Document Type : Original Article(s)

Authors

1 Department of Genetics, College of Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran

2 Cellular and Molecular Research Center, Social Determinants of Health Research Center, Research Institute for Prevention of Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran

Abstract

Background: Malignant melanoma is an aggressive skin cancer whose survival rate is extremely low. Commencing apoptosis is believed to be a significant issue in cancer treatment and targeting the apoptosis and WNT signaling pathways, which is probably a potentially successful strategy to overcome tumor plasticity in melanoma.
Method: We conducted the present in vitro study to investigate antiproliferative and apoptotic effects of Nic-ALA, as a new compound, on A375 melanoma cell line using MTT assay and flow cytometry, respectively. The gene expression profiles of the cancer cells were obtained for Bcl-2 and BAX as the main genes of the apoptosis signaling pathway and WIF1 and beta-catenin genes from the WNT signaling pathway with qRT-PCR.
Results: Nic-ALA’s cytotoxicity on A375 melanoma cell line from MTT assay was obtained with IC50 166.7, 144.2, and 146.1μM. This novel derivative induced 11.3, 46.1, and 85.7% of apoptosis in 24, 48, and 72h time points, respectively. In the treated cells, the expression of BAX, beta-catenin, and WIF1 genes increased, while the expression of Bcl-2 decreased significantly at 200μM concentration and the treated times of 48 and 72h.
Conclusion: The antiproliferation of Nic-ALA at a lower value than what we found in nicotinic acid alone represented the higher bioavailability and transport efficiency of this novel derivative through A375 melanoma cell line. Its antipoetic effects were obtained by increasing the apoptosis rate and expression of the Bax gene and reducing Bcl-2 gene expression. Upregulation of WIF1 and beta-catenin in the WNT signaling pathway emphasized Nic-ALA’s anticancer effect on A375 melanoma cell line.

Keywords

How to cite this article:

Malikhan H, Siasi Torbati E, Majd A, Gheibi N. Anti-cancer properties of Nicotinic Cid-alpha linolenic acid derivative on A375 melanoma cell line: Assessment of apoptosis and WNT signaling pathways. Middle East J Cancer. 2022;13(4):593-606. doi: 10.30476/mejc.2021.89494.1530.

