Abstract
BackgroundThe estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis. In interphase cells, microtubules play an important role in the delivery of proteins to subcellular locations including the focal adhesions. In fact, focal adhesion dynamics and cell migration are in part regulated by microtubules. We hypothesised that novel 2ME2 derivatives can alter cell migration by influencing microtubule dynamics in interphase cells. In this report we describe 2ME2 derivatives that display anti-migratory capabilities in a metastatic breast cancer cell line through their effects on the microtubule network resulting in altered focal adhesion signalling and RhoA activity.MethodsCell migration was assayed using wound healing assays. To eliminate mitosis blockage and cell rounding as a confounding factor cell migration was also assessed in interphase blocked cells. Fluorescence confocal microscopy was used to visualise microtubule dynamics and actin cytoskeleton organisation while western blot analysis was performed to analyse focal adhesion signalling and RhoA activation.Results2ME2 derivatives, ESE-one and ESE-15-one, inhibited cell migration in cycling cells as expected but equally diminished migration in cells blocked in interphase. While no significant effects were observed on the actin cytoskeleton, focal adhesion kinase activity was increased while RhoA GTPase activity was inhibited after exposure to either compound. Microtubule stability was increased as evidenced by the increased length and number of detyrosinated microtubules while at the same time clear disorganisation of the normal radial microtubule organisation was observed including multiple foci.ConclusionsESE-15-one and ESE-one are potent migration inhibitors of metastatic breast cancer cells. This ability is coupled to alterations in focal adhesion signalling but more importantly is associated with severe disorganisation of microtubule dynamics and polarity. Therefore, these compounds may offer potential as anti-metastatic therapies.
Highlights
The estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis
Breast cancer metastasis is the foremost cause of mortality among breast cancer patients due to the impact the secondary tumours have on essential organs
To exclude effects of serum induced proliferation on cell migration and to avoid the known effect of the tested compounds on mitotic arrest which leads to cell rounding that would act as a confounding factor in migration assays, cells were blocked in the G1/S phase of the cell cycle
Summary
The estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis. Successful metastasis depends on a complex sequence of events that allow epithelial cancer cells to alter their adhesion and migratory behaviour leading to invasion of the surrounding stroma This initial step is followed by intravasation of local vasculature, dispersion to distant sites, extravasation from the vasculature, and colonisation and growth within a distant environment to form a metastatic tumour (reviewed in [2]). One of the essential abilities of such metastatic cells is their ability to adhere to and migrate on extracellular matrix (ECM) proteins foreign to normal epithelial cells such as collagen (reviewed in [3]) It follows that inhibition of cell migration is a worthwhile target for therapeutic intervention since this will prevent invasion and metastasis and result in diminished mortality amongst patients
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