Cell lines, reagents, and antibodies
Human prostate cancer LNCaP, 22RV1, PC-3 cells were obtained from ATCC (Manassas, VA, USA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) plus 100 U/ml penicillin/streptomycin and 2 mmol/l L-glutamine. Antibodies for β-Catenin (ab32572), cyclin D1(ab16663), c-Myc (ab32072) were purchased from Abcam (Cambridge, MA, USA). β-actin (MABT825) were purchased from Sigma (Missouri, MO, USA). Alpha-tubulin (11224–1-AP), Lamin A/C (10298–1-AP), and HRP-conjugated goat anti-mouse IgG (SA00001–1) and goat anti-rabbit IgG (SA00001–2) were obtained from Proteintech Group (Chicago, IL, USA). Propidium Iodide (PI) and CCK8 kit were ordered from Beyotime (Shanghai, China).
Bortezomib (HY-10227), MG132 (HY-13259), mitoxantrone (HY-13502A) and Carboxyfluorescein diacetate succinimidyl ester (CFSE) (HY-D0938) were obtained from MCE (New Jersey, NJ). RIPA Buffer (#9806) were obtained from Cell Signaling (Danvers, MA, USA). FBS, RPMI 1640 medium, penicillin/streptomycin, and L-glutamine were obtained from Gibco (by ThermoFisher Scientific, Shanghai, China). Trizol reagent was ordered from Invitrogen (by ThermoFisher Scientific, Shanghai, China).
Western blotting and real-time PCR
Western blotting was performed as previously described [22]. Briefly, cells were lysed using RIPA lysis buffer containing complete protease inhibitor cocktail (Roche, Switzerland). Cytoplasmic and nuclear protein were isolated using the Cytoplasmic and Nuclear Fractionation kit (Beyotime, Shanghai, China). Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transmembrane. The PVDF membranes were incubated with indicated primary antibodies overnight at 4 °C and then incubated with secondary antibody for 1 h at room temperature. Staining was visualized with ECL reagent (Santa Cruz Biotech).
For real-time quantitative PCR, total RNA was extracted using the Trizol reagent according to the manufacturer’s instructions. cDNAs were synthesized was performed using reverse transcription (RT) kit (Applied Biosystems, Foster City, CA). The RT products (0.5 μl) were subjected to real-time PCR using of SYBR Green. 18S rRNA was used as an endogenous control. Quantitative of SYBR Green signal was performed with LightCycler® 480 (ROCHE Diagnostic Spa, Mannheim, Germany). The relative expression level was calculated with the 2[−∆∆Ct] method and expressed as a “change fold”. All data were normalized to endogenous control (18S rRNA) expression. The sequence of primers were designed as follows: 18 s rRNA: sense, 5′-GAG GAT GAG GTG GAA CGT GT-3′ and antisense, 5′- GGA CCT GGC TGT ATT TTC CA-3′; β-Catenin: sense, 5′- GTT CAG TTG CTT GTT CGT GC-3′ and antisense, 5′- GTT GTG AAC ATC CCG AGC TAG-3′; cyclin D1: sense, 5′- CAT CTA CAC CGA CAA CTC CAT C-3′ and antisense, 5′-TCT GGC ATT TTG GAG AGG AAG-3′; c-Myc: sense, 5′-TTC GGG TAG TGG AAA ACC AG-3′ and antisense, 5′- AGT AGA AAT ACG GCT GCA CC-3′; MMP7: sense, 5′- TTC CAA AGT GG TCA CCT ACA G-3′; and antisense, 5′- AGT TCC CCA TAC AAC TTT CCT G-3′; Axin2: sense, 5′- TGT CCA GCA AAA CTC TGA GG-3′; and antisense, 5′- GTG CAA AGA CAT AGC CAG AAC-3′.
