Efecto citotóxico de fosfolipasas A2 del veneno de Crotalus durissus cumanensis de Colombia
Cytotoxic effect of A2 phospholipases of the venom of Crotalus durissus cumanensis from Colombia
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Quintana-Castillo JC, Ávila-Gómez IC, Ceballos-Ruiz JF, Vargas-Muñoz LJ, Estrada-Gómez S. Efecto citotóxico de fosfolipasas A2 del veneno de Crotalus durissus cumanensis de Colombia. Revista Investig. Salud Univ. Boyacá [Internet]. 24 de julio de 2017 [citado 22 de diciembre de 2024];4(1):16-37. Disponible en: https://revistasdigitales.uniboyaca.edu.co/index.php/rs/article/view/194
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Introducción. Los venenos de serpientes representan una fuente importante de proteínas y péptidos, los cuales exhiben diversas actividades biológicas, tales como antibacterianas, antiparasitarias, antivirales, antitumorales, antifúngicas y contra la agregación plaquetaria, entre otras.
Las fosfolipasas A2 presentes en los venenos de serpientes son las proteínas más estudiadas en estos modelos. Se ha demostrado que las fosfolipasas A2, activas e inactivas, poseen actividad catalítica contra células tumorales.
Objetivo. Aislar, purificar y caracterizar la fosfolipasa A2 del veneno de Crotalus durissus cumanensis para evaluar su actividad antitumoral in vitro.
Materiales y métodos. El aislamiento, la purificación y la identificación de la crotoxina B se hizo mediante la cromatografía de exclusión molecular, la cromatografía líquida de alto rendimiento de fase inversa (Reversed Phase High-Performance Liquid Chromatography, RP-HPLC) y la espectrometría de masas. El efecto citotóxico sobre células tumorales (K562) y células normales (células mononucleares de sangre periférica) se determinó utilizando la técnica de MTT.
Resultados. La separación y posterior identificación de la crotoxina B del veneno de C. d. cumanensis de Colombia, permitieron evidenciar que esta fosfolipasa A2 posee efecto citotóxico sobre las células mononucleares de sangre periférica con una dosis de 18,23 ± 0,57 μg/ml, mientras que, para las células K562, fue de 2,34 ± 0,199 μg/ml.
Conclusiones. Los resultados sugieren la posibilidad de utilizar la crotoxina B aislada del veneno de C. d. cumanensis como un posible recurso terapéutico para su aplicación en humanos.
Palabras clave: Crotalus durissus cumanensis; citotoxicidad; fosfolipasas A2; crotoxina B.
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4. Andriao-Escarso SH, Soares AM, Rodrigues VM, Angulo Y, Diaz C, Lomonte B, et al. Myotoxic phospholipases A(2) in bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie. 2000;82:755-63.
5. Cecilio AB, Caldas S, Oliveira RA, Santos AS, Richardson M, Naumann GB, et al. Molecular characterization of Lys49 and Asp49 phospholipases A(2) from snake venom and their antiviral activities against dengue virus. Toxins. 2013;5:1780-98.
6. Muller VD, Russo RR, Cintra AC, Sartim MA, Alves-Paiva RdeM, Figueiredo LT, et al. Crotoxin and phospholipases A(2) from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses. Toxicon. 2012;59:507-15.
7. Murillo LA, Lan CY, Agabian NM, Larios S, Lomonte B. Fungicidal activity of a phospholipase-A2-derived synthetic peptide variant against Candida albicans. Rev Esp Quimioter. 2007;20:330-3.
8. Quintana JC, Chacón AM, Vargas L, Segura C, Gutiérrez JM, Alarcón JC. Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and crotoxin B. Acta Trop. 2012;124:126-32.
9. Six DA, Dennis EA. The expanding superfamily of phospholipase A(2) enzymes: Classification and characterization. Biochim Biophys Acta. 2000;1488:1-19.
10. Evangelista IL, Martins AM, Nascimento NR, Havt A, Evangelista JS, de Noroes TB, et al. Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2. Toxicon. 2010;55:1061-70.
11. Kini RM. Excitement ahead: Structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon. 2003;42:827-40.
12. Landucci EC, de Castro RC, Toyama M, Giglio JR, Marangoni S, De Nucci G, et al. Inflammatory oedema induced by the lys-49 phospholipase A(2) homologue piratoxin-I in the rat and rabbit. Effect of polyanions and p-bromophenacyl bromide. Biochem Pharmacol. 2000;59:1289-94.
