Figure 1.
Inhibition of acute lymphoblastic leukemic cell survival by the SF isothiocyanate.
(A) Phase contrast images of Nalm-6 cells after culture with 10 µM SF for 24 hours. The chemical structure of sulforaphane is shown. (B) Pre-B ALL cells (Nalm-6, REH and RS-4), non-leukemic LCL cells, and T-ALL cells (Jurkat, RPMI, DND41, and KOPTK1) were incubated with SF or vehicle for 48 hours (n = 3). Cytotoxicity was measured using an ATP-based cell viability assay and expressed as a percentage of the vehicle control. The data are representative of three independent experiments (mean and standard deviation). (C) Samples from pediatric patients diagnosed with pre-B ALL (n = 8) and T-ALL (n = 7) were incubated with the specified SF concentrations for 48 hours. Each patient sample was processed in triplicate (mean and standard deviation). (D) The effect of SF (24 h) on PBMC freshly isolated from healthy donors (n = 4, each measured in triplicates) was compared to cell viability in the pre-B and T-ALL patient samples (mean ± S.D.). (E) Nalm-6 cells were cultured in the presence of SF-glutathione, SF-NAC, SF-cysteine, or SF for 48 hours. Cell viability is shown as mean and S.D. (n = 3).
Table 1.
IC50 values in leukemic cell lines, primary lymphoblast cells from pre-B ALL and T-ALL patients.
Figure 2.
SF induces apoptosis selectively in ALL cell lines.
(A) Apoptosis was evaluated by annexin-V and 7-AAD staining of LCL, Nalm-6, Jurkat and KOPTK1 cells cultured in the absence or presence of 7.5 µM SF. The data are representative of three independent experiments. (B) The percentages of annexin-V-positive cells were determined for each cell line cultured in the presence or absence of SF. (C) SF activates the proteolytic cascade of caspases and PARP in leukemic cells. LCL cells (control), pre-B ALL cells (Nalm-6, REH, and RS-4), and T-ALL cells (Jurkat, RPMI, DND41, and KOPTK1) were incubated with 7.5 µM SF for 24 hours and analyzed by immunoblotting. The arrows indicate the cleaved forms of the caspases and PARP. β-actin was used as a loading control. The data represent the mean and standard deviation (n = 3). *** P<0.001 (two-tailed Student’s t-test).
Figure 3.
SF causes G2/M cell cycle arrest in leukemic cells.
(A) The DNA content of LCL, Nalm-6, Jurkat and KOPTK1 cells incubated with 7.5 µM SF or vehicle for 24 hours was analyzed by propidium iodide staining of nuclei and flow cytometry. A representative profile is shown. (B) The cell cycle distribution was calculated as described in the Methods. The data represent the mean and standard deviation (n = 3). * P<0.05, ** P<0.01, *** P<0.001 (two-tailed Student’s t-test).
Figure 4.
Effect of SF on the levels of total and phosphorylated proteins involved in the G2/M cell cycle arrest.
(A) The protein levels of KLF4, p21, p53, cyclin B1, cyclin D1, cdc2, and cdc25C and phosphorylation of cdc2 (Tyr 15) and cdc25C (Ser 216) were evaluated by immunoblots in LCL, pre-B ALL, and T-ALL cell lines incubated with 7.5 µM SF or vehicle for 24 hours. β-actin was used as a loading control. The data are representative of three independent experiments. (B) Detection of phospho-H2AX (pH2AX) in nuclei by flow cytometry in Nalm-6 and Jurkat cells treated with 7.5 µM of SF or Etoposide (ETO) for 24 hours. The data are representative of three independent experiments. (C) Diagram depicting the role of SF in the G2/M cell cycle arrest of ALL cells.
Figure 5.
SF inhibits the AKT/mTOR survival pathway in leukemic cells.
The protein levels of phosphorylated and total AKT and mTOR were examined by immunoblotting. LCL, pre-B ALL, and T-ALL cell lines were incubated with 7.5 µM SF or vehicle for 24 hours. β-actin was used as a loading control. The data are representative of three independent experiments.
Figure 6.
In vivo anti-leukemic effect of SF in ALL xenograft models.
(A) Nalm-6 cells were transduced with the FFluc retrovirus and injected intravenously into NOD/SCID mice. Twenty-four hours later, the mice were treated with SF or vehicle (2 mg i.p. daily) and monitored weekly for the distribution of leukemic cells by bioluminescence imaging (BLI). (B) The Nalm-6-FFluc cells were mixed with a high-protein matrigel and injected into the flank of NOD/SCID mice. One week later, tumor establishment was confirmed by BLI; then, the mice were treated by oral gavage (2 mg/gave, twice daily) for 7 days. The total counts were determined for the control- and SF-treated mice. The data are representative of three independent experiments. The statistical significance was calculated using the two-tailed Student’s t-test.