Cancer cell metastasis forms an almost inevitable endpoint in tumour progression leading to complications and resistance to treatment culminating in patient death. Therefore, a large focus in cancer drug development is and should be on anti-metastatic therapies. 2-Methoxyestradiol (2ME2) is a metabolite of estrogen and was initially identified as an antiangiogenic compound [
13]. Later, it was shown to also have antiproliferative effects mostly due to its ability to bind to microtubules and inhibit their dynamics leading to cell cycle arrest [
13]. Clinical studies precluded further use of 2ME2 as an anticancer compound due to its lack of clinical effect as a result of its rapid metabolism and clearing [
23]. As a result, a number of groups designed compounds that would retain the antiproliferative effects while withstanding clearing and metabolism inside the body. Previously, we designed and described a number of 2ME2 derivatives and their potential antiproliferative effects [
19]. Design was based on the addition of a sulphamoyl moiety to the original structure along with the removal of the 3-methoxy and addition of a 2-ethyl group to make the compounds resistant to metabolic breakdown and allow reversible attachment to red blood cells via the cell surface carbonic anhydrase protein preventing clearing in the liver [
19]. These sulphamoylated compounds did show antiproliferative effects in vitro in different cancer cell lines [
24] although none of the non-sulphamoylated ethyl derivatives showed activity at nanomolar concentrations [
20]. ESE-15-one and ESE-one are two such derivatives that have nanomolar activity in cell proliferation assays. We show here that in a triple negative metastatic cell line, both compounds reduce cell number over a 72-h period alluding to their antimitotic mechanism. This profile of cell number loss can be ascribed to their antimitotic activity which is due to an initial arrest of the cell cycle before eventual induction of apoptosis as has been shown before [
25]. Thus far, all investigations on the activity of 2ME2 and the 2ME2 derivatives generated before have focused on their antiproliferative and antimitotic effect. However, little is known about their anti-metastatic potential. Since these compounds are expected to inhibit microtubule dynamics [
19], we considered it possible that they may influence cell migration. Cancer metastasis depends on the change in adhesive properties of the cell to become able to adhere and migrate along mostly collagen fibres found in the stroma of the connective tissue surrounding most epithelial tissues [
26]. Therefore, 2D migration as measured in vitro using wound healing assays can predict whether interventions can potentially inhibit metastasis. Microtubules have been implicated in cell migration in a number of studies. Their turnover can regulate the activity of actin fibre regulators such as GEF-H1 [
27], they form the delivery channels for membrane proteins needed in focal adhesion assembly [
8], and their contact with focal adhesions has been linked to the disassembly of such structures [
8]. Microtubules undergo a process termed dynamic instability which means that they are continuously turned over, growing until they reach a tipping point after which they rapidly regress. Interfering in this turnover could therefore change the ability of microtubules to contact focal adhesions and would alter focal adhesion signalling and turnover leading to changes in migration. To test whether the compounds ESE-15-one and ESE-one were potential anti-migratory molecules, wound healing assays using metastatic MDA-MB-231 cells were performed and showed a significant decline in cell migration after exposure (Fig.
2). A possible confounding factor was the antimitotic effect of the compounds which meant that cells were arrested at the mitotic border where cells are rounded, poorly attached to the substrate and non-migratory. To eliminate this confounding factor, cells were first blocked in interphase through saturating concentrations of thymidine blocking the DNA synthesis process and arresting cell cycle progression in the G
1/s phase. When migration assays were performed with the interphase blocked cells a reduction in migration was measured in cells exposed to either ESE-15-one or ESE-one (Fig.
3). Therefore, both compounds are
bona fide inhibitors of cancer cell migration independent of their anti-mitotic capabilities. Microtubule dynamics are vitally important for cell migration including regulating focal adhesion turnover and inducing cell polarity meaning that any impact on microtubule dynamics can have consequences for migration [
8,
28]. We tested if microtubule organisation and composition was altered after compound exposure. Indeed, both ESE-15-one and ESE-one caused increases in stabilised microtubule number and length. At the same time, dynamic microtubules were disorganised with multiple centres of origin and parallel tubules found along the cell periphery instead of radiating from a single centre of origin which are the centrosomes (Fig.
5). This suggests that these compounds may inhibit cellular polarity that is dependent on the correct localisation of the centrosome. Furthermore, focal adhesion turnover is regulated in part by the microtubules. Targeting of microtubules to focal adhesions has been shown to lead to their disassembly and therefore the turnover of focal adhesions [
8]. Focal adhesions signal through proteins such as focal adhesion kinase which is activated by phosphorylation [
26]. Moreover, fascin-dependent microtubule stabilisation is associated with increased focal adhesion formation [
29]. In addition to the abovementioned effects on the microtubules we also measured increased FAK phosphorylation and decreased RhoA GTPase activity. While the precise mechanism remains unclear we suggest that the increase in FAK activity may be due to slower turnover of focal adhesions due to a lack of microtubule mediated adhesion disassembly. RhoA GTPase activity is regulated by guanidine exchange factors. One such factor, GEF-H1, associates with the microtubules and upon release through microtubule regress activates RhoA [
30]. Our data shows that the compounds inhibit RhoA activity in correlation with increased microtubule stability suggesting that through the increased binding of GEF-H1, RhoA becomes less active. This results in decreased actin fibre formation and fibre tension. Actin fibre tension is responsible for cell migration as it pulls the cell body towards the focal adhesions. Thus, a lack of tension brought about by reduced RhoA activity could also reduce cell migration.