Circulating neonatal Nav1.5 (nNav1.5) antigen and anti-nNav1.5 antibodies as potential biomarkers for breast cancer metastasis

Neonatal Nav1.5 (nNav1.5) has been known to potentiate breast cancer (BCa) metastasis. The detection of anti-nNav1.5 antibodies (anti-nNav1.5-Ab) reflects the immunogenicity of nNav1.5. However, the presences of circulating nNav1.5 antigen and anti-nNav1.5-Ab in the context of BCa metastasis have not been explored yet. Therefore, the study has attempted to conduct such an investigation using both blood samples from 4T1 orthotopic mice and BCa patients. In the preclinical study, forty female BALB/c mice were divided into three groups: 4T1 orthotopic BCa mice (n=17), control mice (n=20) and positive control mice (n=3). After tumour development, the mice were sacrificed to obtain target organs, whole blood, and serum. Histopathology, cytokine analyses, real-time PCR, and indirect ELISA were performed. Histopathology and cytokine analyses showed the establishment of metastasis in 4T1 orthotopic mice. The concentration of vascular endothelial growth factor (VEGF) was significantly higher in the 4T1 orthotopic mice (P<0.0001****). Circulating nNav1.5 antigen and anti-nNav1.5-Ab were detected in 4T1 orthotopic mice, using real-time PCR and indirect ELISA, respectively. Furthermore, there was an inverse relationship between anti-nNav1.5-Ab and the total metastatic foci (P=0.0485*, r=-0.7306). In the clinical study, 32 BCa patients were grouped based on their stages: early-invasive (n=15) and advanced (n=17) stages. Approximately 3 mL of blood was withdrawn, and only indirect ELISA was conducted. The clinical study showed that BCa patients of advanced-stages portrayed higher expression of anti-nNav1.5-Ab compared to early stages of BCa (P =0.0110*). In conclusion, the detection of nNav1.5 antigen and anti-nNav1.5-Ab was consistent with the presence of BCa metastasis

The roles of circulating neonatal nav1.5 (NNAV1.5) and its antibodies in cancer progression of 4T1 orthotopic mice model and breast cancer patients

The study has aimed to investigate the roles of circulating neonatal Nav1.5 (nNav1.5) and natural antibodies produced against nNav1.5 (anti-nNav1.5-Ab) in the whole blood and serum of 4T1 orthotopic mice model and breast cancer patients. The preclinical research involved three mice groups: control (n=20), 4T1 orthotopic breast cancer model (n=17), and positive control (n=3). Blood samples, target organs and 4T1 tumours were collected. Real-time polymerase chain reaction (qPCR) was conducted to detect the expression of nNav1.5 antigen in the whole blood and an in-house indirect enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of anti-nNav1.5-Ab in the serum. Additionally, lung metastasis clonogenic assay, histology and cytokine analysis were conducted. The clinical study involved 128 participants: healthy participants (n=64) and breast cancer patients (n=64). The sociodemographic details of the participants were analysed. The breast cancer patients were divided based on their treatment status and stages. A total of 6 ml of blood was withdrawn, and similarly, qPCR and ELISA were conducted to detect the circulating nNav1.5 and anti-nNav1.5-Ab, respectively. The cytokine analysis was also conducted in the clinical study. The preclinical study revealed the occurrence of metastasis (via clonogenic assay and histology) and circulating nNav1.5 antigen in 4T1 orthotopic mice. The 4T1 orthotopic mice showed significantly higher absorbance of antinNav1.5- Ab than the control group (P<.001). There was a significant negative correlation between the expression of the nNav1.5 antigen and the absorbance of antinNav1.5- Ab (P<.05, r=-0.549). In the cytokine analyses, there were significant positive correlations between anti-nNav1.5-Ab, IL-6 (P<.05, r=0.643) and VEGF (P<.01, r=0.735). Sociodemographic analysis revealed a significant age difference between healthy participants and breast cancer patients (P<.001). The clinical study showed restricted expression of circulating nNav1.5 antigen, only in five pretreatment patients. The expression of anti-nNav1.5-Ab was detected in both healthy and breast cancer patients, but the absorbance of anti-nNav1.5-Ab was significantly higher in breast cancer patients (P<.001). The pretreatment group portrayed significantly the highest expression of anti-nNav1.5-Ab as compared to the control and ongoing treatment group (P<.001). There was a significant positive correlation between antinNav1.5- Ab and IL-6 (P<.05, r=0.726) in the pretreatment group, followed by a significant negative correlation between anti-nNav1.5-Ab and VEGF (P<.01, r=- 0.842) in the ongoing treatment group. Advanced stage patients exhibited significantly higher expression of anti-nNav1.5-Ab compared to early-invasive patients (P<.05). The presence of anti-nNav1.5-Ab highlights the immunogenicity of nNav1.5. AntinNav1.5- Ab could serve as an immunosurveillance marker for breast cancer metastasis

