Rho GTPase activity assay

Cells were grown in log phase in a 10-cm dish, and were starved in 0.5% serum medium or indicated otherwise for 24 h before lysis in a buffer containing 20 mM Tris HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl2, 1% Nonidet P-40, 10% glycerol, and 1 × protease inhibitor mixture. Lysates were clarified, the protein concentrations were normalized, and the GTP-bound Rac1 in the lysates was measured by an effector domain pull-down assay. For the His6-PAK1 PBD pull-down assay, cell lysates were incubated with Ni2+-agarose-immobilized His6-PAK1 PBD domain (~ 1 μg each) purified from E. coli for 30 min. The Ni2+-agarose co-precipitates were washed twice in the wash buffer and analyzed by immunoblotting with anti-Rac1 monoclonal antibody.

Cell lines

Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 as well as the MCF12A normal mammary epithelial cell line

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.

Reaction Conditions

0 ~ 100 μM; 2 d

Applications

NSC 23766 inhibited cell growth and induced apoptosis. NSC 23766 dose-dependently decreased the viability of MDA-MB-468 and MDA-MB-231 cells, with IC50 of ~ 10 μM, but had little effect on the survival of the MCF12A normal mammary epithelial cells. After 24-h exposure to NSC 23766, MDA-MB-231 cells exhibited an increase from 41% to 65% in G1 phase and a concomitant decrease in S and G2-M phases. 100 μM NSC 23766 induced a six-fold increase of apoptotic MDA-MB-468.

Animal models

C57BL/6 mice

Dosage form

2.5 mg/kg; i.p.

Applications

In the ‘‘poorly mobilizing’’ C57BL/6 mice, intraperitoneal administration of NSC 23766 (2.5 mg/kg) induced a two-fold increase in circulating hematopoietic stem cells/progenitors 6 hr after injection.

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.

NSC-23766 is a specific inhibitor of Rac with IC50 of 50μM.

NSC-23766 blocks the activation of Rac 1 through binding the GEFs including Trio and Tiam 1 [1]. In human dermal microvascular endothelial cells, NSC-23766 decreased trans-endothelial electrical resistance and caused the intercellular gap formation. Inhibition of Rac 1 by NSC-23766 shortly reduced endothelial barrier functions as revealed by measurement of TER and the appearance of intracellular gaps [2]. In the mucous cell of the intestine, inhibition of Rac1 either by NSC-23766 protected cells from TNF-α-induced apoptosis by inhibiting caspase-3, -8 and -9 activities. Inhibition of Rac1 significantly prevented TNF-α-induced activation of JNK1/2, but did not modulate TNF-α-induced ERK1/2, Akt and p38 MAPK activity [3].

References:
[1]. Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A. 2004 May 18;101(20):7618-23.
[2]. Baumer Y, Spindler V, Werthmann RC, Bünemann M, Waschke J. Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown. J Cell Physiol. 2009 Sep;220(3):716-26.
[3]. Jin S1, Ray RM, Johnson LR. Rac1 mediates intestinal epithelial cell apoptosis via JNK. Am J Physiol Gastrointest Liver Physiol. 2006 Dec;291(6):G1137-47.