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

nNav1.5 expression is associated with glutamate level in breast cancer cells Background: 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 gluta- mate 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. Methods: In this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was exam- ined using pharmacological approach ( VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA- nNav1.5). Eec ff t 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. Results: 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 endog- enous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells. Conclusions: An interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation. Keywords: Endogenous glutamate, Exogenous glutamate, Breast cancer, Voltage-gated sodium channels, Neonatal Nav1.5, Invasion Introduction VGSC and glutamate also characterizes neoplastic cells Voltage-gated sodium channels (VGSC) are transmem- [10, 13]. brane protein expressed abundantly in excitable cells Abnormal expression of VGSC in carcinomas (cancer such as neurons and muscle cells. In neurons, its main of the epithelial origin) such as cancer of the breast, pros- role is to propagate action potential critical in gluta- tate, lung, cervical, colon and ovarian had been a conun- matergic neurotransmission i.e. release of signalling drum to physiologist but subsequent ‘proof-of-concept’ molecules, glutamate at pre-synapses [22]. Interestingly, 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 *Correspondence: fatmawati@usm.my 1 [9, 14, 15, 18, 20, 29]. While there are several subtypes Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia of VGSC found upregulated in a number of carcinomas, Full list of author information is available at the end of the article breast cancer in particular, there is distinctively high © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Azahar et al. Biological Research (2022) 55:18 Page 2 of 13 expression of the ‘neonatal’ splice variant of the cardiac human breast cancer cells, MCF-7 which lacks VGSC VGSC isoform, Nav1.5 (nNav1.5) that potentiates motil- expression and non-cancerous human breast epithelial ity, migration, and invasion of aggressive human breast cell, MCF-10A were used in this study (ATCC, USA). The cancer cells in  vitro and metastasis in  vivo [10]. Detect- MDA-MB-231 and MCF-7 cells were cultured in Dulbec- able expression of nNav1.5 in breast tumour tissues co’s modified Eagle’s medium (DMEM) (Nacalai, Japan) positive for lymph node metastasis signified its clinical supplemented with 5% foetal bovine serum and 4  mM importance in the prognosis of breast cancer patients [14, l-glutamine and maintained at 37  °C in a 5% CO and 44]. humidified atmosphere. The DMEM variant, DMEM-F12 In recent years, several mechanical insights for the was used for the MCF-10A supplemented with 5% horse role of Nav1.5 in controlling breast cancer cells capac- serum, 20  ng/ml epidermal growth factor (EGF), 10  µg/ ity to metastasize emerged, mainly involve strong influx ml insulin, 100 µg/ml hydrocortisone, and 10 ng/ml chol- and elevated concentration of Na which interferes with era toxin. 2+ Ca , pH, and gene expression [1]. This mode of interfer - ences has been reported to be associated with the acti- Pharmacology vation of proteases/peptidases activity which enhance Tetrodotoxin (TTX) (Sigma-Aldrich, USA), an estab- degradation of surrounding environment/cells to make lished VGSC-blocker was used to inhibit nNav1.5 way for invasion clearly demonstrated in breast cancer activity and mRNA expression. After purchase, it was [5, 16, 17]. Accordingly, the opening of Nav1.5 allows reconstituted in citrate buffer at 1.0  mM stock concen - strong influx of Na resulted in elevation of intracellular tration. Treatment concentration of 10  µM was used on Na concentration. A more positive intracellular environ- MDA-MB-231 for channel blocking effects [4, 28]. The + + ment leads to activation of Na /H -1 exchanger (NHE1) TTX was stored in − 20 ºC until required. Dehydrated which allows efflux of H resulted in extracellular acidi- riluzole was purchased and reconstituted according to fication from accumulation of proton. Lower pH at the the manufacturer’s instruction (Merck, USA) which was extracellular microenvironment activates cysteine cath- dissolved in DMSO and dH O into a stock concentra- epsins B and S that degrades extracellular matrix, favour- tion of 1.0  mM, before subsequently stored in − 20  °C ing cell invasion [5, 16, 17]. Unfortunately, this type of until required. Treatment concentration used on MDA- mechanical insight data for nNav1.5 in breast cancer is MB-231 was derived from 3-[4, 5-dimethylthiazol- 2-yl]- poorly understood. 2, 5diphenyltetrazolium bromide (MTT) assay. On the same note, elevated glutamate levels in the extracellular environment of rapidly-growing glioblas- MTT assay toma actively kill the surrounding cells to create space 3 × 10 cells were plated in a 96-well plate and incu- for invasion [37]. Glutamate secretion have also been bated for 24  h prior to any treatment (24, 48 and 72  h). observed in cancer cell lines and tumour tissues of non- Each treatment was done in triplicate and the medium neuronal/central nervous system origin [19, 21, 34, 38– was refreshed every 24  h. The medium was removed 40]. Accordingly, elevated glutamate levels in prostate and 100 µl of fresh DMEM and 10 µl of 12 mM of MTT cancer cell lines corresponds to higher serum glutamate were added to each well. Then, the plate was incubated at levels in the majority of prostate cancer patients com- 37 °C for 4 h. After incubation period, 85 µl of MTT solu- pared to benign prostatic hyperplasia tissues and directly tion was removed and 50  µl of 100% dimethyl sulfoxide correlated with aggressiveness [21, 38, 39]. In breast can- (DMSO) was added carefully to the well. The plate was cer, human breast cancer cell line, MDA-MB-231 cells incubated at 37  °C for 10  min and the absorbance was secrete glutamate corresponds to higher tissue glutamate measured at 540 nm using a spectrophotometer. levels [6]. In both prostate and breast cancer, excess glu- tamate contributes to bone metastasis [35]. SiRNA‑mediated nNav1.5 To our knowledge, a connection between glutamate Knockdown was conducted in order to investigate the and VGSCs in breast cancer has never been reported. effects of silencing nNav1.5 expression in MDA-MB-231 This study was designed to preliminary examine if such cells (which expressed the most significant nNav1.5 connection exists. mRNA upregulation) on the concentration of gluta- mate. SiRNA on MDA-MB-231 cells were produced via Methods transient transfection, whereby the siRNA sequences Cell culture against nNav1.5 and controls were acquired commer- The aggressive human breast cancer cells, MDA-MB-231 cially (SMARTpool siRNA reagents from Dharmacon). which overexpresses VGSCs (nNav1.5), less aggressive 3 × 10 cells of MDA were seeded in a well of 24-well A zahar et al. Biological Research (2022) 55:18 Page 3 of 13 Fig. 1 Comparison between nNav1.5 mRNA expression in human breast cancer cell lines; aggressive MDA-MB-231, less aggressive MCF-7 and the −ΔΔCt non-cancerous, MCF-10A. 2-step real-time PCR was conducted to measure the mRNA expression and 2 was used for the semi-quantitative analysis. Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between MCF-7 and MCF-10A versus MDA-MB-231 *** indicate, p < 0.001 plate. The cells were incubated overnight. Before start - RNA extraction and cDNA synthesis ing the treatment on the next day, working concentration Total RNA from the cell lines were extracted using sep- of siRNA from a 1 µM stock resuspension was prepared/ asol-chloroform (Nacalai Tesque, Japan). The purity of by adding 6  µl of it into 44 µl of serum free media in a RNA was assessed by observing the ratio of absorbance tube. A transfection solution was prepared by adding 3 260/280  nm and 260/230  nm in Nanodrop software. µl of transfection reagent (Polyplus-transfection SA, Total isolated RNA (1000 ng)  was transcribed to cDNA France) into 47 µl of serum free media in another tube. using reverse transcription kit (Toyobo, Japan), with Both tubes were slightly vortexed. The transfection solu - genomic DNA remover included by the company as com- tion was later added into the siRNA suspension and was ponent of the reverse transcription solution preparation. vortexed slightly and then left at room temperature for 5  min. The cells that were incubated in the wells over - Quantitative real‑time polymerase chain reaction (qRT‑PCR) night was removed of old media 500 µl of new media Real-time PCR was performed using SensiFAST SYBR added. The siRNA transfection solution was then added Hi-ROX kit according to manufacturer’s protocol (Bio- into the well and incubated at 37  °C for 5  h before the line, UK). Sequence primers used were as follows: β-actin media was changed. To confirm the success of nNav1.5 forward, ATT GCC GAC AGG ATG CAG AAG-3′ and knockdown, the gene in expression in the knock downed reverse, 5′-AGA AGC ATT TGC GGT GGA CG-3′ and cells were measured using qPCR. nNav1.5 forward, 5′-CTG CAC GCG TTC ACT TTC CT-3′ Azahar et al. Biological Research (2022) 55:18 Page 4 of 13 Fig. 2 Comparison between basal exogenous glutamate concentration in human breast cancer cell lines; aggressive MDA-MB-231 and less aggressive MCF-7. The (*) on the MDA-MB-231 results show a significant difference compared to MCF-7 exogenous glutamate. Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between MCF-7 and MCF-10A versus MDA-MB-231 * indicate, p < 0.05 Invasion assay and reverse, 5′-GAC AAA TTG CCT AGT TTT ATA TTT -3. 2 × 10 cells were placed inside wells containing the 3D Quantitative real-time was performed using ABI Prism culture matrix (Cultrex, USA). The plate was centri - 7000 Sequence Detection System (Life Technologies, fuged at 200×g for 3  min in a swing bucket rotor cen- USA) and the amplification conditions were as follows: trifuge (Thermo Fisher, USA) at room temperature. The initial activation for 10 min at 95 °C for one cycle, 10 s at plate was incubated at 37 ºC, 5% CO for 72  h. Image 95  °C and 30  s at 60  °C for 34 cycles. C values of target were taken under a compound microscope (Leica, Ger- genes were normalised to β-actin and the relative mRNA − many) every subsequent 24 h for 7 days. ImageJ imaging expression of target genes were calculated using the 2 ΔΔCt software [32] was used to analyse the size and invasion method [30]. projection.  Glutamate assay Data analysis Glutamate concentration in the cell supernatant (exog- Results are shown as the means ± SEM. Statistical evalu- enous) and endogenous was measured using a fluoro - ations were made using unpaired Student’s t test (Graph- metric assay in a 96-well plate format. Briefly, glutamate Pad Prism 9). 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. A zahar et al. Biological Research (2022) 55:18 Page 5 of 13 Fig. 3 Comparison between basal endogenous glutamate concentration in human breast cancer cell lines; aggressive MDA-MB-231 and less aggressive MCF-7. The (*) on the MDA-MB-231 results show a significant difference compared to MCF-7 endogenous glutamate (p < 0.05). Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between MCF-7 and MCF-10A versus MDA-MB-231* indicate, p < 0.05 Results was 0.004 (p < 0.001) and 0.003 (p < 0.001), respectively Higher expression of nNav1.5 mRNA corresponds (Fig. 1). with elevated exogenous and endogenous glutamate level Fluorometric reading for glutamate in the super- in aggressive human breast cancer cell line MDA‑MB‑231 natant (exogenous glutamate) of MDA-MB-231 at cells 24, 48, and 72  h of culture were higher than MCF-7 The expression of nNav1.5 mRNA in aggressive human and MCF-10A. When calculated using standard breast cancer cell line, MDA-MB-231 was compared to curve, at 24  h, exogenous glutamate level for MDA- less aggressive human breast cancer cell line, MCF-7 and MB-231 was 152.637 ± 33.30  µM (p < 0.05) of gluta- non-cancerous human breast epithelial cell line, MCF- mate, and for MCF-7 and MCF-10A, 70.950 ± 11.25 10A. mRNA expression level of nNav1.5 was significantly (p < 0.05) and 62.232 ± 10.00  µM (p < 0.05), respec- lower in MCF-7 and MCF-10A compared to MDA- tively. After 48  h, supernatant from all three cell −ΔΔCt MB-231. Accordingly, 2 value in MDA-MB-231 was lines showed an increase fluorometric reading and 1.122 ± 0.39 (p < 0.001), while in MCF-7 and MCF-10A MDA-MB-231 distinctively secreted higher exog- enous glutamate, 160.797 ± 33.300  µM (p < 0.05), than Azahar et al. Biological Research (2022) 55:18 Page 6 of 13 MDA-MB- (siRNA- nNav1.5) MCF-7 MCF-10A Fig. 4 Comparison between basal glutamate concentration in