AX is a potent inhibitor in leukemic cell lines without inducing any HSR. (A) K562, KCL22, and HL60 were treated with the indicated (cytotoxic) concentration of AX, NB and AUY922 for 48 hours, and protein lysates were later subjected to immunoblot analysis. AX and NB (C-terminal HSP90 inhibitors) do not induce expression of HSP70, HSP40, and HSP27, whereas AUY922 (an N-terminal HSP90 inhibitor) demonstrates HSR induction by triggering their expression. HSP60 (primarily present in mitochondria) and PDI (primarily present in endoplasmic reticulum) served as controls for the HSR in the cytoplasm, in response to inhibition of HSP90 dimerization via the CTD. (B) K562, KCL22, and HL60 (Mutz-2; data not shown) were treated with AX for 48 hours, and enzymatic activity of caspase-3/7 was later examined by caspase-3/7–dependent Glo assay (absorbance at 405 nm). (C) K562, HL60, KCL22 cells were seeded in methylcellulose medium containing respective compounds at indicated concentration after treatment in liquid medium for 24 hours. Colonies were counted after 14 days. (D) 5 × 105 luciferase-expressing K562 cells were subcutaneously transplanted into NSG mice. Starting the day after transplantation, animals were treated by peritumoral injection (15 µg) of compound AX (0.5 mg/kg dose) or solvent only (DMSO). One control DMSO-treated mouse was sacrificed earlier (on day 16) because of large tumor size. Luminescence was monitored every 3 or 4 days after intraperitoneal injection of 100 µL luciferin, and the final analysis was performed on day 17 (n = 5 mice per group). (E) AX reduced tumor burden with respect to tumor weight 0.24 ± 0.01 g vs vehicle 1.6 ± 0.6 g (P = .04; 1-tailed t test). (F) Immunoblot analysis of tumor samples derived from mice treated with AX revealed downregulation of BCR-ABL1 kinase activity and its associated downstream signaling pathways involving Stat5a and Crkl. (G) Immunoblot analysis of tumor samples derived from mice after treatment with AX. Samples displayed no HSR, as evaluated by expression of HSF-1, HSP70, and HSP27; PDI and HSP60 were used as controls. Columns depict the mean of 3 independent experiments (n = 3). Significance analyses of normally distributed data with variance similar between groups used paired, 2-tailed Student t test. *P < .05, **P < .005, ***P < .001.
