5c and d). effect in DLBCL with mean expression of BCL-2 and myeloid cell leukemia-1 (MCL-1) through upregulating the expression level of BIM and modulating MCL-1 and p-Akt expression. For p53 wild-type DLBCL with high expression of BCL-2, APG-2575 showed strong synergic effect with mouse double minute 2 (MDM2)Cp53 inhibitor APG-115 that can achieve potent antitumor effect and markedly prolong survival in animal models. Collectively, our data provide an effective and precise therapeutic strategy through rational combination of BCL-2 and BTK or MDM2Cp53 inhibitors for DLBCL, which deserves further clinical investigation. for 15 min, the supernatants were collected, and protein concentration was determined by Bradford Protein Assay Kit (Beyotime Institute of Biotechnology, Haimen, China). Cellular extracts (30 g) were then incubated in a 96-well plate with 20 ng of Ac-DEVD-pNA for 2 h at Rabbit polyclonal to ACAP3 37C. Caspase 3 activity was measured by cleavage of the Ac-DEVD-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. Western Blot Analysis Western blot analysis was performed by standard methods as previously described29. The antibodies against BCL-2, BCL-XL, MCL-1, BAX, BAK, cleaved caspase 3, cytochrome c, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Akt, p-Akt, and -tubulin were purchased from Cell Signaling Technology. The antibodies against PARP, BIM, p53, MDM2, and PUMA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against BTK Y-33075 was purchased from ImmunoWay Biotechnology (JiangSu, China). The secondary anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology. AntigenCantibody complexes were detected using Bio-Rad Clarity western ECL substrate, and protein level was quantified by ImageJ (Bio-Rad Laboratory, Hercules, CA, USA). Mitochondrial Cytochrome c Release Assay Cells were pretreated with 20 nmol/l of APG-2575 for 0, 0.5, 1, 3, and 6 h. Cytoplasmic fractionation was isolated using the Cytosol/Mitochondria Fractionation kit (#QIA88; Merck Millipore, Y-33075 Darmstadt, Germany). Cytosolic fractions were isolated from OCI-LY8 cells following the manufacturers instruction. The amount of cytochrome c in cytosol and mitochondria fraction was determined by Western blot analysis as described above. In Vivo Treatment of Xenografts With APG-2575 All animal studies were performed with the approval from the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University Cancer Center (IACUC Y-33075 Approval No. 17040M). To develop OCI-LY8, OCI-LY1, and OCI-LY19 xenograft, 4- to 6-week-old female nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice (Beijing Vital River Laboratory Technology Co. Ltd, Beijing, China) were implanted subcutaneously in the right side of the axillary with 1??107 tumor cells suspended in 100-l volume of PBS containing Matrigel (Corning, Corning, NY, USA) at 1:1 ratio. OCI-LY8 models were used for APG-2575 single-drug efficacy studies; when mean tumor volume reached approximately 100200 mm3, mice were randomized into four groups (six mice per group) with approximately equivalent tumor volume. The mice were treated with vehicle or APG-2575 (25 mg/kg, 50 mg/kg, and 100 mg/kg body weight) daily by oral gavage for 10 days. In OCI-LY1 models applied to study, the combination efficacy of APG-2575 Y-33075 and ibrutinib, mice were randomly divided into four groups (five mice per group) with approximately equivalent tumor volume, and the treatment was started on day 1. Mice were treated with 15 mg/kg ibrutinib or vehicle at day 1 by intraperitoneal injection once a day for 13 consecutive days, while 100 mg/kg APG-2575 administered via oral gavage started at day 8 of six consecutive days. OCI-LY19 models were used to study the combination antitumor effect of APG-2575 and APG-115; mice were randomized into four groups (five mice per group) with approximately equivalent tumor volume. Vehicle, 50 mg/kg APG-2575, 50 mg/kg APG-115, and combination are administered orally once every day for Y-33075 6 days. Tumor sizes were measured by caliper equipment, and animal body weights were recorded two to three times per week. Tumor volume (mm3)?=?1/2??(length??width2). Immunohistochemical Analyses Tumor tissues from the NOD/SCID mice were immunohistochemically stained for Ki-67 using previously reported protocols30. TUNEL staining was performed with an in situ cell death detection kit (Roche Diagnostics Corp., Mannheim, Germany).