human breast cancer cell lines; aggressive MDA-MB-231, less aggressive MCF-7 and the non-cancerous, MCF-10A under immunofluorescence microscopy. A Qualitative measurement of glutamate under fluorescence microscope. B Semi-quantitative measurement of glutamate relative intensity using Leica Application Suite X (LAS X) software. Data presented as mean ± SEM, n = 3 and * indicate, p < 0.05 and ***, p < 0.001 A zahar et al. Biological Research (2022) 55:18 Page 7 of 13 Fig. 4 continued Level of exogenous and endogenous glutamate MCF-7 and MCF-10A, 82.434 ± 11.25 (p < 0.05), and in TTX‑treated MDA‑MB‑231 cells was reduced followed 100.443 ± 10.00  µM (p < 0.05), respectively. Finally, by invasion suppression and downregulation of nNav1.5 at 72  h, exogenous glutamate secreted by MDA- expression MB-231 peaked at 256.368 ± 33.30  µM (p < 0.05), The exogenous glutamate level in MDA-MB-231 cells MCF-7 at 108.961 ± 11.25 (p < 0.05), and MCF-10A at was reduced significantly by 10  µM of TTX after 24  h, 148.641 ± 10.00 µM (p < 0.05) (Fig. 2). with untreated at 120.637 ± 33.30  µM (p < 0.05) but Endogenous glutamate in all cell lines was measured TTX-treated at 98.761 ± 12.33 µM (p < 0.05). After 48 h, using fluorometric assay and fluorescence microscopy. At exogenous glutamate level of untreated MDA-MB-231 the end of the 72 h period from where the culture media was 200.797 ± 33.30  µM (p < 0.05) but TTX-treated, were collected for exogenous glutamate reading, the 172.563 ± 23.34  µM (p < 0.05). Finally, after 72  h, endogenous glutamate were determined whereby again, untreated MDA-MB-231 peaked at 356.368 ± 33.30  µM MDA-MB-231 contained highest significant amount of (p < 0.05) but TTX-treated at 188.341 ± 14.34  µM endogenous glutamate at 112.959 ± 0.39  µM (p < 0.05), (p < 0.05) (Fig . 5A). followed by MCF-7 at 29.540 ± 0.001  µM (p < 0.05), and Similar observation was obtained for endogenous MCF-10A, 73.590 ± 0.003 µM (p < 0.05) (Fig. 3). Presence glutamate with TTX-treated MDA-MB-231, signifi - of endogenous glutamate was clearly visible in a red dye cantly reduced to 43.365 ± 0.24  µM (p < 0.05) from the under fluorescence microscopy at 400× magnification untreated MDA-MB-231, 112.959 ± 0.390  µM (p < 0.05) but almost not visible in MCF-7 and MCF-10A (Fig. 4A). (Fig.  5B). When observed under fluorescence micro - Consequently, when the intensity of the red dye was scope, intensity of the red dye which indicate signal for quantified, presence of glutamate inside MDA-MB-231 endogenous glutamate in TTX-treated MDA-MB-231 was significantly highest at 27.667 ± 1.20 RFU (p < 0.05), was significantly less visible (Fig.  4A) which was MCF-7 at 17.667 ± 2.03 (p < 0.05), 10.667 ± 0.33 (p < 0.05) reduced to 9.333 ± 1.20185 RFU (p < 0.05) at 400× mag- (Fig. 4B). nification compared to untreated MDA-MB-231, 27.667 ± 1.2085 RFU (p < 0.05) (Fig . 6). 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 Azahar et al. Biological Research (2022) 55:18 Page 8 of 13 Fig. 6 Comparison between glutamate concentration in the aggressive human breast MDA-MB-231, under untreated and TTX-treated media under immunofluorescence microscopy with semi-quantitative measurement of glutamate relative intensity using Leica Application Suite X (LAS X) software. Data presented as mean ± SEM, n = 3 and *** indicate, p < 0.001 its peaked in size at day 3 in untreated MDA-MB-231 Fig. 5 Eec ff t of TTX, a known channel-blocking drug, on and later maintained its diameter and projections until endogenous and exogenous glutamate concentration of highly the end at day 7. Whilst for TTX-treated cells diameter metastatic MDA-MB-231 using fluorometric glutamate assay. A maintained throughout the 7 days experiments (Fig. 7B). Exogenous glutamate concentration in aggressive MDA-MB-231 after treated with riluzole, TTX and siRNA-nNav1.5 compared to untreated Suppression of invasion was affirmed after analysis of control measured in culture supernatant after 24 h of treatment. Data nNav1.5 mRNA revealed significant downregulation in presented as mean ± SEM (n = 3) and, statistical analysis used was −ΔΔCt TTX-treated cells. Briefly, 2 for untreated MDA- unpaired Student’s t test where (*) indicate, p < 0.05. The (*) indicate MB-231 cells was 1.122 ± 0.390 whilst for TTX-treated significant difference of TTX, siRNA-nNav1.5 and riluzole compared –3 cells, 4.598 × 10 ± 0.002 (Fig. 8). to untreated control. B Comparison of exogenous glutamate of MDA-MB-231 between untreated and after treated with riluzole, TTX and siRNA-nNav1.5. The (*) on the treated results show a significant Discussion difference compared to untreated endogenous glutamate (p < 0.05). Multiple lines of evidence have indicated clinical sig- Data presented as mean ± SEM n = 3 and, unpaired Student’s t test nificance of elevated serum, plasma and tissue gluta - between untreated versus treated MDA-MB-231* indicate, p < 0.05 mate 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 glutamate- enrich tumours is known [14, 18, 29]. nNav1.5 is a potent metastatic marker for breast cancer [1, 10]. Ele- vated levels of endogenous and exogenous glutamate in aggressive human breast cancer cells, MDA-MB-231 which expresses nNav1.5 compared to the less aggressive A zahar et al. Biological Research (2022) 55:18 Page 9 of 13 Fig. 7 Comparison between nNav1.5 mRNA expression in the aggressive human breast MDA-MB-231, under untreated and after treated −ΔΔCt with riluzole, TTX and siRNA-nNav1.5. 2-step real-time PCR was conducted to measure the mRNA expression and 2 was used for the semi-quantitative analysis. Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between untreated versus treated MDA-MB-231 *** indicate, p < 0.001 Azahar et al. Biological Research (2022) 55:18 Page 10 of 13 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 gluta - mate-induced cell toxicity via VGSC blockade [23]. Con- cordantly, 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 inva - sion 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 , > 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 Fig. 8 The 3D spheroid culture of MDA-MB-231 in the invasion gel membrane which led to suppression of metastatic ability matrix. A The image of MDA-MB-231 spheroid at day 0 and day 7 in of MDA-MB-231 [8]. untreated and TTX treated media. B The graph of the invasion rate of At the meantime, glutamate is a known potent necro- MDA-MB-231 untreated and under the treatment of TTX calculated by ImageJ imaging software. Data presented as mean ± SEM n = 3 sis factor during neuro-excitotoxicity [2, 33] and in and, unpaired Student’s t test between untreated versus treated glioblastoma [25]. Subsequently, tumour aggressiveness MDA-MB-231 indicate, p < 0.05 is exploiting excitotoxicity mechanisms to kill (necro- tising) surrounding cells to create way for metastasis [37]. In the case of breast cancer, metastasis to bone is human breast cancer cells, MCF-7 which lacks nNav1.5 a feature of aggressive breast cancer cells that secrete conforms with the hypothesis that high glutamate and high levels of glutamate [12, 13]. The 3D spheroid inva - expression of nNav1.5 is interconnected in breast can- sion assay was used in this study to replace wound cer. Unexpectedly, MCF-10A had higher glutamate than healing and Matrigel assays to provide more accurate MCF-7 even if it is significantly lower than the sub - insights on the invasion since spheroid have shown to ject of interest, MDA-MB-231. MCF 10A, as reported have higher cell proliferation, migration, and invasion by the American Type Culture Collection (ATCC), is a rate [3]. Regardless, the use of TTX have been shown to non-cancerous cell. But it shows certain tumour mark- reduce the effects of wound healing and Matrigel assays ers such as basal-like phenotype [31]. This is due to the according to previous studies [14, 15]. Besides being process of immortalisation of the cell line from primary used as a preferred in  vitro model for nNav1.5 expres- culture itself. Because of this, no secondary cell line are sion and glutamate secretion, MDA-MB-231 cells is to be considered normal post-immortalisation such as also an established cell line used for development of telomere lengthening process whereby the immortal cells a breast cancer bone metastases model, primarily to tend to derive chromosome abnormality [41] resulting long bones, spine, jaw and lungs [42]. nNav1.5, expres- in results obtained. Other research also points out the sion 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 A zahar et al. Biological Research (2022) 55:18 Page 11 of 13 Author contributions matrix [5, 11, 16]. Interestingly, emerging lines of evi- IIA, NAS and AHMND participated to the technical, experimental works, data dence are also pointing to the pro-metastatic role of analysis and manuscript drafting. ATCH, HJ and SNMN contributed to the cathepsin B in bone metastasis of breast cancer [26]. initial experimental design and review the data and manuscript preparation. NFM conceived the idea and funding for the study, data review and manu- With regards to VGSCs in breast cancer, a wealth of script preparation. data has now confidently demonstrated that Nav1.5 and its splice variant nNav1.5 offers potential value as Funding This study was financially supported by the Ministry of Education, Malaysia metastatic tumour markers in the diagnosis (including (Research University Grant 1001/CIPPM/8012235). prognosis), and therapies of the disease. Previously, the significance of therapeutic values for nNav1.5 and gluta - Availability of data and materials All relevant data are within the paper. mate has been reported separately. nNav1.5 gene and pro- tein expression in breast cancer tissues has been reported Declarations elsewhere [43] and now its value as prognostic and pre- dictive marker is currently being evaluated. In doing so, Ethics approval and consent to participate works on development and characterisation of antibodies Not applicable. against nNav1.5 has been reported. In this regard, a poly- Consent for publication clonal rabbit antibody, NESOpAb was generated to spe- 1. I declare that all data contained are accurate and all statements asserted as cifically recognise the neonatal splice form of Nav1.5 and facts are based on careful research by the author(s). 2. I declare that all authors named have participated in the work in a substan- excluding the adult counterpart [7] that utilise on their tive way and are prepared to take public responsibility for the work. discovery of the 7 divergence of amino acid in the former 3. I declare that the manuscript being submitted to this journal has never, since the divergence is significant enough to have its own except for publications in conference abstracts or thesis, been published in total or in part and is not being submitted for publication elsewhere. specific antibody. Further achievement from our inhouse 4. I declare that results presented in this manuscript, fulfil the ethical/bioethi- project managed to develop a novel monoclonal mouse cal certification given by the Institutional Review Board to carry out the antibody for nNav1.5, 4H8 [36]. This benefits directly for experiments reported by this manuscript. This statement must be signed and dated by all the authors and accompanied future therapeutic development with better specificity on by their printed names. Authors from different countries or institutions may a single epitope, allowing mass production of diagnostic sign separate copies of the same statement. tools for nNav1.5. As for glutamate, including the fact Competing interests that nNav1.5 is correlated with metastatic breast cancer Not applicable. and in other cases also shows significance of blocking nNav1.5 on another molecule such as Major Histocom- Author details Institute for Research in Molecular Medicine (INFORMM), Universiti Sains patibility Complex (MHC) Class I [24]. Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Depar t- ment of Neuroscience, School of Medical Sciences, Universiti Sains Malaysia, Conclusions Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Depar tment of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health, In summary, our study represents a unique connection Kelantan, Malaysia. between nNav1.5 expression and glutamate in aggressive breast cancer cells. 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Neonatal Nav1.5 protein expression in normal adult human tissues compounds for major psychiatric disorders: Evidence from clinical and breast cancer. Pathol - Res Pract. 2017;213(8):900–7. https:// doi. org/ 10. 1016/j. prp. 2017. 06. 003. A zahar et al. Biological Research (2022) 55:18 Page 13 of 13 44. Yang M, Kozminski DJ, Wold LA, Modak R, Calhoun JD, Isom LL, Brackenbury WJ. Therapeutic potential for phenytoin: Targeting Nav1.5 sodium channels to reduce migration and invasion in metastatic breast cancer. Breast Cancer Res Treatment. 2012. https:// doi. org/ 10. 1007/ s10549- 012- 2102-9. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? 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Abstract

