 |
 |
 |
 |
 |
 |
Sains Malaysiana 51(9)(2022):
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)
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
Received: 4
January 2022/Accepted: 14 March 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mL of blood was withdrawn, and only indirect ELISA was conducted.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
References
Adams, J., Carder, P.J., Downey, S., Forbes, M.A.,
MacLennan, K., Allgar, V., Kaufman, S., Hallam, S.,
Bicknell, R., Walker, J.J., Cairnduff, F., Selby,
P.J., Perren, T.J., Lansdown, M. & Banks, R.E.
2000. Vascular endothelial growth factor (VEGF) in breast cancer: Comparison of
plasma, serum, and tissue VEGF and microvessel density and effects of tamoxifen. Cancer
Research 60(11): 2898-2905.
Andrikopoulos, P., Fraser, S.P., Patterson, L., Ahmad, Z., Burcu,
H., Ottaviani, D., Diss, J.K., Box, C., Eccles, S.A. & Djamgoz,
M.B. 2011. Angiogenic functions of voltage-gated Na+ Channels in
human endothelial cells: Modulation of vascular endothelial growth factor
(VEGF) signaling. Journal of Biological
Chemistry 286: 16846-16860.
Brackenbury, W.J., Chioni,
A.M., Diss, J.K.J. & Djamgoz, M.B.A. 2007. The
neonatal splice variant of Nav1.5 potentiates in vitro invasive behavior of MDA-MB-231 human breast cancer cells. Breast Cancer Research and Treatment 101: 149-160.
Brackenbury, W.J. 2012. Voltage-gated sodium
channels and metastatic disease. Channels
(Austin) 6: 352-361.
Catterall, W.A. 2000. From ionic currents to
molecular mechanisms: The structure and function of voltage-gated sodium
channels. Neuron 26: 13-25.
Chang, Q., Bournazou, E.,
Sansone, P., Berishaj, M., Gao, S.P., Daly, L., Wels,
J., Theilen, T., Granitto,
S., Zhang, X., Cotari, J., Alpaugh,
M.L., de Stanchina, E., Manova,
K., Li, M., Bonafe, M., Ceccarelli,
C., Taffurelli, M., Santini, D., Altan-Bonnet,
G., Kaplan, R., Norton, L., Nishimoto, N., Huszar,
D., Layden, D. & Bromberg, J. 2013. The
IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis. Neoplasia 15: 848-862.
Chioni, A.M., Fraser, S.P., Pani, F., Foran, P., Wilkin, G.P., Diss, J.K. & Djamgoz, M.B. 2005. A novel polyclonal antibody specific
for the Na(v)1.5 voltage-gated Na(+)
channel 'neonatal' splice form. Journal
of Neuroscience Methods 147: 88-98.
Diaz, D., Delgadillo, D.M., Hernández-Gallegos, E.,
Ramírez-Domínguez, M.E., Hinojosa, L.M., Ortiz, C.S., Berumen,
J., Camacho, J. & Gomora, J.C. 2007. Functional expression of voltage-gated
sodium channels in primary cultures of human cervical cancer. Journal Cell Physiology 210: 469-478.
Diss, J.K., Fraser, S.P. & Djamgoz,
M.B. 2004. Voltage-gated Na+ channels: Multiplicity of expression,
plasticity, functional implications and pathophysiological aspects. Europe Biophysic Journal 33: 180-193.
Felio, K., Nguyen, H., Dascher, C.C., Choi, H.J.,
Li, S., Zimmer, M.I., Colmone, A., Moody, D.B., Brenner,
M.B. & Wang, C.R. 2009. CD1-restricted adaptive immune responses to
Mycobacteria in human group 1 CD1 transgenic mice. The Journal of Experimental Medicine 206: 2497-2509.
Fraser, S.P., Diss, J.K., Chioni,
A.M., Mycielska, M.E., Pan, H., Yamaci,
R.F., Pani, F., Siwy, Z., Krasowska, M., Grzywna, Z.,
Brackenbury, W.J., Theodorou, D., Koyutürk,
M., Kaya, H., Battaloglu, E., De Bella, M.T., Slade,
M.J., Tolhurst, R., Palmieri, C., Jiang, J., Latchman D.S., Coombes, R.C. & Djamgoz,
M.B. 2005. Voltage-gated sodium channel expression and potentiation of human
breast cancer metastasis. Clinical Cancer
Research 11: 5381-5389.
