The sodium channel-blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis
© Nelson et al.; licensee BioMed Central. 2015
Received: 28 August 2014
Accepted: 22 December 2014
Published: 27 January 2015
Voltage-gated Na+ channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets.
We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen.
This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy.
KeywordsAntiepileptic Breast cancer Metastasis Phenytoin Voltage-gated Na+ channel
Despite recent advances, breast cancer (BCa) is still the leading cause of cancer-related deaths in women . Metastasis, the spread of tumours to secondary sites, is responsible for 90% of these deaths and is rarely curable . Thus, there is an urgent need to identify new molecular targets and curative therapies. Voltage-gated Na+ channels (VGSCs) contain a pore-forming α subunit with smaller β subunits. There are nine α subunits, Nav1.1-Nav1.9, and four β subunits, β1-β4. The β subunits modulate channel function and are cell adhesion molecules (CAMs) . VGSCs transmit electrical activity in cells in the nervous system and regulate neuronal growth and migration during CNS development . VGSCs are clinical targets for a range of disorders, including epilepsy, cardiac arrhythmias, neuropathic pain and depression .
VGSCs are widely expressed in traditionally non-excitable cells, including microglia, astrocytes, immune cells, fibroblasts and cancer cells . In the latter, a number of studies have shown that VGSCs contribute to cellular migration and invasion . Nav1.5 is up-regulated in breast tumours, associating with recurrence, metastasis, and reduced survival [8,9]. Nav1.5 carries a fast inward Na+ current in triple negative (lacking estrogen receptor, progesterone receptor and HER2) MDA-MB-231 cells [9-11]. Pharmacological or genetic ablation of this Na+ current inhibits in vitro cell behaviours associated with the metastatic cascade, including migration, galvanotaxis, and invasion [9-11]. Similar results have been reported in metastatic cell lines from other cancers, suggesting that VGSC expression/activity in cancer may be a general phenomenon [7,12]. Na+ current enhances invasion by promoting cysteine cathepsin activity in caveolae via allosteric regulation of the Na+/H+ exchanger type 1 , and Nav1.5 is a key regulator of a gene network that controls invasion . In addition, the widely used VGSC-blocking Class Ib antiarrhythmic agent and antiepileptic drug (AED) phenytoin (5,5-diphenylhydantoin) reduces the migration and invasion of MDA-MB-231 cells in vitro . Furthermore, we have recently shown that the VGSC β1 subunit is also expressed in BCa specimens, and accelerates tumour growth and metastasis in a mouse model .
Together, these data highlight the potential for VGSCs as novel molecular targets. However, there remains a paucity of clinically relevant in vivo data exploring their potential therapeutic value. The aim of the present study was to study the effect of phenytoin on tumour growth and metastasis in a mouse model of triple negative BCa. We found that systemic phenytoin treatment reduces cellular proliferation, tumour growth, local invasion and metastasis. This is the first in vivo study demonstrating the potential therapeutic value of pharmacologically targeting VGSCs in BCa using an AED.
Phenytoin reduces tumour growth
Phenytoin reduces invasion and proliferation
We found that the prevalence of Ki67-expressing cycling cells was reduced by 62.6% in the tumours of phenytoin-treated animals (P < 0.001; Figure 2C,G). However, the number of apoptotic cells expressing activated caspase-3 was unchanged (Figure 2D,H). Similarly, the phenytoin treatment had no effect on the density of CD31-expressing vascular structures (Figure 2E,I). Together, these data suggest that phenytoin inhibited growth of primary tumours by reducing the number of proliferating cancer cells, rather than by inhibiting angiogenesis or promoting apoptosis. Interestingly, previous studies have indicated that VGSCs do not regulate proliferation of MDA-MB-231 cells in 2D cultures in vitro [9,10]. However, the VGSC blocker tetrodotoxin reduces colony growth in 3D Matrigel matrices . Thus, the contribution of VGSCs to tumour growth in vivo appears complex, and may be dependent on multiple factors, including heterotypic signalling interactions with adjacent cells or the extracellular matrix . In addition, VGSCs may regulate proliferation via reverse Na+/Ca2+ exchange, as has recently been shown in astrocytes after injury .
Phenytoin reduces metastasis
We have previously shown that phenytoin inhibits Na+ current and significantly reduces migration and invasion of BCa cells in vitro . Together with the present data, these findings suggest that targeting VGSC-mediated Na+ current with phenytoin may have therapeutic value. Phenytoin also inhibits migration and secretion in prostate cancer cells [20,21], suggesting that it may have broad utility in other cancers. In support of this, tetrodotoxin has been shown to reduce metastasis in a rat prostate cancer model . In the present study, we provide, for the first time, clinically relevant in vivo data showing that pharmacological targeting of VGSCs with phenytoin significantly reduces tumour growth, local invasion and metastasis in a mouse model of BCa. Indeed, given that the membrane potential (Vm) of cancer cells is relatively depolarized , and that phenytoin displays robust use-dependent and tonic channel block at depolarized voltages , our data suggest that phenytoin may be a highly effective VGSC blocker in tumours.
We propose that VGSCs may be useful molecular targets for BCa therapy, and that repurposing FDA-approved, VGSC-targeting AEDs and Class I antiarrhythmic agents, e.g. phenytoin, carbamazepine, flecainide, to cancer may therefore improve outcome. It is possible that phenytoin may be effective in combination with existing conventional therapies, e.g. in the adjuvant setting, which would need to be tested in a randomised controlled clinical trial. In support of this notion, application of VGSC-targeting local anaesthetics during radical prostatectomy associates with substantially reduced recurrence and metastasis . In addition, the FDA-approved ALS drug, riluzole, which inhibits both metabotropic glutamate receptors and VGSCs, reduces tumour growth . Furthermore, given that VGSCs favour an invasive/metastatic phenotype [9,13-15], it is possible that the adjuvant prescription of AEDs, which cross the blood-brain-barrier, may reduce and/or delay metastasis formation in patients. This would transform the landscape of cancer treatment considerably, with very little added cost, while leading to healthier patients and huge financial savings.
Cell adhesion molecule
Liquid chromatography-mass spectrometry with single reaction monitoring
Voltage-gated Na+ channel
This work was supported by the Medical Research Council [Fellowship G1000508].
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