-3, which phosphorylates and destabilizes cyclinD1 protein. In the present study, DADS inhibits Akt phosphorylation thereby there is increase in GSK-3 which causes the inhibition of Cyclin D1 protein expression which leads to inhibition of cell cycle progression. Thus PI3K/Akt signaling is involved in the regulation of cell cycle progression. In the present study, there was significant decrease in the p-Akt level and also there was significant decrease in the cyclinD1 expression which was correlated to the inhibition of prostate cancer progression. Therefore inhibition of Akt by DADS is a good target for the prostate cancer treatment. Many oncogene proteins function as elements of signaling pathways that stimulate cell proliferation, the gene that encodes cyclinD1 is a potential oncogene which stimulates cell cycle progression. Other oncogenic proteins interfere with cell differentiation and survival oncogene encoding PI3K/Akt inhibits apoptosis. The IGF-IR-induced increase in cyclin D1 synthesis can be mediated through several alternative mechanisms. It may be transcriptionally regulated through the ERK pathway, or it may be mediated through increased mRNA stability in a PI-3 K/Akt-dependent manner. PI-3 K/Akt signaling can also increase cyclin D1 levels through enhanced mTOR-mediated protein translation and inhibition of GSK-3 mediated cyclin D1 phosphorylation. In addition, IGF-IR can also down-regulate the transcription of the cyclin dependent kinase (CDK) inhibitor (CDKI) p27KIP1 or alter its processing and nuclear localization through a PI-3 K/Akt and phosphatase and tensin homologous on chromosome 10 (PTEN)-dependent mechanism (Samani et al., 2007). In the present study, DADS decreases the phosphorylation of Akt as well as cyclinD1 expression. Thus DADS acts as inhibitor for cell proliferation as well as in cell survival. 922 R. Arunkumar et al. / Phytomedicine 19 (2012) 912–923 Previous study from our laboratory shows that diallyl disulfide induces cell cycle arrest (Arunkumar et al., 2006), and also upregulate p21waf/cip1 protein expression and induces apoptosis which was due to the hyperacetylation of histone H3 and H4 (Arunkumar et al., 2007). So it is clearly enlighten that DADS induces cell cycle arrest via PI3K/Akt mediated pathway by inhibiting cyclin D1 and decreasing the phosphorylation of GSK-3. Another major target of Akt is the NFB pathway. Upon activation by an IB kinase, NFB translocates into the nucleus where it regulates gene expression. Many of its target genes have antiapoptotic effects, such as Bcl-2, or regulate the cell cycle, such as c-Myc and cyclin D1. Inhibition of NFB by other anticancer drug has been observed in prostate cancer, breast cancer and other cancers (Pavese et al., 2010). In the present study also DADS inhibits NFB, IKK␣ and  may also partially explain the effects of DADS on cell cycle progression and apoptosis. According to Brooijmans and Kuntz (2003), in-silico molecular docking is one of the most powerful techniques to discover novel ligands for receptors of known structure and thus play a key role in structure-based drug design. Molecular Docking continues to holds great promise in the field of Computer based drug design which screens small molecules by orienting and scoring them in the binding site of a protein. As a result, novel ligands for receptors of known structure are designed and their interaction energies are calculated using the scoring functions (Irwin et al., 2002). According to Johnson and Wolfgang (2000), compounds should possess certain properties to be accepted as drug. Those properties were formulated by Christopher A Lipinski in 1997. It is a rule of thumb to evaluate drug likeness, or to determine if a chemical compound with a certain pharmacological or biological activity has properties that would make it a likely active drug. Lipinski et al., 2001 suggested that any pair wise combination of the following conditions: MW > 500, LOGP > 5, HDO > 5, and HAC > 10, may result in compounds with poor permeability. In this connection, it is very much clear that DADS obeys perfectly with the Lipinski’s Rule of Five and is considered to have a good absorption and permeability. Predicting a drug candidate’s pharmacokinetic and dynamic profile early in the drug development process is the key aspect of ADME testing. Profiling of absorption, distribution, metabolism and excretion (ADME) characteristics earlier in the drug discovery process especially in parallel with screening for activity has become a central focus for many drug discovery groups. ADME/T properties are recognized as key determinants of whether a molecule can be successfully developed as a drug or not. In this context it is more obvious that DADS shows a good absorption, HIA, Aq.Sol.Lev blood–brain barrier penetration without hepato-toxicity. These results confirm that DADS does not required further modifications to develop as drug for cancer. Srinivasan et al. (2011) stated that the most suitable method of evaluating the accuracy of a docking procedure based on energy and libdock score. Libdock score plays a vital role in validating the docking results. A high Libdock score indicates a stronger receptor–ligand binding affinity (Muegge, 2006). The energy value and a high Libdock score obtained in the present docking study confirm that there is a stronger receptor–ligand binding affinity between DADS and IGF receptor. This in-turn proves that the IGF receptor must have been inhibited by DADS which further down regulates Akt and bring concurrent increase in apoptosis by coinciding with the in vitro results. It is therefore essential to perform docking experiments, which can help in validating a target and adds support to the in vitro studies. Conclusion The above research effort shows the parallel running of in vitro results with that of in silico data obtained for DADS, and it is obvious that DADS can serve as an apoptotic negotiator and can be used for further drug discovery approach in the arena of cancer. The present study proved that DADS brings about, the downregulation of IGF signaling and subsequently turning up apoptosis in prostate cancer cells. Since inhibiting this pathway leads to decrease in the cancer cell progression, this inhibition will be very useful for the prostate cancer therapy. Conflict of interest None declared. Acknowledgement The work was supported by grants from Department of Science and Technology (DST) Purse fellowship to Mr. R. Arunkumar. References Amaravadi, R., Thompson, C.B., 2005. The survival kinases Akt and Pim as potential pharmacological targets. Journal of Clinical Investigation 115, 2618–2624. 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