The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model

Abstract

Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC50 of 1 μM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25 mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23 ng/ml levels at 12 h following intraperitoneal (i.p.) administration of 10 to 25 mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms.

Figure 1

Effect of EHop-016 on mammary fat pad tumor growth. Athymic nude mice were inoculated at the mammary fat pad with GFP-MDA-MB-435 cells. Average relative tumor growth from fluorescence in situ images up to 55 days following 0, 10, or 25 mg/kg body weight (BW) EHop-016 (three times a week) was determined. (A) Relative tumor growth as a function of days following EHop-016 administration. (B) Average Relative tumor intensity on day 55 (N = 6, 0 mg/kg BW; N = 8,10 mg/kg BW; N = 4, 25 mg/kg BW). Error bars = SEM, * = p < 0.05. (C) Average mouse weight as a function of days following EHop-016 administration.

Figure 2

Effect of EHop-016 on metastasis. Mammary fat pad tumors were established in athymic nude mice using GFP-MDA-MB-435 cells. Mice were treated with 0, 10, or 25 mg/kg body weight (BW) EHop-016 (three times a week) (N = 5). Lungs, livers, kidneys, and spleens were removed at necropsy and imaged for fluorescent metastatic foci. (A) Representative organs under fluorescence microscopy for 25 mg/kg BW Ehop-016 treatment. (B) Average integrated intensity of fluorescent metastatic foci/organ. Error bars = SEM, * = p < 0.05.

Figure 3

Effect of EHop-016 on angiogenesis. (A) Representative tumors from athymic nude mice inoculated at the mammary fat pad with GFP-MDA-MB-435 cells and treated with 0 or 25 mg/kg body weight (BW) EHop-016 (three times a week) (N = 5). Left, stereoscopic images of representative mammary tumors with blood vessels in red. Right, immunofluorescence micrographs of tissue sections with blood vessels stained for DAPI or CD31 (red fluorescence). (B) Representative micrographs following a tube formation assay using HUVEC cells treated with vehicle or 4 or 8 μM EHop-016 for 24 h (N = 4). Arrows indicate capillaries (tubes). (C) Representative Western blot and quantification of the Rac activity of HUVEC cells treated with 0 or 8 μM EHop-016 for 24 h from a pulldown assay for Rac.GTP (N = 2). Error bars = SEM, * = P < 0.05.

Figure 4

Effect of EHop-016 on apoptosis and Rac/Cdc42/PAK signaling in MDA-MB-435 cells. A) Fold change in caspase 3/7 activity in response to EHop-016. MDA-MB-435 cells treated for 24 h with 0, 5, 10, or 25 μM EHop-016 and subjected to caspase 3/7 activity assays. N = 3 ± SEM, Asterisk = P < .05. B-D. MDA-MB-435 cells treated with 0, 4, or 8 μM EHop-016 were lysed, and equal amounts of protein subjected to Western blotting followed by integrated density of positive bands. N = 2 to 5 ± SEM; Asterisk = P < .05. (B) Representative Western blot stained for c-Myc or Cyclin D and quantification of relative protein expression. (C) Representative Western blot stained for phospho (p)-Akt, Akt, p-JNK, or JNK. (D) Quantification of relative protein phosphorylation.

Figure 5

Rac activity and expression of cell proliferation regulators in response to EHop-016 in PC3 cells. PC3 cells treated for 24 h with 8 μM EHop-016 were subjected to the following assays. (A) Rac activity determined by a pulldown assay for Rac.GTP. Representative Western blot showing positive bands for Rac.GTP and total Rac. Relative Rac activity was quantified from the positive bands from Western blots and represented as the ratio of active Rac/Total Rac. (B) JNK, c-Myc and cyclin D expression in response to 8 μM EHop-016. Representative Western blots and quantification of positive bands for relative protein expression is shown. N = 2–4 ± SEM, Asterisk = P < .05.