CYD0281, a Bcl-2 BH4 domain antagonist, inhibits tumor angiogenesis and breast cancer tumor growth

Chemicals and reagents

Bcl-2-BH4 domain antagonist, BDA-366 (S7849), was purchased from Selleck Chemicals (Selleck, Shanghai, China). CYD0281 was designed and synthesized as well as patented (jointly with Emory University) by Dr. Zhou’s laboratory at the University of Texas Medical Branch (UTMB) and was delivered for biological testing under the mutually signed material transfer agreement (MTA) with approval from the Office of Technology Transfer (OTT) of the UTMB. BDA-366 and CYD0281 were dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich St. Louis, MO, USA). Recombinant Human Bcl-2 Protein (NBP2-34889) was purchased from Novus Biologicals (Littleton, CO, USA). The following primary antibodies were used for immunohistochemical (IHC), immunofluorescence (IF), Western blotting (WB), and immunoprecipitation (IP) assays: rabbit anti-BH3 Domain Specific (AP1303a, diluted at 1:100 for IF and 1:1000 for IP) was purchased from Abgent (San Diego, California, USA), mouse anti-Bcl-2 (15071, diluted at 1:1000 for WB) and rabbit anti-Cytochrome C (4272T, diluted at 1:1000 for WB) were purchased from Cell Signaling Technology (CST, Beverly, MA, USA), rabbit anti-PARP1 (13371-1-AP, diluted at 1:1000 for WB) was purchased from Proteintech, rabbit anti-calnexin (AF2425, diluted at 1:150 for IF) and mouse-anti-Bcl-2 (AG1222, diluted at 1:50 for IF) were purchased from Beyotime (Wuhan, China), rabbit anti-CD31 (ab28364, diluted at 1:100 for IF), rabbit anti-Ki67 (ab15580, diluted at 1:100 for IHC) and rabbit anti-Bcl-2 (ab32124, diluted at 1:50 for co-IP) were purchased from Abcam, and rabbit anti-Mcl1 (PB9132, diluted at 1:1000 for WB), rabbit anti-Bax (AF1270, diluted at 1:1000 for WB), and rabbit anti-Bim (BM4183, diluted at 1:350 for WB) were purchased from Boster (Wuhan, China). Mito-Tracker Red CMXRos (C1035) was purchased from Boster (Wuhan, China). Bcl-2 siRNAs were synthesized by Ribobio Co., Ltd. (Guangzhou, China) and transfected into HUVECs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) at a final concentration of 100 nM.

Cell lines

Primary human umbilical vein vascular endothelium cells (HUVECs, ATCC® PCS-100-010™) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and maintained in endothelial cell growth medium including growth supplements (EGM, CC-3124, Lonza, Walkersville, MD, USA). 4T1 mouse breast cancer cell line and MDA-MB-231 human breast cancer cell line were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The 4T1 cells were maintained in RPIM1640 (Gibco, Carlsbad, CA, USA), and MDA-MB-231 cells were cultured in L-15 (Gibco). Both RPIM1640 and L-15 media contain 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL phytomycin. HUVECs and 4T1 cells were incubated at 37 °C with 5% CO2, and MDA-MB-231 cells were incubated at 37 °C without CO2.

Animals and treatment

Sprague-Dawley rats (SD rats, female, 4 ~ 5 weeks old) and BALB/c mice (female, 5 ~ 6 weeks old) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed in a 12-h light/12-h dark cycle, 24 ± 2 °C, and 50 ~ 60% humidity environmentally condition.

To construct a xenograft tumor model, 4T1 cells (1 × 105 cells/200 μL) were subcutaneously injected into the second right mammary fat pad of the BALB/c mice. After 7 days of tumor cell inoculation, the mice were divided into 3 groups randomly and intraperitoneal injection of DMSO or CYD0281 (10-, 30- and 50-mg/kg body weight) every 2 days for 2 weeks according to the previous report [36]. During tumor growth, the length and width of tumors were measured using calipers, and the tumor volume was calculated as 0.5236 × (length × width2). After drug treatment, the mice were sacrificed by cervical dislocation, and the tumors were harvested and weighed.