Discovering the Triad between Nav1.5, Breast Cancer, and the Immune System: A Fundamental Review and Future Perspectives

Nav1.5 is one of the nine voltage-gated sodium channel-alpha subunit (VGSC-&alpha;) family members. The Nav1.5 channel typically carries an inward sodium ion current that depolarises the membrane potential during the upstroke of the cardiac action potential. The neonatal isoform of Nav1.5, nNav1.5, is produced via VGSC-&alpha; alternative splicing. nNav1.5 is known to potentiate breast cancer metastasis. Despite their well-known biological functions, the immunological perspectives of these channels are poorly explored. The current review has attempted to summarise the triad between Nav1.5 (nNav1.5), breast cancer, and the immune system. To date, there is no such review available that encompasses these three components as most reviews focus on the molecular and pharmacological prospects of Nav1.5. This review is divided into three major subsections: (1) the review highlights the roles of Nav1.5 and nNav1.5 in potentiating the progression of breast cancer, (2) focuses on the general connection between breast cancer and the immune system, and finally (3) the review emphasises the involvements of Nav1.5 and nNav1.5 in the functionality of the immune system and the immunogenicity. Compared to the other subsections, section three is pretty unexploited; it would be interesting to study this subsection as it completes the triad

The phytochemical components of Kelantan grown Annona muricata leaves and its anti-proliferative properties on MCF-7 breast cancer cells

Annona muricata leaves exhibit anti-cancer properties against breast cancer. In this study, the phytochemical components, anti-proliferative activity, and cytotoxicity of Kelantan (Malaysia) grown Annona muricata leaves were evaluated using MCF-7 breast cancer cell line. The preparation of Annona muricata leaves extracts was performed with various organic solvents (ethyl acetate, n-hexane, methanol, and aqueous) using Soxhlet method. Each extract was analysed using gas chromatography-mass spectrometry (GCMS) for phytochemical analysis and characterised with Wiley and NIST library searchers. The effects of all extracts on cell proliferation were analysed using MTT assay. The anti-proliferative activity was determined by evaluating the IC50 value. Based on the lowest IC50 value, further experiments were done to observe the apoptosis effect using the Annexin V-FITC technique. While for cell cycle analysis, Cycletest Plus DNA Reagent Kit was used. Both were further determined using flow cytometry analysis. The ethyl acetate-based Annona muricata extract contained terpenoids, phenolic compounds, and alkaloids. Ethyl acetate extract of Annona muricata exhibited a significant cytotoxic effect on MCF-7 breast cancer cells. The most potent IC50 value obtained was 21.8 ± 3.85 µg/mL. The changes in morphology were more profound after 72 h of treatment with ethyl acetate extract of Annona muricata, including cell shrinkage. Flow cytometry analysis showed that it was induced in early and late apoptosis in vitro and arrest at G0 /G1 cell cycle phase in time-dependant manners. In short, ethyl acetate extract of Annona muricata exhibited a range of phytochemical components, anti-proliferative activity and cytotoxic effect on MCF-7 breast cancer cells