title: ZGDHu-1 and fludarabine have a synergistic effect on apoptosis of chronic lymphocytic leukemia cells.
abstract: Previously, it was demonstrated that the novel proteasome inhibitor N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu‑1) possesses activity against chronic lymphocytic leukemia (CLL). In the present study, we attempted to assess whether this drug has a synergistic effect with fludarabine on the apoptosis of CLL cells. Annexin V/PI staining, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were examined by flow cytometry in short-term cell culture of blood cells from untreated newly diagnosed patients ex vivo. Expression of active caspase-3 and the Bcl-2/Bax ratio for determination of apoptosis were also investigated by flow cytometry and western blot analysis. Our results revealed that the ZGDHu-1 may induce the apoptosis of CLL cells through the mitochondrial pathway and its pro-apoptotic effect is CLL-specific, not affecting normal lymphocytes. Most importantly, a combination of ZGDHu-1 and a non-cytotoxic dose of fludarabine had a synergistic apoptotic effect. To some extent, caspase-3 activation may be involved in the mechanism of the ZGDHu-1 synergistic cytotoxic effect with fludarabine, as well as the cleavage of PARP, consequently leading to apoptosis. Notably, the rate of apoptosis caused by ZGDHu-1 alone or in combination with fludarabine was independent of prognostic markers of CLL disease such as ZAP-70 and CD38 expression or clinical Rai classification stage. In conclusion, ZGDHu-1 exhibited a significant synergistic effect with fludarabine to induce the apoptosis of CLL cells, which implies a possible clinical application.
authors.affiliation: Department of Clinical Laboratory, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang 310004, P.R. China.
authors.forename: Liannv
authors.initials: L
authors.lastname: Qiu
authors.affiliation: Department of Clinical Laboratory, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang 310004, P.R. China.
authors.forename: Jinlin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Clinical Laboratory, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang 310004, P.R. China.
authors.forename: Zhenni
authors.initials: Z
authors.lastname: Wang
authors.affiliation: College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, Zhejiang 310004, P.R. China.
authors.forename: Weixiao
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Hematology, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang 310004, P.R. China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Clinical Laboratory, Zhejiang Provincial People's Hospital, Hangzhou, Zhejiang 310004, P.R. China.
authors.forename: Yonglie
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D004357:Drug Synergism; D005260:Female; D005434:Flow Cytometry; D006573:Heterocyclic Compounds, 1-Ring / Q000008:administration & dosage*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008875:Middle Aged; D017382:Reactive Oxygen Species; D014740:Vidarabine / Q000008:administration & dosage / Q000031:analogs & derivatives*
chemical_list: D006573:Heterocyclic Compounds, 1-Ring; C522668:N,N-di-(m-methylphenyl)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboxamide; D017382:Reactive Oxygen Species; D014740:Vidarabine; C024352:fludarabine