Azidotimidina (AZT) na expressão gênica e na síntese proteíca da telomerase em células de melanoma metastático humano / Azidothymidine (AZT) in gene expression and protein synthesis of telomerase in human metastatic melanoma cells

Celestino Prospero de Souza Sobrinho, Miguel Sabino Neto, Ivan Dunshee de Abranches Oliveira Santos, Andréa Fernandes de Oliveira, Jerônimo Pereira de Franca, Silvana Gaiba, Lydia Masako Ferreira

Abstract


Introdução: As evidências demonstram que o processo de transformação maligna envolve várias alterações genéticas, tanto em melanoma como em outras neoplasias, os quais modificam processos celulares importantes, dentre eles, o encurtamento dos telômeros. A enzima responsável pela síntese do telômero é conhecida como telomerase, a qual é encontrada em diversas linhagens e tumores humanos. A telomerase é reativada pelas células imortalizadas, sugerindo uma ligação com sua progressão e que um agente inibidor dessa enzima, como a azidotimidina (AZT), poderia ser uma droga antitumoral efetiva. Objetivo: Avaliar a ação do AZT na expressão gênica e na síntese proteica da telomerase nas células de melanomas metastático. Método: As células provenientes de amostras de tecidos e células de melanoma Hs839T de pacientes foram utilizadas. A avaliação da proliferação celular foi realizada por microtitulação colorimétrica e a caracterização das células, a marcação para P53, a apoptose e a detecção da telomerase foram realizadas por citometria de fluxo e amplificação gênica da telomerase por PCR RT. Resultados: A ação do AZT diminuiu a expressão gênica da telomerase nas células estudadas com diferença estatisticamente significante, mas não a síntese proteica da enzima. Conclusão: O AZT induziu a diminuição da expressão gênica da telomerase sem promover alteração na síntese dessa enzima em células de melanoma metastático humano.


Keywords


telomerase, melanoma, p53, neoplasias cutâneas, azidotimidina,

References


Andreotti V, Ciribilli Y, Monti P, Bisio A, Lion M, Jordan J, et al. p53 transactivation impact of mutations, cofactors and small molecules using a simplified yeast-based screening system. PLoS One. 2011; 6 (6):1-15.

Aschacher T, Sampl S, Kaser L, Bernhard D, Spittler A, Holzman K et al. The Combined Use of Known Antiviral Reverse Transcriptase Inhibitors AZT and DDI Induce Anticancer Effects at Low Concentrations. Neoplasia. 2012; 14: (1), 44-3.

Bar C, Blasco MA. Telomeres and telomerase as therapeutic targets to prevent and treat age-related diseases. F1000 reserarch.2016; 89:1-11.

Baren NV, Baurain JF, Coulie PG. Lymphoid neogenesis in melanoma. What does it tell us? Oncoimmunol. 2013; 2: (1), 1-2.

Bernal A & Tusell L. J. Mol. Sci. 2018; 19: (294), 1-21.

Bernal A, Abad MM, Dominguez D, Tusell L. Acute telomere deprotection prevents ongoing BFB cycles and rampant instability in p16INK4a-deficient epithelial cells. Oncotarget. 2018; 9:(43), 27151-70.

Blackburn EH, Strahl C. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol Cell Biol. 1996; 16(1): 53- 65.

Brown T, Sigurdson E, Rogatko A, Brocoli D. Telomerase inhibition using azidothymidine in the HT-29 colon cancer cell line. Ann Surg Oncol. 2003; 10(8): 910-15.

Carvalho L, Lipay M, Belfort F, Santos IDAO, Andrade J, Haddad A, et al. Telomerase activity in prognostic histopathologic features of melanoma. J. Plast Reconstr Aesthet Surg. 2006; 59(9): 961-8.

Chudnovsky Y, Khavari PA, Adams AE. Melanoma genetics and the development of rational therapeutics. The J Clin Inv. 2005a; 115(4): 813-24.

