nNav1.5 expression is associated with glutamate level in breast cancer cells

Glutamate and voltage-gated sodium channels, both have been the target of intense investigation for its involvement in carcinogenesis and progression of malignant disease. Breast cancer with increased level of glutamate often metastasize to other organs (especially bone), whilst re-expression of ‘neonatal’ Nav1.5, nNav1.5 in breast cancer is known to promote cell invasion in vitro, metastasis in vivo and positive lymph node metastasis in patients. In this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was examined using pharmacological approach (VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA-nNav1.5). Effect of these agents were evaluated based on endogenous and exogenous glutamate concentration using glutamate fluorometric assay, mRNA expression of nNav1.5 using qPCR and finally, invasion using 3D culture assay. Endogenous and exogenous glutamate levels were significantly higher in aggressive human breast cancer cells, MDA-MB-231 cells compared to less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A. Treatment with TTX to MDA-MB-231 cells resulted in significant reduction of endogenous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells. An interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation.


Introduction
Voltage-gated sodium channels (VGSC) are transmembrane protein expressed abundantly in excitable cells such as neurons and muscle cells. In neurons, its main role is to propagate action potential critical in glutamatergic neurotransmission i.e. release of signalling molecules, glutamate at pre-synapses [22]. Interestingly, VGSC and glutamate also characterizes neoplastic cells [10,13].
Abnormal expression of VGSC in carcinomas (cancer of the epithelial origin) such as cancer of the breast, prostate, lung, cervical, colon and ovarian had been a conundrum to physiologist but subsequent 'proof-of-concept' of their critical role in potentiating metastatic cascades using tetrodotoxin (TTX) and other modulating agents, followed by their detectable expression in patient tumour tissues raised their status as a potent metastatic marker [9,14,15,18,20,29]. While there are several subtypes of VGSC found upregulated in a number of carcinomas, breast cancer in particular, there is distinctively high expression of the 'neonatal' splice variant of the cardiac VGSC isoform, Nav1.5 (nNav1.5) that potentiates motility, migration, and invasion of aggressive human breast cancer cells in vitro and metastasis in vivo [10]. Detectable expression of nNav1.5 in breast tumour tissues positive for lymph node metastasis signified its clinical importance in the prognosis of breast cancer patients [14,44].
In recent years, several mechanical insights for the role of Nav1.5 in controlling breast cancer cells capacity to metastasize emerged, mainly involve strong influx and elevated concentration of Na + which interferes with Ca 2+ , pH, and gene expression [1]. This mode of interferences has been reported to be associated with the activation of proteases/peptidases activity which enhance degradation of surrounding environment/cells to make way for invasion clearly demonstrated in breast cancer [5,16,17]. Accordingly, the opening of Nav1.5 allows strong influx of Na + resulted in elevation of intracellular Na + concentration. A more positive intracellular environment leads to activation of Na + /H + -1 exchanger (NHE1) which allows efflux of H + resulted in extracellular acidification from accumulation of proton. Lower pH at the extracellular microenvironment activates cysteine cathepsins B and S that degrades extracellular matrix, favouring cell invasion [5,16,17]. Unfortunately, this type of mechanical insight data for nNav1.5 in breast cancer is poorly understood.
On the same note, elevated glutamate levels in the extracellular environment of rapidly-growing glioblastoma actively kill the surrounding cells to create space for invasion [37]. Glutamate secretion have also been observed in cancer cell lines and tumour tissues of nonneuronal/central nervous system origin [19,21,34,[38][39][40]. Accordingly, elevated glutamate levels in prostate cancer cell lines corresponds to higher serum glutamate levels in the majority of prostate cancer patients compared to benign prostatic hyperplasia tissues and directly correlated with aggressiveness [21,38,39]. In breast cancer, human breast cancer cell line, MDA-MB-231 cells secrete glutamate corresponds to higher tissue glutamate levels [6]. In both prostate and breast cancer, excess glutamate contributes to bone metastasis [35].
To our knowledge, a connection between glutamate and VGSCs in breast cancer has never been reported. This study was designed to preliminary examine if such connection exists.

Pharmacology
Tetrodotoxin (TTX) (Sigma-Aldrich, USA), an established VGSC-blocker was used to inhibit nNav1.5 activity and mRNA expression. After purchase, it was reconstituted in citrate buffer at 1.0 mM stock concentration. Treatment concentration of 10 µM was used on MDA-MB-231 for channel blocking effects [4,28]. The TTX was stored in − 20 ºC until required. Dehydrated riluzole was purchased and reconstituted according to the manufacturer's instruction (Merck, USA) which was dissolved in DMSO and dH 2 O into a stock concentration of 1.0 mM, before subsequently stored in − 20 °C until required. Treatment concentration used on MDA-MB-231 was derived from 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide (MTT) assay.

