Structural elucidation of EMTAHDCA was performed, as described previously (Niveshika et al., 2016). Three dimensional crystal structures of 11 cancer target receptors with PDBID of 1BIX (Gorman et al., 1997), 1NOW (Mark et al., 2003), 1TE6 (Chai et al., 2004), 2RCW (Madej et al., 2014), 2UVL (Herman et al., 2009), 2VCJ (Brough et al., 2008), 3CRY (Oakley et al., 2010), 3HQU (Chowdhury et al., 2009), 3NMQ (Yun et al., 2011), 5P21 (Pai et al., 1990), and 4B7P (Fogliatto et al., 2013) were retrieved from the protein databank (PDB) (www.rcsb.org/pdb; Table S1). Discovery Studio 3.1 tool was used to optimize 3D models of ligand (EMTAHDCA) as well as 11 cancer related proteins (Gao and Huang, 2011). Preparation of terminus of each proteins were not require to perform. Again discovery studio version 3.1 software was used to remove heteroatom, water molecules, and ligands before docking (Gao and Huang, 2011). MetaPocket 2.0 was used for predicting the active site of a target protein (http://projects.biotec.tu-dresden.de/metapocket/; Huang, 2009; Zhang et al., 2011). An advanced docking tool, Yet Another Scientific Artificial Reality Application (YASARA), was used for docking calculation between receptors and ligand (Krieger and Vriend, 2014). The target protein was set in YASARA to run macro file (dock_run.mcr). YASARA structure provides Autodock and VINA tools to dock ligands with proteins (http://www.yasara.org/docking.htm) using a very simple approach (Morris et al., 1998; Trott and Olson, 2010). AMBER force field was used by docking software YASARA for calculation of energy. AMBER force-field parameters were adjusted iteratively so that the damage done to the structures was minimal (Krieger et al., 2004). Energy minimization was done using default parameter of YASARA Autodock Vina tool (http://www.yasara.org/yamber.htm).

For all experiments, 15–20 weeks old healthy adult mice (AKR strain) were used because these mice are highly susceptible to develop tumor and have a shorter life-span. Under aseptic condition, about 1 × 10⁶ DLA cells were transplanted intraperitoneally in mice. The life-span of DL bearing mice was 20 ± 2 days, however the life-span of healthy mice is about 18 months, as reported by Bharti and Mishra (2008). The success rate of developing cancer using DLA cells is found to be 100% (Goldie and Felix, 1951). Experimental mice were maintained with standard feeding and drinking at the temperature of 25 ± 2°C for 12/12 h light/dark cycle throughout the study period in the animal house facility of the Zoology Department, Institute of Science, Banaras Hindu University, Varanasi, India as per guidelines of the Institutional Animal Ethical Committee (IAEC), Banaras Hindu University (Bharti and Mishra, 2011).

After successful development of the tumor, mice were anesthetized with chloroform for collecting ascites fluids from the peritoneal cavity. The fluid was then centrifuged at 3,000 rpm for 6 min, and cell pellet was taken and re-suspended in 1 mL of 1X Phosphate Buffered Saline (PBS). Next, 100 μL of cell suspension was mixed with 100 μL of 2X trypan blue (Sigma) to check viable cell density using hemocytometer. The formula used to count viable cells is as follows: No. of cells × dilution factor/ volume (0.4 mm³ i.e., 4 squares were counted, each with area of 1 mm², and depth of counting chamber of 0.1 mm).

A preliminary study of DLA cell viability under different concentrations of EMTAHDCA (100, 250, 500, 750, and 1,000 ng/mL) were performed. The 1 mg/mL concentration of EMTAHDCA was prepared by dissolving 1 mg of purified EMTAHDCA in 1 mL of 1% DMSO. Different concentrations of EMTAHDCA were prepared separately using 1 mg/mL concentration as stock solution by formula C₁V₁ = C₂V₂ where C₁ represent the stock concentration i.e., 1 mg, C₂ represent the working concentrations i.e., 100, 250, 500, 750, and 1,000 ng, V₁ represent the required volume of stock concentration to make working concentration in desired volume (V₂).

Approximately, 1 × 10⁶ cells were seeded per well in 6-well plate using complete growth medium (RPMI+10% FBS), then cells were treated with different concentrations of EMTAHDCA and incubated for 24 h inside the CO₂ incubator with 5% CO₂. Cells treated with only medium served as a control group. Cell imaging was done under EVOS FLoid Cell Imaging Station.

Cytotoxicity of EMTAHDCA at various concentrations (100, 250, 500, 750, and 1,000 ng/mL) were assessed by using MTT assay in DLA cells and normal mouse bone marrow cells (BMC). BMC were isolated from femur bones of 15–20 weeks old adult mice. The mice was anesthetized using chloroform for collecting bone marrow. After that with the help of 24-gauge needle, bone marrow was flushed with pre-warmed PBS and single cells suspension was prepared. The cells were suspended in RPMI-1640 medium supplemented with antibiotic solution and 10% FBS. Cells were seeded in culture plates and maintained in 5% CO₂ at 37°C for 24 h (Prasad and Koch, 2014).