13. Barbosa PS, Martins AM, Havt A, Toyama DO, Evangelista JS, Ferreira DP, et al. Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom. Toxicon. 2005;46:376-86.
14. Kini RM, Evans HJ. Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2. J Biol Chem. 1987;262:14402-7.
15. de Moura AA, Kayano AM, Oliveira GA, Setubal SS, Ribeiro JG, Barros NB, et al. Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells. Biomed Res Int. 2014;195356.
16. Nelson J, Barlow K, Beck DO, Berbert A, Eshenroder N, Francom L, et al. Synergistic effects of secretory phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus with cancer chemotherapeutic agents. Biomed Res Int. 2013;565287.
17. Samel M, Vija H, Kurvet I, Kunnis-Beres K, Trummal K, Subbi J, et al. Interactions of PLA2-s from Vipera lebetina, Vipera berus berus and Naja naja oxiana venom with platelets, bacterial and cancer cells. Toxins. 2013;5:203-23.
18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.
19. Gutiérrez JM, Ávila C, Rojas E, Cerdas L. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon. 1988;26:411-3.
20. Habermann E, Hardt KL. A sensitive and specific plate test for the quantitation of phospholipases. Analyt Biochem. 1972;50:1963-5.
21. Lomonte B, Gutiérrez JM, Romero M, Nunez J, Tarkowski A, Hanson LA. An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies. J Immunol Methods. 1993;161:231-7.
22. Bouchier C, Ducancel F, Guignery-Frelat G, Bon C, Boulain JC, Menez A. Cloning and sequencing of cDNAs encoding the two subunits of Crotoxin. Nucleic Acid Res. 1988;16:9050.
23. Azevedo-Marques MM, Cupo P, Coimbra TM, Hering SE, Rossi MA, Laure CJ. Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil. Toxicon. 1985;23:631-6.
24. Cupo P, Azevedo-Marques MM, Hering SE. Clinical and laboratory features of South American rattlesnake (Crotalus durissus terrificus) envenomation in children. Trans R Soc Trop Med Hyg. 1988;82:924-9.
25. Martins AM, Toyama MH, Havt A, Novello JC, Marangoni S, Fonteles MC, et al. Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys. Toxicon. 2002;40:1165-71.
26. Oshima-Franco Y, Hyslop S, Prado-Franceschi J, Cruz-Hofling MA, Rodrigues-Simioni L. Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin. Toxicon. 1999;37:1341-57.
27. Costa AF, Dantas RT, Toyama MH, Diz Filho E, Zara FJ, Rodrigues MG, et al. Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and L-amino acid oxidase. Toxicon. 2010;55:795-804.
28. Murakami MT, Arruda EZ, Melo PA, Martínez AB, Calil-Elias S, Tomaz MA, et al. Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin. J Mol Biol. 2005;350:416-26.
29. Faure G, Xu H, Saul FA. Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric beta-neurotoxin. J Mol Biol. 2011;412:176-91.
30. Faure G, Bon C. Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus. Toxicon. 1987;25:229-34.
31. Hendon RA, Fraenkel-Conrat H. Biological roles of the two components of crotoxin. Proc Natl Acad Sci U S A. 1971;68:1560-3.
32. Fagundes FH, Oliveira M, Huancahuire-Vega S, Romero-Vargas FF, Ponce-Soto LA, Marangoni S. cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake. Braz J Med Biol Res. 2010;43:262-70.
33. Ponce-Soto LA, Baldasso PA, Romero-Vargas FF, Winck FV, Novello JC, Marangoni S. Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom. Protein J. 2007;26:39-49.
34. Ponce-Soto LA, Lomonte B, Rodrigues-Simioni L, Novello JC, Marangoni S. Biological and structural characterization of crotoxin and new isoform of crotoxin B PLA(2) (F6a) from Crotalus durissus collilineatus snake venom. Protein J. 2007;26:221-30.
35. Costa LA, Miles H, Araujo CE, González S, Villarrubia VG. Tumor regression of advanced carcinomas following intra- and/ or peri-tumoral inoculation with VRCTC- 310 in humans: Preliminary report of two cases. Immunopharmacol Immunotoxicol. 1998;20:15-25.
36. Costa LA, Miles HA, Diez RA, Araujo CE, Coni CM, Cervellino JC. Phase I study of VRCTC- 310, a purified phospholipase A2 purified from snake venom, in patients with refractory cancer: Safety and pharmacokinetic data. Anticancer Drugs. 1997;8:829-34.
