PF-05212384 (PKI-587)

Cell experiment:

Cell proliferation is measured using the MTT dye reduction method. In brief, tumor cells (2×103/100 μL/well) are plated into each well of 96-well plates in RMPI-1640 with 10% FBS and incubated for 24 hours. Culture medium containing several concentrations of GSK2126458 (0-3 μM) or Gedatolisib (PKI-587) (0-3 μM) are added to each well, and the cells are incubated for 24 to 72 hours. Next, 50 μL of MTT (2 mg/mL) is added to each well, and cells are incubated for 2 hours at 37°C. The medium containing MTT solution is removed, and the dark blue crystals are dissolved by adding 100 μL DMSO. The absorbance is measured with a microplate reader at test and reference wavelengths of 550 and 630 nm, respectively. The percentage growth is determined relative to untreated controls. Each experiment is performed at least three times, each with triplicate samples[2].

Animal experiment:

Mice[2] Suspensions of 5×106/0.2 mL 5-8F cells are injected subcutaneously into the right hindlimbs of 5- to 7-week-old female BALB/c-nu/nu nude mice. When tumor volumes reach 200 mm3, mice are randomly assigned to control and treated groups (5 mice per group). The treated groups receive 300 μg/kg GSK2126458, 25 mg/kg PKI-587, IR, 300 μg/kg GSK2126458 combined with IR, or 25 mg/kg Gedatolisib (PKI-587) combined with IR. GSK2126458 is administered by intragastric administration once daily for 5 consecutive days each week and Gedatolisib (PKI-587) is administered by intravenous injection via tail vein once per 5 days. Mice in the IR groups are irradiated with 2 Gy every other day for four treatments. In combination therapy, GSK2126458 or Gedatolisib (PKI-587) is administered 2 hours before IR exposure. Tumor sizes are calculated every 2 days. Tumor regrowth delay is expressed as the time in days for xenografts treated with GSK2126458, Gedatolisib (PKI-587), or radiation to grow from 200 to 1,000 mm3 in volume minus the time in days for untreated tumors to reach the same size.

References:

[1]. Venkatesan AM, et al. Bis(morpholino-1,3,5-triazine) derivatives: potent adenosine 5'-triphosphate competitive phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibitors: discovery of compound 26 (PKI-587), a highly efficacious dual inhibitor. J Med Chem. 2010, 53(6), 2636-2645.
[2]. Freitag H, et al. Inhibition of mTOR's Catalytic Site by PKI-587 Is a Promising Therapeutic Option for Gastroenteropancreatic Neuroendocrine Tumor Disease. Neuroendocrinology. 2017;105(1):90-104.
[3]. Liu T, et al. Dual PI3K/mTOR inhibitors, GSK2126458 and PKI-587, suppress tumor progression and increase radiosensitivity in nasopharyngeal carcinoma. Mol Cancer Ther. 2015 Feb;14(2):429-39.