Correlation between inhibition of CaMKIIγ kinase activity and inhibition of leukemia cell proliferation by berbamine analogs. (A) Berbamine (BBM) and 2-methylbenzoyl berbamine (BBD24) inhibited phosphorylation of CaMKIIγ of leukemia cells. BBM or BBD24 treatment reduced phosphor-CaMKIIγ protein level, but not total CaMKIIγ protein level of K562 cells. Leukemia cells were treated with BBM or BBD24 at the indicated concentrations for 48 hours, and then total proteins were extracted for Western blot analysis for phosphor and total CaMKIIγ proteins. BBD5 was served as an inactive berbamine analog. (B) Correlation between inhibition of CaMKIIγ kinase and inhibition of cell proliferation by analogs of berbamine (BBM, BBD3, BBD12, BBD15, BBD24, and CP15; R2 = 0.9867). (C) Overexpression of CaMKII γ attenuated berbamine-induced growth inhibition of leukemia cells in vitro. Control or CaMKII γ overexpression cells were treated with BBM at the indicated concentrations for 48 hours, and then collected for analysis of cell viability by MTT assay, and phosphor and total CaMKIIγ proteins by Western blot. β-actin was used as loading control (* P < .01). (D) Overexpression of CaMKII γ reduced berbamine-induced growth inhibition of xenograft tumors in a dose-dependent manner (*P < .01). (E-F) hematoxylin-eosin (E) and PCNA (F) stain of xenograft tumors from NOD-SCID mice dosed with vehicle (top), BBM 50 mg/kg (middle), and BBM 100mg/kg (bottom). Left: A representative of control groups. Right: A representative of CaMKIIγ overexpression groups.