MTT assay
3 × 10 4 cells were plated in a 96-well plate and incubated for 24 h prior to any treatment (24, 48 and 72 h). Each treatment was done in triplicate and the medium was refreshed every 24 h. The medium was removed and 100 µl of fresh DMEM and 10 µl of 12 mM of MTT were added to each well. Then, the plate was incubated at 37 °C for 4 h. After incubation period, 85 µl of MTT solution was removed and 50 µl of 100% dimethyl sulfoxide (DMSO) was added carefully to the well. The plate was incubated at 37 °C for 10 min and the absorbance was measured at 540 nm using a spectrophotometer.

SiRNA-mediated nNav1.5
Knockdown was conducted in order to investigate the effects of silencing nNav1.5 expression in MDA-MB-231 cells (which expressed the most significant nNav1.5 mRNA upregulation) on the concentration of glutamate. SiRNA on MDA-MB-231 cells were produced via transient transfection, whereby the siRNA sequences against nNav1.5 and controls were acquired commercially (SMARTpool siRNA reagents from Dharmacon). 3 × 10 4 cells of MDA were seeded in a well of 24-well plate. The cells were incubated overnight. Before starting the treatment on the next day, working concentration of siRNA from a 1 µM stock resuspension was prepared/ by adding 6 µl of it into 44 µl of serum free media in a tube. A transfection solution was prepared by adding 3 µl of transfection reagent (Polyplus-transfection SA, France) into 47 µl of serum free media in another tube. Both tubes were slightly vortexed. The transfection solution was later added into the siRNA suspension and was vortexed slightly and then left at room temperature for 5 min. The cells that were incubated in the wells overnight was removed of old media 500 µl of new media added. The siRNA transfection solution was then added into the well and incubated at 37 °C for 5 h before the media was changed. To confirm the success of nNav1.5 knockdown, the gene in expression in the knock downed cells were measured using qPCR.

RNA extraction and cDNA synthesis
Total RNA from the cell lines were extracted using sepasol-chloroform (Nacalai Tesque, Japan). The purity of RNA was assessed by observing the ratio of absorbance 260/280 nm and 260/230 nm in Nanodrop software. Total isolated RNA (1000 ng) was transcribed to cDNA using reverse transcription kit (Toyobo, Japan), with genomic DNA remover included by the company as component of the reverse transcription solution preparation.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Real-time PCR was performed using SensiFAST SYBR Hi-ROX kit according to manufacturer's protocol (Bioline, UK). Sequence primers used were as follows: β-actin forward, ATT GCC GAC AGG ATG CAG AAG-3′ and reverse, 5′-AGA AGC ATT TGC GGT GGA CG-3′ and nNav1.5 forward, 5′-CTG CAC GCG TTC ACT TTC CT-3′ and reverse, 5′-GAC AAA TTG CCT AGT TTT ATA TTT -3. Quantitative real-time was performed using ABI Prism 7000 Sequence Detection System (Life Technologies, USA) and the amplification conditions were as follows: initial activation for 10 min at 95 °C for one cycle, 10 s at 95 °C and 30 s at 60 °C for 34 cycles. C t values of target genes were normalised to β-actin and the relative mRNA expression of target genes were calculated using the 2 − ΔΔCt method [30].

Glutamate assay
Glutamate concentration in the cell supernatant (exogenous) and endogenous was measured using a fluorometric assay in a 96-well plate format. Briefly, glutamate standard and the samples were prepared according to manufacturer's protocol (Abcam, USA). The samples were diluted to several dilutions to ensure the readings fall within the standard value range. The measurement was made using a flourescence microplate reader at Ex/ Em = 540/590 nm.

Invasion assay
2 × 10 3 cells were placed inside wells containing the 3D culture matrix (Cultrex, USA). The plate was centrifuged at 200×g for 3 min in a swing bucket rotor centrifuge (Thermo Fisher, USA) at room temperature. The plate was incubated at 37 ºC, 5% CO 2 for 72 h. Image were taken under a compound microscope (Leica, Germany) every subsequent 24 h for 7 days. ImageJ imaging software [32] was used to analyse the size and invasion projection.

Data analysis
Results are shown as the means ± SEM. Statistical evaluations were made using unpaired Student's t test (Graph-Pad Prism 9).   (Fig. 2).
Reduction of endogenous and exogenous glutamate level by TTX diminished the ability of the cells to invade the surrounding matrix in the invasion assay (Fig. 7A, B). Briefly, high invasion spike was observed from day 1 until  (Fig. 7B).