1.Wan Q, Jin L, Wang Z. Comprehensive analysis of cancer hallmarks in cutaneous melanoma and identification of a novel unfolded protein response as a prognostic signature. Aging (Albany NY). 2020;12(20):20684-701. doi: 10.18632/aging.103974.
2.Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66(2):115-32. doi: 10.3322/caac.21338.
3.Trotter SC, Sroa N, Winkelmann RR, Olencki T, Bechtel M. A global review of melanoma follow-up guidelines. J Clin Aesthet Dermatol. 2013;6(9):18-26.
4.Erdmann F, Lortet-Tieulent J, Sch¸z J, Zeeb H, Greinert R, Breitbart EW, et al. International trends in the incidence of malignant melanoma 1953-2008--are recent generations at higher or lower risk? Int J Cancer. 2013;132(2):385-400. doi: 10.1002/ijc.27616.
5.Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364(26):2507-16. doi: 10.1056/NEJMoa1103782.
6.Gogas HJ, Kirkwood JM, Sondak VK. Chemotherapy for metastatic melanoma. Cancer. 2007;109(3):455-64. doi: 10.1002/cncr.22427.
7.Berrocal A, Cabañas L, Espinosa E, Fernández-de-Misa R, Martín-Algarra S, Martínez-Cedres JC, et al. Melanoma: Diagnosis, staging, and treatment. Consensus group recommendations. Adv Ther. 2014;31(9):945-60. doi: 10.1007/s12325-014-0148-2.
8.Arendse, Lyle. The modulating effect of conjugated linoleic acid (CLA) on cancer cell survival in vitro. [dissertation] Cape Town: University of the Western Cape; 2014. 158 p.
9.Asghari H, Chegini KG, Amini A, Gheibi N. Effect of poly and mono-unsaturated fatty acids on stability and structure of recombinant S100A8/A9. Int J Biol Macromol. 2016;84(2):35-42. doi: 10.1016/j.ijbiomac. 2015.11.065.
10.Zajdel A, Wilczok A, Tarkowski M. Toxic effects of n-3 polyunsaturated fatty acids in human lung A549 cells. Toxicol In Vitro. 2015;30(1):486-91. doi: 10.1016/j.tiv.2015.09.013.
11.Das UN. Essential fatty acids: biochemistry, physiology and pathology. Biotechnol J. 2006;1(4):420-39. doi: 10.1002/biot.200600012.
12.Hussain G, Schmitt F, Loeffler JP, Gonzalez De Aguilar J. Fatting the brain: a brief of recent research. Front Cell Neurosci. 2013;7:144. doi: 10.3389/fncel. 2013.00144.
13.Giordano C, Plastina P, Barone I, Catalano S, Bonofiglio D. n-3 polyunsaturated fatty acid amides: new avenues in the prevention and treatment of breast cancer. Int J Mol Sci. 2020;21(7):2279. doi: 10.3390/ijms21072279.
14.Ljungblad LM, Johnsen JI, Wickström M, Kogner P, Gleissman H. Abstract 3275: A novel approach to treat medulloblastoma: The omega-3 fatty acids DHA and EPA reduce medulloblastoma tumor growth in vitro and in vivo. Cancer Res. 2015;75(15):3275. doi:10.1158/1538-7445.AM2015-3275.
15.Das UJ. Tumoricidal action of cis-unsaturated fatty acids and their relationship to free radicals and lipid peroxidation. Cancer Lett. 1991;56(3):235-43. doi: 10.1016/0304-3835(91)90008-6.
16.Moniri NH, Farah Q. Short-chain free-fatty acid G protein-coupled receptors in colon cancer. Bioch Pharmacol. 2021;186:114483. doi: 10.1016/j.bcp. 2021.114483.
17.Bogan KL, Brenner C. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition. Annu Rev Nutr. 2008;28(3):115-30. doi: 10.1146/annurev. nutr.28.061807.155443.
18.Pan L, Yu G, Chen X, Li X. Nicotinic acid inhibits angiogenesis likely through cytoskeleton remodeling. Organogenesis. 2017;13(4):183-91. doi: 10.1080/ 15476278.2017.1364829.
19.Dubrall D, Pflock R, Kosinska J, Schmid M, Bleich M, Himmerkus N, et al. Do dimethyl fumarate and nicotinic acid elicit common, potentially HCA2 -mediated adverse reactions? A combined epidemiological-experimental approach. Br J Clin Pharmacol. 2021. doi: 10.1111/bcp.14787.
20.Norbury CJ, Hickson ID. Cellular responses to DNA damage. Annu Rev Pharmacol Toxicol. 2001;41(1) :367-401. doi: 10.1146/annurev.pharmtox. 41.1.367.
21.Berke GJC. The CTL's kiss of death. Cell. 1995;81(1):9-12. doi: 10.1016/0092-8674(95)90365-8.
22.Trisciuoglio D, Del Bufalo D. New insights into the roles of antiapoptotic members of the Bcl-2 family in melanoma progression and therapy. Drug Discov Today. 2021;S1359-6446(21)00059-3. doi: 10.1016/j.drudis.2021.01.027.
