Cell lines, reagents, antibodies, and mice
Human prostate cancer LNCaP, 22RV1, PC-3 cells were obtained from ATCC (Manassas, VA). 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). β-actin (MABT825) were purchased from Sigma. HRP-conjugated goat anti-mouse IgG (SA00001-1) and goat anti-rabbit IgG (SA00001-2) were obtained from Proteintech Group` (Chicago, IL).
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). FBS, RPMI 1640 medium, penicillin/streptomycin, and L-glutamine were obtained from Gibco (by ThermoFisher Scientific, Shanghai, China).
Immunocompromised mice (SCID Beige) were acquired from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).
Western blotting and Real time PCR
Western blotting was performed as previously described . Briefly, cells were lysed using RIPA lysis buffer containing complete protease inhibitor cocktail (Roche, Switzerland), followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and trarsmembrane. 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 (Invitrogen) 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). 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: 18s 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'.
Apoptosis analysis and cell proliferation
Apoptosis was evaluated using a Dead Cell Apoptosis Kit (ThermoFisher Scientific, catalog #V13242) as previously described. Briefly, 5 x 105 cells treated with indicated drugs were incubated with 5 µl FITC-conjugated Annexin-V antibody and 5 µl propidium iodide (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. CFSE-labbed 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).
Immunohistochemistry and Scoring
The Immunohistochemistry (IHC) staining procedure and scoring in our publications. Briefly, tissues were fixed in 4% formalin. Paraffin-embedded tissue sections (4µm) 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). IHC scoring was perform as previously described [20, 22, 23].
Proteasome activity assay
Proteasome activity was measured as previously described[24, 25]. 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 hour at 30℃ and the free AMC fluorescence was determined by Cytation-i5 Cell Imaging Reader (Biotek, USA).
To construct the mice Xenograft model, prostate cancer cells were implanted subcutaneously into the flanks of male SCID mice. Two week after injection, mice were treated with BZM (1 mg/kg, intraperitoneally, twice weekly), MTX (3mg/kg, intraperitoneally, every day), or combination (0.5 mg/kg BZM, twice weekly; 1.5 mg/kg MTX, every day). Tumor diameter was assessed every 3 days using a caliper. Tumor growth and animal survival rate were monitored every day. Tumor volume were calculated using the following formula: [(length) x (width)2]/2. Mice bearing a tumor volume exceeding 1,500 mm3 were euthanized. For survival cure, animal were monitored up to 65 day.