3.1 ChemicalsBM-1197 was kindly provided by Professor Yang Dajun.BEZ235 and ABT-263 were purchased from Selleckchem (Houston, TX). Gemcitabine hydrochloride was purchased from Jiangsu Haosen Company and Doxorubicin hydrochloride was purchased from Pharmacia Co., Ltd. Vinblastine sulfate was purchased from Shenzhen Wanle Pharmaceutical Co., Ltd. For in vitro assay, all compounds were dissolved in dimethylsulfoxide (DMSO; Sigma Aldrich, MO, USA) at a stock concentration of 40 mM, stored at -20 ℃. The final concentration of DMSO to dilute compound in culture media did not exceed 0.1%. 3.2 Cell lines and cell culture
Cell lines used in this study include OCI-ly1, OCI-ly8, OCI-ly19, Su-DHL-4 and Nu-DHL-1 cells that are from diffuse large B cell lymphoma (DLBCL). Raji, Ramos and Namalwa cells are from Burkitt lymphoma. Jurkat cells are from human peripheral blood leukemia T cells. All cells were cultured at 37℃ with 5% CO2. OCI-ly1, OCI-ly8,OCI-ly19,Raji,Ramos,Namalwa cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, while Su-DHL-4 and Nu-DHL-1 cells were cultured in RPMI-1640 medium containing 20% fetal bovine serum. All culture media contain 100 IU/ml penicillin and 100 mg/ml streptomycin. All experiments were performed in the logarithmic growth phase of the cells.
3.3 Cell viability assay
Cell viability was determined by CCK-8 kit. Lymphoma cells at the logarithmic growth phase were inoculated into 96-well culture plates with 180 μl media (cell concentration 5000/well). After incubation for 24 h, 20 μl BM-1197 at different concentration was added. After incubation for 68 h, 10 μl of CCK-8 was added to each well and incubated for 3-4 h. OD value was measured at 450 nm using a microplate reader. All experiments were performed in triplicates.
The combination effect of BM-1197 and other chemotherapy drugs were determined by combined index value (CI value). After OCI-ly8 cells incubation for 24 h, BM-1197 combined with Adriamycin, Gemcitabine and Vincristine at indicated ratio and concentration. After incubation for 68 h, 10 μl of CCK-8 was added to each well and incubated for another 3-4 h. The OD value was read at 450 nm using a microplate reader. The inhibitory rate was calculated according to the above formula. CompuSyn software (Chou-Talalay formula) was used to calculate the combined index value (CI value). CI value less than 0.1 indicated a strong synergistic effect of two drugs; CI value less than 0.9 indicated synergistic effects of two drugs; CI value greater than 0.9 and less than 1.1 indicated a weak synergistic effect; CI values greater than 1.1 indicates antagonistic effect of two drugs.3.4 Hoechst 33258 staining for detection of apoptotic morphological changes OCI-ly8 cells were treated with BM-1197 at different concentrations (0, 0.5, 1.0, 2.0 μM) for 24 h. The cells were collected and fixed with 0.5 ml fixative at 4℃ overnight. After fixation cells were washed twice with PBS, each 3 min. Cells were fixed in a glass slide and 0.5 ml Hoechst 33258 staining solution was added and stained for 5 min. After wash in PBS, anti- quenching solution was added followed with covering slips. Blue nucleus staining was observed under a fluorescence microscopy with the excitation wavelength of about 350 nm, emission wavelength of about 460 nm.3.5 Annexin V / PI double staining for detection of early apoptosis
OCI-ly8 cells in logarithmic growth phase were inoculated in 6-well plate at 3 × 105 per well, and incubated for 2-4 hours. After treated with BM-1197 at different concentrations (0, 0.5, 1.0, 2.0 μM) for 24 h, the cells were collected. Cells were washed with PBS twice and 20 μl Annexin-Ⅴ and PI staining solution was added. Cells were incubated for 15 min at room temperature in the dark. Stained cells were analyzed within 1 h with a flow cytometer. The negative control group was divided into three groups with binding buffer, one was Annexin Ⅴ and another was single stained PI. The other one was used as standard sample for flow cytometry.
3.6 Detection of Caspase-3/ Caspase-9activity
The activity of caspase-3/caspase-9 was determined using the Caspase-3/ caspase-9 activity kit (Beyotime Institute of Biotechnology, Haimen, China). After treatment of BM-1197, cell lysates were prepared by incubating 2 × 106 cells/ml in extraction buffer (25 mM Tris–HCl, pH 7.5, 20 mM MgCl2, and 150 mM NaCl, 1% Triton X-100, 25 μg/ml leupeptin, and 25 μg/ml aprotinin) for 30 minute on ice. Lysates were centrifuged at 12,000×g for 15 min, the supernatants collected and protein concentration determined by Bradford Protein Assay Kit (Beyotime Institute of Biotechnology, Haimen, China). Cellular extracts (30 μg) were then incubated in a 96-well plate with 20 ng Ac-DEVD-pNA/ Ac-LEHD-pNA for 2h at 37 °C. Caspase-3 activity and caspase-9 were respectively measured by cleavage of the Ac-DEVD-pNA and Ac-LEHD-pNA substrate to pNA, the absorbance of which was measured at 405 nm. Relative caspase activity was calculated as a ratio of emission of treated cells to untreated cells.
