Cell culture
HeLa, MCF-7, A549, L929 and B16F10 were purchased from the cell repository of National Centre for Cell Sciences (NCCS), Pune, India. The multi-drug resistant EMT6/AR1 cell line was purchased from Sigma, St. Louis, MO, USA. HeLa and MCF-7 were maintained using Eagle’s Minimal Essential Medium, L929 and B16F10 cells were cultured in Dulbecco’s Modified Eagle’s Medium whereas A549 cells were cultured in F-12K nutrient medium, supplemented with 10% (v/v) Fetal bovine serum and 1% (v/v) antibiotic-antimycotic solution as described earlier [1]. EMT6/AR1 cell line was cultured as described previously [2]. All the cells were grown in optimized conditions in a humidified incubator in 5% CO2 at 37 °C (Sanyo, Tokyo, Japan).
Screening of combretastatin analogues
Cell-based screening assay was performed to check the potency of the synthesized combretastatin (1-21) analogues. The stocks of the combretastatin analogues were prepared in 100% cell culture grade DMSO. The compounds (1-21) were screened in HeLa cells using Sulforhodamine B assay [3]. Briefly, HeLa cells (10,000 cells/well) were seeded in 96-well cell culture plates and incubated for 24 hours for attachment. Fresh media was then added containing either vehicle (0.1% DMSO) or 25 nM of each of the compounds. After 24 hours, cells were fixed using 50% TCA for 1 hour at 4°C. The plates were then washed and dried completely. Sulforhodamine B (0.4% in 1% acetic acid) dye was added to the wells, incubated for 1 hour and later washed with 1% glacial acetic acid. When the plates dried completely, Tris chloride (10 mM, pH 10.0) was added to the wells and incubated for 30 minutes at room temperature. Absorbance readings were taken at 520 nm and percentage inhibition of cell proliferation was calculated using Spectramax M2e. Three independent sets of experiments were performed.
Determination of Half-maximal inhibitory concentration (IC50)
A549 cells (10,000 cells/well) were seeded into 96-well cell culture plate and incubated for 24 hours for attachment. Different concentrations of the 7 active compounds were added and incubated for 24 hours and further processed for sulforhodamine B assay.
The IC50 values were calculated using GraphPad software version 6.0 (Graph Pad Software, CA, USA) by fitting the values in an equation from non-linear regression
Y=Bottom+ ((Top-Bottom))/((1+10^((X-Log IC_50 ))))
where, Y is the response , X is the logarithmic concentration of the compound, Bottom is the minimum response, Top is the maximum response and IC50 is the concentration of the compound that gives a response mid-way between Top and Bottom.
The IC50 values for both C-13 and C-21 in L929 and B16F10 and only C-13 in HeLa, MCF-7, EMT6/AR1 cells were obtained similarly on incubating the cells for one cell cycle. All IC50 values were determined three times independently for each of the cell lines.
Determination of Dissociation constant (Kd) for binding of C-13 to tubulin
Tubulin was isolated from goat brain using two cycles of polymerization and depolymerization as described earlier [4] and the protein concentration was determined using Bradford’s method [5]. Tubulin (2 μM) was incubated in the absence and presence of different concentrations (0.5,1,2,5,10,15,20,25,30 and 40 μM) of C-13 in 25 mM PIPES buffer (pH 6.8) for 30 min at 25°C. The tryptophan fluorescence of tubulin was monitored in the absence and presence of C-13 using an excitation wavelength of 295 nm in a 0.3 cm path length cuvette (FP-6500 spectrofluorometer JASCO, Tokyo, Japan). Inner filter effect correction was done for the measured fluorescence intensities using the formula [2]
F_corrected=F_observed × anti log (A_excitation+A_emission )/2
where, Fcorrected is the corrected fluorescence intensity, Fobserved is the observed fluorescence intensity, Aexcitation is the absorbance of the compound at excitation wavelength (295 nm) and Aemission is the absorbance of the compound at emission wavelength (335 nm).
