Reagents and apparatus
All reagents and chemicals used during experimental work were analytical grade. The N- N-acetyl amino acids, (+)-Dehydroabietylamine, dehydroabietylamine, dichloromethane, dicyclohexyl carbodiimide, 4-dimethyl amino pyridine, ethanol, methanol and ethyl acetate purchased from Sigma–Aldrich (St. Louis, MO, USA). DMSO was obtained from Applichem Biochemica (Darmstadt, Germany). Analytical thin-layer chromatography was performed on precoated silica gel plates (Merck PF254, Darmstadt, Germany) and detection of spots were made by UV light and/or iodine vapors. Silica gel 60 (Merck, particle, size 0.040–0.063 mm, 230–240 mesh) was used for preparative column chromatography.
IR spectra were recorded on a Thermo-Nicolet 5700 Fourier transform infrared (FTIR) spectrometer (Fitchburg, WI) with KBr pellets. NMR spectra were recorded at room temperature on Bruker AM instrument operating at 400 and 500 MHz (1H). Residual solvent signals are internally referenced. Chemical shifts δ is referred in terms of ppm, coupling constants J are given in Hz. Following abbreviations classify the multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or unresolved, br = broad signal. Infrared spectra were recorded on a Schimadzu system and reported in cm−1. Samples were prepared in thin film technique. Mass spectra were done using the facilities in laboratories of HEJ Research institute of Chemistry, University of Karachi.
General procedure for the preparation of Amides from amino acids (DAAD 1–7)
To a solution of dehydroabietylamine (1 mol) in dichloromethane, dicyclohexyl carbodiimide (DCC, 1 mol), 4-dimethyl amino pyridine (DMAP, 0.5 mol) and N-acetyl amino acids (1 mol) were added, the resulting mixture was stirred at room temperature for 2 h. After completion of reaction 50 ml of ethyl acetate was added and filter. Filtrate washed with water (20 ml×2), and dried over anhydrous Na2SO4 and the solvent removed by evaporation. The formations of DAAD (1–7) were monitored by TLC; the compound was extracted from reaction mixture and characterized by applying different spectroscopic techniques. Formation of amide was completed in 2 to 2.5 h. N-acetyl-α-amino acid conjugates of (+)-Dehydroabietylamine (DAA) were prepared using Glycine, L-Cysteine, L-Methionine, L-Tyrosine, L-Aspartic acid, L-Phenylalanine and L-Alanine to furnish amides 1–7 respectively. All amides were obtained in good yields. The structure confirmation of compounds 1–7 was carried out (Additional file 1: Suppl. data Chemistry and Additional file 2: Characterization and Synthesis) using spectroscopic techniques including ESI-MS, FTIR, 1H-NMR, 1D & 2D, 13C-NMR, COSY, HMBC, HSQC and NOESY spectroscopy.
Cell lines care and cytotoxicity screening protocol
Ten HCC cell lines including Huh7, Hep3B, Hep3B-TR, HepG2, Hep40, SNU449, Mahlavu, PLC/PRF/5, SNU387, SNU475 and one breast cancer line MCF7 cryogenically stored in a liquid nitrogen Tank. Sulforhodamine B (SRB) method has been implemented for in vitro primary screening [21, 22]. Initially, a HCC cell line Huh7 and one breast cancer line MCF7 were used for primary screening of trial drugs (seven derivatives of synthesized DAA). For both Huh7 and MCF7 cell lines, 2000 cells/well were cultured in 96 well plate in incubator at 37 °C with 5% CO2 in complete medium (DMEM, 10% FBS, 1% NEA, 1% L-Glutamine and 1% P/S) and incubated for 24 h. All stock solutions were prepared in 100% DMSO with a concentration of 20 mM. Further dilutions were made with the help of respective media for each cell line. After 24 h, all trial drugs were introduced in two different (50 μM and 10 μM) concentrations in triplicate for each sample and plates were further incubated for next 72 h. After 72 h, media discarded and cells were washed once by using 1XPBS.
