In Vitro

Kinase Assay:The PI3K inhibitor PIK-75 is dissolved at 10 mM in dimethyl sulfoxide and stored at –20°C until use. PI3K enzyme activity is determined in 50 μL of 20 mM HEPES, pH 7.5, and 5 mM MgCl2 containing 180 μM phosphatidyl inositol, with the reaction started by the addition of 100 μM ATP (containing 2.5 μCi of [γ-32P]ATP). After a 30-minute incubation at room temperature, the enzyme reaction is stopped by the addition of 50 μL of 1 M HCl. Phospholipids are then extracted with 100 μL of chloroform/methanol [1:1 (v/v)] and 250 μL of 2 M KCl followed by liquid scintillation counting. Inhibitors are diluted in 20% (v/v) dimethyl sulfoxide to generate a concentration versus inhibition of enzyme activity curve, which is then analyzed with the use of Prism version 5.00 for Windows to calculate the IC50. For kinetic analysis, a luminescent assay measuring ATP consumption is used. PI3K enzyme activity is determined in 50 μL of 20 mM HEPES, pH 7.5, and 5 mM MgCl2 with PI and ATP at various concentrations. After a 60-minute incubation at room temperature, the reaction is stopped by the addition of 50 μL of Kinase-Glo followed by a further 15-minute incubation. Luminescence is then read using a Fluostar plate reader. Results are analyzed using Prism.

Cell Assay: Mitochondrial activity is assessed after stimulation with TGFβ with or without inhibitors for 48 hours using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Harvested washed cells are resuspended in DMEM-lO% FCS and aliquoted (500 μL) into 24-well cluster plates prior to serial dilution (1:2) in duplicates. To each well, 100 μL of an appropriate MTT concentration (dissolved in PBS and filtered through a 0.2 μm filter before use to remove any blue formazan product) is added immediately after diluting the cells, which are then incubated for 3.5 hours at 37 °C. The resulting blue formazan product is solubilized overnight (16 hours) at 37 °C by the addition of 500 μL of 10% sodium dodecyl sulfate　(SDS) in 0.01 M HCl to each well. A sample (150　μL)　from each duplicate well is transferred to a 96-well　microplate, and the optical density determinedby automated spectrophotometry against a reagent blank (no　cells).　Absorbance is measured at a test wavelength of 570 nm and a reference wavelength of 690 nm. For each primary cell culture, results from three to six wells from each treatment are averaged, and data are expressed as absorbance 570 to 690 nm. Cells: A2780, A2780/cp70, 2780AD, HCT116, HT29, WIL, CALU-3, MCF7, PC3 and HS852 cells.

PIK-75 shows the impressive potency and isoform selectivity at p110α while the corresponding IC50 values are 1300 nM, 76 nM and 510 nM for other PI3K isoforms, p110β, -γ, and -δ, respectively. Furthermore, when binding to purified p110α, PIK-75 is a noncompetitive inhibitor with respect to ATP with Ki of 36 nM and competitive with respect to the substrate PI with Ki of 2.3 NM. PIK-75 also shows potent inhibition of DNA-PK. PIK-75 (1 μM) reduces cell survival by significantly decreasing mitochondrial activity in unstimulated nonasthmatic airway smooth muscle (ASM) cells, asthmatic ASM cells, and lung fibroblasts. While in TGFβ-stimulated ASM cells, PIK75 only decreases mitochondrial activity in asthmatic cells without effects in nonasthmatic cells. A recent study shows that PIK-75 (10 nM) inhibits TNF-α-induced CD38 mRNA expression and significantly attenuates of TNF-α-induced ADP-ribosyl cyclase activity in human airway smooth muscle cells.

In Vivo

In the ErbB3WT tumor model, PIK-75 reduces in vitro chemotactic response to HRGβ1 and lowers pAkt levels by 40%. Besides, PIK-75 significantly reduces tumor cell motility and in vivo invasion in ErbB3WT primary tumors. In the CD1 male mice, PIK-75 leads to serious impairments in the insulin tolerance test (ITT) and glucose tolerance test (GTT), and an increase in glucose production during a pyruvate tolerance test (PTT).

Animal model

RAMTLn3 cells are injected into the right fourth mammary fat pad from the head of female severe-combined immunodeficient/NCr mice.

Formulation & Dosage

Dissolved in DMSO and then diluted in PBS.; ≤1 μM; i.p.

References

[1] Zheng Z, et al. Mol Pharmacol. 2011, 80(4), 657-664.;[2] Smirnova T, et al. Oncogene. 2012, 31(6), 706-715.

RF2-knockdown reduces the proliferation of pancreatic cancer AsPC-1 cells. Int J Oncol. 2014 Mar;44(3):959-69.

PIK-75 reduces NRF2 transcriptional activity in pancreatic cancer cells.

PIK-75 induces the proteasome-mediated degradation of NRF2. Int J Oncol. 2014 Mar;44(3):959-69.

PIK-75 potentiates gemcitabine-induced cytotoxicity in pancreatic cancer cells. Int J Oncol. 2014 Mar;44(3):959-69.

PIK-75 inhibits the proliferation and survival of pancreatic cancer cells through apoptotic cell death. Int J Oncol. 2014 Mar;44(3):959-69.

PIK-75 enhances gemcitabine-induced apoptotic cell death and reduces MRP5 expression. Int J Oncol. 2014 Mar;44(3):959-69.