Jung Hyun Kim Seo Hyun Moon Min Young Kim


The present study aimed to assess the effect of NO on melanoma A375 cell growth and apoptotic cell death. Trypan blue exclusion assay was employed to detect the cytotoxicity induced by controlled steady-state concentrations (given in µM • min) of NO. The characteristics of the cellular cell cycle and apoptosis in NO-treated A375 cells were also analyzed by Annexin V/PI and DNA fragmentation assays. Western blotting was applied to detect the expression of apoptosis-related proteins (p53, Bax, Fas, DR5, caspase-3 and -9, and PARP). When exposed to preformed 100% NO for 8 h reactor system, a cumulative dose of 3360 μM • min reduced the viability by 22% 24 h after treatment and promoted apoptosis, 2.9- and 12.2-folds 24 and 48 h after treatment higher than the argon control, respectively. Cell cycle analysis 48 h after treatment revealed S-phase arrest in cells treated with 3360 μM • min NO. It was also observed that the expression of p53, DR5, caspase 9 and PARP increased significantly upon NO treatment. In addition, the present study assessed the inhibitory effects of endogenous NO on the proliferation of human melanoma cells by employing specific (AMG, 1400W and/or SMTC) and nonspecific (NMA) NO synthase (NOS) inhibitors resulting in melanoma cell growth inhibition; the highest cytotoxic effect was seen when inducible NOS inhibition by 1 mM 1400W treatment. Collectively, the present data suggest that NOis involved in a key mechanism limiting melanoma proliferation and apoptosis, which may play in improving the efficacy of melanoma treatment.




