PP2 (AG 1879)

PP2 is a reversible and ATP-competitive Src family kinases inhibitor with IC50s of 4 and 5 nM for Lck and Fyn, respectively.

Signaling Pathways >> Protein Tyrosine Kinase/RTK >> Src

Research Areas >> Cancer

IC50: 4 nM (Lck), 5 nM (Fyn)[1]

At 10 μM, the effect of PP2 on cellular proliferation is not significant, indicating that, at this low concentration, the effect of PP2 on Gemcitabine cytotoxicity does not simply reflect a direct antiproliferative effect, but rather a potentiation of Gemcitabine-induced cytotoxicity. Above 20 μM, growth is increasingly suppressed, a finding consistent with reports in other human cancer cell lines. Although 10 μM PP2 is used in our study, at higher concentrations PP2 is reported to inhibit other intracellular kinases[2]. PP2 is the most widely used commercially available Src family kinase inhibitor. PP2 inhibits Src family kinase activity with IC50 of ~5 nM in vitro, concentrations to 10 μM are often necessary to achieve complete Src family kinase inhibition in cell culture[3].

The tumor growth inhibition rate is 25% in the PP2 treatment group and 5% in the Gemcitabine treatment group (P>0.05). When administered in combination, PP2 and Gemcitabine produce a tumor growth inhibition rate of 98% (P<0.05). Hepatic metastasis occurred in 100% of control and Gemcitabine-treated groups; 88% of the PP2-treated group developed liver metastases. There are no detectable metastases in the group treated with PP2 and Gemcitabine in combination (P<0.05)[2].

Cell growth is determined by MTT assay and confirmed by cell counting. Results of the MTT assay have been shown to correlate well with [3H]thymidine incorporation in pancreatic cancer cell lines. Logarithmically growing cells are plated at 5×103 cells/well in 96-well plates, allowed to adhere for 24 h, and cultured in the presence or absence of PP2 and Gemcitabine. Cell proliferation is determined after 96 h. Plates are read using a Vmax microplate spectrophotometer at a wavelength of 570 nm corrected to 650 nm and normalized to controls. Each independent experiment is performed three times, with 10 determinations for each condition tested. The IC50 of Gemcitabine is calculated from these results. At identical time points, cells are trypsinized to form a single cell suspension. Intact cells, determined by trypan blue exclusion, are counted using a Neubauer hemocytometer, and the number of cells per mL is calculated and compared with the control group to confirm the MTT results[2].

Mice[2] Male athymic nu/nu mice (age, 5 weeks; weight, 20-22 g; specific pathogen free) are anesthetized with i.p. ketamine (200 mg/kg) and xylazine (10 mg/kg) and inoculated with 106 Gemcitabine-resistant PANC1GemRes cells in 20 μL of PBS by surgical orthotopic implantation into the pancreas. After inoculation, mice are randomized to three treatment groups: (a) treatment group 1 (n=8) receive 2 mg/kg PP2 in 1% DMSO by i.p. injection three times a week; (b) treatment group 2 (n=8) receive Gemcitabine (100 mg/kg) in the same volume of 1% DMSO vehicle as received by group 1, three times a week; and (c) treatment group 3 (n=8) receive 2 mg/kg PP2 and 100 mg/kg Gemcitabine in the same volume of DMSO as groups 1 and 2, three times a week. The control group receive the same volume of 1% DMSO vehicle as the other groups, three times a week. Treatment is commenced 1 day after implantation, and necropsy is performed 4 weeks after implantation. Primary tumors are identified, weighed, and normalized to total body mass. Tumor growth inhibition rate is calculated using the following formula: tumor growth inhibition rate (%)=(1−MT/MC)×100, where MT and MC are the mean normalized tumor masses of treatment and control groups, respectively. Liver metastases are counted and confirmed histologically.

[1]. Hanke JH, et al. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck-and FynT-dependentT cell activation. J Biol Chem. 1996 Jan 12;271(2):695-701.

[2]. Duxbury MS, et al. Inhibition of SRC tyrosine kinase impairs inherent and acquired Gemcitabine resistance in human pancreatic adenocarcinoma cells. Clin Cancer Res. 2004 Apr 1;10(7):2307-18

[3]. Summy JM, et al. AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes. Mol Cancer Ther. 2005 D

[4]. Inoue A, et al. Phosphorylation of NMDA receptor GluN2B subunit at Tyr1472 is important for trigeminal processing of itch. Eur J Neurosci. 2016 Oct;44(7):2474-2482.

Saracatinib | A 419259 trihydrochloride | DCC-2036 (Rebastinib) | SU6656 | 1-Naphthyl PP1 | WH-4-023 | Pelitinib | Src Inhibitor-1 | ENMD-2076 | TC-S 7003 | PD173955 | TG 100572 (Hydrochloride) | XL228 | A-196 | pp1