Squalene suppresses Jam-A, Claudin 5, and Occludin by accelerating cell death and reducing neuronal interaction in the neuroblastoma cell line SH-SY5Y

Abstract

Although squalene is an isoprenoid compound that plays a role in cholesterol biosynthesis, studies on its molecular mechanism and biological activities on cells have been limited. However, in recent years, it has been found as a functional ingredient in nutrition and taken from various sources due to its anticancer effects. In this study, we evaluated the effect of Squalene on tight junction proteins and cell apoptosis, which are effective on metastasis in neuroblastoma. Squalene was applied at a 1-100 µg/mL concentration to SH-SY5Y human neuroblastoma cell line. Cell viability, colony formation, wound healing, and Annexin-V binding were evaluated with various doses of squalene. In addition, immunofluorescence staining determined junctional adhesion molecule-A (Jam-A), Claudin-5 (Cldn5), and Occludin (Ocln) levels. As a result, squalene in SH-SY5Y cells at 25 µg/mL concentration increases cell death, suppresses colony formation and migration levels, triggers apoptosis, and suppresses Cldn5, Ocln, and Jam-A levels significantly. The mechanism of action of squalene, taken as a supplement, is essential in preserving neurological functions and detecting drug interactions in tumors such as neuroblastoma, which can metastasize and are common in early childhood.

Keywords

• Apoptosis
• caspases
• neuroblastoma
• squalene
• tight junction

References

1. [1] Cheung NK.V, Dyer MA. Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer. 2013;13(6):397–411. [CrossRef]
2. [2] Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003;3(3):203–216. [CrossRef]
3. [3] Das B, Yeger H, Baruchel H, Freedman MH, Koren G, Baruchel S. In vitro cytoprotective activity of squalene on a bone marrow versus neuroblastoma model of cisplatin-induced toxicity: Implications in cancer chemotherapy. Eur J Cancer. 2003;39(17):2556–2565. [CrossRef]
4. [4] Reddy LH, Couvreur P. Squalene: A natural triterpene for use in disease management and therapy. Adv Drug Deliv Rev. 2009;61(15):1412–1426. [CrossRef]
5. [5] Ghimire GP, Thuan NH, Koirala N, Sohng JK. Advances in Biochemistry and Microbial Production of Squalene and Its Derivatives. J Microbiol Biotechnol. 2016;26(3):441–451. [CrossRef]
6. [6] Smith TJ. Squalene: potential chemopreventive agent. Expert Opin Investig Drugs. 2000;9(8):1841–1848. [CrossRef]
7. [7] Costa MA. Biophysical characterization of asolectin-squalene liposomes. Colloids Surf B Biointerfaces. 2018;170:479–487. [CrossRef]
8. [8] Erdogan O. Green synthesis of silver nanoparticles via Cynara scolymus leaf extracts: The characterization, anticancer potential with photodynamic therapy in MCF7 cells. PLoS One. 2019;14(6). [CrossRef]

