Cells and culture
SUDHL-4 and DB cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). NU-DUL-1 and OCI-LY8 cells were obtained by Professor Xiaoyan Zhou (Department of Pathology, Fudan University of Shanghai Cancer Center, Shanghai, China). TMD8 and U2932 cells were kindly provided by Professor Dongsheng Xu (Shanghai Tenth People's Hospital, Tongji University of Medical, Shanghai, China). And GCB cell lines mainly includes SUDHL-4, DB, OCI-LY1 and OCI-LY8 and the NU-DUL-1, U2932 and TMD8 are belong to ABC subtypes in the DLBCL cell lines. SUDHL-4, DB, NU-DUL-1, TMD8 and PBMCs were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) containing 10% FBS (FBS; Gibco, BRL, USA) and 1% PS (PS; Gibco, Carlsbad, CA, USA). U2932 was cultured in Dulbecco’s Modified Eagle’s Medium/Low Glucose (Gibco, Carlsbad, CA, USA), supplemented with 10% FBS and 1% PS. OCI-LY1 and OCI-LY8 were cultured in Iscove’s Modified Dulbecco’s Medium (Gibco, Carlsbad, CA, USA) containing 10% FBS and 1% PS. All cells were incubated in a humidified atmosphere at 37 °C, 5% carbon-dioxide.
Reagents
DCZ0014 stock solution was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) and stored at − 20 °C. Antibodies for phospho-PI3K, cleaved caspase-3, cleaved Caspase-8, Caspase-9, poly ADP-ribose polymerase (PARP), Bax, Bad, B cell lymphoma-2 (Bcl-2), Bcl-xl, C-myc, Mcl-1 and β-actin (for western blot) were purchased from Cell Signaling Technology (Danvers, MA, USA). Lyn, phospho-Lyn, Syk, phospho-Syk, Akt, phospho-Akt, STAT3, phospho-STAT3, STAT1, phospho-STAT1, ATM, phospho-ATM, ATR, phospho-ATR, CHK2, phospho-checkpoint kinase2 (p-CHK2), CHK1, phospho-checkpoint kinase1 (p-CHK1), cell division cycle 25A (cdc25A), CDK4, CDK6 and cyclinD1 antibodies were obtained from Abcam (Cambridge, MA, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan), the Annexin-V/ propidium iodide (PI) apoptosis detection kit from BD Pharmingen (Franklin Lakes, NJ, USA) and the JC-1 Kit from Beyotime Institute of Biotechnology (Haimen, China).
Cell viability assay
DLBCL cell lines (SUDHL-4, OCI-LY8, OCI-LY1, NU-DUL-1, TMD8, U2932 and DB) and PBMCs were seeded into 96-well plates in 95µL complete media at a density of 2 × 105 cells/mL and treated with different concentrations of DCZ0014 (0, 0.5, 1, 2, 4 and 8 µM) for 48 h. Cell proliferation was evaluated by 10µL of Cell Counting Kit-8 (CCK8, Dojindo, Kumamoto, Japan) adding into each well of the plate. Half maximal inhibitory concentration (IC50) values were evaluated by using CalcuSyn software.
Clonogenic assay
OCI-LY8 and NU-DUL-1 cells were seeded in six-well plates at 1000 cells per well and incubated at 37 °C incubator for 2 weeks. Cell colonies were stained with 0.1% crystal violet for 30 minutes. Colonies with at least 50 cells were counted.
Analysis of cell cycle
OCI-LY8 and NU-DUL-1 cells were cultured in 12-well plates at a density of 2 × 105 cells/mL and treated with DCZ0014 (0 and 2 µM) and incubated for 12, 24 or 48 h. Then cells were collected and washed in PBS and fixed with ice cold 70% ethanol overnight. After washed in PBS, cells were incubated with propidium iodide (PI) (BD Pharmingen, Franklin Lakes, NJ, USA) at room temperature for 15 min and analyzed by flow cytometry.