For MTT assay, 1 × 10⁶ cells/mL DLA and BMC (positive control) were seeded per well in 96-well plate and treated with different concentrations (100–1,000 ng/mL) of EMTAHDCA along with the control (only medium) and vehicle control (1% DMSO). After 24 h, supernatant was removed from each well, washed twice with PBS and MTT solution (5 mg ml⁻¹ in PBS) with 100 μL of medium was added. After incubation for another 4 h, resultant formazan crystals were dissolved in dimethyl sulfoxide (100 μL), and the absorbance was measured by a microplate reader (Bio-RAD 680, USA) at 590 nm with a reference wavelength of 620 nm (Prasad and Koch, 2014) and IC₅₀ value of EMTAHDCA was calculated using GraphPad Prism 7 software. All experiments were performed in quadruplicate, and the relative cell viability (%) was expressed as a percentage relative to control group.

In case of time dependent study, cells were exposed to 250, 500, and 750 ng/mL of EMTAHDCA along with control and vehicle control and incubated for 12 and 24 h, respectively, and the cell viability test was performed as described above.

Overall survival time and body weight of DL bearing mice after treatment with EMTAHDCA were evaluated to check anti-cancer and cytotoxic effect of EMTAHDCA in DL induced mice.

DL transplanted mice were randomly divided into two groups, DL group (DL) and DL+ EMTAHDCA treated group. EMTAHDCA treated groups were further divided into two subgroups, 5 and 10 mg/kg B.W. groups, and were administered with respective doses at 6th, 12th, and 18th day of DL transplantation. For the survival time analysis, the percent of mean survival time of EMTAHDCA treated mice (T) over mean survival time of the control group (C) mice were calculated followed by the evaluation of antitumor activities of EMTAHDCA by computing the T/C value of respective groups. The DL group mice were taken as control group for survival time analysis. The body weight was measured at day 0, 3, 6, 9, 12, 15, and 18 because an increase in body weight in ascites tumor model represents tumor mass as explained by Sharma and Koch (2016). Briefly, male mice of 22 g B.W. were taken for the experiment and considered as blank. The graph was plotted after subtracting the body weight of DL mice and 5 and 10 mg/kg B.W. of EMTAHDCA treated DL mice with that of the healthy mice. Kaplan–Meier survival curve was plotted and analyzed by GraphPad Prism7 software using log-rank analysis to examine the level of significance, and p < 0.05 was obtained by comparing between normal mice represented as control, DL induced mice and EMTAHDCA treated DL mice groups.

Statistical analysis were performed using software SPSS (version 16.0). For each experiment, six adult male mice were taken in each group (n = 6). Each experiments were done 4 times independently. Results were expressed as mean ± standard error of mean (SEM). The IC₅₀ value of compound was calculated using GraphPad Prism 7 software. Data were analyzed by chi-square test and One-way ANOVA followed by Tukey post-hoc test. A p < 0.05 was considered as statistically significant.

Molecular docking was successfully performed between the selected ligand (EMTAHDCA) and 11 cancer protein targets (PDB ID: 1BIX, 1NOW, 1TE6, 2RCW, 2UVL, 2VCJ, 3CRY, 3HQU, 3NMQ, 5P21, and 4B7P; Table S1) using YASARA software. The active site of cancer protein targets was determined using Metapocket. Summary of molecular docking results of selected ligand with cancer target protein is represented in Table S2. EMTAHDCA interacted efficiently with the cancer target 3CRY with binding energy of 9.2 and dissociation constant of 169507.6. Contracting receptor residues involved in their interaction through the formation of electrostatic bond, van der Waals force, covalent bond, and hydrogen bond were Gly²³, Ser²⁴, Asn²⁵, Arg³⁰, Arg³⁴, Gly⁵⁸, Lys⁵⁹, Thr⁶⁰, Ser⁶¹, Gln⁶², Thr⁶³, Trp⁶⁴, Ile⁶⁸, Gln⁹⁷, Gln⁹⁸, Met¹⁰⁴, Tyr¹⁰⁵, and Tyr¹³⁹ (Figure 1).

Moreover, EMTAHDCA efficiently interacted with the cancer target 2VCJ with a good binding energy of 8.5 and less dissociation constant of 567576.2. In this case, active site 1 residues viz. Leu⁴⁸, Asn⁵¹, Ser⁵², Ala⁵⁵, Lys⁵⁸, Tyr⁶¹, Glu⁶², Asp⁹³, Ile⁹⁶, Gly⁹⁷, Met⁹⁸, Asp¹⁰², Leu¹⁰⁷, Gly¹⁰⁸, Phe¹³⁸, Val¹⁵⁰, Thr¹⁸⁴, and Val¹⁸⁶ were found to be involved in the interaction. Residues interacted through electrostatic, van der Waals, covalent, and hydrogen bonding (Figure 2).

