Various concentrations of WF-210 or PAC-1 are added to procaspase-3 in buffer containing 50 mM HEPES, 0.1% CHAPS, 10% glycerol, 100 mM NaCl, 0.1 mM EDTA, 10 mM DTT pH 7.4,and incubated for 12 h at 37°C. The final volume is 10 mL and the final concentration of procaspase-3 is 1 mM. Then 40 mL of the substrate Ac-DEVD-pNA (final concentration 0.4 mM) in buffer containing 50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA disodium salt, 0.10% CHAPS, 10% glycerol is added and the absorbance of the plate is read at 405 nm for a total of 1 h. The slope of the linear portion for each well is determined as the enzyme activity^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell viability is measured using the MTT method or the Cell Titer-Glo luminescent assay. For the MTT assay, the cells (1×10⁵ cells/mL) are seeded into 96- well culture plates. After overnight incubation, cells are treated with various concentrations of agents (PAC-1, WF-210 or other agents) for 24 or 72 h. Then 10 mL MTT solution (2.5 mg/mL in PBS) is added to each well, and the plates are incubated for an additional 4 h at 37°C. After centrifugation (2500 rpm, 10min), the medium containing MTT is aspirated, and100mL DMSO is added. The optical density of each well is measured at 570 nm with a Biotek Synergy HT Reader. The Cell Titer-Glo kit is used to determine the relative levels of intracellular ATP as a biomarker for live cells^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
To determine the in vivo anti-tumor activity of WF-210, viable human gallbladder cancer GBC-SD cells (5×10⁶/100 mL PBS per mouse), human breast cancer MDA-MB-435 cells (1×10⁷/100 mL PBS per mouse), human liver cancer Hep3B cells (5×10⁶/100 mL PBS per mouse) and human breast cancer MCF-7 cells (1×10⁷/100 mL PBS per mouse) are subcutaneously (s.c.) injected into the right flank of 7- to 8-week old male SCID mice or Balb/c nude mice. Cell numbers are confirmed by trypan blue staining prior to injection. Specially, MCF-7 xenograft mice are also administered with the hormone 17-beta-estradiol (3 mg/kg) on alternate days. When the average s.c. tumor volume reached 100 mm³, mice are randomly divided into various treatment and control groups (eight mice per group). Tumor size is measured once every two days with a caliper (calculated volume=shortest diameter²×longest diameter/2). Body weight, diet consumption and tumor size are recorded once every two days. After two or four weeks, mice are sacrificed and tumors are excised and stored at -80°C until further analysis.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Wang F, et al. A novel small-molecule activator of procaspase-3 induces apoptosis in cancer cells and reduces tumor growth in human breast, liver and gallbladder cancer xenografts. Mol Oncol. 2014 Dec;8(8):1640-52.

  [2]. Seervi M, et al. ERO1α-dependent endoplasmic reticulum-mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1). Cell Death Dis. 2013 Dec 19;4:e968.

  [3]. Putt KS, et al. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol. 2006 Oct;2(10):543-50.

392.49

C₂₃H₂₈N₄O₂

315183-21-2

O=C(N/N=C/C1=CC=CC(CC=C)=C1O)CN2CCN(CC3=CC=CC=C3)CC2

Room temperature in continental US; may vary elsewhere