37. Finn R. Snake venom protein paralyzes cancer cells. J Natl Cancer Inst. 2001;93:261-2.
38. He JK, Wu XS, Wang Y, Han R, Qin ZH, Xie Y. Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice. Acta Pharmacol Sin. 2013;34:295- 300.
39. Kim DS, Jang YJ, Jeon OH, Kim DS. Saxatilin, a snake venom disintegrin, suppresses TNF-alpha-induced ovarian cancer cell invasion. J Biochem Mol Biol. 2007;40:290-4.
40. Yan CH, Yang YP, Qin ZH, Gu ZL, Reid P, Liang ZQ. Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells. Acta Pharmacol Sin. 2007;28:540-8.
41. Newman RA, Vidal JC, Viskatis LJ, Johnson J, Etcheverry MA. VRCTC-310--a novel compound of purified animal toxins separates antitumor efficacy from neurotoxicity. Invest New Drugs. 1993;11:151-9.
42. Vadas P. Group II phospholipases A2 are indirectly cytolytic in the presence of exogenous phospholipid. Biochim Biophys Acta. 1997;1346:193-7.
43. Lomonte B, Angulo Y, Calderón L. An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon. 2003;42:885-901.
2. Weinberg R. The biology of cancer. Second edition. EE.UU: Taylor and Francis Group; 2013. 960 p.
3. Riss TL, Moravec RA. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev Technol. 2004;2:51-62.
4. Andriao-Escarso SH, Soares AM, Rodrigues VM, Angulo Y, Diaz C, Lomonte B, et al. Myotoxic phospholipases A(2) in bothrops snake venoms: Effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie. 2000;82:755-63.
5. Cecilio AB, Caldas S, Oliveira RA, Santos AS, Richardson M, Naumann GB, et al. Molecular characterization of Lys49 and Asp49 phospholipases A(2) from snake venom and their antiviral activities against dengue virus. Toxins. 2013;5:1780-98.
6. Muller VD, Russo RR, Cintra AC, Sartim MA, Alves-Paiva RdeM, Figueiredo LT, et al. Crotoxin and phospholipases A(2) from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses. Toxicon. 2012;59:507-15.
7. Murillo LA, Lan CY, Agabian NM, Larios S, Lomonte B. Fungicidal activity of a phospholipase-A2-derived synthetic peptide variant against Candida albicans. Rev Esp Quimioter. 2007;20:330-3.
8. Quintana JC, Chacón AM, Vargas L, Segura C, Gutiérrez JM, Alarcón JC. Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and crotoxin B. Acta Trop. 2012;124:126-32.
9. Six DA, Dennis EA. The expanding superfamily of phospholipase A(2) enzymes: Classification and characterization. Biochim Biophys Acta. 2000;1488:1-19.
10. Evangelista IL, Martins AM, Nascimento NR, Havt A, Evangelista JS, de Noroes TB, et al. Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A2. Toxicon. 2010;55:1061-70.
11. Kini RM. Excitement ahead: Structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon. 2003;42:827-40.
12. Landucci EC, de Castro RC, Toyama M, Giglio JR, Marangoni S, De Nucci G, et al. Inflammatory oedema induced by the lys-49 phospholipase A(2) homologue piratoxin-I in the rat and rabbit. Effect of polyanions and p-bromophenacyl bromide. Biochem Pharmacol. 2000;59:1289-94.
13. Barbosa PS, Martins AM, Havt A, Toyama DO, Evangelista JS, Ferreira DP, et al. Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom. Toxicon. 2005;46:376-86.
14. Kini RM, Evans HJ. Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2. J Biol Chem. 1987;262:14402-7.
15. de Moura AA, Kayano AM, Oliveira GA, Setubal SS, Ribeiro JG, Barros NB, et al. Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells. Biomed Res Int. 2014;195356.
16. Nelson J, Barlow K, Beck DO, Berbert A, Eshenroder N, Francom L, et al. Synergistic effects of secretory phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus with cancer chemotherapeutic agents. Biomed Res Int. 2013;565287.
17. Samel M, Vija H, Kurvet I, Kunnis-Beres K, Trummal K, Subbi J, et al. Interactions of PLA2-s from Vipera lebetina, Vipera berus berus and Naja naja oxiana venom with platelets, bacterial and cancer cells. Toxins. 2013;5:203-23.
18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.
19. Gutiérrez JM, Ávila C, Rojas E, Cerdas L. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon. 1988;26:411-3.
20. Habermann E, Hardt KL. A sensitive and specific plate test for the quantitation of phospholipases. Analyt Biochem. 1972;50:1963-5.
