Figure 6.

5b is effective against human leukemic cell lines without inducing any HSR. (a) Comparative cytotoxicity assessment of different subgroups of leukemic cell lines (K562, KCL22, SUPB15, HL60, MOLT4, DND41, TALL1, and HPBALL), imatinib-resistant cell lines (K562-IMr, KCL22-IMr, SUPB15-IMr), and the bortezomib-resistant cell line (HL60-BTZr) after 72 h of exposure to 5b. The IC₅₀ data was plotted as a clustered heat map, followed by unsupervised hierarchical clustering. The vertical axis of the dendrogram exemplifies the dissimilarity between clusters, whereas the color of the individual cell is related to its position along a log IC₅₀ (μM) gradient. (b) The treatment of K562 cells with 5b and respective controls (AUY922 and NB) for 48 h resulted in the downregulation of BCR-ABL1⁺ and subsequent downstream signaling pathways including phosphorylated and unphosphorylated Stat5a, Crkl, Akt, S6 (mTOR), and cMyc. (c) K562 cells were treated with the indicated (cytotoxic) concentration of 5b, NB, and AUY922 for 48 h, and later, protein lysates were subjected to immunoblot analysis. As expected, 5b and NB did not induce expression of Hsp70, Hsp40, and Hsp27, whereas AUY922 led to HSR induction. Hsp60 (primarily present in mitochondria) and PDI (endoplasmic reticulum) served as a control. (d) (upper) description of the experimental rationale; (middle) representative image of a xenotransplanted zebrafish embryo at 32 hpf [scale bar, 250 μm; note that human T-ALL cells (green) were distributed in the yolk, brain, and hematopoietic tissue (arrows)]; (lower) fold-change of labeled cells normalized to the average percentage of labeled cells in the DMSO-treated group. Each dot represents three embryos pooled as one biological sample. Data are mean ± standard deviation. The p-values were calculated with the Mann–Whitney test.