ZGDHu-1 promotes apoptosis of chronic lymphocytic leukemia cells

Abstract

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. We aimed to examine the proapoptotic effects of a novel proteasome inhibitor, N,N'-di-(m-methylphenyi)-3,6- dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), on CLL cells. B lymphocytes were isolated from CLL patients and normal healthy controls, and treated with various concentrations of ZGDHu-1 for different days. CLL cell viability was detected by MTT assay. The apoptosis, mitochondrial membrane potential (∆ψm) and reactive oxidative species (ROS) were examined by flow cytometry. The expression of caspase-3 and Bcl-2/Bax ratio was detected by western blotting. ZGDHu-1 significantly reduced the viability of CLL cells and induced apoptosis in comparison to the control cells (both P<0.05). Normal peripheral B cells were resistant to the apoptosis-inducing effects of ZGDHu-1. Apoptosis induced by ZGDHu-1 was accompanied with generation of ROS, loss of ∆ψm, downregulation of Bcl-2 and increase of caspase-3 cleavage. Results of this study indicate that ZGDHu-1 is a promising specific treatment for CLL in the clinic.

ZGDHu-1 and fludarabine have a synergistic effect on apoptosis of chronic lymphocytic leukemia cells

“…Then the T lymphocytes were removed using anti-CD3 Dynabeads ® (Dynal, Merseyside, UK). The B lymphocytes were further purified by flow cytometric sorting based on CD19 antibody staining (Immunotech, Coulter, uSA) as previously reported (12). B lymphocytes were then counted in a Neubauer Counting Chamber with trypan blue to exclude dead cells.…”

“…It has been identified as a potential proteasome inhibitor (11). It was demonstrated that ZGDHu-1 induces the apoptosis of B lymphocytes from CLL patients (12). In the present study, we investigated ZGDHu-1 used alone or combined with fludarabine in regards to the ex vivo apoptotic effects on CLL cells and normal lymphocytes derived from peripheral blood.…”

“…B lymphocytes were then counted in a Neubauer Counting Chamber with trypan blue to exclude dead cells. The cells were then resuspended in RPMI-1640 medium with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin G and 0.1 mg/ml streptomycin (Sigma-Aldrich) in 75 cm 3 flasks at a density of 1-4x10 6 /ml and cultured at 37˚C in 5% CO 2 as previously reported (12). The cells were further divided into 4 groups.…”

“…All experiments were performed in triplicate and repeated at least three times. The cell viability was expressed as a percentage of the DMSO-treated control samples as previously reported (12).…”

“…In the present study, we investigated ZGDHu-1 used alone or combined with fludarabine in regards to the ex vivo apoptotic effects on CLL cells and normal lymphocytes derived from peripheral blood. We examined the apoptosis of CLL cells, loss of mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation across the plasma membrane (13,14) and accumulation of reactive oxygen species (ROS) (15 (12). CLL cells were stained with the following mAbs: anti-CD19-PerCP CY 5.5 (ID3) and anti-CD5-APC (53.-7.3) (both from BD Pharmingen, San Diego, CA, uSA), anti-ZAP-70 Alexa Fluor ® 488 (SBZAP) and anti-CD38-FITC (T16; both from Immunotech), anti-active caspase-3-PE (C92-605; BD Pharmingen), anti-Bax-FITC (SC20067; Santa Cruz Biotechnology, CA, uSA); and anti-Bcl-2-PE (Bcl-2/100; BD Pharmingen).…”