9. [9] Kelly GS. Squalene and its potential clinical uses. Altern Med. 1999;4(1):29–36.
10. [10] Ikekawa T, Otagiri K, Tanaka S. Intensification of host’s immunity by squalene in sarcoma 180 bearing ICR mice. J Pharmacobiodyn. 1983;6(2):148–151. [CrossRef]
11. [11] Skopinska-Rózewska E. Inhibitory effect of shark liver oil on cutaneous angiogenesis induced in Balb/c mice by syngeneic sarcoma L-1, human urinary bladder and human kidney tumour cells. Oncol Rep. 1999;6(6):1341–1344.
12. [12] Smith TJ, Yang GY, Seril DN, Liao J, Kim S. Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis by dietary olive oil and squalene. Carcinogenesis. 1998;19(4):703–706. [CrossRef]
13. [13] Rao CV, Newmark HL, Reddy BS. Chemopreventive effect of squalene on colon cancer. Carcinogenesis. 1998;19(2):287–290. [CrossRef]
14. [14] Murakoshi M. Inhibition by squalene of the tumor-promoting activity of 12-O-tetradecanoylphorbol-13-acetate in mouse-skin carcinogenesis. Int J Cancer. 1992;52(6):950–952. [CrossRef]
15. [15] Sasaki K. Modulation of the neurotransmitter systems through the anti-inflammatory and antidepressant-like effects of squalene from Aurantiochytrium sp. PLoS One. 2019;14(6). [CrossRef]
16. [16] Shin K, Fogg VC, Margolis B. Tight junctions and cell polarity. Annu Rev Cell Dev Biol. 2006;22:207–235. [CrossRef]
17. [17] Haynes MD, Martin TA, Jenkins SA, Kynaston HG, Matthews PN, Jiang WG. Tight junctions and bladder cancer (review). Int J Mol Med. 2005;16(1):3–9.
18. [18] Ikenouchi J, Matsuda M, Furuse M, Tsukita S. Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci. 2003;116(Pt 10):1959–1967. [CrossRef]
19. [19] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol. 2001;2(4):285–293. [CrossRef]
20. [20] Park MW, Kim CH, Cheong JH, Bak KH, Kim JM, Oh SJ. Occludin expression in brain tumors and its relevance to peritumoral edema and survival. Cancer Res Treat. 2006;38(3):139. [CrossRef]
21. [21] Beeman N, Webb PG, Baumgartner HK. Occludin is required for apoptosis when claudin-claudin interactions are disrupted. Cell Death Dis. 2012;3(2). [CrossRef]
22. [22] Rachow S. Occludin is involved in adhesion, apoptosis, differentiation and Ca2+-homeostasis of human keratinocytes: implications for tumorigenesis. PLoS One. 2013;8(2). [CrossRef]
23. [23] Aijaz S, Balda MS, Matter K. Tight junctions: molecular architecture and function. Int Rev Cytol. 2006;248:261–298. [CrossRef]
24. [24] Karnati H. Down regulated expression of Claudin-1 and Claudin-5 and up regulation of β-catenin: association with human glioma progression. CNS Neurol Disord Drug Targets. 2014;13(8):1413–1426. [CrossRef]
25. [25] Ma SC. Claudin-5 regulates blood-brain barrier permeability by modifying brain microvascular endothelial cell proliferation, migration, and adhesion to prevent lung cancer metastasis. CNS Neurosci Ther. 2017;23(12):947–960. [CrossRef]
26. [26] Stamatovic S, Keep R, Andjelkovic A. Brain endothelial cell-cell junctions: how to ‘open’ the blood brain barrier. Curr Neuropharmacol. 2008;6(3):179–192. [CrossRef]
27. [27] Luissint AC. Guanine nucleotide-binding protein Gαi2: a new partner of claudin-5 that regulates tight junction integrity in human brain endothelial cells. J Cereb Blood Flow Metab. 2012;32(5):860–873. [CrossRef]
28. [28] Kojima S, Rahner C, Peng S, Rizzolo LJ. Claudin 5 is transiently expressed during the development of the retinal pigment epithelium. J Membr Biol. 2002;186(2):81–88. [CrossRef]
29. [29] Escudero-Esparza A, Jiang WG, Martin TA. The Claudin family and its role in cancer and metastasis. Front Biosci (Landmark Ed). 2011;16(3):1069–1083. [CrossRef]
30. [30] Williams AF, Barclay AN. The immunoglobulin superfamily--domains for cell surface recognition. Annu Rev Immunol. 1988;6:381–405. [CrossRef]
31. [31] Solimando AG. JAM-A as a prognostic factor and new therapeutic target in multiple myeloma. Leukemia. 2018;32(3):736–743. [CrossRef]
32. [32] Xu PP. JAM-A overexpression is related to disease progression in diffuse large B-cell lymphoma and downregulated by lenalidomide. Sci Rep. 2017;7(1). [CrossRef]
33. [33] McSherry EA. JAM-A expression positively correlates with poor prognosis in breast cancer patients. Int J Cancer. 2009;125(6):1343–1351. [CrossRef]
34. [34] Naik MU, Naik TU, Suckow AT, Duncan MK, Naik UP. Attenuation of junctional adhesion molecule-A is a contributing factor for breast cancer cell invasion. Cancer Res. 2008;68(7):2194–2203. [CrossRef]