Background: 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 gluta- mate 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. Methods: In this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was exam- ined using pharmacological approach ( VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA- nNav1.5). Eec ff t 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. Results: 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 endog- enous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells. Conclusions: An interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation. Keywords: Endogenous glutamate, Exogenous glutamate, Breast cancer, Voltage-gated sodium channels, Neonatal Nav1.5, Invasion Introduction VGSC and glutamate also characterizes neoplastic cells Voltage-gated sodium channels (VGSC) are transmem- [10, 13]. brane protein expressed abundantly in excitable cells Abnormal expression of VGSC in carcinomas (cancer such as neurons and muscle cells. In neurons, its main of the epithelial origin) such as cancer of the breast, pros- role is to propagate action potential critical in gluta- tate, lung, cervical, colon and ovarian had been a conun- matergic neurotransmission i.e. release of signalling drum to physiologist but subsequent ‘proof-of-concept’ molecules, glutamate at pre-synapses [22]. Interestingly, 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 *Correspondence: fatmawati@usm.my 1 [9, 14, 15, 18, 20, 29]. While there are several subtypes Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia of VGSC found upregulated in a number of carcinomas, Full list of author information is available at the end of the article breast cancer in particular, there is distinctively high © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Azahar et al. Biological Research (2022) 55:18 Page 2 of 13 expression of the ‘neonatal’ splice variant of the cardiac human breast cancer cells, MCF-7 which lacks VGSC VGSC isoform, Nav1.5 (nNav1.5) that potentiates motil- expression and non-cancerous human breast epithelial ity, migration, and invasion of aggressive human breast cell, MCF-10A were used in this study (ATCC, USA). The cancer cells in  vitro and metastasis in  vivo [10]. Detect- MDA-MB-231 and MCF-7 cells were cultured in Dulbec- able expression of nNav1.5 in breast tumour tissues co’s modified Eagle’s medium (DMEM) (Nacalai, Japan) positive for lymph node metastasis signified its clinical supplemented with 5% foetal bovine serum and 4  mM importance in the prognosis of breast cancer patients [14, l-glutamine and maintained at 37  °C in a 5% CO and 44]. humidified atmosphere. The DMEM variant, DMEM-F12 In recent years, several mechanical insights for the was used for the MCF-10A supplemented with 5% horse role of Nav1.5 in controlling breast cancer cells capac- serum, 20  ng/ml epidermal growth factor (EGF), 10  µg/ ity to metastasize emerged, mainly involve strong influx ml insulin, 100 µg/ml hydrocortisone, and 10 ng/ml chol- and elevated concentration of Na which interferes with era toxin. 2+ Ca , pH, and gene expression [1]. This mode of interfer - ences has been reported to be associated with the acti- Pharmacology vation of proteases/peptidases activity which enhance Tetrodotoxin (TTX) (Sigma-Aldrich, USA), an estab- degradation of surrounding environment/cells to make lished VGSC-blocker was used to inhibit nNav1.5 way for invasion clearly demonstrated in breast cancer activity and mRNA expression. After purchase, it was [5, 16, 17]. Accordingly, the opening of Nav1.5 allows reconstituted in citrate buffer at 1.0  mM stock concen - strong influx of Na resulted in elevation of intracellular tration. Treatment concentration of 10  µM was used on Na concentration. A more positive intracellular environ- MDA-MB-231 for channel blocking effects [4, 28]. The + + ment leads to activation of Na /H -1 exchanger (NHE1) TTX was stored in − 20 ºC until required. Dehydrated which allows efflux of H resulted in extracellular acidi- riluzole was purchased and reconstituted according to fication from accumulation of proton. Lower pH at the the manufacturer’s instruction (Merck, USA) which was extracellular microenvironment activates cysteine cath- dissolved in DMSO and dH O into a stock concentra- epsins B and S that degrades extracellular matrix, favour- tion of 1.0  mM, before subsequently stored in − 20  °C ing cell invasion [5, 16, 17]. Unfortunately, this type of until required. Treatment concentration used on MDA- mechanical insight data for nNav1.5 in breast cancer is MB-231 was derived from 3-[4, 5-dimethylthiazol- 2-yl]- poorly understood. 2, 5diphenyltetrazolium bromide (MTT) assay. On the same note, elevated glutamate levels in the extracellular environment of rapidly-growing glioblas- MTT assay toma actively kill the surrounding cells to create space 3 × 10 cells were plated in a 96-well plate and incu- for invasion [37]. Glutamate secretion have also been bated for 24  h prior to any treatment (24, 48 and 72  h). observed in cancer cell lines and tumour tissues of non- Each treatment was done in triplicate and the medium neuronal/central nervous system origin [19, 21, 34, 38– was refreshed every 24  h. The medium was removed 40]. Accordingly, elevated glutamate levels in prostate and 100 µl of fresh DMEM and 10 µl of 12 mM of MTT cancer cell lines corresponds to higher serum glutamate were added to each well. Then, the plate was incubated at levels in the majority of prostate cancer patients com- 37 °C for 4 h. After incubation period, 85 µl of MTT solu- pared to benign prostatic hyperplasia tissues and directly tion was removed and 50  µl of 100% dimethyl sulfoxide correlated with aggressiveness [21, 38, 39]. In breast can- (DMSO) was added carefully to the well. The plate was cer, human breast cancer cell line, MDA-MB-231 cells incubated at 37  °C for 10  min and the absorbance was secrete glutamate corresponds to higher tissue glutamate measured at 540 nm using a spectrophotometer. levels [6]. In both prostate and breast cancer, excess glu- tamate contributes to bone metastasis [35]. SiRNA‑mediated