Apoptosis, Cell proliferation, Human melanoma cells, Nitric oxide

Baldelli, I., Mangialardi, M.L., Salgarello, M. & Raposio, E. (2020). Surgical reconstruction following wide local excision of malignant melanoma of the scalp. Plastic and Reconstructive Surgery, 8(8), e3059. doi.org/10. 1097/GOX.0000000000003059.
Chen, Y., Fang, L., Zhou, W., Chang, J., Zhang, X., He, C., Chen, C., Yan, R., Yan, Y., Lu, Y., Xu, C. & Xiang, C (2021) Nitric oxide-releasing micelles with intelligent targeting for enhanced anti-tumor effect of cisplatin in hypoxia. Journal of Nanobiotechnology, 19, 246. doi.org/10.1186/s12951-021-00989-z
D'Este1, F., Pietra, E.D., Pazmay, G.V.B., Xodo, L.E. & Rapozzi, V. (2020). Role of nitric oxide in the response to photooxidative stress in prostate cancer cells. Biochemical Pharmacology, 182, 114205. doi.org/10.1016/j.bcp.20 20.114205
Dind, Z., Ogata, D., Roszik, J., Qin, Y., Kim, S.H., Tetzlaff, M.T., Lazar, A.J., Davies, M.A., Ekmekcioglu, S. & Grimm, E.A. (2021). iNOS associates with poor survival in melanoma: a role for nitric oxide in the PI3K-AKT pathway stimulation and PTEN S-nitrosylation. Frontiers in Oncology, 11, 631766. doi.org/10.3389/fonc.2021.631766
Drača, D., Edeler, D., Saoud, M., Dojčinović, B., Dunđerović, D., Đmura, g., Maksimović-Ivanić, D., Mijatović, S. & Kaluđerović, G.N. (2021). Antitumor potential of cisplatin loaded into SBA-15 mesoporous silica nanoparticles against B16F1 melanoma cells: in vitro and in vivo studies, Journal of Inorganic Biochemistry, 217, 111383. doi.org/10.1016/j.jinorgbio.2021.111383
Ekmekcioglu, S., Ellerhorst, J.A., Prieto, V.G., Johnson, M.M., Broemeling, L.D. & Grimm, E.A. (2006). Tumor iNOS predicts poor survival for stage III melanoma patients. International Journal of Cancer, 119, 861-866. doi.org/10.1002/ijc.21767
Gonçalvesa, D.A., Xistoc, R., Gonçalvesa, J.D., da Silvac, D.B., Soaresc, J.P.M., Icimotod, M.Y., Annac, C.S., Gimenezc, M., de Angelisc, K., Llesuye, S., Fernandesf, D.C., Laurindof, F., Jasiulionisa, M.G. & de Melo, F.H.M. (2019). Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human melanoma development, International Journal of Biochemistry and Cell Biology, 115, 105592. doi.org/10.1016/j.biocel.2019.105592
Hanly, A., Gibson, F., Nocco, S., Rogers, S., Wu, M. & Alani, R.M. (2022). Drugging the epigenome: Overcoming resistance to targeted and immunotherapies in Melanoma. JID Innovations, 2(2), 100090. doi.org/10.1016/j.xjidi.2021.100090
Kim, J.H. & Kim, M.Y. (2016) Effect of dose and dosing rate on the mutagenesis of nitric oxide in supF shuttle vector. Tropical Journal of Pharmaceutical Research, 15, 2587. doi.org/10.4314/tjpr.v15i12.8
Kim, M.Y. (2017) Intracellular and extracellular factors influencing the genotoxicity of nitric oxide and reactive oxygen species. Oncology Letters,13, 1417. doi.org/10.3892/ol.2017.5584
Kim, M.Y., Lim, C.H., Trudel, L.J., Deen, W.M. & Wogan, G.N. (2012) Delivery method, target gene structure, and growth properties of target cells impact mutagenic responses to reactive nitrogen and oxygen species. Chemical Research in Toxicology, 25, 873. doi.org/10.1021/tx2004882
Kim, M.Y., Trudel, L.J. & Wogan, G.N. (2009) Apoptosis induced by capsaicin and resveratrol in colon carcinoma cells requires nitric oxide production and caspase activation. Anticancer Research, 29, 3733-3740.
Kim, S.S., Sumner W.A., Miyauchi, S., Cohen, E.E.W., Califano, J.A. & Sharabi, A.B. (2021) Role of B Cells in Responses to Checkpoint Blockade Immunotherapy and Overall Survival of Cancer Patients. Clinical Cancer Research, 27(22), 6075-6082. doi.org/10.1158/1078-0432.CCR-21-069
Li, C.Q. & Wogan, G.N. (2005) Nitric oxide as a modulator of apoptosis. Cancer Letters, 226, 1-15. doi.org/10.1016/j.canlet.2004.10.021
Li, J., Sun, Y., Yan, R., Wu, X., Zou, H. & Meng, Y. (2022) Urea transporter B downregulates polyamines levels in melanoma B16 cells via p53 activation, BBA - Molecular Cell Research, 1869, 119236. doi.org/10.1016/j.bbamcr.20 22.119236
Liu, R., Sun, X., Hu, Z., Peng, C. & Wu, T. (2022) Knockdown of long non-coding RNA MIR155HG suppresses melanoma cell proliferation, and deregulated MIR155HG in melanoma is associated with M1/M2 balance and macrophage infiltration. Cells & Development, 170, 203768. doi.org/10.1016/j.cdev.2022.203768
Mazurek, M. & Rola, R. (2021) The implications of nitric oxide metabolism in the treatment of glial tumors. Neurochemistry International, 150, 105172. doi.org/10.1016/j.neuint.2021.105172
Medzhitov, R. (2008) Origin and physiological roles of inflammation. Nature, 454(7203), 428-435. doi.org/10.1038/nature07201.
Melo, F.H.M. de, Gonçalves, D.A., Sousa, R.X. de, Icimoto, M.Y., Fernandes, D.C., Laurindo, F.R.M. & Jasiulionis, M.G. (2021) Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry. International Journal of Molecular Sciences, 22(17), 9556. doi.org/10.3390/ijms22179556
Miranda, K.M., Ridnour, L.A., McGinity, C.L., Bhattacharyya, D. & Wink, D.A. (2021) Nitric Oxide and Cancer: When to Give and When to Take Away? Inorganic Chemistry, 60(21), 15941-15947. doi.org/10.1021/acs.inorgchem.1c02434
Monteiroa, H.P., Rodriguesb, E.G., Reisc, A.K.C.A., Longo, L.S., Ogataa, F.T., Morettie, A.I.S., da Costaa, P.E., Teodoroa, A.C.S., Toledoa, M.S., Stern, A. (2020) Nitric oxide and interactions with reactive oxygen species in the development of melanoma, breast, and colon cancer: A redox signaling perspective. Nitric Oxide, 89, 1-13. doi.org/10.1016/j.niox.2019.04.009
Obrador, E., Salvador, r., Lopez-Blanch, R., Jihad-Jebbar, A., Alcacer, J., Benlloch, M., Pellicer, J. & Estrela, J.M. (2021) Melanoma in the liver: Oxidative stress and the mechanisms of metastatic cell survival. Seminars in Cancer Biology, 71, 109-121. doi.org/10.1016/j.semcancer.20 20.05.001
Özenvera, N. & Efferth, T. (2020) Small molecule inhibitors and stimulators of inducible nitric oxide synthase in cancer cells from natural origin (phytochemicals, marine compounds, antibiotics), Biochemical Pharmacology, 176, 113792. doi.org/10.1016/j.bcp.2020.113792
Shi, M., Zhang, J., Wang, Y., Peng, C., Hu, H., Qiao, M., Zhao, X. & Chen, D. (2022) Tumor-specific nitric oxide generator to amplify peroxynitrite based on highly penetrable nanoparticles for metastasis inhibition and enhanced cancer therapy. Biomaterials, 283, 121448.
Skudalski, L., Waldman, R., Kerr, P.E., & Grant-Kels, J.M. (2022) Melanoma: An update on systemic therapies. Journal of the American Academy of Dermatology, 86(3), 515-524. doi.org/10.1016/j.jaad.2021.09.075
Sun, K., Lu, X., Yin, C. & Guo, J. (2022) In vitro antitumor activity of nano-pulse stimulation on human anaplastic thyroid cancer cells through nitric oxide-dependent mechanisms. Bioelectrochemistry, 145, 108093. doi.org/10.10 16/j.bioelechem.2022.108093
Tang, L. & Wang, K. (2016) Chronic inflammation in skin malignancies. Journal of Molecular Signaling, 11(2), 1-13. doi.org/10.5334/1750-2187-11-2
Tripathi, D.N., Chowdhury, R., Trudel, L.J., Tee, A.R., Slack, R.S., Walker, C.L. & Wogan, G.N. (2013) Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2–mediated suppression of mTORC1. Proceedings of the National Academy of Sciences of the United States of America, 110, E2950. doi.org/10.1073/pnas.1307736110
Research Articles

How to Cite

Modulation of A375 human melanoma cell proliferation and apoptosis by nitric oxide. (2022). Journal of Applied and Natural Science, 14(2), 320-325. https://doi.org/10.31018/jans.v14i2.3365