7,8-Dihydroxy-3-arylcoumarin Induces Cell Death Through S-Phase Arrest in MDA-MB-231 Breast Cancer Cells

Materials and Methods

Chemicals. F12K and DMEM-F12 media, penicillin-streptomycin anti-biotic solution (100×), fetal bovine serum (FBS), trypsin-EDTA solution (1×), phosphate buffered saline (PBS), 50% glutaraldehyde, crystal violet, IGEPAL CA-630, propidium iodide, tamoxifen, docetaxel and RNase were obtained from Sigma-Aldrich company (St. Louis, MO, USA). Total protein cell lysis buffer was obtained from AMRESCO (Solon, OH, USA). The EZ Block protease inhibitor cocktail (BioVision, Milpitas, CA, USA) was obtained from VWR International (Suwanee, GA, USA). p21^{Waf1/Cip1(12D1)} rabbit mAb 2947, CDK2 rabbit mAb 2556, cyclin A2 mouse mAb 4639, cyclin B1 rabbit mAb 12231, cyclin E2 Ab 4132, CDK 4 rabbit mAb 12790, and CDK6 mouse mAb 3136 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell line maintenance. The human lung (A549), breast (MDA-MB-231) and prostate (PC-3) cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as per the guidelines supplied. The cells were maintained in F12K (A549, PC-3) and DMEM-F12 (MDA-MB-231) media containing 100 units of penicillin/ml, 100 μg of streptomycin/ml, 2 mM L-glutamine and 10% FBS in T-75 cm² flasks at 37°C in a 5% CO₂ incubator.

Treatment and measurement of cell viability. The cells (MRC-9, MDA-MB-231 or PC-3) were plated at a density of 5×10⁴ cells per well in a polystyrene, flat bottom 24-well microtiter plate (Corning Costar, Rochester, NY, USA) in cell type pertaining medium containing 10% FBS and allowed to stabilize overnight in a 5% CO₂ incubator at 37°C. Afterwards, the cells were treated with compounds (7a-i) at different concentrations of (0, 10, 25, 50, 75 and 100 μM) in a final volume of 1 ml per well in triplicate wells for each treatment for 48 h at 37°C in a 5% CO₂ incubator. All studies were repeated at least twice. At the end of incubation period, the viability was evaluated by crystal violet dye uptake assay and cytotoxic concentration (CC₅₀) was calculated according to our previously reported method (6). Glutaraldehyde (400 μl of 0.25%) was added to each well and incubated for 30 min at room temperature (RT) to fix the cells. The glutaraldehyde (0.07% final concentration in the well) in the crystal violet dye staining assay procedure fixed the viable cells after treatment with the compound. The plates were rinsed with water to wash off the dead cells and dried under airflow inside a laminar hood for 5-10 min. Crystal violet (400 μl of 0.1%) was added to each well, incubated for 15 min, washed and dried. To solubilize the dye, 1 ml of 0.05 M sodium phosphate solution (monobasic) in 50% ethyl alcohol was added to each well and the plates were read at 540 nm in a plate reader (Bio-Tek EL800 Plate Reader, Winooski, VT, USA). The mean absorbance value of control samples was considered as 100% and the treated sample percentages were calculated by comparing the treated samples absorbance with the control.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

The cytotoxic activity of the most active compound 7h. The cells were treated with compound 7h for 48 h and the viability was measured by crystal violet binding assay.

Cell cycle analysis. Cell cycle analysis was carried out according to our previous report (6) by treating 1.3×10⁶ MDA-MB-231 cells / T-25 flask in complete medium, in triplicate flasks for each treatment with 7h (0, 5, 10 μM) for 24 h. At the end of incubation, cells were trypsinized, pelleted, washed with PBS and resuspended in 1 ml of Vindelov's reagent (PBS 1× containing ribonuclease A (10 μg/ml), propidium iodide (7.5 μg/ml) and IGEPAL CA- 630 (1 μl/ml). The cells were stained at 4°C overnight and analyzed using a flow cytometer for cell cycle analysis at different phases, at a low flow rate of ~150 cells/sec or less.