Ligand EMTAHDCA also interacted very efficiently with the cancer protein target 1TE6 with a good binding score of 8.4 and lowest dissociation constant [pM] of 647439.1. Active site 1 residues viz. Lys²⁷, Gly²⁸, Leu²⁹, Phe³⁰, Arg³¹, Ala³², Lys¹¹⁹, Ala¹²², Ala¹²³, Glu¹²⁶, Leu¹²⁷, Pro¹²⁸, Glu³⁷⁶, Asp³⁷⁷, Thr³⁷⁸, and Phe³⁷⁹ were involved in their interaction (Figure 3).

In case of interaction between EMTAHDCA and 2RCW, the binding score was found to be 8.4, whereas the dissociation constant was 689160.3. Amino acid residues involved in this interaction were Lys⁴², Val¹¹², Ser¹¹⁵, Leu¹¹⁶, Gly¹¹⁹, Gly¹²⁰, Ser¹²¹, Val¹³¹, Glu¹³⁴, Lys¹³⁵, Lys¹³⁷, Arg¹⁸⁰, Glu¹⁸¹, Gly¹⁸², Glu¹⁸³, Arg¹⁸⁶, Ser²¹³, Gln²¹⁴, and Ile³³³. In addition, 2D view of ligand and protein interaction depicted very clearly that residues interacted through electrostatic, van der Waals, covalent, and hydrogen bonding (Figure 4).

Interaction between EMTAHDCA and 1BIX secured binding score of 8.2 and dissociation constant of 1017760.4. Amino acid residues involved in their interaction were Glu⁹⁶, Tyr¹²⁸, Arg¹⁵⁶, Tyr¹⁷¹, Asn¹⁷⁴, Gly¹⁷⁶, Arg¹⁷⁷, Gly¹⁷⁸, Arg¹⁸¹, Asn²²², Asn²²⁶, Asn²²⁹, Phe²⁶⁶, Trp²⁶⁷, Thr²⁶⁸, Met²⁷¹, Ala²⁷³, Val²⁷⁸, Gly²⁷⁹, and Trp²⁸⁰. These residues were interacted through electrostatic, van der Waals, covalent, and hydrogen bonding (Figure 5).

EMTAHDCA interacted with 1NOW at its active site with a high binding score of 8.2 and low dissociation constant of 1024654.8. Active site residues were Ile¹⁴³, Ser¹⁴⁴, Ser¹⁴⁵, Asp¹⁴⁶, Glu¹⁴⁷, Ser¹⁴⁸, Ser¹⁹⁷, Pro¹⁹⁸, Arg¹⁹⁹, Phe²⁰⁰, Ser²⁰¹, Arg²⁰³, Pro⁴⁴², Leu⁴⁶⁹, Gln⁴⁷⁵, Lys⁴⁷⁸, Gln⁴⁷⁹, Leu⁴⁸⁰, Phe⁴⁸¹, Ile⁴⁸², and Lys⁵¹⁷ (Figure 6).

In case of EMTAHDCA and 5P21 interaction, binding score was 8.2 and dissociation constant was 1608019.0. Active site 1 residues were Gly¹², Gly¹³, Val¹⁴, Gly¹⁵, Lys¹⁶, Ser¹⁷, Ala¹⁸, Val²⁹, Asp³⁰, Glu³¹, Tyr³², Asp³³, Pro³⁴, Gln⁶¹, Asn⁸⁵, Asn⁸⁶, Thr⁸⁷, Lys⁸⁸, Lys¹¹⁷, and Thr¹²⁴. The 2D view clearly mentioned that residues interacted through electrostatic, van der Waals, covalent, and hydrogen bonding (Figure 7).

In case of 3NMQ, EMTAHDCA interacted with a binding score of 7.8 and dissociation constant of 1955781.4. Active site residues viz. Asn⁵¹, Ala⁵⁵, Lys⁵⁸, Ile⁹⁶, Gly⁹⁷, Met⁹⁸,Asp¹⁰², Leu¹⁰⁷, Ile¹¹⁰, Ser¹¹³, Gly¹¹⁴, Phe¹³⁴, Gly¹³⁵, Val¹³⁶, Phe¹³⁸, Val¹⁵⁰, His¹⁵⁴, Thr¹⁸⁴, and Val¹⁸⁶ interacted through electrostatic, van der Waals, covalent, and hydrogen bonding (Figure 8).

