Reagents and antibodies
Demethylzeylasteral (T-96, molecular formula: C29H36O6, molecular weight: 480, T3418), Z-VAD-FMK (T6013), Rapamycin (T1537), Chloroquine (T8689) and Cisplatin (T1564) were purchased from TOPSCIENCE (Shanghai, China).The reagents including 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Penicillin-Streptomycin, propidium iodide (PI) were purchased from Sigma-Aldrich (MO, USA).Cell culture medium including DMEM (SH30022.01B) and F12K (SH30526.01) were ordered from HyClone (Logan, USA) while fetal bovine serum (FBS) was purchased from Natocor (Cordoba, Argentina). All the primary antibodies involved in present study were bought from Cell Signaling Technology (MA, USA) and the secondary antibodies were purchased from LI-COR Biosciences (NE, USA).
Cell culture
Human CaP cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC, VA, USA). Both cell lines were mycoplasma-free and have been authenticated using STR profiling. Cells were cultured in DMEM/HIGH glucose culture medium and F12K supplemented with 10% fetal bovine serum, 1%Penicillin-Streptomycin at 37 °C in a humidified incubator containing 5% CO2.
Cell viability assay
DU145 and PC3 cells were plated into 96-well plates (3000 cells/well) and incubated with various concentrations of T-96, cisplatin, T96 and cisplatin for designed times at 37 °C. Then, 1% MTT was added (20 μL/well) and incubated at 37 °C for 4 h. After the crystals dissolution in DMSO, the absorbance value at 570 nm was detected in a microplate reader (Bio-Tek, VT, USA), and analyzed by GraphPad Prism 7.0. All experiments were performed in triplicates independently.
Colony formation assay
DU145 and PC3 cells were plated into six-well plates (500 cells/well) and cultured for 7 days under designed drug conditions. Colonies were washed twice with PBS (Phosphate Buffered Saline), fixed with 4% PFA (Paraformaldehyde) for 15 min at room temperature and then stained with 1% crystal violet for 30 min. All statistical measurements were performed in triplicates independently.
5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
Proliferation of human CaP cells was investigated using BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075S, Beyotime, Shanghai, China) according to the manufacturer’s protocols. Briefly, logarithmic growth stage cells were seeded into 24-well plate prior to treatment with different concentrations of T-96 for 48 h. DU145 and PC3 cells were incubated with 10μMEdU working solution in the dark room for 2 h at 37°C. Next, the labeled cells were fixed with 4% PFA for 20 min, permeabilized with 0.1% Triton-X100 for 10 min,and then stained with click reaction solution in the dark room for 30min. In addition, cells were incubated with 1 mg/mL DAPI-PBS for 30 min. The images were captured with fluorescence microscope (Olympus, Tokyo, Japan)and the percentage of EdU-positive cells was calculatedby GraphPad Prism 7.0.
Flow cytometry analysis
DU145 and PC3 cells during the logarithmic growth phase were harvested and transferred into six-well plates with 30% cell density. After exposure to the indicated concentrations of T-96, Z-VAD-FMK, T-96 and Z-VAD-FMK, rapamycin, T-96 and rapamycin, chloroquine, T-96 and chloroquine, cisplatin, and T-96 and cisplatin for 48 h respectively, cells were collected and employed in flow cytometry analysis. For cell cycle assay, cells were harvested, fixed with 70% ethanol in 4 °C for 24 h, and then washed three times with PBS. Subsequently, PBS containing 50μg/ml PI and 100μmg/ml RNase (Sigma Aldrich, USA) were added to the cells for 0.5 h incubation at 37 °C. Finally, the stained cells were analyzed using BD AccuriTM C6 flow cytometry (BD Biosciences, USA) and FlowJo 7.6 software. For cell apoptosis assay, the collected cells were subjected to an AnnexinV-FITC/PI apoptosis assay kit (C1062S, Beyotime, China) according to the manufacturer's protocols. The fluorescence-positive cells were analyzed using a flow cytometer within 1 h to assess the proportion of apoptotic cells, and the apoptotic rate was visualized using FlowJo 7.6.
Western blotting analysis
The harvested DU145 and PC3 cells were lysed in RIPA buffer (P0013B, Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktail (P1046, Beyotime, Shanghai, China) at 4 °C for 30 min, and then the protein concentration was quantified using a BCA Protein Assay Kit (P0010S, Beyotime, Shanghai, China). Protein lysates (30 μg/lane) were loaded on appropriate SDS gels, resolved by electrophoresis and transferred to PVDF membranes (IPVH00010, Millipore, Billerica, MA, USA). After incubation with 5% BSA in TBST for 2 h at room temperature, the membranes were incubated with the indicated primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with the corresponding IRDye 800CW donkey anti-mouse IgG (H + L) or IRDye 680LT donkey anti-rabbit IgG (H + L) secondary antibody, and immunoreactivity was visualized by an odyssey two-color infrared fluorescence imaging system (LI-COR Biosciences, NE, USA). b-tubulin was used as the loading control.
Determination of reactive oxygen species (ROS) formation
Intracellular ROS level was measured using a ROS Assay Kit (S0033S, Beyotime, Shanghai, China) according to the manufacturer's protocols. Briefly, after treatment with or withoutT-96, the collected CaP cells were incubated with serum-free DMEM or F12K containing DCFH-DA (10 μM) for 20 min. Subsequently, cell suspensions were centrifuged and washed three times with serum-free DMEM, and then visualized by a fluorescence microscope (IX53/DP80, Olympus Corporation, Japan).
Ca2+ signals measurement
The signals of intracellular Ca2+were determined with Fluo-4 AM (S1060, Beyotime, Shanghai, China) according to manufacturers’ protocols. Briefly, CaP cells were collected and washed three times with PBS, and then incubated with 1 μMFluo-4 AM in PBS for 30 min at 37 °C. Next, cells loading with Fluo-4 AM were washed with PBS and incubated for an additional 20 min to ensure that Fluo-4 AM was completely transformed into Fluo-4.The fluorescent intensity was captured by a fluorescence microscope (IX53/DP80, Olympus Corporation, Japan).
Autophagy analysis
For autophagy analysis, DU145 and PC3 cells stably expressed GFP-LC3B and mCherry-EGFP-LC3B via a lentivirus infection process were used. In brief, the lentivirus packaging vectors (Pspax2, pMD2G) and GFP-LC3B/mCherry-GFP-LC3B were co-transfected into HEK293T cells by the Lipo8000 transfection reagent (C0533,Beyotime, Shanghai, China) according to the manufacturer’s protocols. Viral particles were collected after 48 h transfection and then infected into the CaP cells in the assistance of Polybrene (10 μg/mL). Subsequently, cells were screened with puromycin (10 μg/mL) to obtain the stable cells expressing GFP-LC3B or mcherry-EGFP-LC3B. After treatment with designed concentrations of T-96, the transgenic cells were analyzed using the high content analysis system-operetta CLSTM (PerkinElmer, Waltham, MA, USA).
Statistical analysis
All experiments were performed in triplicates independently. GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis. Data are presented as the mean ±Standard deviation, and the ANOVA method was used to compare differences between groups. P<0.05 was considered to indicate a statistically significant difference.