21. Lomonte B, Gutiérrez JM, Romero M, Nunez J, Tarkowski A, Hanson LA. An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies. J Immunol Methods. 1993;161:231-7.
22. Bouchier C, Ducancel F, Guignery-Frelat G, Bon C, Boulain JC, Menez A. Cloning and sequencing of cDNAs encoding the two subunits of Crotoxin. Nucleic Acid Res. 1988;16:9050.
23. Azevedo-Marques MM, Cupo P, Coimbra TM, Hering SE, Rossi MA, Laure CJ. Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil. Toxicon. 1985;23:631-6.
24. Cupo P, Azevedo-Marques MM, Hering SE. Clinical and laboratory features of South American rattlesnake (Crotalus durissus terrificus) envenomation in children. Trans R Soc Trop Med Hyg. 1988;82:924-9.
25. Martins AM, Toyama MH, Havt A, Novello JC, Marangoni S, Fonteles MC, et al. Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys. Toxicon. 2002;40:1165-71.
26. Oshima-Franco Y, Hyslop S, Prado-Franceschi J, Cruz-Hofling MA, Rodrigues-Simioni L. Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin. Toxicon. 1999;37:1341-57.
27. Costa AF, Dantas RT, Toyama MH, Diz Filho E, Zara FJ, Rodrigues MG, et al. Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and L-amino acid oxidase. Toxicon. 2010;55:795-804.
28. Murakami MT, Arruda EZ, Melo PA, Martínez AB, Calil-Elias S, Tomaz MA, et al. Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin. J Mol Biol. 2005;350:416-26.
29. Faure G, Xu H, Saul FA. Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric beta-neurotoxin. J Mol Biol. 2011;412:176-91.
30. Faure G, Bon C. Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus. Toxicon. 1987;25:229-34.
31. Hendon RA, Fraenkel-Conrat H. Biological roles of the two components of crotoxin. Proc Natl Acad Sci U S A. 1971;68:1560-3.
32. Fagundes FH, Oliveira M, Huancahuire-Vega S, Romero-Vargas FF, Ponce-Soto LA, Marangoni S. cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake. Braz J Med Biol Res. 2010;43:262-70.
33. Ponce-Soto LA, Baldasso PA, Romero-Vargas FF, Winck FV, Novello JC, Marangoni S. Biochemical, pharmacological and structural characterization of two PLA2 isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom. Protein J. 2007;26:39-49.
34. Ponce-Soto LA, Lomonte B, Rodrigues-Simioni L, Novello JC, Marangoni S. Biological and structural characterization of crotoxin and new isoform of crotoxin B PLA(2) (F6a) from Crotalus durissus collilineatus snake venom. Protein J. 2007;26:221-30.
35. Costa LA, Miles H, Araujo CE, González S, Villarrubia VG. Tumor regression of advanced carcinomas following intra- and/ or peri-tumoral inoculation with VRCTC- 310 in humans: Preliminary report of two cases. Immunopharmacol Immunotoxicol. 1998;20:15-25.
36. Costa LA, Miles HA, Diez RA, Araujo CE, Coni CM, Cervellino JC. Phase I study of VRCTC- 310, a purified phospholipase A2 purified from snake venom, in patients with refractory cancer: Safety and pharmacokinetic data. Anticancer Drugs. 1997;8:829-34.
37. Finn R. Snake venom protein paralyzes cancer cells. J Natl Cancer Inst. 2001;93:261-2.
38. He JK, Wu XS, Wang Y, Han R, Qin ZH, Xie Y. Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice. Acta Pharmacol Sin. 2013;34:295- 300.
39. Kim DS, Jang YJ, Jeon OH, Kim DS. Saxatilin, a snake venom disintegrin, suppresses TNF-alpha-induced ovarian cancer cell invasion. J Biochem Mol Biol. 2007;40:290-4.
40. Yan CH, Yang YP, Qin ZH, Gu ZL, Reid P, Liang ZQ. Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells. Acta Pharmacol Sin. 2007;28:540-8.
41. Newman RA, Vidal JC, Viskatis LJ, Johnson J, Etcheverry MA. VRCTC-310--a novel compound of purified animal toxins separates antitumor efficacy from neurotoxicity. Invest New Drugs. 1993;11:151-9.
42. Vadas P. Group II phospholipases A2 are indirectly cytolytic in the presence of exogenous phospholipid. Biochim Biophys Acta. 1997;1346:193-7.
43. Lomonte B, Angulo Y, Calderón L. An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon. 2003;42:885-901.
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