Our previous studies demonstrated that EHop-016, at concentrations < 10 μM, inhibits the Rac activity of metastatic breast cancer cells MDA-MB-435 and MDA-MB-231 , as well as the SKBR3 cell line (data not shown). To determine the potential of EHop-016 as a general Rac inhibitor, we also tested the effect of EHop-016 in the metastatic prostate cancer cell line PC3; a cell line that has been shown to be dependent on Rac/Vav signaling for migration/invasion . Figure 5A demonstrates that 8 μM EHop-016 inhibits the Rac activity of PC3 cells by 50%. To understand the mechanisms by which EHop-016 may reduce cell survival
and induce apoptosis, we investigated the effect of EHop-016 on known Rac/Cdc42/PAK signaling pathway molecules, which have been implicated in controlling cell survival and proliferation. Activated Rac and Cdc42 mTOR inhibitor may affect cell cycle progression via up-regulation NVP-BKM120 chemical structure of the oncogenes Cyclin D and c-Myc , , ,  and . Rac/PAK signaling also regulates cell growth via signaling to Akt, ERK, JNK, and p38 MAPK  and . Figure 4 and Figure 5 show that in both MDA-MB-435 and PC3 cells, EHop-016 significantly reduced the expression of the oncogenic cell cycle regulators c-Myc and Cyclin D expression
by ~ 25% to 60%. Next, we investigated the effect of EHop-016 on MAPK activity and expression. EHop-016 did not affect ERK activity or expression (data not shown). However, EHop-016 significantly reduced the JNK activity of MDA-MB-435 cells by ~ 30%. In PC3 prostate cancer cells, p-JNK levels were decreased but total JNK levels were also reduced to a similar extent, indicating that JNK expression is also down-regulated in this cell line. Moreover, in the MDA-MB-435 cells, EHop-016 reduced Akt activity by 40% at 4 and 8 μM, without affecting
the Akt activity of PC3 cells (data not shown). These differences may be attributed to disparate cancer types of the two different cell lines. Studies have also linked Akt activity L-gulonolactone oxidase and thus, the regulation of the anti-apoptotic protein BAD with Rac action , and may account of the observed reduction in caspase activity in the MDA-MB-435 cell line, where a parallel 1.4-fold calculated increase in caspase activity is observed at 8 μM, when Akt activity is decreased by − 1.4-fold (Figure 4, A and B). Therefore, EHop-016 may reduce cell viability and tumor growth via a number of Rac-regulated pathways that control cell survival and death. We have demonstrated that EHop-016 is a viable tool for blocking Rac activity via inhibition of the Vav/Rac interaction and thus, metastatic breast cancer cell migration In Vitro at μM concentrations . Following this publication, the utility of EHop-016 as a Rac inhibitor has also been demonstrated in leukemia and melanoma cells  and .