TUNEL assay
OCI-LY8 and NU-DUL-1 cells were exposed to DCZ0014 for 48 h, collected, fixed in 4% paraformaldehyde for 20 min, ruptured with 0.1% Triton X-100, stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 15 min and then TUNEL (Roche, Basel, Switzerland)) at 37 °C for 1 h. The cells were imaged under a fluorescence microscope.
EdU assay
OCI-LY8 and NU-DUL-1 cells were exposed to DCZ0014 and treated with DCZ0014 (0 and 2 µM) for 48 h and collected. The incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was measured using an EdU kit (RiboBio, Guangzhou, China) according to the manufacturer's instruction.
Cell apoptosis analysis
OCI-LY8 and NU-DUL-1 cells were cultured in 12-well plates at a density of 2 × 105 cells/mL and treated with DCZ0014 (0, 1, 2 and 4 µM) and incubated for 12, 24, 36, 48 or 72 h. Then according to the manufacturer's protocol. Then cells were collected and washed in PBS, and then stained with annexin V-FITC and PI (BD Pharmingen, Franklin Lakes, NJ, USA) with room temperature for 15 min incubating to detect apoptosis by a BD FASCCanto II flow cytometer (BD BioScience, San Jose, CA, USA).
MMP analysis
OCI-LY8 and NU-DUL-1 cells were cultured in 24-well plates at a density of 2 × 105 cells/mL and treated with DCZ0014 (0 and 2 µM) and incubated for 48 h, and incubated with 2 µM JC-1 at 37 °C, in 5% CO2 for 20 minutes. Then cells were collected and washed in PBS and analyzed with flow cytometry.
Western blot analysis
Cells treated with different concentrations of DCZ0014 and Total proteins were extracted with lysis buffer (100 mM Tris-HCL, PH 6.8, 4% SDS, 20% glycerol). Cytosolic proteins (30 µg per lane) were electrophoretically separated on a 6% or 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE Bio-Rad, CA, USA) and transferred electrophoretically onto polyvinylidene difluoride or nitrocellulose membranes, blocked in 5% non-fat milk or 5% BSA for 1 h, and incubated with the relevant primary antibodies overnight at 4 °C. Membranes were washed with Phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) three times and incubated with the appropriate secondary antibodies (anti-rabbit or anti-mouse IgG) for 1 h at room temperature. membranes were subsequently detected by the Odyssey two-color infrared laser imaging system (LI-COR, Lincoln, NE, USA).
Cell transfection
The small interfering RNAs (siRNAs) targeting human Lyn,the negative control siRNA were designed and constructed by RiboBio (Guangzhou, China) and the overexpression of Lyn plasmid were transfected into OCI-LY8 and NU-DUL-1 cells cultured in Opti-MEM (Gibco) by using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) up to a final concentration of 50 nM. The siRNA sequences (5′–3′) were as follows: GCTGGAGCTTTCCTTATTA.
Tumor xenograft model
2 × 106 OCI-LY8 cells in 100µL serum-free culture medium were inoculated subcutaneously into the BALB/C nude mice (Shanghai Laboratory Animal Center, Shanghai, China). When the tumors were measurable, mice were randomly divided into treatment groups receiving 15 mg/kg DCZ0014 or into a control group receiving vehicle (DMSO and saline). DCZ0014 was dissolved into 200µL of vehicle. Mice were injected intraperitoneally with vehicle or DCZ0014 for 18 days. Tumor size and the body weight of mice were measured every other day. Tumor volume was determined as 1/2 ×(width/2)2 × length. At the end of the experiment, mice were sacrificed. The tumors underwent hematoxylin-eosin (H&E) staining, Ki67, cleaved-caspase 3, TUNEL, and immunohistochemical staining. The study protocol was approved by the Animal Care and Use Committee of The Tenth People's Hospital of Shanghai (ID: SYXK 2014-0026) and Tongji University (Shanghai, China).
Statistical analysis
The data were expressed as mean ± standard deviation (SD). Student's t-test was performed as appropriate using SPSS v20.0 statistical analysis software (IBM, Armonk, NY, USA). p < 0.05 was considered significant.