Dissociation constant (Kd) was calculated using GraphPad software version 6.0 (Graph Pad Software, CA, USA) by fitting the change in fluorescence in an equation [6]
ΔF =∆Fmax×(([P0]+[L0]+Kd)-√(〖([P0]+[L0]+Kd)〗^2-4[P0][L0]))/(2[P0])
where, ΔF is the change in fluorescence intensity on binding with the compound, ΔFmax is the maximum difference in the fluorescence intensity when the compound saturates the binding site of tubulin, P0 is the concentration of tubulin, L0 is the concentration of the compound. A similar experiment was carried out with C-21. The experiment was carried out five times for both C-13 and C-21.
Determination of binding site of C-13 on tubulin
Tubulin (5 μM) was incubated without and with C-13 (2,5,10,20,30,50,60 and 70 μM) in 25 mM PIPES buffer (pH 6.8) for 15 minutes at 37 °C. Subsequently, colchicine (10 μM) was added to the reaction mixture and incubated at 37°C for 45 minutes. Fluorescence spectra (410-500 nm) were monitored using excitation wavelength of 340 nm in a 0.3 cm path length cuvette (FP-6500 spectrofluorometer JASCO, Tokyo, Japan). The experiment was done four times. Inhibition constant (Ki) was calculated using GraphPad software version 6.0 (Graph Pad Software, CA, USA) by fitting the change in fluorescence in an equation [7]
K_i= (EC_50)/(1+ □(L/K_d ))
where, Ki is the inhibition constant, EC50 is the value at which the fluorescence intensity was reduced to half in the presence of the compound, L is the concentration of the compound and Kd is the dissociation constant of binding of tubulin to colchicine [8] A similar experiment was carried out with C-21. The experiment was carried out four times for both C-13 and C-21.
Competition assay with Compound 12
Compound 12 was found to bind at the colchicine-binding site on tubulin [6]. Tubulin (2 μM) was incubated without and with C-13 (2,5,10,15,20,30,50 and 60 μM) in 25 mM PIPES buffer (pH 6.8) for 20 minutes at 37 °C. Later, Compound 12 (5 μM) was added into the reaction milieu and incubated for 10 minutes at room temperature. Fluorescence spectra (410-500 nm) were measured using excitation wavelength of 350 nm in a 0.3 cm path length cuvette (FP-6500 spectrofluorometer JASCO, Tokyo, Japan). Inner filter effect correction was done [2] and percentage inhibition of compound 12 binding was determined using GraphPad software version 6.0 (Graph Pad Software, CA, USA) by fitting the change in fluorescence in the above described equation. The experiment was done thrice.
Immunofluorescence assay
HeLa or A549 cells (2.5 × 104 cells/well) were seeded onto glass coverslips and incubated for 24 hours. HeLa cells were treated either with vehicle (0.1% DMSO) or with 100 and 200 nM of C-13 and 35 and 70 nM of C-21 while A549 cells were treated with vehicle (0.1% DMSO) or with 100 and 200 nM of C-13 and 300 and 600 nM of C-21. Immunostaining was performed by staining the cells with α-tubulin antibody (1:400) or phospho-histone H3 (Serine 10) antibody (1:400), diluted in 2% BSA in PBS for 3 hours at room temperature or overnight at 4ºC. FITC conjugated IgG secondary antibody (1:400 dilution in 2% BSA in PBS) was later added to the wells and incubated for 1 hour at room temperature [6]. DNA was stained with Hoechst 33258 (10 µg/ml). Images were taken using Eclipse TE 2000U microscope (Nikon, Tokyo, Japan) at a magnification of 60X and processed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).
Effect of C-13 on Taxol-induced tubulin polymerization
Tubulin (18 μM) was incubated without and with different concentrations (30,60,100 and 150 μM) of C-13 in PEM buffer on ice for 10 minutes. Subsequently, Taxol and GTP was added to the reaction mixture to a final concentration of 10 µM Taxol and 1mM GTP. The assembly kinetics of tubulin was monitored by taking the absorbance of the reaction mixture at 350 nm (37 °C) using Spectramax M2e. Three independent experiments were performed.