Damsky WE, Bosenberger. Melanocytic nevi and melanoma: unraveling a complex relationship. Oncogene.2017; 36 (42) 5771-92.

Datta A, Bellon M, Datta US,Bazarbachi A, Lepelletier Y, Canioni D, Thomas et al. Persistent inhibition of telomerase reprograms adult Tcell leukemia to p53 dependent senescence. Blood. 2006; 108(3): 1021-9.

Fadri-Moskwik M, Zhou Q, Chai W. Beyond Telomerase: Telomere Instability as a Novel Target for Cancer Therapy. J Mol Genet Med. 2013; 7(4): 1-14.

Falchetti A, Franchi A, Bordi C, Mavilia C, Mais L, Cioppi F, Recenti et.al., Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture. J Bone Miner Res. 2005; 20(3):410-18.

Ferreira LM, Gomes EF, Silva EF, Silvia MSB, Araújo RP, Sales A. Metástase tonsilar de melanoma maligno. Rev Bras Otorrinolaringol. 2006; 72: (6)851.

Figueiredo LC, Cordeiro LN, Arruda AP, Carvalho MDF, Ribeiro EM, et al. Câncer de pele: Estudo dos principais marcadores moleculares do melanoma cutâneo. Rev Bras Cancerologia. 2003; 49(3): 179-83.

Flach EH, Rebecca VW, Herlyn M, Smalley KSM, Anderson ARA. Fibroblasts contribute to melanoma tumour growth and drug resistance. Mol Pharm. 2011;8(6):2039-49.

Gandini S, Montella M, Ayala F, Benedetto L, Rossi CR, Vecchiatto A, et al. Sun exposure and melanoma prognostic factors. Oncol Lett. 2016; 11(4): 2706–14.

Ganesan K & Baojun X. Telomerase Inhibitors from Natural Products and Their Anticancer Potential. Int. J. Mol Sci. 2018; 19 (1): 13.

Gomez DA, Armando RG, Alonso DF. AZT as telomerase inhibitor. Font Oncol. 2012; 2(113): 1-5.

Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 1989; 119(2):203-10.

Hayflick L. The cell biology of aging. J Invest Dermatol. 1979; 73(1):8-14.

Humer J, Ferko B, Waltenberg, Rapberger R, Pehamberger H, Muster T. Azidothymidine inhibts melanoma cell grownth in vitro and in vivo. Melanoma Res. 2008; 18(5): 314-21.

Jafri MA, Ansari SA, Algahtani MH, Shay JW. Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Med. 2016; 8: 1-18.

Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994; 266:2011-5.

Lima JM, Serafim PVP, Silva IDCG, Forones NM. Estudo do polimorfismo genético no gene p53 (códon 72) em câncer colorretal. Gastroenterol. 2006; 43(1): 8-13.

Mergny JL, Riou JF, Mailliet P, Fichou MPT, Gilson E. Natural and pharmacological regulation of telomerase. Nucleic Acid Res. 2002. 30(4): 839-65.

Mo Y, Gan Y, Song S, Johnston X, Wientjes MG, Jessie LS. Simultaneous targeting of telomeres and telomerase as cancer therapeutic approach. Câncer Res. 2003; 63:579-85.

Nam KW, Bae YC, Nam SB, Kim JH, Kim HS, Choi YJ. Characteristics and Treatment of Cutaneous Melanoma of the Foot Arch Plast Surg. 2016; 43(1): 59-65.

Nguyen THD, Tam J, Wu RA, Greber BJ, Toso D, Nogales E, Collins K. Cryo-EM structure of substrate-bound human telomerase holoenzyme. Nature.2018; 557 (7704): 190-5.

Oliveira AF, Gragnani A, Oliveira Filho RS, Santos IDAO, França SG, Enokihara MMSS, Ferreira LM. Modelo experimental de cultura primária de melanoma metastático por punção aspirativa de agulha fina. Acta Cir Bras. 2005; 20(5): 390-03.

