The MDA-MB-231 cell line is isolated from the pleural effusion of human metastatic breast carcinoma. The carcinoma cells lack the expression levels of ER-α, PR and amplified HER-2, and represent an experimental model for TNBC (12,13). This cell line was obtained from the American Type Culture Collection (ATCC), and the cells were maintained in RPMI-1640 medium with L-glutamine and 5% fetal bovine serum (Life Technologies; Thermo Fisher Scientific, Inc.) following the protocol a recommended by ATCC.

A non-fractionated aqueous extract of PC was prepared by boiling 20 g of seeds of PC provided by the author GYCW in 200 ml of deionized distilled water followed by sequential boiling and centrifugation to obtain a concentrated extract at a concentration of 20 g extract in 20 ml deionized distilled water (7,8,10). This stock solution was subsequently diluted in the culture medium to obtain a concentration of 1 mg extract/1 ml. This diluted stock solution was subsequently used on the cell cultures at the µg/ml concentration range.

The dose response effect of PC was determined by examining cell viability using the Cell Titre Glo assay (Promega Corporation) as recommended in the protocol provided by the manufacturer. This assay measures relative luminescent units (RLUs) as a quantitative end point. Briefly, 5.0x10³ cells were seeded in 96-well plates and treated with PC at a 1-100 µg/ml concentration range for 6 days at 37˚C in a CO₂ incubator. The medium containing PC was replenished every 72 h. Untreated cultures maintained in the culture medium represented the control. Cell viability was measured on day 7 using a Fluoroskan plate reader (Thermo Fisher Scientific, Inc.), and was expressed as RLUs. The data obtained from the dose response experiment were extrapolated to identify the minimally effective (IC₂₅), half maximum (IC₅₀) and maximally cytostatic (IC₉₀) inhibitory concentrations for the cells treated with PC.

This assay was performed following the optimized protocol. Briefly, MDA-MB-231 cell suspensions, at a density of 5x10⁵ per ml prepared in 0.33% agar with a concentration range of 1-80 µg/ml PC. The untreated cell suspension represented the control. These cell suspensions were overlaid on a basement matrix of 0.6% agar. The cultures were incubated at 37˚C in a CO₂ incubator for 21 days. The AI colonies were fixed in 4% phosphate-buffered formaldehyde (cat. no. 100494, Sigma-Aldrich; Merck KGaA, stained with 0.005% crystal violet (cat. no. C-6158, Sigma-Aldrich; Merck KGaA), overnight at room temperature. The colony counts were determined at x10 magnification. The data were expressed as the AI colony number. These data were extrapolated to identify the IC₂₅, IC₅₀ and IC₉₀ inhibitory concentrations.

For the cell cycle analysis, 5x10⁵ cells were seeded in T-25 flasks and treated for 24 h after seeding with 3, 6 and 12 µg/ml of PC for 48 h. The cells were harvested by trypsinization, pelleted at 500 x g, and washed twice with cold PBS. The cells were then fixed with cold 70% ethanol, washed with cold PBS, and stained with 50 µg/ml propidium iodide in PBS (Sigma-Aldrich; Merck KGaA), followed by the addition of 10 µg/ml ribonuclease (Sigma-Aldrich; Merck KGaA), and incubation at 37˚C for 20 min and a 4-h incubation of the mixture at 4˚C in the dark. Cell cycle progression was monitored following an optimized protocol that included the fluorescence-assisted sorting of PI-stained cells, with the use of a 488 nm excitation filter and a 520 nm band pass filter, and gating of the sorted cells on a forward versus side scatter. The DNA content was analyzed using a BD FACScan Flow Cytometer (BD Biosciences) and analyzed by FCS Express software version 306 (De Novo Software). The flow cytometry-based analyses determined the distribution of individual cell populations in the G₁ (quiescent), S, G₂/M (proliferative) and sub G₀ (apoptotic) phases of the cell cycle. These primary data were expressed as the G₁: S + G₂/M ratio and the percentage sub G₀ population to assess altered cell cycle progression and cellular apoptosis, respectively.