Gao, R., Shen, Y., Cai, J., Lei, M. & Wang, Z.
2010. Expression of voltage-gated sodium channel subunit in human ovarian
cancer. Oncology Reports 23:
1293-1299.
Gao, R., Cao, T., Chen, H., Cai, J., Lei, M. &
Wang, Z. 2019. Nav1.5-E3 antibody inhibits cancer progression. Translational Cancer Research 8: 44-50.
Gillet, L., Roger, S., Besson, P., Lecaille, F., Gore, J., Bougnoux,
P., Lalmanach, G. & Le Guennec,
J.Y. 2009. Voltage-gated sodium channel activity promotes cysteine
cathepsin-dependent invasiveness and colony growth of human cancer cells. The Journal of Biological Chemistry 284:
8680-8691.
Giuliano, A.E., Edge, S.B. & Hortobagyi, G.N. 2018. Eighth edition of the AJCC cancer
staging manual: Breast cancer. Annal of Surgical
Oncology 25: 1783-1785.
Greenfield, E.A. 2020. Standard immunization of
mice, rats, and hamsters. Cold Spring Harbour Protocol doi:10.1101/pdb.prot100297
Jones, D., Pereira, E.R. & Padera,
T.P. 2018. Growth and immune evasion of lymph node metastasis. Frontiers in Oncology 8: 36.
Kamarulzaman, N.S., Dewadas, H.D., Leow,
C.Y., Yaacob, N.S. & Mokhtar, N.F. 2017. The role
of REST and HDAC2 in epigenetic dysregulation of Nav1.5 and nNav1.5 expression
in breast cancer. Cancer Cell
International 17: 74.
Kujawski, M., Kortylewski, M., Lee, H., Herrmann, A.,
Kay, H. & Yu, H. 2008. Stat3 mediates myeloid cell-dependent tumor
angiogenesis in mice. The Journal of Clinical
Investigation 118(10): 3367-3377.
Luo, Q., Wu, T., Wu, W., Chen, G., Luo, X., Jiang,
L., Tao, H., Rong, M., Kang, S. & Deng, M. 2020. The functional role of
voltage-gated sodium channel Nav1.5 in metastatic breast cancer. Frontiers in Pharmacology 11: 1111.
Madu, C.O., Wang, S., Madu, C.O. & Lu, Y.
2020. Angiogenesis in breast cancer progression, diagnosis, and treatment. Journal of Cancer 11(15): 4474-4494.
Masjedi, A., Hashemi, V., Hojjat-Farsangi, M., Ghalamfarsa, G., Azizi, G., Yousefi,
M. & Jadidi-Niaragh, F. 2018. The significant
role of interleukin-6 and its signaling pathway in the immunopathogenesis and
treatment of breast cancer. Biomedicine
& Pharmacotherapy 108: 1415-1424.
Mishra, P., Pandey, C.M., Singh, U., Gupta, A., Sahu, C. & Keshri, A. 2019.
Descriptive statistics and normality tests for statistical data. Annals of Cardiac Anaesthesia 22(1): 67-72.
Nelson, M., Yang, M., Millican-Slater,
R. & Brackenbury, W.J. 2015. Nav1.5 regulates breast tumor growth and
metastatic dissemination in vivo. Oncotarget 6(32):
32914-32929.
Norsa'adah, B. 2013. Univariable Analyses using IBM SPSS Statistics Version 20.0.
Kota Bharu, Malaysia: Universiti Sains Malaysia.
Okuda, T., Shimizu, K., Hasaba,
S. & Date, M. 2019. Induction of specific adaptive immune responses by
immunization with newly designed artificial glycosphingolipids. Scientific Reports 9: 18803.
Onganer, P.U. & Djamgoz, M.B. 2005. Small-cell
lung cancer (human): Potentiation of endocytic membrane activity by
voltage-gated Na(+) channel expression in vitro. The Journal of Membrane Biology 204(2): 67-75.
Onkal, R., Mattis, J.H., Fraser, S.P., Diss, J.K., Shao, D., Okuse, K. & Djamgoz, M.B.
2008. Alternative splicing of Nav1.5: An electrophysiological comparison of
'neonatal' and 'adult' isoforms and critical involvement of a lysine residue. Journal
of Cellular Physiology 216(3): 716-726.
