The sulforhodamine B (SRB) cytotoxicity assay is used. Briefly, optimal cell density for cytotoxicity assay, 2×10⁴ to 3×10⁴ cells per well, is determined by growth curve analysis. HDMECs are seeded at 2.5×10⁴ per well in a 96-well plate and allowed to adhere overnight. Drug or control is diluted in EGM2-MV and layered onto cells, which are allowed to incubate for times as indicated in the figures. Alternatively, HDMECs are coincubated with TW-37 and 0 to 100 ng/mL recombinant human VEGF (rhVEGF)₁₆₅ or 0 to 100 ng/mL recombinant human CXCL8. Cells are fixed on the plates by addition of cold trichloroacetic acid (10% final concentration) and incubation for 1 hour at 4°C. Cellular protein is stained by addition of 0.4% SRB in 1% acetic acid and incubation at room temperature for 30 minutes. Unbound SRB is removed by washing with 1% acetic acid and the plates are air dried. Bound SRB is resolubilized in 10 mM unbuffered Tris-base and absorbance is determined on a microplate reader at 560 nm. Test results are normalized against initial plating density and drug-free controls. Data are obtained from triplicate wells per condition and are representative of at least three independent experiments^[1].

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

Mice^[1]
Porous poly L-lactic acid scaffolds (6×6×1 mm) with an average pore diameter of 180 μm are fabricated. Just before implantation, scaffolds are seeded with 1×10⁶ HDMECs in a 1:1 Matrigel/EGM2-MV mix. Male severe combined immunodeficient (SCID) mice (CB.17.SCID) are anesthetized with ketamine and xylazine, and two scaffolds are implanted s.c. in the dorsal region of each mouse. At 10 days after transplantation, six mice per treatment are treated with 3 mg/kg or 30 mg/kg TW-37 (in vehicle: PBS/Tween 80/ethanol) or vehicle alone i.v. for 5 consecutive days. At the end of the treatment period, mice are euthanized, and the scaffolds are retrieved, fixed overnight in 10% buffered formaldehyde at 4°C, and mounted on glass slides. Immunohistochemistry is done for Factor VIII and microvessels are counted in 6 fields per scaffold and 12 scaffolds per treatment at ×200 magnification. Alternatively, sections are stained with H&E and occluded blood vessels are counted.

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

• [1]. Zeitlin BD, et al. Antiangiogenic effect of TW37, a small-molecule inhibitor of Bcl-2. Cancer Res. 2006 Sep 1;66(17):8698-706.

  [2]. Mohammad RM, et al. Preclinical studies of TW-37, a new nonpeptidic small-molecule inhibitor of Bcl-2, in diffuse large cell lymphoma xenograft model reveal drug action on both Bcl-2 and Mcl-1. Clin Cancer Res. 2007 Apr 1;13(7):2226-35.

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877877-35-5

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