nNav1.5 To our knowledge, a connection between glutamate Knockdown was conducted in order to investigate the and VGSCs in breast cancer has never been reported. effects of silencing nNav1.5 expression in MDA-MB-231 This study was designed to preliminary examine if such cells (which expressed the most significant nNav1.5 connection exists. mRNA upregulation) on the concentration of gluta- mate. SiRNA on MDA-MB-231 cells were produced via Methods transient transfection, whereby the siRNA sequences Cell culture against nNav1.5 and controls were acquired commer- The aggressive human breast cancer cells, MDA-MB-231 cially (SMARTpool siRNA reagents from Dharmacon). which overexpresses VGSCs (nNav1.5), less aggressive 3 × 10 cells of MDA were seeded in a well of 24-well A zahar et al. Biological Research (2022) 55:18 Page 3 of 13 Fig. 1 Comparison between nNav1.5 mRNA expression in human breast cancer cell lines; aggressive MDA-MB-231, less aggressive MCF-7 and the −ΔΔCt non-cancerous, MCF-10A. 2-step real-time PCR was conducted to measure the mRNA expression and 2 was used for the semi-quantitative analysis. Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between MCF-7 and MCF-10A versus MDA-MB-231 *** indicate, p < 0.001 plate. The cells were incubated overnight. Before start - RNA extraction and cDNA synthesis ing the treatment on the next day, working concentration Total RNA from the cell lines were extracted using sep- of siRNA from a 1 µM stock resuspension was prepared/ asol-chloroform (Nacalai Tesque, Japan). The purity of by adding 6  µl of it into 44 µl of serum free media in a RNA was assessed by observing the ratio of absorbance tube. A transfection solution was prepared by adding 3 260/280  nm and 260/230  nm in Nanodrop software. µl of transfection reagent (Polyplus-transfection SA, Total isolated RNA (1000 ng)  was transcribed to cDNA France) into 47 µl of serum free media in another tube. using reverse transcription kit (Toyobo, Japan), with Both tubes were slightly vortexed. The transfection solu - genomic DNA remover included by the company as com- tion was later added into the siRNA suspension and was ponent of the reverse transcription solution preparation. vortexed slightly and then left at room temperature for 5  min. The cells that were incubated in the wells over - Quantitative real‑time polymerase chain reaction (qRT‑PCR) night was removed of old media 500 µl of new media Real-time PCR was performed using SensiFAST SYBR added. The siRNA transfection solution was then added Hi-ROX kit according to manufacturer’s protocol (Bio- into the well and incubated at 37  °C for 5  h before the line, UK). Sequence primers used were as follows: β-actin media was changed. To confirm the success of nNav1.5 forward, ATT GCC GAC AGG ATG CAG AAG-3′ and knockdown, the gene in expression in the knock downed reverse, 5′-AGA AGC ATT TGC GGT GGA CG-3′ and cells were measured using qPCR. nNav1.5 forward, 5′-CTG CAC GCG TTC ACT TTC CT-3′ Azahar et al. Biological Research (2022) 55:18 Page 4 of 13 Fig. 2 Comparison between basal exogenous glutamate concentration in human breast cancer cell lines; aggressive MDA-MB-231 and less aggressive MCF-7. The (*) on the MDA-MB-231 results show a significant difference compared to MCF-7 exogenous glutamate. Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between MCF-7 and MCF-10A versus MDA-MB-231 * indicate, p < 0.05 Invasion assay and reverse, 5′-GAC AAA TTG CCT AGT TTT ATA TTT -3. 2 × 10 cells were placed inside wells containing the 3D Quantitative real-time was performed using ABI Prism culture matrix (Cultrex, USA). The plate was centri - 7000 Sequence Detection System (Life Technologies, fuged at 200×g for 3  min in a swing bucket rotor cen- USA) and the amplification conditions were as follows: trifuge (Thermo Fisher, USA) at room temperature. The initial activation for 10 min at 95 °C for one cycle, 10 s at plate was incubated at 37 ºC, 5% CO for 72  h. Image 95  °C and 30  s at 60  °C for 34 cycles. C values of target were taken under a compound microscope (Leica, Ger- genes were normalised to β-actin and the relative mRNA − many) every subsequent 24 h for 7 days. ImageJ imaging expression of target genes were calculated using the 2 ΔΔCt software [32] was used to analyse the size and invasion method [30]. projection.  Glutamate assay Data analysis Glutamate concentration in the cell supernatant (exog- Results are shown as the means ± SEM. Statistical evalu- enous) and endogenous was measured using a fluoro - ations were made using unpaired Student’s t test (Graph- metric assay in a 96-well plate format. Briefly, glutamate Pad Prism 9). 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. A zahar et al. Biological Research (2022) 55:18 Page 5 of 13 Fig. 3 Comparison between basal endogenous glutamate concentration in human breast cancer cell lines; aggressive MDA-MB-231 and less aggressive MCF-7. The (*) on the MDA-MB-231 results show a significant difference compared to MCF-7 endogenous glutamate (p < 0.05). Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between MCF-7 and MCF-10A versus MDA-MB-231* indicate, p < 0.05 Results was 0.004 (p < 0.001) and 0.003 (p < 0.001), respectively Higher expression of nNav1.5 mRNA corresponds (Fig. 1). with elevated exogenous and endogenous glutamate level Fluorometric reading for glutamate in the super- in aggressive human breast cancer cell line MDA‑MB‑231 natant (exogenous glutamate) of MDA-MB-231 at cells 24, 48, and 72  h of culture were higher than MCF-7 The expression of nNav1.5 mRNA in aggressive human and MCF-10A. When calculated using standard breast cancer cell line, MDA-MB-231 was compared to curve, at 24  h, exogenous glutamate level for MDA- less aggressive human breast cancer cell line, MCF-7 and MB-231 was 152.637 ± 33.30  µM (p < 0.05) of gluta- non-cancerous human breast epithelial cell line, MCF- mate, and for MCF-7 and MCF-10A, 70.950 ± 11.25 10A. mRNA expression level of nNav1.5 was significantly (p < 0.05) and 62.232 ± 10.00  µM (p < 0.05), respec- lower in MCF-7 and MCF-10A compared to MDA- tively. After 48  h, supernatant from all three cell −ΔΔCt MB-231. Accordingly, 2 value in MDA-MB-231 was lines showed an increase fluorometric reading and 1.122 ± 0.39 (p < 0.001), while in MCF-7 and MCF-10A MDA-MB-231 distinctively secreted higher exog- enous glutamate, 160.797 ± 33.300  µM (p < 0.05), than