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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 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. Methods 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. 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 endogenous 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.

Background: Insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP) are the major components in the IGF axis that play an important role in the development and progression of cancer. However, little is known about the circulatory levels of these proteins with recurrence and metastasis in breast carcinoma (BC). Methods: One hundred and twenty-eight (128) breast cancer patients who underwent mastectomy were purposively taken from a prospective study. Clinicopathological information was collected from patients' medical records. Anthropometric and biochemical parameters were measured by standard methods. Growth and metabolic factors were estimated by enzyme-linked immunosorbent assay (ELISA), and compared for patients' recurrence and metastasis. Results: The mean age at diagnosis was 44 years. Most of the tumors (94%) were ductal cell carcinoma. Two thirds were moderately differentiated tumor grade and lymph node positive [70% and 67% respectively]. Circulatory levels of IGFBP1 were significantly higher (p<0.05) in patients who received adjuvant chemotherapy; and patients with recurrence and metastasis while IGF1 was found to be significantly increased in only metastatic breast cancer (p<0.05). IGFBP-1 levels were correlated with SGPT, ALP, hemoglobin, WBC, ESR, CA15.3 and contraceptive use, whereas, IGF-1 was associated with insulin, HOMA %B and ESR. Conclusions: High serum levels of IGFBP-1 and IGF-1 may be associated with breast cancer recurrence and metastasis. These results need to be confirmed in larger breast cancer survivor cohorts. Legal entity responsible for the study: TWAS, Ministry of Education (MoE) Bangladesh.

Background: Detectable neonatal Nav1.5 (nNav1.5) expression in tumour breast tissue positive for lymph node metastasis and triple-negative subtype serves as a valid tumour-associated antigen to target and prevent breast cancer invasion and metastasis. Therapeutic antibodies against tumour antigens have become the predominant class of new drugs in cancer therapy because of their fewer adverse effects and high specificity. Objective: This study was designed to investigate the therapeutic and anti-metastatic potential of the two newly obtained anti-nNav1.5 antibodies, polyclonal anti-nNav1.5 (pAb-nNav1.5) and monoclonal anti-nNav1.5 (mAb-nNav1.5), on breast cancer invasion and metastasis. Methods: MDA-MB-231 and 4T1 cells were used as in vitro models to study the effect of pAb-nNav1.5 (59.2 µg/ml) and mAb-nNav1.5 (10 µg/ml) (24 hours treatment) on cell invasion. 4T1-induced mammary tumours in BALB/c female mice were used as an in vivo model to study the effect of a single dose of intravenous pAb-nNav1.5 (1 mg/ml) and mAb-nNav1.5 (1 mg/ml) on the occurrence of metastasis. Real-time PCR and immunofluorescence staining were conducted to assess the effect of antibody treatment on nNav1.5 mRNA and protein expression, respectively. The animals' body weight, organs, lesions, and tumour mass were also measured and compared. Results: pAb-nNav1.5 and mAb-nNav1.5 treatments effectively suppressed the invasion of MDA-MB-231 and 4T1 cells in the 3D spheroid invasion assay. Both antibodies significantly reduced nNav1.5 gene and protein expression in these cell lines. Treatment with pAb-nNav1.5 and mAb-nNav1.5 successfully reduced mammary tumour tissue size and mass and prevented lesions in vital organs of the mammary tumour animal model whilst maintaining the animal's healthy weight. mRNA expression of nNav1.5 in mammary tumour tissues was only reduced by mAb-nNav1.5. Conclusion: Overall, this work verifies the uniqueness of targeting nNav1.5 in breast cancer invasion and metastasis prevention, but more importantly, humanised versions of mAb-nNav1.5 may be valuable passive immunotherapeutic agents to target nNav1.5 in breast cancer.

‘Neonatal’ Nav1.5 (nNav1.5) is a potent tumour metastasis marker found especially in aggressive human breast cancer cells in vitro, in tumour tissues of in vivo metastatic animal models and in patients positive for lymph-node metastasis. Its expression has been recently described in human brain neuroblastoma and astrocytoma. However, a thorough understanding of nNav1.5’s role in cancers has been limited by the lack of specific antibodies against it. Here, a mouse monoclonal antibody, 4H8 mAb-nNav1.5, was obtained and characterised concerning its efficacy in detecting nNav1.5 using indirect ELISA, surface plasmon resonance (SPR), Western blotting and immunofluorescence microscopy. 4H8 mAb-nNav1.5 was selected from a panel of hybridoma clones raised against nNav1.5 specific peptide (15 mers). The antibody exhibited linear association against nNav1.5 specific-linear peptide in indirect ELISA and was supported by SPR. The antibody also demonstrated strong immunoreactivity in immunofluorescence imaging of nNav1.5-abundant cells, human and mouse aggressive breast cancer cells, MDA-MB-231 and 4T1, respectively, which was not observed in nNav1.5-deficient cells, human less aggressive breast cancer cells, MCF-7 and non-cancerous breast epithelial cells, MCF-10A. This study demonstrates the initial description of 4H8 mAb-nNav1.5, which could serve as a beneficial tool to enhance future studies on nNav1.5 expression and function in cancers.