Oren, M. Decision making by p53: life, death and cancer. Cell Death and Differentiation.2003; 10 (4): 431-42.

Parreiras FC, Wainstem AJA, Morete M, Geo LS. Prevalência de dor em pacientes com melanoma. Rev Dor. São Paulo. 2016.17(1): 39-2.

Parsons HA. Telômeros, telomerase e câncer. Rev Fac Ciênc Méd Sorocaba. 2003; 5(1): 54-9.

Piatyszek MA, Kim NW, Weinrich SL, Hiyama K, Hiyama E, Wright WE, Shay JW. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods in Cell Science. 1995; 17: 1-15.

Ramirez RD, Wright E, Shay JW, Taylor RS. Telomerase activity concentrates in the mitotically active segments of human hair follicles. J Invest Dermatol. 1997; 108: 113-17.

Rouaud F, Tekaya NH, Cerezo M, Abbe P, Zangari J, Hofman V, Ohanna M, et al.Cell Death & Disease. 2018; 5:527.

Rowe SP, Luber B, Makell M, Brothers P, Santmyer J, Schollenberger MD, Quinn H, et al. From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real-world setting. Molecular Oncology. 2018; 12: 1661-72.

Santos IDAO, Brunstein F, Minami E, Carvalho C, Filho EFA, Ferreira LM. Neoplasias malignas de pele: Análise epidemiológica de 1.242 pacientes operados. JBM 1996; 71(2): 61- 63.

Souza Sobrinho CP, Gragnani A, Santos IDAO, Oliveira AF, Garcia JE, Ferreira LM. Melanoma Cutâneo e Telomerase. Applied Cancer Research. 2009; 29(2): 1-18.

Souza Sobrinho CP, Gragnani A, Santos IDAO, Oliveira AF, Lipay MVN, Ferreira LM. AZT na atividade da telomerase e na proliferação de células de melanoma HS 839.T. Acta Ciru Bras. 2012; 27(12): 855-60.

Taylor RS, Ramirez RD, Ogoshi M, Chaffins M, Piatyszek MA, Shay JW. Detection of telomerase activity in malignant and nonmalignant skin condition. J Invest Dermatol. 1996; 106(4): 759-65.

Tucci-Viegas, Hochman B, França JP, Ferreira LM. Keloid explant culture: A model for keloid fibroblasts isolation and cultivation based on the biological differences of its specific regions. Int Wound J. 2010;7(5):339-48.

Umemoto T, Yamato M, Nishida K, Yang J, Tano Y, Okano T. Limbal epithelial side- population cells have stem cell–like properties, including quiescent state. Stem Cell. 2006; 24:86-94.

Vazquez VL, Silva TB, Vieira MA, Oliveira ATT, Lisboa MV, Andrade DAP, et al., Melanoma characteristics in Brazil: demographics, treatment, and survival analysis. BMC Res Notes. 2015; 8: 4.

Vozza A, Borriello A, Criniti V, Vozza G, Ragioneà FD. New established melanoma cell lines: Genetic biochemical characterization of cell division cycle. J. Eur Acad of Derm. and Venereol. 2003; 17(1): 37–41.

Wu RA, Upton HE, Vogan JM, Collins K. Telomerase Mechanism of Telomere Synthesis. Annu Rev. Biochem.2018;86:439-60.

Zaccagnini G, Gaetano C, Pietra L, Nanni S, Grasselli A, Mangoni A, et al. Telomerase mediates vascular endothelial growth factor-dependent responsiveness in a rat model of hind limb ischemia. J Biol Chem. 2005; 280(15):14790-98.

Zhang F, Cheng D, Wang S, Zhu J. Human Specific Regulation of the Telomerase Reverse Transcriptase Gene. Genes Basel. 2016; 28 (7):1-7.




DOI: https://doi.org/10.34117/bjdv8n5-415