Azahar et al. Biological Research (2022) 55:18 Page 6 of 13 MDA-MB- (siRNA- nNav1.5) MCF-7 MCF-10A Fig. 4 Comparison between basal glutamate concentration in human breast cancer cell lines; aggressive MDA-MB-231, less aggressive MCF-7 and the non-cancerous, MCF-10A under immunofluorescence microscopy. A Qualitative measurement of glutamate under fluorescence microscope. B Semi-quantitative measurement of glutamate relative intensity using Leica Application Suite X (LAS X) software. Data presented as mean ± SEM, n = 3 and * indicate, p < 0.05 and ***, p < 0.001 A zahar et al. Biological Research (2022) 55:18 Page 7 of 13 Fig. 4 continued Level of exogenous and endogenous glutamate MCF-7 and MCF-10A, 82.434 ± 11.25 (p < 0.05), and in TTX‑treated MDA‑MB‑231 cells was reduced followed 100.443 ± 10.00  µM (p < 0.05), respectively. Finally, by invasion suppression and downregulation of nNav1.5 at 72  h, exogenous glutamate secreted by MDA- expression MB-231 peaked at 256.368 ± 33.30  µM (p < 0.05), The exogenous glutamate level in MDA-MB-231 cells MCF-7 at 108.961 ± 11.25 (p < 0.05), and MCF-10A at was reduced significantly by 10  µM of TTX after 24  h, 148.641 ± 10.00 µM (p < 0.05) (Fig. 2). with untreated at 120.637 ± 33.30  µM (p < 0.05) but Endogenous glutamate in all cell lines was measured TTX-treated at 98.761 ± 12.33 µM (p < 0.05). After 48 h, using fluorometric assay and fluorescence microscopy. At exogenous glutamate level of untreated MDA-MB-231 the end of the 72 h period from where the culture media was 200.797 ± 33.30  µM (p < 0.05) but TTX-treated, were collected for exogenous glutamate reading, the 172.563 ± 23.34  µM (p < 0.05). Finally, after 72  h, endogenous glutamate were determined whereby again, untreated MDA-MB-231 peaked at 356.368 ± 33.30  µM MDA-MB-231 contained highest significant amount of (p < 0.05) but TTX-treated at 188.341 ± 14.34  µM endogenous glutamate at 112.959 ± 0.39  µM (p < 0.05), (p < 0.05) (Fig . 5A). followed by MCF-7 at 29.540 ± 0.001  µM (p < 0.05), and Similar observation was obtained for endogenous MCF-10A, 73.590 ± 0.003 µM (p < 0.05) (Fig. 3). Presence glutamate with TTX-treated MDA-MB-231, signifi - of endogenous glutamate was clearly visible in a red dye cantly reduced to 43.365 ± 0.24  µM (p < 0.05) from the under fluorescence microscopy at 400× magnification untreated MDA-MB-231, 112.959 ± 0.390  µM (p < 0.05) but almost not visible in MCF-7 and MCF-10A (Fig. 4A). (Fig.  5B). When observed under fluorescence micro - Consequently, when the intensity of the red dye was scope, intensity of the red dye which indicate signal for quantified, presence of glutamate inside MDA-MB-231 endogenous glutamate in TTX-treated MDA-MB-231 was significantly highest at 27.667 ± 1.20 RFU (p < 0.05), was significantly less visible (Fig.  4A) which was MCF-7 at 17.667 ± 2.03 (p < 0.05), 10.667 ± 0.33 (p < 0.05) reduced to 9.333 ± 1.20185 RFU (p < 0.05) at 400× mag- (Fig. 4B). nification compared to untreated MDA-MB-231, 27.667 ± 1.2085 RFU (p < 0.05) (Fig . 6). 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 Azahar et al. Biological Research (2022) 55:18 Page 8 of 13 Fig. 6 Comparison between glutamate concentration in the aggressive human breast MDA-MB-231, under untreated and TTX-treated media under immunofluorescence microscopy with semi-quantitative measurement of glutamate relative intensity using Leica Application Suite X (LAS X) software. Data presented as mean ± SEM, n = 3 and *** indicate, p < 0.001 its peaked in size at day 3 in untreated MDA-MB-231 Fig. 5 Eec ff t of TTX, a known channel-blocking drug, on and later maintained its diameter and projections until endogenous and exogenous glutamate concentration of highly the end at day 7. Whilst for TTX-treated cells diameter metastatic MDA-MB-231 using fluorometric glutamate assay. A maintained throughout the 7 days experiments (Fig. 7B). Exogenous glutamate concentration in aggressive MDA-MB-231 after treated with riluzole, TTX and siRNA-nNav1.5 compared to untreated Suppression of invasion was affirmed after analysis of control measured in culture supernatant after 24 h of treatment. Data nNav1.5 mRNA revealed significant downregulation in presented as mean ± SEM (n = 3) and, statistical analysis used was −ΔΔCt TTX-treated cells. Briefly, 2 for untreated MDA- unpaired Student’s t test where (*) indicate, p < 0.05. The (*) indicate MB-231 cells was 1.122 ± 0.390 whilst for TTX-treated significant difference of TTX, siRNA-nNav1.5 and riluzole compared –3 cells, 4.598 × 10 ± 0.002 (Fig. 8). to untreated control. B Comparison of exogenous glutamate of MDA-MB-231 between untreated and after treated with riluzole, TTX and siRNA-nNav1.5. The (*) on the treated results show a significant Discussion difference compared to untreated endogenous glutamate (p < 0.05). Multiple lines of evidence have indicated clinical sig- Data presented as mean ± SEM n = 3 and, unpaired Student’s t test nificance of elevated serum, plasma and tissue gluta - between untreated versus treated MDA-MB-231* indicate, p < 0.05 mate 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 glutamate- enrich tumours is known [14, 18, 29]. nNav1.5 is a potent metastatic marker for breast cancer [1, 10]. Ele- vated levels of endogenous and exogenous glutamate in aggressive human breast cancer cells, MDA-MB-231 which expresses nNav1.5 compared to the less aggressive A zahar et al. Biological Research (2022) 55:18 Page 9 of 13 Fig. 7 Comparison between nNav1.5 mRNA expression in the aggressive human breast MDA-MB-231, under untreated and after treated −ΔΔCt with riluzole, TTX and siRNA-nNav1.5. 