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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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PAC-1 is a potent procaspase-3 activator with EC50 of 0.22 μM. |
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Targets
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Procaspase-3 |
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IC50 |
0.22 μM [1] |
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In Vitro
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PAC-1 activates procaspase-7 in a less efficient manner with EC50 of 4.5 μM. Elevated caspase 3 level in cancer cell lines allows PAC-1 to selectively induce apoptosis in a manner proportional to procaspase-3 concentration with IC50 of 0.35 μM for NCI-H226 cells to ~3.5 μM for UACC-62 cells. PAC-1 induces apoptosis in the primary cancerous cells with IC50 values of 3 nM to 1.41 μM, more potently than in the adjacent noncancerous cells with IC50 of 5.02 μM to 9.98 μM, which is also directly related to the distinct procaspase-3 concentration. [1] PAC-1 activates procaspase-3 by chelating zinc ions, thus relieving the zinc-mediated inhibition and allowing procaspase-3 to auto-activate itself to caspase-3. [2] PAC-1 is capable to induce cell death in Bax/Bak double-knockout cells and Bcl-2 and Bcl-xL-overexpressing cells with the same efficacy as its wild-type counterpart in a delayed manner. PAC-1 induces cytochrome c release in a caspase-3 independent manner, which subsequently triggers downstream caspase-3 activation and cell death. PAC-1 can not induce cell death and caspase-3 activation in Apaf-1 knockout cells, suggesting that apoptosome formation is essential for caspase-3 activation by PAC-1-mediated cell death. [3] |
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In Vivo
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Administration of PAC-1 at 5 mg with low and steady releasing significantly inhibits the growth of ACHN renal cancer xenograft in mice. Oral administration of PAC-1 (50 or 100 mg/kg) significantly retards tumor growth of NCI-H226 lung cancer xenograft in a dose-dependent manner, and markedly prevents the cancer cells from infiltrating the lung tissue. The in vivo anti-tumor effect of PAC-1 is ascribed to procaspase-3 activation and subsequently apoptosis induction consistent with the activity in vitro. [1] |
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Clinical Trials
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Features
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PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3 |
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Protocol
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Kinase Assay
[1]
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| In vitro procaspase-3 activation |
Procaspase-3 is expressed and purified in Escherichia coli. Various concentrations of PAC-1 are added to 90 μL of a 50 ng/mL of procaspase-3 in caspase assay buffer in a 96-well plate, The plate is incubated for 12 hours at 37 °C. A 10 μL volume of a 2 mM solution of caspase-3 peptidic substrate acetyl Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNa) in caspase assay buffer is then added to each well. The plate is read every 2 minutes at 405 nm for 2 hours in a Spectra Max Plus 384 well plate reader. The slope of the linear portion for each well is determined, and the relative increase in activation from untreated control wells is calculated. |
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Cell Assay
[1]
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| Cell Lines |
U-937, HL-60, CRL-1872, ACHN, NCI-H226, Hs888Lu, Hs578Bst, MCF-10A, SK-MEL-5, BT-20, MDA-MB-231, UACC-62, SK-N-SH, B16-F10 , Hs 578t, and PC-12 |
| Concentrations |
Dissolved in DMSO, final concentrations ~100 μM |
| Incubation Time |
72 hours |
| Methods |
Cells are exposed to various concentrations of PAC-1 for 72 hours. Cell death is quantified by the addition of MTS/PMS CellTiter 96 Cell Proliferation Assay reagent. The plates are incubated at 37 °C for approximately 1 hour (until the colored product formed), and the absorbance is measured at 490 nm. |
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Animal Study
[1]
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| Animal Models |
Ovariectomized female athymic BALB/c (nude, nu/nu) mice injected subcutaneously with ACHN cells, male athymic BALB/c nude mice injected subcutaneously with NCI-H226 cells, and male athymic BALB/c–/– mice injected intravenously with NCI-H226 cells |
| Formulation |
Mixed with cholesterol and pelleted into a 3-mm-diameter 20-mg (total weight) pellet, or dissolved in a mixture of 24:1 vegetable oil/DMSO |
| Doses |
~100 mg/kg |
| Administration |
Pellet implantation subcutaneously or oral gavage |
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References |
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[1] Putt KS, et al. Nat Chem Biol, 2006, 2(10), 543-550.
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[2] Peterson QP, et al. J Mol Biol, 2009, 388(1), 144-158.
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[3] Seervi M, et al. Cell Death Dis, 2011, 2, e207.
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