Effect of C-13 on MAP-rich tubulin polymerization
MAP-rich tubulin was purified as described earlier [9]. MAP-rich tubulin (2 mg/ml) was incubated in the absence and presence of different concentrations (5,10,20,50,100 and 200 μM) of C-13 in PEM (25 mM PIPES pH 6.8, 3 mM MgCl2, 1 mM EGTA) buffer for 10 minutes on ice. Subsequently, 1 mM GTP was added to the reaction mixture and assembly kinetics was monitored using spectrofluorometer by taking absorbance at 350 nm (37 °C) [10]. The experiment was carried out four times.
Effect of C-13 on generation of ROS
Production of intracellular ROS was quantified using DCFDA (2′,7′-dichloro fluorescein diacetate) dye. HeLa cells (2 × 105 cells/ml) were seeded in 24-well cell culture plates and treated either with vehicle (0.1% DMSO) or with C-13 (75 and 200 nM) and incubated for 6 hours. CA-4 (20 nM) was used for comparison while H2O2 was used as a positive control. The cells were collected by centrifugation at 2500 rpm for 10 minutes and washed twice with PBS. Counting of cells was carried out using trypan blue dye. Cells were then incubated with DCFDA dye (25 μM) in dark at 37 °C for 1 hour. Fluorescence spectra (510-600 nm) was monitored using excitation wavelength of 488 nm by FP-6500 spectrofluorometer JASCO, Tokyo, Japan. Fluorescence intensity per cell at 525 nm was calculated and statistical significance was deduced using students t-test. Three independent sets of experiments were performed.
Determination of level of soluble and polymeric tubulin in the cells after C-13 treatment
Western blot analysis was carried out to determine the effect of C-13 on polymerized mass of microtubules in HeLa cells [11]. HeLa cells (in T-25 flasks) were incubated in the absence and presence of 120 and 240 nM C-13 for 24 hours. After 24 hours, cell pellet was collected and to the pellet PEM buffer with 25% glycerol and 0.5% Triton X-100 was added without disturbing the pellet. It was then incubated for 2 minutes at 37 ºC and the supernatant was removed gently from the top of the cell pellet. The supernatant represents soluble fraction of tubulin. From the remaining cell pellet, lysates were prepared [10] by incubating the cells with lysis buffer (Tris 20 mM, NaCl 200 mM, Triton X-100 – 0.1%, DTT 1mM at pH 7.2) for 1 hour followed by centrifugation and collection of supernatant representing the polymeric fraction of tubulin. Protein concentration was measured by Bradford’s assay [5]. 20 µg of protein from both soluble and polymeric fraction was taken and subjected to SDS-PAGE. The protein band was transferred onto the PVDF membrane via electro-blotting. Immunoblotting was carried out using α-tubulin monoclonal antibody and the intensity of the bands was analyzed using ImageJ software.
Scratch wound healing assay
A549 cells were seeded on coverslips (1×105 cells or upto 90% confluency). After the attachment of the cells, a wound was created on the coverslip by sterile 10 µl micropipette tip [6]. The coverslips were then treated with 0, 100 and 200 nM of C-12 and DIC images were taken at different time intervals (0, 6 and 10 hours or till the wound heals) to see the effect of C-13 on wound closure. The percentage of wound healed was calculated using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).
PI staining
HeLa cells (2.5 × 104 cells/well) were seeded onto glass coverslips and allowed to attach for 24 hours. The cells were then treated either with vehicle (0.1% DMSO) or with 75 and 200 nM C-13 and 20 nM CA-4 for 24 hours. The plates were then centrifuged at 2500 rpm for 15 minutes and washed with PBS. Live/dead cell staining with FITC Annexin V apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was carried out [2]. Cells were incubated with 5 µl of PI solution (50 µg/ml) and incubated for 15 minutes at room temperature in dark. Images were taken using Eclipse TE 2000U microscope (Nikon, Tokyo, Japan) at a magnification of 60X and processed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).