Apoptosis analysis, cell proliferation, and cell cycle
Apoptosis was evaluated using a Dead Cell Apoptosis Kit (ThermoFisher Scientific, catalog #V13242) as previously described [23]. Briefly, 5 × 105 cells treated with indicated drugs were incubated with 5 μl FITC-conjugated Annexin-V antibody and 5 μl PI for 10 min according to manufacturer’s instructions. The data was measured by flow cytometry (Beckman CytoFLEX, Germany) and analysed using the CytExpert software (Beckman Coulter, Brea, CA, USA).
Proliferation was detected by CFSE assay and CCK8 assay. CFSE is cleaved by esterase in live cells. Cleaved CFSE produces green fluorescence. The fluorescent in cells will reduce due to divide equally into daughter cells during cell division. Therefore, proliferation of cells can be tracked by fluorescent of cleaved CFSE [24]. CFSE-labelled prostate cancer cells were treated with indicated treatments for 24 h. CFSE was determined by flow cytometry. Mean fluorescence intensity (MFI) was determined by flow cytometric analysis (Beckman CytoFLEX, Germany).
CCK-8 Assay was performed as previously described. Briefly, 1 × 105 cells were incubated in a 96-well culture plate. After incubation for 24 h, the cell viability was measured by a Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) according to the manufacture’s protocol. The absorbance at 450 nm was determined by CytExpert software (Beckman Coulter, Brea, CA, USA).
To evaluate cell cycle, cells were stained with PI solution. Cells were fixed with cold ethanol overnight at °C followed by RNA digestion using RNase A at 37 °C for 30 min. PI fluorescence was determined by flow cytometry (Beckman CytoFLEX, Germany). Cell cycle was determined by modfitLT software (Verity Software House, Topsham, ME).
Immunohistochemistry and scoring
The immunohistochemistry (IHC) staining procedure and scoring in our publications [22]. Briefly, tissues were fixed in 4% formalin. Paraffin-embedded tissue sections (4 μm) were subjected to dewaxing and rehydration, followed by inactivation of endogenous peroxidase activity and antigen retrieval. Tissue sections were incubated with indicated primary antibodies. Immunosignals were visualized with a DAKO LSAB System (Dako, Carpenteria, CA, USA). IHC scoring was perform as previously described [22, 25, 26].
Proteasome activity assay
Proteasome activity was measured as previously described [27, 28]. Briefly, cells were lysed with the lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 mM ATP, 1 mM DTT and 10% glycerol). Equal amount of proteins was incubated with the substrate (LLVY-AMC as chymotrypsin-like activity) for 1 h at 30 °C and the free AMC fluorescence was determined by Cytation-i5 Cell Imaging Reader (Biotek, USA).
Animal experiments
Severe combined immunocompromised (SCID Beige) mice were acquired from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were housed in specific pathogen-free (SPF) conditions. To construct the mice Xenograft model, prostate cancer cells were implanted subcutaneously into the flanks of 6-week-old male SCID mice. Two week after injection, mice were randomly divided into four groups and treated with vehicle, BZM (1 mg/kg, intraperitoneally, twice weekly), MTX (3 mg/kg, intraperitoneally, every day), or combination (0.5 mg/kg BZM, twice weekly; 1.5 mg/kg MTX, intraperitoneally, every day) (n = 14, per group). Tumor diameter was assessed every 3 days using a caliper. Testing order was randomized and blinded. Tumor growth and animal survival rate were monitored every day. Tumor volume were calculated using the following formula: [(length) x (width)2]/2 (n = 10, per group). When maximum tumor volume was close to 1500 mm3 were euthanized via CO2 inhalation. Tumors were removed from mice for IHC (i.e., Ki67 and TUNEL staining) (n = 4, per group). For survival cure, animal were monitored up to 65 day (n = 10, per group).
Statistical analysis
Data were expressed as means ± s.e.m. Statistical significance between two groups was analyzed with unpaired Student’s t test. Differences of multiple groups were determined by one-way ANOVA analysis. Comparisons between tumor volumes were determined by two-way ANOVA analysis. Survival curves were measured with Kaplan–Meier analysis. Statistical analyses were performed with SPSS 20.0 software (Chicago, IL). p < 0.05 were considered significant.