3.7 Western Blot AnalysisOCI-ly8 cells were treated with different concentrations of BM-1197 (0, 0.25, 0.5, 1, 2 μM) for 24 h or treated with 2 μM BM-1197 for 0, 1, 3, 6, 12 and 24 h. After that, cells were collected and washed twice with cold PBS and lysed in 1 × cell lysis buffer which was diluted from 10 × cell lysis buffer (Cell Signaling Technology). 1x proteinase inhibitor cocktail was added in the lysis buffer. Lysates were centrifuged at 12000 g at 4 °C for 20 min. Supernatants were collected and stored at -80℃ until used. The protein concentration of the supernatants was determined using the BCA protein assay reagents. The relevant primary antibodies used included: Bcl-2, Bax (6A7) and PARP-1 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Mcl-1, PUMA, Bcl-xl, caspase 3 , Caspase 9, cytochrome c (cyt c), GAPDH monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA). COXⅣ polyclonal antibody was purchased from Biyun Tian Biotechnology Research Institute. Mouse and rabbit secondary antibodies were from Cell Signaling Technology (Danvers, MA). Antigen-antibody complexes were detected using PhototopeTM-HRP Chemiluminescent Substrate system (Cell Signaling Technology) as per manufacturer’s instructions.
3.8 Mitochondrial cytochrome c release assay
OCI-ly8 cells were pretreated with 2μM BM-1197 for 0h, 1h, 3h, 6h. Cytoplasmic fractionation was isolated using the Cytosol/Mitochondria Fractionation kit (#QIA88: Merck Millipore, Darmstadt, Germany). Cytosolic fractions were isolated from OCI-ly8 cells following the instruction. The amount of cytochrome c in cytosol and mitochondria fraction were determined by western blot analysis as described above.
3.9 Immunoprecipitation
OCI-ly8 cells were incubated with 2 μM BM-1197 or 0.1% DMSO for 6 h. The cells (1×107) were then harvested and lysed in CHAPS lysis buffer 500 μl (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 1% CHAPS, 1 mM EDTA, 1 mM EGTA, protease inhibitors, PhosSTOP [Roche], and 20 mM MG132) on ice for 30 min. Whole cell lysates were obtained, precleared with protein A/G-Sepharose, and incubated overnight with 1μg of the specific antibody (Bak, Bax, Bcl-2, Bcl-xl, PUMA, Bim) at 4℃. Immuno-complexes were captured with either protein A-Sepharose or protein G-Agarose. The beads were pelleted, washed 3 times, and boiled in SDS sample buffer. The presence of immuno-complexes was determined by western blot analysis.
3.10 Gene knockdown using siRNA
The siRNAs to Mcl-1 or control siRNA were all purchased from GenePharma (Shanghai). OCI-Ly8 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were incubated for 8 h and then refresh the culture medium for further treatment.
3.11 In vivo xenograft studies
In the xenograft cancer model, male nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice were purchased from Charles River (Beijing, China), housed in groups of five and given three days to acclimate to the housing facility. Each of the mice was about 4 weeks with the body weight around 18g before cancer cell implanted. The mice were feed in constant temperature (21℃ ±2℃) and humidity (55% ±10%) and specific pathogen free (SPF) rooms in Sun Yat-sen university laboratory animal center with a 12:12 light:dark cycle with lights on at 07:00 and off at 19:00. During housing, animals were monitored twice daily for health status. No adverse events were observed. All the materials were autoclaving sterilized before contacted with mice.
OCI-Ly8 cells were implanted subcutaneously in the right side of axillary with 1×107 tumor cells suspended in 200 μl volume of PBS. For drug efficacy studies, when mean tumors volume reached approximately 100~200 mm3, mice were randomized into vehicle control group (1% Klucel LF/ 0.1% Tween 80) or BM-1197 treatment group (10 mg/kg; QOD), with five mice per group. The randomization was performed with randomized table. Tail intravenous injection was performed in both groups. Tumor sizes were measured by caliper equipment and animal body weights were recorded 2~3 times per week. Tumor volume (mm3) =1/2 × (length×width2). The mice were killed by cervical dislocation method. All animal experiments were carried out under the guide of Sun Yat-sen University Committee for Use and Care of Laboratory Animals and approved by the animal experimentation ethics committee.
3.12 Statistical analysis
All experiments were performed at least three times. Statistical analysis was carried out using Microsoft Excel 2001 and data are presented as mean± standard error of the mean (SEM) combining three experimental repeats. P<0.05 was considered of significant difference.