Cancer target protein 4B7P interacted with EMTAHDCA with positive energy of 7.8 and low dissociation constant of 1975688.0. Residues such as Arg⁴⁶, Glu⁴⁷, Ser⁵⁰, Asn⁵¹, Ser⁵³, Asp⁵⁴, Asp⁵⁷, Arg⁶⁰, Lys¹¹², Gly¹³², Gln¹³³, Gly¹³⁵, Val¹³⁶, Gly¹³⁷, Phe¹³⁸, Phe²¹³, Ile²¹⁴, Gly²¹⁵, and Tyr²¹⁶ were involved in this interaction (Figure 9).

Interaction between EMTAHDCA and 2UVL secured a binding score of 7.8 and dissociation constant of 2081812.8. Active site amino acid residues such as Arg²⁴⁴, Thr²⁴⁶, Val²⁴⁷, Tyr²⁸², Tyr²⁸³, Val²⁸⁴, Gly²⁸⁵, Asn²⁸⁶, Asp²⁸⁹, Lys²⁹¹, Gly²⁹⁸, Leu²⁹⁹, and Arg³⁰⁰ were involved in their interaction. Electrostatic, van der Waals, covalent, and hydrogen bond were involved in this interaction as clearly mentioned in the 2D view (Figure 10).

Interaction between EMTAHDCA and 3HQU occurred with positive energy of 7.6 and low dissociation constant of 2645629.8. Amino acid residues such as Leu¹⁸⁸, Pro¹⁸⁹, Ala¹⁹⁰, Leu¹⁹³, Tyr¹⁹⁷, Val²¹⁰, Asp²¹¹, Asp²¹², Phe²¹³, Leu²¹⁴, Gly²¹⁵, Lys²¹⁶, Glu²¹⁷, Thr²¹⁸, Ser²⁷⁵, Asp²⁷⁸, and Leu²⁷⁹ were involved in their interaction using electrostatic, van der Waals, covalent, and hydrogen bonding (Figure 11).

In order to test the anticancer activity of EMTAHDCA, DLA cells were initially cultured with different concentrations of EMTAHDCA i.e., 100, 250, 500, 750, and 1,000 ng/mL for 24 h (Figures 12A–F) and observed to have cytotoxic effect. Cytotoxic effects of different concentrations of EMTAHDCA on DLA cells and normal BMC were also examined through MTT assay. The percent (%) viability of DLA cells were observed to be 100, 99.8, 97.7, 92.5, 51.2, 46.6, 40.2 at control, vehicle control, 100, 250, 500, 750, and 1,000 ng/mL, respectively whereas percent (%) viability in case of normal BMC were found to be 100, 99.7, 98.9, 98.9, 98.8, 98.7, and 98.7 at control, vehicle control, 100, 250, 500, 750, and 1,000 ng/mL, respectively (Figure 13A, Table S3). The calculated IC₅₀ value was found to be 372.4 ng/mL EMTAHDCA against DLA cells. Further, in order to determine time dependent cytotoxicity of EMTAHDCA, MTT assay was performed using 250, 500, and 750 ng/mL concentration of EMTAHDCA at 12 and 24 h time points. The cell viability of DLA cells treated with 500 ng EMTAHDCA was observed 85% after 12 h and 52.5% after 24 h of incubation as compared to the control (100%). However, treatment with 250 ng/mL EMTAHDCA showed 95 and 94% cell viability after 12 and 24 h, respectively. Cell viability of DLA cells treated with 750 ng/mL EMTAHDCA decreased from 89% after 12 h to 45.6% after 24 h (Figure 13B, Table S4).

In vivo results showed a significant inhibition of tumor growth in mice of EMTAHDCA treated group as compared to the DL group. More importantly, median life span/survival days of tumor bearing mice treated with 5 and 10 mg/kg B.W. of EMTAHDCA significantly increased to 190 and 181%, respectively as compared to DL group (Figure 14A). Increased longevity of EMTAHDCA treated mice as compared to the DL mice was further verified by Kaplan's Meier survival curve using log-rank statistics which showed a significant increase in survival, ~1.9- and 1.81-fold with doses of 5 and 10 mg/kg B.W. of EMTAHDCA respectively as compared to DL mice (Figure 14B). An increase in body weight of DL mice was observed 6 days after the intraperitoneal injection of DLA cells until the death of mice. In contrast, a significant decrease in body weight was observed in DL mice treated with 5 and 10 mg/kg B.W. of the compound on 6th, 12th, and 18th days as compared to DL group (Figure 15). On 9th day, the body weight decreased to 5.8 and 5.7 g after the treatment with 5 and 10 mg/kg B.W. of the compound, respectively, as compared to DL group (7.2 g body weight). On 18th day, the weight decreased to 15 and 17 g after the treatment with 5 and 10 mg/kg B.W. of the compound, respectively, as compared to DL group (21 g body weight). We also found that the administration of EMTAHDCA in healthy mice at a same dose did not cause any major side effects.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.