Adaphostin has significant and selective activity against chronic and acute myeloid leukemia cells

Abstract

Adaphostin is a tyrphostin that was designed to inhibit Bcr/Abl tyrosine kinase by altering the binding site of peptide substrates rather than that of adenosine triphosphate, a known mechanism of imatinib mesylate (IM). However, it has been shown that adaphostin-mediated cytotoxicity is dependent on oxidant production and does not require Bcr/Abl. We have tested adaphostin against both Philadelphia chromosome (Ph)-positive (K562, KBM5, KBM5-R [IM resistant KBM5], KBM7, and KBM7-R [IM-resistant KBM7]) and Ph-negative (OCI/AML2 and OCI/AML3) cells, and against cells from patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Adaphostin significantly inhibited growth of all cell lines (50% inhibition of cell proliferation [IC50] 0.5-1 microM) except K562 (IC50 13 microM). Ph-positive IM-resistant cell lines showed significant cross resistance to adaphostin. Simultaneous or sequential treatment with adaphostin and IM did not exert a synergistic effect in any KBM line. Adaphostin induced superoxide and apoptosis in a dose-dependent and time-dependent fashion in both Ph-positive and Ph-negative cells. Adaphostin selectively inhibited colony growth of cells from CML (IM-sensitive and IM-resistant) and AML patients. Analysis of tyrosine phosphorylated proteins after treatment with adaphostin revealed alternate effects in different cells consistent with the modulation of multiple targets. In conclusion, adaphostin showed significant and selective activity against CML and AML cells and its development for clinical testing is warranted.

Figure 1

Antiproliferative/cytotoxic activity of adaphostin against chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines. Cells were incubated for 72 h with adaphostin and were then assessed for viability using MTS dye. (A) The effect of adaphostin on CML lines (imatinib‐sensitive) K562, KBM5 and KBM7, and AML lines AML2 and AML3. (B,C) The effect of adaphostin on imatinib‐sensitive (KBM5 and KBM7) and imatinib‐resistant (KBM5‐R and KBM7‐R) cell lines. Results shown are a summary of four independent experiments, each done in triplicate. ^XStatistically significant difference (P < 0.03).

Figure 2

(A) Induction of apoptosis in AML2 and KBM7 cells by adaphostin. Cells were cultured with adaphostin for the times indicated. Apoptosis was then assessed using the caspase 3/7 activity fluorogenic assay. Results shown are representative of two independent experiments and are expressed as relative fluorescence light units (RFLU). (B) Induction of apoptosis in KBM5 cells by adaphostin treatment. KBM5 cells were incubated in the presence of different concentrations of adaphostin for 24 h. Changes in mitochondrial membrane potential (CMXRos/MTGreen), level of caspase 3 and caspase 3‐like activities (PhiPhiLux/PI), and in plasma membrane (annexin V/PI) were evaluated. Results shown are representative of three independent experiments.

Figure 3

Induction of apoptosis after imatinib or adaphostin exposure in Ph‐positive cell lines. KBM5 and KBM7 cells were assessed for annexin V binding after 48 h incubation in the presence of (A) imatinib or (B) adaphostin. Results shown are representative of four independent experiments.

Figure 4

Effect of imatinib and adaphostin combination treatment on chronic myeloid leukemia (CML)‐derived cell lines. (A) Imatinib‐sensitive KBM5 and KBM7, and imatinib‐resistant KBM5‐R and KBM7‐R cells were cultured in the presence of fixed dose of imatinib (IC₂₀ for corresponding cell line) and increasing concentrations of adaphostin. Effect on survival was evaluated after 72 h by MTS assay. Result shown are a summary of three independent experiments, each carried out in triplicate. (B) Different CML cell lines were cultured with adaphostin and imatinib for 24 h, stained with propidium iodide and assessed for DNA fragmentation. The doses used were: K562, 10 µM imatinib and 5 µM adaphostin; KBM5 and KBM7, 0.05 µM imatinib and 0.5 µM adaphostin; KBM5‐R and KBM7‐R, 5 µM imatinib and 1 µM adaphostin. Result shown are a summary of three independent experiments, each carried out in triplicate (except for K562, which is representative of two independent experiments).

Figure 5

Effects of adaphostin on the growth and survival of primary chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells. (A,B) Mononuclear cells from CML and AML patients, as well as normal donors, were plated in methylcellulose with the indicated concentrations of adaphostin. After 8 days, colonies containing more than 50 cells were counted. Data represent the percentage of colonies (compared with the number of colonies formed by untreated cells). (C) In addition, mononuclear cells from AML patients were incubated for 72 h with the indicated concentrations of adaphostin and were then assessed for their viability using MTS dye.

Figure 6

Effect of adaphostin on the phosphorylation of tyrosine polypeptides in leukemic cells. KBM5 (Philadelphia chromosome [Ph]‐positive) and AML3 (Ph‐negative) cells were incubated with adaphostin at concentrations of 2 or 5 µM for 3 or 6 h, lysed and cell lysates were examined by western blot analysis with antiphosphotyrosine antibody. Numbers on arrows indicate the specific molecular weights of the proteins.

Figure 7

Effect of adaphostin on Bcl‐2, Crk‐L and Cbl. KBM5 and AML3 cells were exposed to adaphostin for 0, 3 or 6 h. Lysates were obtained and exposed to western blotting using antibodies directed against Bcl‐2, Crk‐L or Cbl. Neither KBM5 nor AML3 cell lines expressed Crk‐L or Cbl, suggesting that adaphostin‐mediated cytotoxicity observed in both cell lines was not mediated via inhibition of Bcr‐Abl tyrosine kinase activity. K562 cell lysates were used as a positive control for Crk‐L and Cbl expression. No significant variation in the expression of Bcl‐2 was noted in any cell line at the dose range tested. Lines 1, 4, 7 and 10 are control, no drug exposure, and samples; lines 2, 5, 8 and 11 are samples exposed to 2 µM of adaphostin; lines 3, 6, 9 and 12 are samples exposed to 5 µM of adaphostin; line 13 is negative control and line 14 is positive control (K562 cells).

Figure 8

Effect of adaphostin on superoxide production in leukemic cells. (A) AML3 and (B) KBM5 cells were incubated with 2 or 5 µM adaphostin alone or in combination with 24 mM N‐acetyl‐cysteine (NAC) for the time indicated. Superoxide production was assessed using dihydroethidium (DHE) and expressed as percentage of untreated control. Results shown are a summary of three independent experiments.