Figure 1.
Dose- and time-dependent effect of ASHD on viability of leukemic cells.
A. Chemical structure of azaspiro hydantoin derivative, ASHD. B–D. MTT assay showing effect on cell proliferation following treatment with ASHD. After 48 and 72 h of treatment with ASHD (10, 50, 100 and 250 µM), cells were incubated with MTT to determine cell proliferation. Bar diagram showing percentage of cell proliferation of Reh (B), K562 (C) and 8E5 (D). Error bars represented in each panel is based on three independent batches of experiments.
Table 1.
IC50 value of ASHD calculated at different time points.
Figure 2.
Effect of ASHD on cell cycle progression in leukemic cells.
A Tritiated thymidine incorporation assay to determine the effect of ASHD on cell proliferation. The incorporation of tritiated thymidine was measured after 48 h of compound treatment. Incorporation of [3H] Thymidine after addition of ASHD to Reh (a) and K562 (b) cells following addition of ASHD. Error bars represented in each panel is based on three independent batches of experiments. B–C. Reh and K562 cells were harvested following incubation with ASHD (10, 50, 100 and 250 µM) for 72 h, stained with propidium iodide and sorted using FACS. Results are presented as histograms and are representative of two independent experiments with similar findings. Histogram resulting from the FACS analysis of Reh cells (B) and K562 cells (C). In each panel, a and b are histogram of control cells and cells treated with DMSO, respectively. c, d, e and f represent cells treated with ASHD (10, 50, 100 and 250 µM, respectively). D. Histograms showing the quantification of effect of ASHD treatment on specific cell cycle stages of Reh and K562. Comparison of G1 phase (a), S phase (b), G2/M phase (c) and G0/G1 phase (d) are shown. Error bars represented in each panel is based on two independent batches of experiments. E. Expression levels of cell cycle related proteins upon ASHD treatment. Western blotting studies showing expression profile of p-Histone 3, PCNA, Cyclin B1and CDK2 following treatment of Reh cells with ASHD (100 μM). F. The quantification of the western blots for each protein shown in panel E.
Figure 3.
Detection of apoptosis induced by ASHD using flow cytometry and confocal microscopy.
Reh (A) and K562 (B) cells were cultured with ASHD (50 and 250 μM) for 72 h and processed for Annexin V- FITC/PI double-staining. The cells were then quantitatively or qualitatively monitored. In panels A and B, lower left quadrant shows cells which are negative for both Annexin V-FITC and PI, lower right shows Annexin V positive cells which are in the early stage of apoptosis, upper left shows only PI positive cells which are dead, and upper right shows both Annexin V and PI positive, which are in the stage of late apoptosis or necrosis. The values mentioned in the quadrants show the percentage of cells positive for both the Annexin V and PI (Top) or Annexin V alone (Bottom). In both panels A and B, cells treated with DMSO (a), ASHD, 50 μM (b), and ASHD 250 μM (c) are shown. In both panels, bar diagram showing comparison of early and late apoptotic cells at different doses of ASHD treatment are presented (d). (C) and (D) shows confocal microscopy visualization of Reh or K562 cells, following treatment with ASHD. Cells incubated with DMSO alone (a), or ASHD 50 μM (b, c) and 250 μM (d, e) respectively are used for the study.
Figure 4.
Effect of ASHD on mitochondrial transmembrane permeability (Δψm).
Reh cells incubated with ASHD at different concentrations for 24 (A), 48 (B) and 72 h (C) were stained with JC-1 dye and loss of mitochondrial membrane potential was assessed with the signal from monomeric and J-aggregate JC-1 fluorescence by flow cytometry. DMSO treated cells were taken as control. 2,4-DNP was used as a positive control. Dot plots show that Δψm increases in a dose- and time-dependent manner. Bar diagram showing percentage of apoptotic and nonapoptotic cells are shown on the right side of each panel.
Figure 5.
ASHD alters the expression of apoptotic proteins in Reh and K562 cells.
A–C. Cell lysates were prepared from Reh cells after incubating with ASHD (100 µM) for 24, 48 and 72 h. DMSO treated cell lysate was used as control. Approximately, 40 µg of protein per sample was resolved on SDS-PAGE and transferred to a PVDF membrane. The membrane was probed for the expression of BAD, BCL2 (A), PARP (B) and caspase 3, caspase 9 and caspase 8 (C) with specific primary antibodies and appropriate secondary antibodies. D–E. Cell lysates were prepared from K562 cells after incubating with ASHD (30 µM) for 48 h and used for western blotting. The proteins studied are BAD, BAX, PARP (D), and caspase 8, caspase 9 and cytochrome C (E). The α-tubulin was used as an internal loading control in all the panels. F–G. Effect of pancaspase inhibitor (z-VAD-FMK) on Reh cells treated with ASHD. Approximately 0.75×105 cells/ml were cultured and incubated with 30 µM ASHD, with or without 50 µM z-VAD-FMK. DMSO treated cells were used as vehicle control. F. MTT assay showing effect of ASHD on cell proliferation following treatment with z-VAD-FMK at 24 and 48 h. G. Trypan blue assay showing cell viability. For other details refer Fig. 1 legend. Error bars in panels, F and G are based on three independent batches of experiments.
Figure 6.
Detection of DNA strand breaks and repair proteins in Reh and K562 cells following treatment with ASHD.
ASHD treated Reh (A) or K562 (B) cells (72 h) were subjected to DNA damage analysis by comet assay. In both panels, cells were treated with (a) vehicle control, (b, c) ASHD 50 μM and (d, e), ASHD 250 μM. (C, D). Agarose gel profiles showing DNA fragmentation. The chromosomal DNA was extracted from Reh (C) and K562 (D) cells, following treatment with different concentrations of ASHD. The purified DNA was then resolved on a 1% agarose gel. In both panels, Lane 1, DMSO, Lane 2–4, 50, 100 and 250 μM of ASHD, respectively. “M” is Marker. (E–G) Altered expression of p53 and KU70/80 following treatment with ASHD in Reh (E,F) and K562 (G) cells studied using immunoblotting. For other details refer Fig. 6 legend.
Figure 7.
Proposed model for the mechanism of ASHD induced cytotoxicity through apoptosis.
ASHD treatment can induce apoptosis through one of the following pathways. First, the DNA damages induced by ASHD could lead to the activation of p53, which can block the expression of BCL2. Downregulation of BCL2 probably leads to loss of mitochondrial membrane permeability and further to the activation of the initiator caspase 9. This results in the activation of the apoptotic protein cascade wherein caspase 3 gets cleaved and activated which can further either induce PARP cleavage or cleave the inhibitor of the caspase-activated DNase which finally results in fragmentation and degradation of the cellular DNA. In addition, ASHD activates p38 MAPK pathway, upregulation of proapoptotic proteins and BAD, which can directly result in apoptosis.