Preparation of samples for western blot. Approximately 4.4×10⁶ MDA-MB-231 cells were plated in T-175 cm² flasks in complete DMEM-F12 medium and allowed to stabilize overnight. The cells were treated with 7h at different concentrations (0, 20 and 40 μM) and incubated for 24 h in a 5% CO₂ incubator at 37°C. At the end of incubation, the cells were trypsinized and pelleted by centrifuging at 2,500 rpm for 5 min. The control and treated cell pellets were suspended in 100 μl of total protein cell lysis buffer (AMRESCO, Solon, OH, USA) containing EZBlock protease inhibitor cocktail (BioVision, Milpitas, California, USA) and incubated on ice for 30 min with periodic vortexing. The tubes were centrifuged at 14,000 g for 20 min at 4°C. The supernatant was transferred to a fresh tube and stored at a −80°C freezer till further use.

Protein estimation. The protein concentration of cell lysates was quantitatively measured using the Pierce BCA Protein Assay (Life technologies, Grand Island, NY, USA). Different concentrations of albumin standard (BSA) and working reagent were prepared according to the kit's manual. Each standard concentration and 1:10 diluted cell lysate (25 μl) were pipetted in triplicate wells in a 96-well plate and 200 μl of working reagent was added to each well. The plate was incubated at 37°C for 30 min and then read at 540 nm in a plate reader (Bio-Tek EL800 Plate Reader, Winooski, VT, USA). The protein concentration of cell lysate was determined using the generated standard curve by the KC Junior plate reader software.

Western blot analysis. The protein (30 μg) from each sample in 1X SDS gel loading buffer was loaded into 15% SDS PAGE, run at 80 V and electro-transferred to a nitrocellulose membrane at 30 V for 16 h. The western blot analysis was performed according to manual instructions by Cell Signaling Technology. The nitrocellulose membrane was washed with 25 ml of Tris buffered saline with 0.5% Tween 20 (TBS/T) buffer for 5 min at RT. The membrane was incubated in 10 ml of blocking buffer (5% Non-fat milk in TBS/T buffer) for 1 h at RT, and incubated with a primary antibody (1:1,000 dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. Later, the membrane was washed with 15 ml of TBS/T for 5 min each (3×), incubated with the appropriate HRP-conjugated secondary antibody (1:2,000) in 10 ml of blocking buffer with gentle agitation for 1 h at RT and then washed for 5 min each (3×) with 15 ml of TBS/T. The membrane was then incubated with 10 ml LumiGLO® (Cell Signaling Technology, Danvers, MA, USA) (0.5 ml 20× LumiGLO®, 0.5 ml 20× Peroxide and 9.0 ml Milli-Q water) with gentle agitation for 5 min at RT. The excess developing solution was drained from the membrane, and the membrane was wrapped in plastic wrap followed by imaging using ChemiDoc machine. The density of the bands on the blot was quantified using the Un-Scan-It get TM program (Silk scientific, Inc., Orem, UT, USA).

Statistical analysis. The data were presented as mean±standard deviation (SD, n=3). All treated cells data were presented as percentage values in comparison to the untreated control (100%). The data were analyzed for significance by one-way ANOVA, and then compared by Dunnett's multiple comparison tests, using the GraphPad Prism Software, version 3.00 (GraphPad Software, Inc., San Diego, CA, USA). Differences from the respective untreated control were considered statistically significant when p<0.05. The viability and CC₅₀ graphs were plotted in Prizm 3.00 software (GraphPad Software, Inc., San Diego, CA, USA). The CC₅₀ value was deduced from the point of the graph where the live and dead cells lines meet (the dose at which 50% of the cells dies) using the Prizm 5 software.