PIK-75

Kinase experiment:

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 starts by the addition of 100 μM ATP (containing 2.5 μCi of [γ-32P]ATP). After a 30-min 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 (e.g., PIK75) ) are diluted in 20% (v/v) DMSO 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[2].

Cell experiment:

MIA PaCa-2 cells are maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (HI-FBS), 2.5% horse serum (HS) and 100 U/mL Penicillin/Streptomycin. AsPC-1 cells are cultured in RPMI-1640 media supplemented with 20% HI-FBS, 100 U/mL Penicillin/Streptomycin and 1 mM sodium pyruvate. A total of 2,000 human pancreatic cancer cells (MIA PaCa-2 or AsPC-1) per well are plated in 96-well flat-bottom plates and then treated with either Gemcitabine, PIK-75 alone (0.1 μM, 0.3 μM and 1 μM) or in combination of both drugs with indicated concentrations. At the indicated times, 20 μL of 1 mg/mL MTT in PBS is added to each well and further incubated for ~4 h. After centrifugation and removal of the medium, 150 μL of DMSO is added to each well to dissolve the formazan crystals. The absorbance is measured at 562 nm using an ELx808 absorbance microplate reader. Absorbance of untreated cells is designated as 100%, and the relative viable cells are expressed as a percentage of this value[3]

Animal experiment:

Mice[3]MIA PaCa-2 cells (~1.7×106 cells/mouse) mixed with Matrigel are injected subcutaneously into the flank of male athymic nude (Foxn1nu) mice aged 6-weeks. Gemcitabine (50 mg/mL) is dissolved in PBS and PIK-75 (20 mg/mL) is dissolved in DMSO. Injection solution is made as 10% of Cremophor EL and 3% of poly(ethylene glycol) 400 in sterile water. Before administration of compounds, Gemcitabine is further diluted in PBS and DMSO or PIK-75 is further diluted in the injection solution and sterilized by 0.2 μm filter unit. These diluents are mixed with 1:1 ratio and administered into peritoneal cavity of the mouse. Gemcitabine (20 mg/kg) or Gemcitabine (20 mg/kg)/PIK-75 (2 mg/kg) combination is administered twice per week and vehicle control and PIK-75 (2 mg/kg) are administered 5 times per week. The body weights and tumor sizes are measured 3 times per week. Tumor volumes are calculated.

References:

[1]. Knight ZA, et al. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006 May 19;125(4):733-47.
[2]. Duong HQ, et al. Inhibition of NRF2 by PIK-75 augments sensitivity of pancreatic cancer cells to gemcitabine. Int J Oncol. 2014 Mar;44(3):959-69.
[3]. Zheng Z, et al. Isoform-selective inhibition of phosphoinositide 3-kinase: identification of a new region of nonconserved amino acids critical for p110α inhibition. Mol Pharmacol. 2011 Oct;80(4):657-64.