Cell viability assay

Cell viability was assessed using a cell counting kit-8 (CCK-8) kit (C0039, Beyotime, Shanghai, China) and BS350B (Biosharp, Hefei, China)). Briefly, HUVECs cells were seeded in 96-well plates at a density of 2 × 103 cells/well (200 μL). Then, the indicated concentrations of BDA-366 and CYD0281 were added to the cells 24 h later. After 48 h incubation, 20 μL CCK8 reagent was added to each well and incubated for 3 h. The optical density was measured at 450 nm using a multimode enzyme plate analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

In vitro HUVECs cell migration assay

HUVECs cells were treated with the indicated concentrations of DMSO, BDA-366, and CYD0281 for 48 h. Then, the cells were re-suspended at the concentration of 1.5 × 105 cells/mL in the serum-free EBM-2 medium containing DMSO, BDA-366, or CYD0281, and added to the upper chamber of the Transwell culture system, and then placed to the lower chamber that contained EBM-2 with growth supplements. After 20 h incubation, the cells migrated to the bottom side of the membrane and were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet solution for 15 min. Then, cells on the upper surface of the membrane were removed. The membranes were photographed under an inverted microscope and the migrated cells were counted.

In vitro HUVECs tube formation assay

The pre-treated HUVECs cells were re-suspended in EBM-2 with half-content growth supplements and the indicated concentration of DMSO, BDA-366, or CYD0281, and then added to a Matrigel pre-coated 96-well plate at the density of 3 × 104 cells/well. The cells were further maintained for 5 h at 37 °C with 5% CO2 to form a vascular tube. The cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature and the tubes were photographed under an inverted microscope and the length of the tubes was measured.

In vitro rat aortic ring assay

Aortas were isolated from Sprague-Dawley rats (4 ~ 5 weeks old, female; the Guangdong Medical Laboratory Animal Center, Guangzhou, China) and removal of the outer connective fibro adipose tissue surrounding the aortas and cut into approximately 1 mm thick rings. The rings were randomly put into 48-well plates, which were pre-coated with the growth factor reduced Matrigel, and further sealed with Matrigel. EBM-2 with growth supplements and the indicated concentration of DMSO, BDA-366, or CYD0281 was added to the wells and incubated at 37 °C with 5% CO2 for 8 days. The formed microvessel that sprouts from aortic rings was fixed using 4% paraformaldehyde and photographed using an inverted microscope. Microvessel outgrowth was quantified using Image-Pro Plus 6.0 image analysis system (IPP 6.0, Media Cybernetics, Inc., Rockville MD, USA).

Chick embryo chorioallantoic membrane (CAM) assay

Fertilized eggs of white leghorn chicken were obtained from the Avian Farm of South China Agriculture University (Guangzhou, China). According to the previous report, the eggshells were cleaned with 75% ethanol and incubated under the condition of 60% humidity and 37 ± 1 °C for 7.5 days and randomly divided into DMSO, BDA-366, and CYD0281 groups [37]. Then, a small window (1 cm diameter) was drilled above the air chamber of well-developed eggs with a dental drill, and the shell was peeled off using ophthalmic forceps. The indicated concentrations of compounds (below ~ 0.1% concentration of xenograft tumor model treatment) were prepared by dissolving them in 30 μL of phosphate-buffered saline (PBS, pH 7.4), and were placed directly on CAM of the live chick embryo through the window. After 48 h incubation, the eggshells were sacrificed, and the CAM vasculature around the windows was cut off. The CAMs were inverted and photographed under a stereomicroscope (Olympus SZX16). The percentage of blood vessel area in the total drug-treated area was calculated as microvessel density (MVD) using the IPP 6.0 image analysis system.

Chick embryo yolk sac membrane (YSM) assay

After 3 days of incubation, the well-developed chick embryos were transferred into sterile dishes, and the vessels were faced upward. Then, the marked silastic rings were placed onto the top of the vessel regions of the yolk sac membrane, and DMSO or indicated concentrations of CYD0281 were added in the center of silastic rings, respectively. The YSM vasculature in the rings was photographed and the MVD was quantified using IPP 6.0 at 0, 12, and 24 h after drug treatment.

Experimental breast cancer assay

MDA-MB-231 cells were re-suspended at the concentration of 1 × 108 cells/mL, and 50 μL cell suspension was added in the middle of the silastic rings that were placed on the CAM of chick embryos that were prepared as CAM assay indicated above. After 30 h of incubation, the chicken embryos were randomly grouped and treated with DMSO and indicated concentrations of BDA-366 or CYD0281. The drugs were added in the middle of the rings once daily for 3 days. After treatment, the macroscopic tumor could be seen on the CAM in successfully established models and the CAMs were peeled off and photographed. The length and width of the tumor were measured and tumor volume on CAM was calculated as 0.5236 × (length × width2). The MVD was calculated using IPP 6.0. Then, the tumors were fixed with 4% polymethylene formaldehyde and embedded with paraffin for hematoxylin and eosin (H&E) staining.