Cells were seeded in 10-cm dishes at 70% confluence 1 day prior to treatment. Cells were treated with 2, 4 and 6 µg/ml of PC and the cultures were incubated for 48 h in a CO₂ incubator at 37˚C. Cells were harvested and immediately lysed with RIPA buffer containing protease inhibitors (Sigma-Aldrich; Merck KGaA), and centrifuged at 4˚C for 15 min at 10,000 x g. The protein content of the lysate was quantified using the Bradford method, and an equal quantity of protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories) and was blocked at 4˚C overnight with 5% non-fat dry milk followed by incubation with primary and secondary antibodies (Table I). The chemiluminescent signal was developed with ECL-plus reagent (Bio-Rad, Laboratories, Inc.) and detected by autoradiography. The antibody for β-actin was used as the reference protein for western blot analysis. The signal intensity in the western blots was quantified as arbitrary scanning units (ASU) using Molecular Imager GS800 and Quantity One software (Bio-Rad Laboratories, Inc.)

Caspase-3/7 activity in the MDA-MB-231 cells was measured using a Caspase-Glo assay kit (Promega Corporation). Briefly, the cells treated with PC were homogenized by sonication in homogenization buffer (25 mmol/l HEPES, pH 7.5, 5 mmol/l MgCl₂, and 1 mmol/l EGTA) and protease inhibitors (all from Sigma-Aldrich; Merck KGaA). The homogenate was centrifuged at 6,500 x g at 4˚C for 15 min and the supernatant was used for experiments. To 10 µl of the supernatant, equal volume of the assay reagent was added, and the mixture was incubated at room temperature for 2 h. The luminescence was measured using a luminometer (Thermo Fisher Scientific, Inc.). The data were expressed as RLUs.

The data were obtained from replicate experiments for the dose response, AI growth, cell cycle progression and caspase-3/7 activity assays and are expressed as the mean ± SD, n=3 per treatment group. The comparisons of statistically significant differences between the common control and multiple treatment groups were analyzed using one-way analysis of variance with Dunnett's multiple comparison test as a post-hoc with a threshold of α=0.05. P<0.05 was considered to indicate a statistically significant difference.

The data on the growth inhibitory effects of PC are presented in Table II. These data exhibited a dose-dependent inhibitory response in MDA-MB-231 cells. As indicated by the RLU values, treatment with 6, 12 and 24 µg/ml PC for 7 days resulted in a decrease of 67.7% (P=0.020), in a decrease of 72.3% (P=0.014) and in a decrease of 84.8% (P=0.001), respectively, relative to the untreated control. These data identified the IC₂₅ as 2.4±0.3 µg/ml, the IC₅₀ as 4.4±1.0 µg/ml and the IC₉₀ as 25.5±1.8 µg/ml, respectively, relative to the untreated control.

The data on inhibition of AI colony formation by PC are presented in Tables III and SI. A 21-day treatment with PC identified the IC₂₅ as 5 µg (P=0.050), the IC₅₀ as 10 µg (P=0.037) and the IC₉₀ as 80 µg (P=0.014), respectively, relative to the untreated control.

The effect of PC on cell cycle progression is presented in Tables IV, SII and SIII. The data expressed as the G₁: S + G₂/M ratio demonstrated that treatment with PC at 6 µg/ml exhibited a 2.5-fold increase (P=0.020) and treatment with PC at 12 µg/ml exhibited a 3.5-fold (P=0.014) increase respectively, relative to the untreated control.

The data presented in Fig. 1 are the results of the analyses of the effects of PC on the RB signaling. The proteins, cyclin D1, CDK4, CDK6 and p-RB, involved in this pathway exhibited a dose-dependent decrease, relative to the untreated control.

The data presented in Tables V and SIV are the results of the analyses of the effect of PC on cellular apoptosis by quantifying the status of cells in sub G₀ (apoptotic) phase of the cell cycle and the pro-apoptotic caspase-3/7 activity. Treatment with PC resulted in a dose-dependent increase in the sub G₀ phase, as well as an increase in the caspase activity, relative to the untreated control.