2.1 Cell culture
MCF7 and MDAMB231 breast cancer cells were provided by the Pathology Department of Dalian Medical University (Dalian, China). MCF7 cells were maintained in minimal essential medium (MEM) (HyClone™; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone™), and MDAMB231 cells were cultured in Dulbecco’s modified Eagle’s medium DMEM/F12 (HyClone™, Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (HyClone™, Thermo Fisher Scientific, Inc.) at 37ºC in a humidified atmosphere of 95% air and 5% CO2.
2.2 Cell Viability Assay
A Cell Counting Kit8 (CCK8) (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China) assay was used to examine cell viability. CCK8 assays are convenient to use because of Dojindo’s highly watersoluble tetrazolium salt. WST8 [2(2methoxy4nitrophenyl)3(4nitrophenyl)5(2,4disulfophenyl)2Htetrazolium, monosodium salt] produces a watersoluble formazan dye upon reduction in the presence of an electron mediator, 1methoxy phenazinium methylsulfate (PMS). WST8 is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. MCF7 and MDAMB231 cells were plated at a density of 5×103 cells per well in 96well plates and treated with different concentrations of parthenolide (purchased from SigmaAldrich, Co; now Merck Millipore, Burlington, MA, USA) for 24 and 48 h, respectively. Thereafter, 10 µl CCK solution and 100 µl fresh medium was added to each well and incubated in the dark at 37ºC for 2 h. After incubation, the absorbance of each well was measured at a wavelength of 452 nm. All experiments were repeated at ≥ 3 times. The halfmaximal lethal concentration (LC50) values were calculated according to the concentration of parthenolide required to inhibit cell viability by 50% compared with untreated cells.
2.3 4',6‑Diamidino‑2‑phenylindole (DAPI) staining
Nuclear fragmentation and chromatin condensation were analyzed through DAPI (eBioscience; Thermo Fisher Scientific, Inc.) staining. MCF7 and MDAMB231 cells were treated with parthenolide at different concentrations (0, 2, 4, and 8 µM; and 0, 2.5, and 5 µM) for 24 h. Cells were fixed with 3.7% formaldehyde, and subsequently stained with DAPI. The plates were then washed with PBS three times prior to viewing under a fluorescence microscope (Olympus Corporation, Tokyo Japan).
2.4 Annexin V/propidium Iodide Apoptosis Assay
Apoptosis assay was performed using a commercially available kit, as per the manufacturer’s protocol. MCF7 and MDAMB231 cells (0.1×106 cells) were plated in each well of 6well plates. After 24 h, parthenolide was added in different concentrations for 24 h. After 24 h treatment, cells were harvested by trypsinization and suspended in PBS. The cells were subsequently suspended in 500 µl binding buffer (Nanjing KeyGen Biotech. Co., Ltd.). Later, 5µl annexinV fluorescein isothiocyanate (FITC) (Nanjing KeyGen Biotech. Co., Ltd.) and 5µl propidium iodide (PI) (both purchased from Nanjing KeyGen Biotech. Co., Ltd.) were added, and the cells were incubated for 20 min in the dark. Finally, the samples were analyzed using flow cytometric analysis.
2.5 Western Blot Analysis
Antibodies raised against PI3K, AKT, phosphorylated (p)AKT, PTEN, mTOR, ATG13, ATG14, Beclin1, P62, caspase3 and Bcl2 were purchased from Proteintech Group, Inc. MCF7 and MDAMB231 cells were treated with parthenolide at different concentrations for 24 h. Total protein was extracted using a protein extraction kit (Nanjing KeyGen Biotech. Co., Ltd.), according to the manufacturer’s protocol, and then quantified with a bicinchoninic acid (BCA) kit (Nanjing KeyGen Biotech. Co., Ltd.). Protein lysates were heated at 100ºC for 5 min, subsequently separated with sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE), and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). Membranes were blocked with 5% nonfat milk in Trisbuffered saline/Tween 20 (TBST) for 1 h. PVDF membranes were incubated with the primary antibodies at 4ºC overnight, and then washed with TBST three times, followed by incubation with the secondary antibodies at 37ºC for 1 h. Protein bands were detected using an enhanced chemiluminescence (ECL) kit (Advansta Inc., Menlo Park, CA, USA). The density of each band was quantified with ImageJ software, and corrected by reference to the expression value for GAPDH.
2.6 Immunofluorescence Analysis
Cells were seeded in a 24well plate (1×104 cells/well) and allowed to attach overnight. After treatment with parthenolide for an additional 24 h, the cells were fixed with methanol for 10 min, permeabilized with 0.1% Triton X100 for 10 min and blocked with 5% BSA (SigmaAldrich; now Thermo Fisher Scientific, Inc.) for 1 h, after which cells were incubated overnight with primary antibody (antiBeclin 1). Cells were washed and incubated with AlexaFluor 596 secondary antibody(Invitrogen; Thermo Fisher Scientific, Inc.) on the following day. After washing, DNA was counterstained with DAPI, and images were captured using a fluorescence microscope.
2.7 Statistical Analysis
Prism 5.0 software (GraphPad Software Inc, San Diego, CA, USA) were used for all statistical analyses. Results are presented as the mean ± standard deviation (SD) or standard error of the mean. Statistical significance was determined using Student’s ttest. P < 0.05 was considered to indicate a statistically significant value.