Sains Malaysiana 51(9)(2022): 2967-2984

http://doi.org/10.17576/jsm-2022-5109-18

 

Circulating Neonatal Nav1.5 (nNav1.5) Antigen and Anti-nNav1.5 Antibodies as Potential Biomarkers for Breast Cancer Metastasis

(Peredaran Antigen dan Antibodi Neonatal Nav1.5 (nNav1.5) Sebagai Penanda Biologi Berpotensi untuk Metastasis Kanser Payu Dara)

 

HARISHINI RAJARATINAM1, NUR SYAHMINA RASUDIN1, MAYA MAZUWIN YAHYA2,3, WAN ZAINIRA WAN ZAIN2, SABREENA SAFUAN1, NURUL ASMA-ABDULLAH1, NOOR FATMAWATI MOKHTAR4 & WAN EZUMI MOHD FUAD1*

 

1School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

2Department of Surgery, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

3Breast Cancer Awareness and Research (BestARi) Unit, Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

4Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

 

Diserahkan: 4 Januari 2022/Diterima: 14 Mac 2022

 

Abstract

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.

 

Keywords: Breast cancer patients; in vivo; metastasis; Neonatal Nav1.5; orthotopic; 4T1

 

Abstrak

Neonatal Nav1.5 (nNav1.5) telah dikenal pasti mampu mendorong metastasis kanser payu dara. Pengesanan antibodi anti-nNav1.5 (anti-nNav1.5-Ab) mencerminkan nNav1.5 bersifat immunogen. Walau bagaimanapun, peredaran antigen neonatal Nav1.5 dan anti-nNav1.5-Ab di dalam konteks kanser payu dara (KP) yang bermetastasis masih belum dikaji. Oleh itu, penyelidikan ini telah dijalankan untuk mengkaji perkara tersebut dengan menggunakan sampel darah daripada tikus ortotopik 4T1 dan pesakit KP. Dalam kajian praklinikal, empat puluh ekor tikus BALB/c betina dibahagikan kepada tiga kumpulan: tikus KP ortotopik 4T1 (n=17), tikus kawalan (n=20) dan tikus kawalan positif (n=3). Selepas perkembangan tumor, tikus dikorbankan untuk mendapatkan organ sasaran, darah dan serum. Histopatologi, analisis sitokin, PCR masa-nyata dan ELISA tidak langsung telah dijalankan. Histopatologi dan analisis sitokin menunjukkan berlakunya pembentukan metastasis pada tikus ortotopik 4T1. Kepekatan faktor pertumbuhan endothelium vaskular (VEGF) adalah lebih tinggi secara signifikan pada tikus ortotopik 4T1 (P<0.0001****). Peredaranantigen nNav1.5 dan anti-nNav1.5-Ab telah dikesan pada tikus ortotopik 4T1, masing-masing menggunakan PCR masa-nyata dan ELISA tak langsung. Tambahan pula, terdapat hubung kait songsang antara anti-nNav1.5-Ab dan jumlah fokus metastatik (P=0.0485*, r=-0.7306). Melalui kajian klinikal pula, 32 pesakit KP telah dikumpulkan berdasarkan peringkat kanser: peringkat awal invasif (n=15) dan lanjutan (n=17). Sebanyak, 3 mL darah telah diambil dan hanya ELISA tak langsung telah dijalankan. Kajian klinikal ini membuktikan bahawa pesakit KP peringkat lanjutan menunjukkan ekspresi anti-nNav1.5-Ab yang lebih tinggi berbanding pesakit KP peringkat awal (P=0.0110*). Kesimpulannya, pengesanan antigen nNav1.5 dan anti-nNav1.5-Ab adalah konsisten dengan kehadiran metastasis KP.

 

Kata kunci: in vivo; metastasis; neonatal Nav1.5; ortotopik; pesakitkanser payu dara; 4T1

 

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