For fixation, 50 μL of ice cold 10% TCA was added into each well and kept in dark at 4 °C for 1 h. After fixation, the TCA was removed by tapping and plates were washed 4–5 times with ~200 μL dH2O. Plates were left over night for drying under hood. Finally, 50 μL of 0.4% SRB in 1% acetic acid solution was added to each well and left at room temperature for 10 min. Excessive SRB dye was removed and the plates washed 4–5 times with 1% acetic acid before air drying. Bound SRB dye was solubilized with 100 μL of 10 mM un-buffered chilled tris-base solution and plates were left on a plate shaker for at least 1–2 min. Absorbance was recorded using μ-Quant microplate reader with a wave length range of 405–515 nm. The test OD values were defined as the absorbance of each sample. Mean values were determined and standard deviation was found satisfactory ranging in between 0.001 to 0.25 in all respective samples from triplicates wells which were calculated automatically using MS Office Excel 2007 (v14.0) software for Windows.
Measurement of cell morphology
Six-well plates were used for photographs and 3 × 105 cells were maintained in each well, after 24 h drug was administered. Photographs of most sensitive (Hep3B) cell line were taken during three consecutive intervals i.e., 24, 48 and 72 h and compared with DMSO controls. All photographs were taken at 10X and 20X using a light microscope.
Cell proliferation assay
Cells were plated in 10 cm2 Petri plates at 3–4 × 105 per plate. After drug treatment, cells were harvested in different intervals by trypsinization and washed with PBS. Cells were fixed in ice-cold 70% ethanol, washed, and resuspended in 3 mL of 70% ethanol for storage at 4 °C; fixed cells treated with RNase A; and stained with propidium iodide (PI) for 45 min at room temperature. The stained cells were analyzed by flow cytometry using BD FACScalibur™.
Comet assay 
Hep3B cell line was used and around 200,000 cells/well were seeded in six well plates. Adriamycin (1 μM), and Camptothecin (5 μM) were used as positive controls. DAAD2 was administered in two different concentrations (5uM and 10 μM). Drugs were given to cells after 24 h of seeding. Both treated and untreated samples were collected after 24,48 and 72 h from plates through scraper with whole media after centrifugation at 1000 rpm for 10 min at +4 °C.
Freshly prepared 100 mL 1% normal melting agarose poured into a jar and kept in a water bath at 50–55 °C. The slides were placed into jar vertically and kept for about 15–20 min at room temperature. 15 ml of 0.7% low melting agarose (LMA) was also prepared and kept it at 4 °C. Both controls and treated cells were taken from incubator and washed with 1X cold PBS once. 2 mL of 1XPBS for each well of 6 wells plate added and cells were transferred directly in to 15 mL falcon tubes. The cells were centrifuged at 1000 rpm for 6 min at 4 °C. Supernatant was discarded and pallet of the cells re-suspend with cold PBS (700 μL) without bubbling. 5–10 μL cell suspension was put on already prepared 1% agarose coated slide, and coated with 70–80 μL LMA with cover slip (24 × 50 mm) onto the cells. Cells were counted under the microscope to validate the desired cell number and stored for 12–15 min at 4 °C to allow LMA layer to soluble. For third layer, again 70–80 μL of LMA covered with cover slip was used for coating, and the slides were kept at 4 °C for about 12–15 min. Slides were pour in to tank for at least 1 h containing lysis buffer and importantly temperature was maintained at 4 °C. Slides were taken out and washed with neutralization buffer thrice. In the next step, freshly prepared electrophoresis buffer (0.555 g Na-EDTA + 10 g NaOH cold 1.5 L dH2O maintained at 4 °C) was poured into electrophoresis tank and the tank was covered with ice to maintain the temperature before use. The washed slides were placed without any gap into the tank as writing place of slides on positive side of the electrode. Power supply for electrolysis tank was maintained on 25 V and maximum up to 280 A. Incubation of the slides was done in dark with electrophoresis buffer for 20 min to denature DNA and then DNA was run at 25 V per 260–280 A per 20 min. At last, slides were taken out and washed with neutralization buffer thrice gently and kept slides into the buffer until DAPI staining. For DAPI staining, 45 μL, (5 μg/mL DAPI) with 24 × 50 mm coverslip onto the slides was applied and kept into the humidity chamber at (papers with water) 4 °C before taking the pictures on fluorescent microscope.