In vivo Matrigel plug angiogenesis assay

The widely used in vivo Matrigel plug angiogenesis assays for quantifying the angiogenic ability are more easily than chick embryo angiogenesis assays [38]. BALB/c mice (female, 5 ~ 6 weeks old) were purchased from Guangdong Medical Laboratory Animal Center. The mice were randomly divided into two groups and were subcutaneously injected with 0.5 mL of Matrigel (356230, Corning, Bedford, MA, USA) supplemented with 150 ng/mL of fibroblast growth factor-2 (FGF-2, 3139-FB/CF, R&D systems, Minneapolis, MN, USA), 60 Units/mL of heparin (Cisen pharmaceutical company, Shandong, China), and DMSO or CYD0281 (50 μg/mL) according to the previous report [39]. The Matrigel quickly polymerized to form a solid gel and incubated for 7 days in vivo. Then, the mice were killed and the Matrigel plugs were collected and photographed. The angiogenesis in the plugs was analyzed by detecting the expression of CD31, a vascular marker, using an immunofluorescence assay.

Histological, immunofluorescence, and immunohistochemistry assays

The fixed tumor tissues in CAM and BALB/c mice and Matrigel plugs were embedded in paraffin and cut into 3 μm sections. The sections from tumor tissues from CAM were stained with H&E staining for histological analyses. The sections of tumor tissues in BALB/c mice were incubated with CD34 primary antibody and HRP-conjugated secondary antibody for immunohistochemistry assay to detect tumor angiogenesis. The sections of Matrigel plugs were incubated with CD31 primary antibody to detect angiogenesis in vivo and HUVECs cells were incubated with Bcl-2 (BH3-specific) primary antibody to detect the conformations change in the exposure of the BH3 domain, and then incubated with Alexa Fluor 555-conjected secondary antibody for immunofluorescence assay. DAPI was used to stain the cell nuclei. The MVD was analyzed using the IPP 6.0 image analysis system.

Western blotting assay

Total proteins from HUVECs that were treated with DMSO, BDA-366, or CYD0281 were harvested using RIPA lysis buffer. Proteins of mitochondrial and cytosolic were extracted using the Cell Mitochondria Isolation Kit (C3601, Wuhan, China) according to the manufacturer’s instructions. All the proteins were examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins on the gel were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with relevant primary antibodies and HRP-conjugated secondary antibodies. Then, protein signals on the membranes were visualized using enhanced chemiluminescence (ECL) chemiluminescent system (#4AW011-200, 4A Biotech, Beijing, China). The intensity of protein bands was quantified using Quantity One software (version 4.6.2 for Windows; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a protein loading control. The signals were visualized using an ECL chemiluminescent system.

Immunoprecipitation assay

Recombinant Human Bcl-2 Protein was treated with CYD0281 in 1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) lysis buffer at 4 °C for 2 h, followed by immunoprecipitation (IP) using anti-Bcl2/BH3 domain antibody at 4 °C overnight. Then, protein A/G magnetic beads (25 µL) were added and incubated at room temperature for 1 h. The protein complex was washed 3 times and released from the beads by incubating with eluent (100 μL) for 10 min at room temperature. The eluted proteins were analyzed by western blot using the Bcl-2 antibody indicated above.

Apoptosis analysis by flow cytometry

HUVECs were treated with DMSO, BDA-366, or CYD0281 and then collected after 24 h. The apoptosis cells were detected using the Annexin V-FITC Apoptosis Detection kit (#C1062M, Beyotime Shanghai, China) according to the manufacturer’s introduction. The collected cells were gently resuspended by adding Annexin V-FITC (5 μL), propidium iodide (10 μL), and Annexin V-FITC binding solution (195 μL) for 20 min at room temperature in the dark, and then assessed by fluorescence-activated cell sorter (FACS) analysis on a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The percentage of apoptotic cells was analyzed using FlowJo software (FlowJo LLC; Ashland, OR, USA). All data are represented as the mean ± standard deviation (SD).

Statistical analyses

All data are represented as the mean ± standard deviation (SD). The statistical significance of differences between the two groups was determined with a two-sided Student’s t-test at P < 0.05 and analyzed using GraphPad Prism 5 (San Diego, CA, USA).

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