Paschall, A.V. & Liu, K. 2016. An orthotopic mouse model of spontaneous
breast cancer metastasis. Journal of
Visualized Experiments 114: 54040.
Patel, F. & Brackenbury, W.J. 2015. Dual roles
of voltage-gated sodium channels in development and cancer. The International Journal of Developmental
Biology 59(7-9): 357-366.
Potdar, P.D. & Lotey, N.K. 2015. Role of
circulating tumor cells in future diagnosis and therapy of cancer. Journal Cancer Metastasis Treat 1:
44-56.
Pulaski, B.A. & Ostrand-Rosenberg,
S. 2001. Mouse 4T1 breast tumor model. Current
Protocols in Immunology 20: 22.
Rajaratinam, H., Rasudin, N.S., Al Astani,
T., Mokhtar, N.F., Yahya, M.M., Zain, W., Asma-Abdullah, N. & Fuad, W. 2021. Breast cancer therapy affects the expression
of antineonatal Nav1.5 antibodies in the serum of
patients with breast cancer. Oncology
Letters 21(2): 108.
Roger, S., Besson, P. & Le Guennec,
J.Y. 2003. Involvement of a novel fast inward sodium current in the invasion
capacity of a breast cancer cell line. Biochimica et Biophysica Acta 1616(2): 107-111.
Rook, M.B., Evers, M.M., Vos, M.A. & Bierhuizen, M.F. 2012. Biology of cardiac sodium channel
Nav1.5 expression. Cardiovascular
Research 93(1): 12-23.
Sauter, B.V., Martinet, O., Zhang, W.J., Mandeli, J. & Woo, S.L. 2000. Adenovirus-mediated gene
transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth
and metastases. Proceedings of the
National Academy of Sciences of the United States of America 97(9):
4802-4807.
Schmittgen, T.D. & Livak, K.J. 2008. Analyzing
real-time PCR data by the comparative C(T) method. Nature Protocols 3(6): 1101-1108.
Stuelten, C.H., Parent, C.A. & Montell, D.J. 2018.
Cell motility in cancer invasion and metastasis: Insights from simple model
organisms. Nature Reviews Cancer 18(5): 296-312.
Tao, K., Fang, M., Alroy,
J. & Sahagian, G.G. 2008. Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer 8: 228.
Tsukamoto, H., Fujieda,
K., Senju, S., Ikeda, T., Oshiumi, H. &
Nishimura, Y. 2018. Immune-suppressive effects of interleukin-6 on
T-cell-mediated anti-tumor immunity. Cancer
Science 109(3): 523-530.
Uddback, I.E.M., Pedersen, L.M.I., Pedersen, S.R., Steffensen,
M.A., Holst, P.J., Thomsen, A.R. & Christensen, J. 2016. Combined local and
systemic immunization is essential for durable T-cell mediated heterosubtypic
immunity against influenza A virus. Scientific
Reports 6: 20137.
Yamaci, R.F., Fraser, S.P., Battaloglu, E., Kaya,
H., Erguler, K., Foster, C.S. & Djamgoz, M. 2017. Neonatal Nav1.5 protein expression in
normal adult human tissues and breast cancer. Pathology, Research and Practice 213(8): 900-907.
Yang, M., James, A.D., Suman, R., Kasprowicz, R.,
Nelson, M., O'Toole, P.J. & Brackenbury, W.J. 2020. Voltage-dependent
activation of Rac1 by Nav 1.5 channels promotes cell migration. Journal of Cellular Physiology 235(4):
3950-3972.
Yang, M., Kozminski,
D.J., Wold, L.A., Modak,
R., Calhoun, J.D., Isom, L.L. & Brackenbury, W.J. 2012. Therapeutic
potential for phenytoin: Targeting Na(v)1.5 sodium channels to reduce migration
and invasion in metastatic breast cancer. Breast
Cancer Research and Treatment 134(2): 603-615.
Ye, Q., Ling, S., Zheng, S. & Xu, X. 2019.
Liquid biopsy in hepatocellular carcinoma: Circulating tumor cells and
circulating tumor DNA. Molecular Cancer 18(1): 114.
*Corresponding author; email: wanezumi@usm.my
|
|||
|
