Rac Activity Assays

Rac activity is determined from lysates of the MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines. Cancer cells in culture medium (DMEM, 10% FBS, pH 7.5) are treated with vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μM) for 24 h. Rac1 activity is determined using the G-LISA Rac1 activation assay kit.

Cell lines

MDA-MB-435 and MDA-MB-231 metastatic cancer cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

0–10 μM for 24 h

Applications

EHop-016 inhibited Rac activity and reduced Rac-directed lamellipodia formation in both MDA-MB-435 and MDA-MB-231 metastatic cancer cells. Moreover, EHop-016 decreased Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed metastatic cancer cells migration.

Animal models

KITD814V-bearing mice model

Dosage form

2.5 μM; i.v. injection, for 45 days;

Applications

Treatment of KITD814V-bearing cells with EHop-016 significantly enhanced leukemic mice survival and delayed disease progression.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

EHop-016 is a novel and specific inhibitor of Rac1 and Rac3 activity (IC50= 1.1 μm).

Rac is a member of the RAS superfamily of small GTPase. It functions in various cellular processes including regulation of secretory processes, membrane ruffles, cell polarization and phagocytosis of apoptotic cells etc. It is contributed to malignant progression.

Ehop-016 inhibited Rac activity in metastatic cancer cell MDA-MB-435 which overexpressed Rac and inhibited high endogenous Rac activity as well. In metastatic breast cancer cell MDA-MB-231, Ehop-016 blocked the Rac activity and reduced Rac-mediated lamelipodia formation. It also attenuated the effects of Rac downstream protein p21-activated kinase 1 and directed migration of metastatic cancer cells. [1]

In C3H/HeJ mice carrying KITD814V following overnight treatment of 2.5 μM EHop-016 or 25 μM NSC23766, EHop-016 reduced spleen size and PB counts thus significantly promoted the survival of leukemic mice compared with NSC23766. [2]

References:
1.  Montalvo-Ortiz BL, Castillo-Pichardo L, Hernández E et al. Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase. J Biol Chem. 2012 Apr 13;287(16):13228-38.
2.  Martin H, Mali RS, Ma P et al. Pak and Rac GTPases promote oncogenic KIT-induced neoplasms. J Clin Invest. 2013 Oct;123(10):4449-63.