Figure 1.
DIM induces apoptosis, caspase activation, downregulation of Mcl-1, upregulation of p21, inactivation of Akt, and activation of JNK in U937 human leukemia cells in dose- and time-dependent manners.
U937 cells were treated with various concentrations of DIM as indicated for 12 h and 24 h or treated with 80 µM DIM for 1, 3, 6, 9, 12, and 24 h. (a) Cells were washed twice with PBS and stained with Annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations. The values obtained from annexin V/PI assays represent the mean ± SD for three separate experiments. (b–d) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated.
Figure 2.
DIM induces apoptosis in U937, Jurkat, and HL-60 cells, and in AML blast samples, but not in normal bone marrow mononuclear cells.
(a) U937, Jurkat, and HL-60 cells were treated with 80 µM DIM for 24 h, after which apoptosis was determined by annexin V/PI staining and flow cytometry. ** Values for cells treated with DIM were significantly increased compared to values in control cells by Student's t-test, p<0.01. (b) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (c–d) Blasts from 15 patients with AML and normal bone marrow mononuclear cells were treated with 80 µM DIM for 24 hours, apoptosis was determined by annexin V/PI staining and flow cytometry.
Figure 3.
Effects of inhibition of caspases by Z-VAD-FMK on apoptosis, expression of Mcl-1 and p21, and phosphorylation of Akt and JNK.
U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (20 µM) for 1 h, followed by treatment with 80 µM DIM for 12 h and 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and Z-VAD-FMK were significantly reduced compared to values obtained for DIM alone by Student's t-test, p<0.01. (b–d) Total protein extracts were prepared and subjected to Western blot assay using antibodies as indicated.
Figure 4.
Effects of PI3K inhibitor, LY294002 (LY) and genetic activation of Akt on apoptosis induced by DIM.
U937 cells were pretreated with 20 µM of LY for 1 h, followed by the addition of 40 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and LY were significantly greater than those for cells treated with DIM alone by Student's t-test; p<0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (d) U937 cells were stably transfected with an empty vector (pcDNA3.1), Akt-CA, and Akt-DN. Cells were treated with 80 µM of DIM for 24 h, after which apoptosis was analysed using Annexin V/PI assay. **Values for Akt-CA cells treated with DIM were significantly decreased compared to those for pcDNA3.1 cells by Student's t-test, p<0.01. (e–f) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated.
Figure 5.
Inhibition of JNK significantly protect cells from DIM-induced apoptosis.
U937 cells were pretreated with 10 µM of JNK inhibitor, SP600125 (SP), for 1 h, followed by the addition of 80 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and SP were significantly less than those obtained for cells treated with DIM alone by Student's t-test, p<0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated. (d) U937 cells were transfected with JNK1 siRNA oligonucleotides or controls and incubated for 24 h at 37°C, after which cells were treated with 80 µM of DIM for 24 h. Apoptosis was determined using the Annexin V/PI assay. **Values for cells treated with DIM after transfection with JNK1 siRNA were significantly decreased compared to those for control cells treated with DIM by Student's t-test; p<0.01. Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against JNK1.
Figure 6.
In vivo antileukemic activity of DIM in U937 xenografts.
20 NOD/SCID mice were inoculated with U937 cells (2×106 cells/mouse, i.p.) and randomly divided into two groups (10/group) for treatment with DIM (50 mg/kg, i.p., daily, five times per week) or with vehicle control solvent. (a) Average tumor volume in vehicle control mice and mice treated with 50 mg/kg DIM. P<0.01, significantly different compared with vehicle control by Student's t-test. (b) body weight changes of mice during the 20 days of study. (c) At the 20 days after DIM treatment, the tumors were excised and subjected to H&E staining for determination of pathological evaluation, TUNEL assay for determination of apoptosis, and immunohistochemical staining to determine Cleavage-caspase-3 immunoreactivity. Original magnification ×400. (d) After treatment with DIM, tumor tissues were sectioned and subjected to immunohistochemistry using anbibodies as indicated.