23.Tessoulin B, Papin A, Gomez-Bougie P, Bellanger C, Amiot M, Pellat-Deceunynck C, et al. BCL2-family dysregulation in B-cell malignancies: From gene expression regulation to a targeted therapy biomarker. Front Oncol. 2019;8:645. doi: 10.3389/fonc.2018.00645.
24.Liu YL, Yang HP, Gong L, Tang CL, Wang HJ. Hypomethylation effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin on WIF-1 promoter in non-small cell lung cancer cell lines. Mol Med Rep. 2011;4(4):675-9. doi:10.3892/mmr.2011.473.
25.Paul S, Dey AJN. Wnt signaling and cancer development: therapeutic implication Minireview. Neoplasma. 2008;55(3):165-76.
26.Xiong X, Xu W, Gong J, Wang L, Dai M, Chen G, et al. miR-937-5p targets SOX17 to modulate breast cancer cell cycle and cell proliferation through the Wnt signaling pathway. Cell Signal. 2021;77:109818. doi:10.1016/j.cellsig.2020.109818.
27.Hsieh JC, Kodjabachian L, Rebbert ML, Rattner A, Smallwood PM, Samos CH, et al. A new secreted protein that binds to Wnt proteins and inhibits their activites. Nature. 1999;398(6726):431-6. doi:10.1038/18899.
28.Mazieres J, He B, You L, Xu Z, Lee AY, Mikami I, et al. Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Res. 2004;64(14):4717-20. doi: 10.1158/0008-5472.CAN-04-1389.
29.Jalilvand A, Soltanpour MS. Promoter hypermethylation of Wnt/β-catenin signaling pathway inhibitor WIF-1 gene and its association with MTHFR C677T polymorphism in patients with colorectal cancer. Oman Med J. 2020;35(3):e131. doi: 10.5001/omj.2020.49.
30.Freitas RD, Campos MM. Protective effects of omega-3 fatty acids in cancer-related complications. Nutrients. 2019;11(5):945. doi:10.3390/nu11050945.
31.Ma Y, Wang J, Li Q, Cao B. The Effect of omega-3 polyunsaturated fatty acid supplementations on anti-tumor drugs in triple negative breast cancer. Nut. Cancer. 2021;73(2):196-205. doi:10.1080/01635581. 2020.1743873.
32.Jing K, Song KS, Shin S, Kim N, Jeong S, Oh HR, et al. Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53. Autophagy. 2011;7(11):1348-58. doi:10.1080/ 01635581.2020.1743873.
33.Jamali Z, Rezaei Behbehani G, Zare K, Gheibi N. Effect of chrysin omega-3 and 6 fatty acid esters on mushroom tyrosinase activity, stability, and structure. J Food Biochem. 2019;43(3):e12728. doi: 10.1111/ jfbc.12728.
34.Gheibi N, Taherkhani N, Ahmadi A, Haghbeen K, Ilghari D. Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase. Iran J Basic Med Sci. 2015;18(2):122-9.
35.Kiesewetter B, Mayerhoefer ME, Lukas J, Zielinski CC, M¸llauer L, Raderer M. Rituximab plus bendamustine is active in pretreated patients with extragastric marginal zone B cell lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma). Ann Hematol. 2014;93(2):249-53. doi:10.1007/s00277-013-1865-3.
36.He YC, He L, Khoshaba R, Lu FG, Cai C, Zhou FL, et al. Curcumin nicotinate selectively induces cancer cell apoptosis and cycle arrest through a P53-mediated mechanism. Molecules. 2019;24(22):4179. doi: 10.3390/molecules24224179.
37.Hu Jn, Zou Xg, He Y, Chen F, Deng ZY. Esterification of quercetin increases its transport across human Caco-2 cells. J Food Sci. 2016;81(7):H1825-H1832. doi: 10.1111/1750-3841.13366.
38.Benavente CA, Jacobson MK, Jacobson EL. NAD in skin: therapeutic approaches for niacin. Curr Pharm Des. 2009;15(1):29-38. doi: 10.2174/1381612 09787185760.
39.Luo H, Rankin GO, Li Z, DePriest L, Chen YC. Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway. Food Chem. 2011;128(2):513-9. doi: 10.1016/j.foodchem. 2011.03.073.
40.Mou S, Zhou Z, He Y, Liu F, Gong L. Curcumin inhibits cell proliferation and promotes apoptosis of laryngeal cancer cells through Bcl-2 and PI3K/Akt, and by upregulating miR-15a. Oncol Lett. 2017;14(4):4937-42. doi: 10.3892/ol.2017.6739.
41.Sheng L, Wei R. Long non-coding RNA-CASC15 promotes cell proliferation, migration, and invasion by activating Wnt/β-Catenin signaling pathway in melanoma. Pathobiology. 2020;87(1):20-9. doi: 10.1159/000502803.
42.Fakhrabadi HG, Rabbani-Chadegani A, Ghadam P, Amiri S. Protective effect of bleomycin on 5-azacitidine induced cytotoxicity and apoptosis in mice hematopoietic stem cells via Bcl-2/Bax and HMGB1 signaling pathway. Toxicol Appl Pharmacol. 2020;396:114996. doi: 10.1016/j.taap.2020.114996.