Discussion
Multiple lines of evidence have indicated clinical significance of elevated serum, plasma and tissue glutamate in the prognosis of prostate, pancreatic, lung and breast cancer [6,19,21,40]. In separate studies, VGSC expression in respective cell lines of these glutamateenrich tumours is known [14,18,29]. nNav1.5 is a potent metastatic marker for breast cancer [1,10]. Elevated levels of endogenous and exogenous glutamate in aggressive human breast cancer cells, MDA-MB-231 which expresses nNav1.5 compared to the less aggressive  human breast cancer cells, MCF-7 which lacks nNav1.5 conforms with the hypothesis that high glutamate and expression of nNav1.5 is interconnected in breast cancer. Unexpectedly, MCF-10A had higher glutamate than MCF-7 even if it is significantly lower than the subject of interest, MDA-MB-231. MCF 10A, as reported by the American Type Culture Collection (ATCC), is a non-cancerous cell. But it shows certain tumour markers such as basal-like phenotype [31]. This is due to the process of immortalisation of the cell line from primary culture itself. Because of this, no secondary cell line are to be considered normal post-immortalisation such as telomere lengthening process whereby the immortal cells tend to derive chromosome abnormality [41] resulting in results obtained. Other research also points out the relevance of MCF-10A as non-tumorous breast epithelial cell-line model and it was due to increased karyotyping in the cell lines causing certain proteins to be expressed higher [27,31].
In examining the mode of connection between these two, MDA-MB-231 cells was treated with TTX (specific blocker for VGSC), a common 'tool' used to study VGSC in excitable cells and in the study of 'proof-of-concept' for the role of VGSC in cancers. Earlier, the use of TTX has helped researchers to understand the critical method role of VGSC in regulating excitotoxic glutamate release in several ischemic induced in vitro and in vivo models where TTX exerted neuroprotective effects against glutamate-induced cell toxicity via VGSC blockade [23]. Concordantly, endogenous, and exogenous glutamate level was significantly reduced in TTX-treated MDA-MB-231 cells, revealing for the first time the role of nNav1.5 expression in regulating glutamate level in aggressive breast cancer. Treatment with TTX also led to significant downregulation of nNav1.5 gene expression followed by inhibition of the cell invasion. The observed cell invasion suppression in this study is unrelated to any effects on cell viability as the TTX concentration used (10 µM) was sub-lethal-20× lower than the IC 50 , > 200 µM. These observations are in accordance to study that showed TTX blockade of the channel's pore, prevented the amount of Na + entering the cells thus interfered with the positive autoregulation of the channels expression at the plasma membrane which led to suppression of metastatic ability of MDA-MB-231 [8].
At the meantime, glutamate is a known potent necrosis factor during neuro-excitotoxicity [2,33] and in glioblastoma [25]. Subsequently, tumour aggressiveness is exploiting excitotoxicity mechanisms to kill (necrotising) surrounding cells to create way for metastasis [37]. In the case of breast cancer, metastasis to bone is a feature of aggressive breast cancer cells that secrete high levels of glutamate [12,13]. The 3D spheroid invasion assay was used in this study to replace wound healing and Matrigel assays to provide more accurate insights on the invasion since spheroid have shown to have higher cell proliferation, migration, and invasion rate [3]. Regardless, the use of TTX have been shown to reduce the effects of wound healing and Matrigel assays according to previous studies [14,15]. Besides being used as a preferred in vitro model for nNav1.5 expression and glutamate secretion, MDA-MB-231 cells is also an established cell line used for development of a breast cancer bone metastases model, primarily to long bones, spine, jaw and lungs [42]. nNav1.5, expression and activity in these cells has been demonstrated to regulate the pH-dependent activity of cathepsins B and S, a type of proteinases that degrade extracellular matrix [5,11,16]. Interestingly, emerging lines of evidence are also pointing to the pro-metastatic role of cathepsin B in bone metastasis of breast cancer [26].
With regards to VGSCs in breast cancer, a wealth of data has now confidently demonstrated that Nav1.5 and its splice variant nNav1.5 offers potential value as metastatic tumour markers in the diagnosis (including prognosis), and therapies of the disease. Previously, the significance of therapeutic values for nNav1.5 and glutamate has been reported separately. nNav1.5 gene and protein expression in breast cancer tissues has been reported elsewhere [43] and now its value as prognostic and predictive marker is currently being evaluated. In doing so, works on development and characterisation of antibodies against nNav1.5 has been reported. In this regard, a polyclonal rabbit antibody, NESOpAb was generated to specifically recognise the neonatal splice form of Nav1.5 and excluding the adult counterpart [7] that utilise on their discovery of the 7 divergence of amino acid in the former since the divergence is significant enough to have its own specific antibody. Further achievement from our inhouse project managed to develop a novel monoclonal mouse antibody for nNav1.5, 4H8 [36]. This benefits directly for future therapeutic development with better specificity on a single epitope, allowing mass production of diagnostic tools for nNav1.5. As for glutamate, including the fact that nNav1.5 is correlated with metastatic breast cancer and in other cases also shows significance of blocking nNav1.5 on another molecule such as Major Histocompatibility Complex (MHC) Class I [24].

Conclusions
In summary, our study represents a unique connection between nNav1.5 expression and glutamate in aggressive breast cancer cells. Importantly, our findings raise to new understandings and opportunity to a new therapeutic strategy to combat metastasis which is the major cause of mortality for breast cancer patients.