Apoptozole

Kinase experiment:

Stock solutions of malachite green (0.081% w/v), polyvinyl alcohol (2.3% w/v), and ammonium heptamolybdate tetrahydrate (5.7% w/v in 6 M HCl) are prepared and stored at 4°C. Three solutions are mixed with water in the ratio of 2 : 1 : 1 : 2 to prepare the malachite green reagent. For the determination of the ATPase activity of Hsc70, a master mixture of an ATPase domain of Hsc70 is prepared in assay buffer (100 mM Tris-HCl, 20 mM KCl, and 6 mM MgCl2, pH 7.4) as the final concentration of 1 mM. An aliquot (10 mL) of this mixture is added into each well of a 96-well plate. To this solution is added each compound (including Apoptozole) in assay buffer, and the plate is incubated for 30 min at room temperature. To start the reaction, 1 mL of 4 mM ATP is added to the solution. The final concentrations are 1 mM protein and 200 mM ATP in 20 mL of assay buffer. After 3 h incubation at 37°C, 80 mL of the malachite green reagent is added into each well. The samples are mixed thoroughly and incubated at 37°C for 15 min, and 10 mL of 34% sodium citrate is added to stop the nonenzymatic hydrolysis of ATP. The absorbance is determined at 620 nm on a SpectraMax 340 PC 384[1].

Cell experiment:

Cells (5 × 105 per well) are plated in triplicate in 96-well plates in 0.1 mL of culture media with 10% FBS. After 24 hr, cells are treated with various concentrations of Apoptozole (0-15 μM) in culture media with 3% FBS (final volume: 0.2 mL per well) for 18, 48, and 72 hr before treatment with MTT. Absorbance at 570 nm is measured using a UV microplate reader[3].

Animal experiment:

Male nude mice are housed in a pathogen-free room under controlled temperature and humidity. Mice aged 4 weeks are injected with tumor cells for the xenograft experiments. Viable A549 and RKO cells (5 × 106) and HeLa cells (5 × 106) are injected subcutaneously into the flank of mice. The A549 and RKO cell xenograft mice are immediately and randomly assigned to two groups. The first group (n = 10) is used as a control group and receives vehicle only. The second group (n = 10) receives intraperitoneal injections of Apoptozole (4 mg/kg/day) every other day for 2 weeks. The HeLa cell xenograft mice are immediately and randomly assigned to four groups. The first group (n = 10) is a control group receiving vehicle only. The second group (n = 10) receives intraperitoneal injections of Apoptozole (4 mg/kg/day) every other day for 2 weeks. The third group (n = 10) receives intraperitoneal injections of doxorubicin (15 mg/kg/day) every other day for 2 weeks. The fourth group (n = 10) receives intraperitoneal injections of Apoptozole (4 mg/kg/day) and doxorubicin (15 mg/kg/day) every other day for 2 weeks. Tumors in all mice are measured in two dimensions with calipers every 3 days and tumor volumes are calculated using the formula volume = w × l2/2, where w is the width at the widest point of the tumor and l is the length perpendicular to w. The results from individual mice are plotted as average tumor volumes versus time[3].

References:

[1]. Williams DR, et al. An apoptosis-inducing small molecule that binds to heat shock protein 70. Angew Chem Int Ed Engl. 2008;47(39):7466-9.
[2]. Cho HJ, et al. Probing the effect of an inhibitor of an ATPase domain of Hsc70 on clathrin-mediated endocytosis. Mol Biosyst. 2015 Oct;11(10):2763-9.
[3]. Ko SK, et al. A small molecule inhibitor of ATPase activity of HSP70 induces apoptosis and has antitumor activities. Chem Biol. 2015 Mar 19;22(3):391-403.