Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy


Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.

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Figure 1: PAC-1 activates procaspase-3 in vitro.
Figure 2: PAC-1 induces apoptosis in HL-60 cells.
Figure 3
Figure 4: PAC-1 induces death in cells isolated from freshly resected colon tumors.
Figure 5: PAC-1 significantly retards tumor growth in three mouse models of cancer.


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This work was supported by the National Science Foundation (NSF CAREER award to P.J.H., CHE-0134779), the University of Illinois (to P.J.H.) and the National Institutes of Health (Grant CA77355 to W.G.H. and AG024387 to W.G.H. and D.R.D.). K.S.P. is supported by a fellowship from the American Chemical Society Division of Medicinal Chemistry. M.S.H. was supported by National Institute of Environmental Health Sciences Training Grant 5T32ES007326-05. J.T.K., S.K.H. and H.J. are recipients of a Brain Korea 21 fellowship. M.H.C. is supported by a Korea Science and Engineering Foundations (KOSEF), Ministry of Science and Technology (MOST) grant (550-20060062). We thank P. Tender and L. Tangen (Carle Foundation Hospital) for the primary colon cancer samples. We thank R. Hoffman (University of Illinois-Chicago Cancer Center) for the generous gift of human bone marrow. We thank G. Salvesen (Burnham Institute) for the gift of the procaspase-3 and procaspase-7 expression vectors. We thank D. Goode and A. Sharma (University of Illinois) for synthesis of the peptidic caspase substrate. We acknowledge the assistance of the Flow Cytometry Facility of the Biotechnology Center at the University of Illinois.

Author information




K.S.P. and P.J.H. were responsible for designing the experiments and writing the manuscript. K.S.P. conducted the high-throughput screen, all in vitro and all cell culture experiments with PAC-1, and all experiments with the primary colon tumors. G.W.C. and J.M.P. assisted with the high-throughput screen. J.S.S. was responsible for the synthesis of PAC-1 and its derivatives. M.S.H. and W.G.H. evaluated PAC-1 in the ACHN xenograft model. J.T.K., S.K.H., H.J. and M.H.C. evaluated PAC-1 in the NCI-H226 xenograft models. D.R.D. and M.I.C. determined the concentrations of PAC-1 in mouse serum.

Corresponding author

Correspondence to Paul J Hergenrother.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Western blot showing PAC-1 efficacy at 5 μM. (PDF 66 kb)

Supplementary Fig. 2

Flow cytometry data showing PAC-1 efficacy at 5 μM. (PDF 75 kb)

Supplementary Fig. 3

Graph showing that Z-VAD-FMK inhibits cell death induced by PAC-1. (PDF 22 kb)

Supplementary Fig. 4

Graph showing that PAC-1 does not inhibit PARP-1. (PDF 19 kb)

Supplementary Fig. 5

Graph showing etoposide-induced cell death does not correlate with cellular procaspase-3 concentrations. (PDF 20 kb)

Supplementary Fig. 6

Primary data showing procaspase-3 concentrations in cancerous and noncancerous colon tissue taken from 23 people. (PDF 312 kb)

Supplementary Fig. 7

Graph showing the concentration of PAC-1 in serum. (PDF 19 kb)

Supplementary Methods

All chemical and biological protocols. (PDF 202 kb)

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Putt, K., Chen, G., Pearson, J. et al. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol 2, 543–550 (2006).

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