43.Qin XY, Wang YN, Liu HF, Luo ZH, Zhang PL, Li-Fang H, et al. Anti-cancer activities of metal-based complexes by regulating the VEGF/VEGFR2 signaling pathway and apoptosis-related factors Bcl-2, BAX, and caspase-9 to inhibit angiogenesis and induce apoptosis. Metallomics. 2020;12(1):92-103. doi: 10.1039/ c9mt00248k.
44.Mungamuri SK, Yang X, Thor AD, Somasundaram K. Survival signaling by Notch1: mammalian target of rapamycin (mTOR)-dependent inhibition of p53. Cancer Res. 2006;66(9):4715-24. doi: 10.1158/0008-5472.CAN-05-3830.
45.Proud C. Role of mTOR signalling in the control of translation initiation and elongation by nutrients. In: Thomas G, Sabatini D.M., Hall MN, editors. TOR. Current topics in microbiology and immunology. vol 279. Springer: Berlin, Heidelberg; 2014. doi:10.1007/ 978-3-642-18930-2_13.
46.James RG, Bosch KA, Kulikauskas RM, Yang PT, Robin NC, Toroni RA, et al. Protein kinase PKN1 represses Wnt/β-catenin signaling in human melanoma cells. J Biol Chem. 2013;288(48):34658-70. doi: 10.1074/jbc.M113.500314.
47.Kaur A, Webster MR, Weeraratna AT. In the Wnt-er of life: Wnt signalling in melanoma and ageing. Br J Cancer. 2016;115(11):1273-9. doi: 10.1038/ bjc.2016.332.
48.Uka R, Britschgi C, Krättli A, Matter C, Mihic D, Okoniewski MJ, et al. Temporal activation of WNT/β-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth. Oncogene. 2020;39(20):4132-54. doi: 10.1038/s41388-020-1267-7.
49.Rahmani B, Asl DH, Farivar TN, Azad M, Sahmani M, Gheibi N. The effects of omega-3 PUFA (ALA) on WT1 gene expression in pancreatic cancer cell line (MIA PaCa-2). Middle East J Fam Med. 2018;99(5832):1-5. doi: 10.3892/mmr.2016.5639.
50.Li J, Fang R, Wang J, Deng L. NOP14 inhibits melanoma proliferation and metastasis by regulating Wnt/≤-catenin signaling pathway. Braz J Med Biol Res. 2019;52(1):e7952. doi:10.1590/1414-431x20187952.
51.Yin X, Yu XW, Zhu P, Zhang YM, Zhang XH, Wang F, et al. Endogenously synthesized n-3 fatty acids in fat-1 transgenic mice prevent melanoma progression by increasing E-cadherin expression and inhibiting β-catenin signaling. Mol Med Rep. 2016;14(4):3476-84. doi: 10.3892/mmr.2016.5639.
52.Brembeck FH, Rosário M, Birchmeier W. Balancing cell adhesion and Wnt signaling, the key role of β-catenin. Curr Opin Genet Dev. 2006;16(1):51-9. doi: 10.1016/j.gde.2005.12.007.
53.Takayama T, Shiozaki H, Shibamoto S, Oka H, Kimura Y, Tamura S, et al. Beta-catenin expression in human cancers. Am J Pathol. 1996;148(1):39-46.
54.Kim J, You L, Xu Z, Kuchenbecker K, Raz D, He B, et al. Wnt inhibitory factor inhibits lung cancer cell growth. J Thorac Cardiovasc Surg. 2007;133(3):733-7. doi: 10.1016/j.jtcvs.2006.09.053.
55.Kovacs D, Migliano E, Muscardin L, Silipo V, Catricalá C, Picardo M, et al. The role of Wnt/β-catenin signaling pathway in melanoma epithelial-to-mesenchymal-like switching: evidences from patients-derived cell lines. Oncotarget. 2016;7(28):43295. doi: 10.18632/ oncotarget.9232.
56.Zimmerman ZF, Kulikauskas RM, Bomsztyk K, Moon RT, Chien AJ. Activation of Wnt/β-catenin signaling increases apoptosis in melanoma cells treated with trail. PLoS One. 2013;8(7):e69593. doi: 10.1371/journal.pone.0069593.
57.Biechele TL, Kulikauskas RM, Toroni RA, Lucero OM, Swift RD, James RG, et al. Wnt/β-catenin signaling and AXIN1 regulate apoptosis triggered by inhibition of the mutant kinase BRAFV600E in human melanoma. Sci Signal. 2012;5(206):ra3-ra3. doi: 10.1126/scisignal.2002274.
58.Atkinson JM, Rank KB, Zeng Y, Capen A, Yadav V, Manro JR, et al. Activating the Wnt/β-catenin pathway for the treatment of melanomañapplication of LY2090314, a novel selective inhibitor of glycogen synthase kinase-3. PLoS One. 2015;10(4):e0125028. doi: 10.1371/journal.pone.0125028.
59.Chien AJ, Moore EC, Lonsdorf AS, Kulikauskas RM, Rothberg BG, Berger AJ, et al. Activated Wnt/β-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model. Proc Nat Acad Sci. 2009;106(4): 1193-8. doi: 10.1073/pnas.0811902106.
60.Sarma A, Gajan A, Kim S, Gurdziel K, Mao G, Nangia-Makker P, et al. RAD6B loss disrupts expression of melanoma phenotype in part by inhibiting WNT/β-catenin signaling. Am J Pathol. 2021;191(2):368-84. doi: 10.1016/j.ajpath.2020.10.015.