DOX and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO, USA). DOX was dissolved in DMSO at a concentration of 40 mM, and stored at −20°C. PD was purchased from Chengdu Best Reagent Co., LTD (Chengdu, Sichuan, China), and dissolved in DMSO at a concentration of 40 mM and stored at −20°C. 3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) were purchased from Molecular Probes (Eugene, OR, USA). RPMI 1640 medium, fetal bovine serum (FBS) and antibiotics were purchased from Gibco (Carlsbad, CA, USA). Primary antibodies [i.e., poly (ADP-ribose) polymerase (PARP) and β-actin], and secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).
Cell line and culture
MCF-7 (ATCC number: HTB-22) and MDA-MB-231 (ATCC number: HTB-26) human breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in a RPMI 1640 medium supplemented with 10% (v/v) FBS and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin). The cells were grown in a 5% CO2 incubator at 37°C.
The effects of DOX (0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, and 5 μM), PD (1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM), and DOX + PD (0.3125 μM DOX + 1.25 μM PD, 0.625 μM DOX + 2.5 μM PD, 1.25 μM DOX + 5 μM PD, 2.5 μM DOX + 10 μM PD, and 5 μM DOX + 20 μM PD) on MCF-7 and MDA-MB-231 cell proliferation were examined by the MTT assay . Exponentially growing MCF-7 and MDA-MB-231 cells were seeded onto 96-well plates. Upon reaching approximately 70% to 80% confluence, the cells were incubated with the indicated compounds for 48 h. Cell viability was determined by incubating the cells in a medium containing 1 mg/mL MTT for 4 h. Then, 100 μL of DMSO was added to solubilize the formazan by shaking for 10 min in the dark. The absorbance at 570 nm was recorded with a microplate reader (Perkin Elmer, 1420 Multilabel Counter Victor3, Wellesley, MA, USA).
Observation of morphological changes
Exponentially growing MCF-7 and MDA-MB-231 cells were seeded onto 6-well plates. After adhesion, cells were treated with 2.5 μM DOX, 10 μM PD or 2.5 μM DOX + 10 μM PD for 24 h. Then, the cellular morphology was observed with an AxioCam HRC CCD camera (Carl Zeiss, Germany).
Western blot analysis
MCF-7 and MDA-MB-231 cells were treated with and without 2.5 μM DOX, 10 μM PD, and 2.5 μM DOX + 10 μM PD for 24 h. Total protein was extracted with a radioimmunoprecipitation lysis buffer containing 1% phenylmethanesulfonyl fluoride and 1% protease inhibitor cocktail for 25 min. The protein concentrations were determined with the BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane followed by blocking in 5% non-fat dried milk for 1 h. The membrane was incubated with specific primary antibodies against PARP (1:1000) and β-actin (1:2000) followed by incubation with the corresponding secondary antibodies. The specific protein bands were visualized with an ECL advanced western blot analysis detection kit (BD Biosciences, Bedford, MA, USA).
Mitochondrial membrane potential assay by JC-1 staining
The mitochondrial membrane potential (MMP) assay of intact cells was stained with JC-1 before visual determination . JC-1 probe is a dual-emission fluorescent dye that can reflect the changes in MMP. It forms aggregates that result in a red emission in normal polarized mitochondria, while it forms monomers that emit green fluorescence on the depolarized mitochondrial membrane, the MMP depolarized is an early phenomenon of apoptosis . MCF-7 and MDA-MB-231 cells were seeded onto 96-well plates. After a 24 h incubation for adhesion, the cells were treated with the indicated compounds for 4 h. The medium was removed and the cells were incubated with JC-1 probe for 30 min. Then, the medium with the probe was removed and the cells were rinsed with phosphate-buffered saline (PBS). The cells were observed with a fluorescent microscope, and images were obtained with an Axiovert 200 fluorescent inverted microscope (Carl Zeiss) and an AxioCam HRc CCD camera (Carl Zeiss).
DOX uptake detection
The intracellular uptake of DOX was examined as previously described . Briefly, MCF-7 and MDA-MB-231 cells were seeded onto 12-well plates. After adhesion, the cells were treated with and without the indicated compounds for 1 h. The cells were then washed with PBS three times and re-suspended in PBS. The mean fluorescence intensities of the cells were determined with a fluorescence-activated cell sorting (FACS) cytometer (FACSCalibur, Becton Dickinson, San Jose, CA, USA).
All experiments were repeated at least three times. The mean ± standard deviation (SD) was determined for each group. Statistical analysis was performed with one-way analysis of variance (one-way ANOVA) and Tukey’s test. Differences were considered statistically significant when P < 0.05, and the exact P values were shown unless P < 0.001. The concentration dependence was visually determined from the graphs.