C O M M U N I C A T I O N S
Figure 1. Induction of death in U-937 cells by library members at 100
µM.
Figure 3. Cancerous U-937 cells are almost completely killed by 13-D,
whereas non-cancerous white blood cells show virtually no death. (A) Cells
were treated with 500 µM 13-D for 72 h. The IC50 of 13-D for U-937 cells
is 44 µM, whereas it is greater than 1000 µM for the splenocytes. (B) The
T-cell population from the mouse splenocytes was purified and stimulated
to grow, treated with 500 µM 13-D for 72 h, and stained with propidium
iodide. Flow cytometry analysis of the nontreated (green) and treated (red)
samples indicates a viable cell population (R1) of 86 and 87%, respectively.
death was assessed after 72 h. Again, 13-D had virtually no effect
on these purified non-cancerous cells (Figure 3B).
In summary, we have identified a small molecule that selectively
induces apoptosis in cancerous white blood cells but is nontoxic
toward non-cancerous white blood cells. In the process, a facile
synthetic route for the synthesis of natural product 1 and a library
of its derivatives has been developed. Further studies on the
biological basis of this selectivity will be reported in due course.
Acknowledgment. We are grateful to the National Science
Foundation (NSF-CAREER Award to P.J.H.) for funding this
research. P.J.H. is the recipient of a Research Innovation Award
(from the Research Corporation) and a Beckman Young Investigator
Award.
Figure 2. Induction of apoptosis by 13-D. (A) Caspase-3 activity of U-937
cells treated with 100 µM 13-D. (B) Flow cytometry of U-937 cells treated
with 100 µM 13-D and stained with JC-9 after 72 h; nontreated and
etoposide-treated cells are shown as controls.
Supporting Information Available: Full experimental protocols
and characterization data (PDF). This material is available free of charge
Further experiments assessed whether the observed death from
12-D, 13-D, and 15-D was due to apoptosis or necrosis. In this
regard, compound 13-D appeared to be a fairly potent apoptotic
inducer. Cells treated with this molecule showed several hallmarks
of apoptotic cell death, including strong induction of caspase-3
activity (Figure 2A) and staining with the apoptotic-specific dye
JC-9 (Figure 2B) as assessed by flow cytometry; JC-9 provides a
sensitive readout on mitochondrial depolarization, and dyes of this
class are commonly used to quantitate apoptosis.11 In this case,
100 µM 13-D had induced apoptosis in over 70% of the cell
population after 72 h (as quantitated from Figure 2B). Additional
evidence of apoptosis was provided by microscopy, which showed
membrane blebbing and cell shrinkage in the 13-D treated samples
(see Supporting Information).
Finally, the selectivity of 13-D for cancerous white blood cells
over non-cancerous white blood cells was determined. For this
experiment, the spleen from a euthanized mouse was harvested,
and the splenocytes were isolated.12 The T-cells were then
stimulated to grow by the addition of concanavalin A. Remarkably,
as the images in Figure 3A show, high concentrations (500 µM) of
compound 13-D showed virtually no toxicity toward the actively
dividing splenocytes, while almost completely killing the cancerous
U-937 cells. The IC50 of 13-D was 44 µM for the cancerous U-937
cells, but greater than 1000 µM for the non-cancerous splenocytes.
Additionally, in a separate experiment T-cells were purified from
the heterogeneous splenocyte mixture, stimulated to grow with
concanavalin A (in the presence or absence of 500 µM 13-D), and
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