Propidium iodide staining 
Hep3B cells were maintained in 5 cm2 Petri plates and cover slips were added at the time of splitting. After 24 h, DAAD2 (2 μM & 5 μM) was supplemented. While, media with less than 0.1% DMSO were also changed for negative controls. After 72 h of incubation, cover slips from both control and sample were taken out and as per given protocol cover slips were covered with annexin V/PI staining solution for 15 min. Photographs were taken at fluorescent microscope with detection range of 515–565 nm (green).
Microarray gene-expression analyses [25, 26]
Affymetrix Human Genome U133 Plus 2.0 Gene-Chips were used for whole-genome gene expression profiling experiments. Isolated total RNAs of treated and non-treated DAAD2-sensitive HEP3B and DAAD2-resistant SNU449 cells processed according to manufacturer’s instructions. Quality control analyses of microarray data performed using the BRB Array Tools V 4.2.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html). Triplicate samples from each sample type (12 samples in total) were used for the rest of the analyses. Normalizations of the raw data obtained from Gene-Chip Operating Software performed using the BRB Array Tools.
Lists of differentially expressed genes were determined using the Class Comparison Tool of the BRB Array Tools software. Genes differing more than 1.5 fold between control and DAAD-2 treated Hep3B cells, as well as control and DAAD-2 treated SNU449 cells were determined in separate lists.
Lists of differentially expressed genes were further analyzed via Connectivity Map (cmap) Tool  to further investigate molecular mechanisms responsible for resistance and sensitivity to DAAD-2 treatments. Gene expression signatures of drug-sensitive and drug-resistant HCC cells were used to determine similarities with signatures of previously characterized chemicals in the cmap database.
Immuno-peroxidase assay 
Hep3B cells were plated for 24 h in 12 well/plates at 50,000 cells on cover-slips for per each well. After DAAD2 treatment (2 μM & 5 μM) of 48 h, medium was aspirated. Cells were fixed with acetone and methanol mix solution (1:1) at −20 °C for 10 min. After aspiration of fixation solution, three times washing with 1XPBS performed for each plate. Hydrogen peroxide was added and hold for 10 min at room temperature in dark to block endogenous per oxidase activity. Washing step was repeated thrice again. Blocking was done using 10% FBS and 0.3% Triton X-100 in 1X PBS solution for 1 h in dark at room temperature. Anti human Caspase-3 (cleaved) was diluted in blocking solution (1:1000) and 100 μl of primary antibody was added on top of the cover slip drop by drop. After overnight incubation at 4 °C, cover-slips were washed with 1X PBS-T solution. 80 mL of Dako Envision secondary antibody was added on the top of the coverslips drop by drop and incubated in dark for 1 h at room temperature. Washing with 1X PBS-T solution performed twice. DAB solution was prepared and used as per manufacture guidelines. Counterstaining with hematoxylin solution was done for 5 min. at room temperature. After vigorous washing, coverslips were stained with blueing solution (0.1% Sodium Bicarbonate in distilled water) for 1 min at room temperature and rinsed with water. Finally, 85% of glycerol was added on top of each coverslip and put upside down on glass slide.
Western blotting 
To determine the protein level of differentially expressed genes of interest, cells were treated with 2 μM dose of DAAD2 for 72 h and the lysed with a Radioimmunoprecipitation Assay (RIPA) Buffer. Concentrations of protein lysates were measured by the conventional Bradford assay utilizing spectrophotometer at 595 nm. Sample protein concentrations were normalized in accordance with bovine serum albumin (BSA) standard curve. Around 30 μg of total proteins were subjected to gel electrophoresis using NuPAGE system with MES and/or MOPS buffers. Proteins were wet-transferred onto HyBond ECL nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5% BSA in TBS-T. Membranes were incubated with the primary antibodies either at room temperature or at 4 °C for overnight. Following primary antibody incubations and extensive washing with TBS-T, secondary antibodies conjugated with horse-radish peroxidase (HRP) incubated 1 h at room temperature. After an additional wash of half an hour with TBSt-T, chemiluminescent reaction was recorded using ECL prime western blot detection kit (Thermoscientific), as per manufacturer’s guidelines. X-ray films were exposed to the emitted chemiluminescence.
The 19th version of SPSS (SPSS, Chicago, IL, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations. Data were collected and expressed as the mean ± standard deviation of three independent experiments. Statistical analysis was performed by correlation of determination (R2) and probability test.