Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro

Abstract

Aim: To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro.

Methods: 32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells.

Results: C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC₅₀ at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC₅₀ at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells.

Conclusion: C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

Figure 1

C817 inhibits proliferation of imatinib-resistant CML cells overexpressing wild-type or mutant Bcr-Abl. (A) Chemical structure of C817. (B, C) Proliferation of 32D, 32D-T315I, 32D-Q252H, and 32D-Y253F cell lines in the presence of escalating concentrations of (B) imatinib mesylate (0–30 000 nmol/L) or (C) C817 (0–30 000 nmol/L). Cell growth was assessed by MTT-based viability assay. (D) Bcr-abl gene copies analyzed by FISH in K562 or K562/G01 cells. (E, F) Proliferation of K562 or K562/G01 cells in the presence of escalating concentrations of (E) imatinib mesylate (0–32 000 nmol/L) or (F) C817 (0–10 000 nmol/L). Cell growth was assessed by MTT-based viability assay.

Figure 2

C817 inhibits wild-type or mutant Abl kinase in vitro or Bcr-Abl initiating signaling in imatinib-resistant cells. (A–D) Wild-type or T315I/Q252H/Y253F mutant Abl kinase assays were performed at different ATP concentration as described in Materials and methods by using Kinase-Glo Luminescent Kinase Assay Platform. (E) C817 inhibits tyrosine phosphorylation of Bcr-Abl and downstream targets STAT-5 and CrkL. K562 or K562/G01 cells (1×10⁶) were incubated for 24 h in 20 mL media in the presence of C817 (0, 0.5, or 1 μmol/L). Total cell lysates were prepared and separated by SDS-PAGE on 10% gels (30 μg protein/lane). Western blotting analysis for phospho-Bcr-Abl, phospho-STAT5, phospho-CrkL, and eIF4E (protein loading control) was performed by using a Pathscan Bcr/Abl Multiplex Western Detection Kit (Cell Signaling Technology).

Figure 3

Effect of C817 on induction of apoptosis and cell cycle arrest in imatinib-sensitive or -resistant CML cells. (A) K562 and K562/G01 cells were cultured in the presence of C817 at indicated concentrations for 24 h, harvested, then dually stained with annexin V and PI and subsequently analyzed by flow cytometry. (B) C817 arrests the cell cycle at the G₂ phase in both imatinib-sensitive and -resistant CML cells. K562 and K562/G01 cells were cultured in the presence of C817 at the indicated concentrations for 24 h, harvested, and then stained with PI and subsequently analyzed by flow cytometry, with quantitation using FlowJo software.

Figure 4

C817 triggers mitochondrial pathway of apoptosis in imatinib-sensitive or -resistant CML cells. (A) Treatment of K562 or K562/G01 cells with C817 at indicated concentrations for 24 h significantly led to reduction of MMP examined by JC-1 staining. (B) Treatment of imatinib-sensitive or -resistant K562 cells with C817 for 24 h activated the stream of caspase 9, 7, and 3 pathway analyzed by Western blotting. Tubulin serves as a control for the loading of protein levels.

Figure 5

Effects of C817 on CML progenitor/stem cells survival from human bone marrow. (A) Colony formation of CML CD34⁺ cells. CML (n=3) or normal CD34⁺ cells were isolated and cultured with low growth factors, either without inhibitors (control), or in the presence of graded concentrations of C817 for 24 h and then plated in methylcellulose progenitor culture for 14 d. Data are presented as the percentage of suppression of CFUs of CML or normal CD34⁺ cells compared to untreated controls. Significant differences between CML and normal CD34⁺ cells (unpaired t tests; ^cP<0.01) are indicated. (B) Colony formation of imatinib-resistant CML CD34⁺ cells. Data are presented as the percentage of suppression of CFUs compared to untreated controls. No significant differences between CML and imatinib-resistant CML CD34⁺ cells (unpaired t tests; ^aP>0.05) are indicated. (C) Effects of C817 on LTC-IC cells self-renew from CML bone marrow. MNCs from CML patients were exposed to C817 at the concentrations indicated for 24 h. Cells were then assayed for primitive progenitors (LTC-IC) by counting CAFC. The percentage inhibition of primitive progenitor growth after C817 or imatinib treatment relative to untreated controls is shown. Results represent the mean plus or minus SEM based on quinplicate wells (CML, n=3). Error bars represent SEM.