2-step real-time PCR was conducted to measure the mRNA expression and 2 was used for the semi-quantitative analysis. Data presented as mean ± SEM n = 3 and, unpaired Student’s t test between untreated versus treated MDA-MB-231 *** indicate, p < 0.001 Azahar et al. Biological Research (2022) 55:18 Page 10 of 13 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 gluta - mate-induced cell toxicity via VGSC blockade [23]. Con- cordantly, 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 inva - sion 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 , > 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 Fig. 8 The 3D spheroid culture of MDA-MB-231 in the invasion gel membrane which led to suppression of metastatic ability matrix. A The image of MDA-MB-231 spheroid at day 0 and day 7 in of MDA-MB-231 [8]. untreated and TTX treated media. B The graph of the invasion rate of At the meantime, glutamate is a known potent necro- MDA-MB-231 untreated and under the treatment of TTX calculated by ImageJ imaging software. Data presented as mean ± SEM n = 3 sis factor during neuro-excitotoxicity [2, 33] and in and, unpaired Student’s t test between untreated versus treated glioblastoma [25]. Subsequently, tumour aggressiveness MDA-MB-231 indicate, p < 0.05 is exploiting excitotoxicity mechanisms to kill (necro- tising) surrounding cells to create way for metastasis [37]. In the case of breast cancer, metastasis to bone is human breast cancer cells, MCF-7 which lacks nNav1.5 a feature of aggressive breast cancer cells that secrete conforms with the hypothesis that high glutamate and high levels of glutamate [12, 13]. The 3D spheroid inva - expression of nNav1.5 is interconnected in breast can- sion assay was used in this study to replace wound cer. Unexpectedly, MCF-10A had higher glutamate than healing and Matrigel assays to provide more accurate MCF-7 even if it is significantly lower than the sub - insights on the invasion since spheroid have shown to ject of interest, MDA-MB-231. MCF 10A, as reported have higher cell proliferation, migration, and invasion by the American Type Culture Collection (ATCC), is a rate [3]. Regardless, the use of TTX have been shown to non-cancerous cell. But it shows certain tumour mark- reduce the effects of wound healing and Matrigel assays ers such as basal-like phenotype [31]. This is due to the according to previous studies [14, 15]. Besides being process of immortalisation of the cell line from primary used as a preferred in  vitro model for nNav1.5 expres- culture itself. Because of this, no secondary cell line are sion and glutamate secretion, MDA-MB-231 cells is to be considered normal post-immortalisation such as also an established cell line used for development of telomere lengthening process whereby the immortal cells a breast cancer bone metastases model, primarily to tend to derive chromosome abnormality [41] resulting long bones, spine, jaw and lungs [42]. nNav1.5, expres- in results obtained. Other research also points out the sion 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 A zahar et al. Biological Research (2022) 55:18 Page 11 of 13 Author contributions matrix [5, 11, 16]. Interestingly, emerging lines of evi- IIA, NAS and AHMND participated to the technical, experimental works, data dence are also pointing to the pro-metastatic role of analysis and manuscript drafting. ATCH, HJ and SNMN contributed to the cathepsin B in bone metastasis of breast cancer [26]. initial experimental design and review the data and manuscript preparation. NFM conceived the idea and funding for the study, data review and manu- With regards to VGSCs in breast cancer, a wealth of script preparation. data has now confidently demonstrated that Nav1.5 and its splice variant nNav1.5 offers potential value as Funding This study was financially supported by the Ministry of Education, Malaysia metastatic tumour markers in the diagnosis (including (Research University Grant 1001/CIPPM/8012235). prognosis), and therapies of the disease. Previously, the significance of therapeutic values for nNav1.5 and gluta - Availability of data and materials All relevant data are within the paper. mate has been reported separately. nNav1.5 gene and pro- tein expression in breast cancer tissues has been reported Declarations elsewhere [43] and now its value as prognostic and pre- dictive marker is currently being evaluated. In doing so, Ethics approval and consent to participate works on development and characterisation of antibodies Not applicable. against nNav1.5 has been reported. In this regard, a poly- Consent for publication clonal rabbit antibody, NESOpAb was generated to spe- 1. I declare that all data contained are accurate and all statements asserted as cifically recognise the neonatal splice form of Nav1.5 and facts are based on careful research by the author(s). 2. I declare that all authors named have participated in the work in a substan- excluding the adult counterpart [7] that utilise on their tive way and are prepared to take public responsibility for the work. discovery of the 7 divergence of amino acid in the former 3. I declare that the manuscript being submitted to this journal has never, since the divergence is significant enough to have its own except for publications in conference abstracts or thesis, been published in total or in part and is not being submitted for publication elsewhere. specific antibody. Further achievement from our inhouse 4. I declare that results presented in this manuscript, fulfil the ethical/bioethi- project managed to develop a novel monoclonal mouse cal certification given by the Institutional Review Board to carry out the antibody for nNav1.5, 4H8 [36]. This benefits directly for experiments reported by this manuscript. This statement must be signed and dated by all the authors and accompanied future therapeutic development with better specificity on by their printed names. Authors from different countries or institutions may a single epitope, allowing mass production of diagnostic sign separate copies of the same statement. tools for nNav1.5. As for glutamate, including the fact Competing interests that nNav1.5 is correlated with metastatic breast cancer Not applicable. and in other cases also shows significance of blocking nNav1.5 on another molecule such as Major Histocom- Author details Institute for Research in Molecular Medicine (INFORMM), Universiti Sains patibility Complex (MHC) Class I [24]. Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Depar t- ment of Neuroscience, School of Medical Sciences, Universiti Sains Malaysia, Conclusions Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia. Depar tment of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health, In summary, our study represents a unique connection Kelantan, Malaysia. between nNav1.5 expression and glutamate in aggressive breast cancer cells. 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Biological ResearchSpringer Journals

Published: Apr 29, 2022

Keywords: Endogenous glutamate; Exogenous glutamate; Breast cancer; Voltage-gated sodium channels; Neonatal Nav1.5; Invasion

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