Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Abstract\ud \ud Background\ud Phospholipases A2 (PLA2s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA2 isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines.\ud \ud \ud Methods\ud The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I.\ud \ud \ud Results\ud It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %).\ud \ud \ud Conclusions\ud These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.The authors would like to thank the financial support provided by the State\ud of São Paulo Research Foundation (FAPESP, grants n. 2010/03243-43 and\ud 2011/23236-4), the Coordination for the Improvement of Higher Education\ud Personnel (CAPES) and the National Council for Scientific and Technological\ud Development (CNPq process n. 476932/2012-2). We are also grateful to\ud Fabiana Rosseto Morais, from FCFRP-USP, for the technical assistance in the\ud flow cytometry analyses. Thanks are also due to the Center for the Study of\ud Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication\ud of this special collection (CNPq process 469660/2014-7)

Evaluation of damage signaling pathways and induction of inflammatory cytokines in the presence of BthTX-I, isolated from Bothrops jararacussu snake venom

As fosfolipases são bastante estudadas e podem ser encontradas de forma abundante nas peçonhas. Algumas fosfolipases que sofrem modificação no resíduo 49 com a substituição do aminoácido Asp por Lys, o que provoca perda do seu sítio catalítico, são classificadas como miotoxinas. A miotoxina BthTX-I foi isolada da peçonha de Bothrops jararacussu, possui 121 resíduos de aminoácidos, pI 8,2 e massa molecular de 13kDa. O objetivo do presente projeto foi avaliar a ação da BthTX-I, quanto à sua ação citotóxica, indutora de morte celular, interferência na cinética celular e expressão de genes responsáveis pela morte celular em quatro linhagens tumorais, HL-60 (leucemia promielocítica), HepG-2 (hepatocarcinoma humano), PC-12 (feocromacitoma murino) e B16F10 (melanoma murino). Também foi analisada a indução de citocinas inflamatórias IL-8 e TNF-? em linhagens humanas HL-60 e HepG2. A citotoxicidade, avaliada pela metodologia do MTT, apresentou valores citotóxicos em torno de 62, 53, 53 e 63%, respectivamente para HepG2, HL-60, PC12 e B16F10. A toxina mostrou ação moduladora do ciclo celular na fase S em células HepG2; na fase G2/M em células HL-60. Nas linhagens B16F10 e PC-12 a toxina provocou atraso na fase G0/G1 e redução de células na fase S e G2/M. O perfil eletroforético em gel de agarose a 1,5% mostrou fragmentação do conteúdo do DNA, indicando possível apoptose, que foi confirmada pelo ensaio de morte celular por citometria de fluxo. O ensaio revelou que a linhagem B16F10 tem maior índice apoptótico (~40%), seguido da HepG2 (~35%), PC-12 (~25%) e HL-60 (~4%). A indução de citocinas pela metodologia de ELISA apresentou valores elevados de IL-8 para linhagem HL-60 (~400 pg/mL) e HepG2 (~400 pg/mL), sugerindo uma possível quimiotaxia/migração de células de defesa. TNF- ? também apresentou alteração em seus níveis representando cerca de 150 pg/mL na linhagem HepG2. A expressão dos genes Bax, Bcl-2 e p53 demonstrou que o gene p53 se altera somente na linhagem HepG2, todavia a expressão dos genes Bax e Bcl-2 mostrou ser diferente para cada linhagem. Em células B16F10 a toxina aumentou os níveis de Bax e diminuiu os níveis de Bcl-2 enquanto que, as células PC-12 exibiram aumento nos níveis de ambos. Em HepG2 houve redução da expressão do gene antiapoptótico Bcl-2 e valor inalterado de Bax, ao passo que a linhagem HL-60 apresentou resultado contrário ao de células HepG2, com aumento na expressão de Bax e valores inalterados de Bcl-2. Assim a toxina mostra diferente ação na expressão dos genes responsáveis pela apoptose em diferentes linhagens celulares, mostrando que a BthTX-I influencia a expressão desses genes, ou promove apenas a alteração de dessses, levando assim à apoptose celular das linhagens tratadas com a miotoxina. Diante dos dados obtidos ficou evidenciado o potencial antitumoral da BThTX-I levando a busca de outros mecanismos de atuação da toxina, abrindo perspectivas de sua aplicação biotecnológica para a produção de novo fármaco antitumoral e tornando-se uma terapia alternativa a essa enfermidade.Phospholipases are widely studied and can be found in abundance in several animal venoms. Some phospholipases, classified as myotoxins, present a modification at residue 49, with replacement of the amino acid Asp by Lys, causing loss of their catalytic site. BthTX-I was isolated from the venom of Bothrops jararacussu and is a myotoxin with 121 amino acid residues, pI 8.2 and a molecular mass of 13kDa. The aim of this study was to evaluate BthTX-I regarding its cytotoxic action, induction of cell death, interference with cell kinetics and expression of genes responsible for cell death in four tumor cell lines: HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma). The induction of inflammatory cytokines (IL-8 and TNF-?) in the human cell lines HepG2 and HL-60 was also analyzed. Cytotoxicity, as assessed by the MTT methodology, showed values around 62, 53, 53 and 63% for HepG2, HL-60, PC12 and B16F10, respectively. The toxin showed modulating action of the cell cycle in the S phase in HepG2 cells, in the G2/M phase in HL-60 cells and delay in the G0/G1 phase. The toxin also caused a delay in the G0/G1 phase and reduced the number of cells in the S and G2/M phases in B16F10 and PC-12 cell lines. The electrophoretic profile on 1,5% agarose gel showed fragmentation of DNA content, possibly indicating apoptosis, which was confirmed by flow cytometry cell death assays. This assay revealed that B16F10 cells presented a higher apoptotic rate (~40%), followed by HepG2 (~35%), PC-12 (~25%) and HL-60 (~4%) cells. Induction of cytokines analyzed by ELISA showed high levels of IL-8 in HL-60 (~400 pg/mL) and HepG2 (~400 pg/mL) cells, suggesting a possible chemotaxis and migration of immune cells. The levels of TNF-? also showed changes, representing about 150 pg/mL in HepG2 cells and 14 pg/mL in HL-60 cells. Gene expression of Bax, Bcl-2 and p53 showed that the p53 gene did not change only in HepG2 cells, with the gene expression of Bax and Bcl-2 being different for each cell line. The toxin increased the levels of Bax and decreased the levels of Bcl-2 in B16F10 cells, while PC-12 cells showed increased levels of both genes. Regarding HepG2 cells, a decrease in the expression of the antiapoptotic gene Bcl-2 was observed, with unchanged values for Bax, while opposite results were observed for HL-60 cells, with increased expression of Bax and unchanged values of Bcl-2. Thus, the toxin showed different actions on the expression of genes responsible for apoptosis in different cell lines, showing that BthTX-I influences the expression of both genes, promoting changes in one or another apoptotic gene, thus leading the tumor cell lines treated with this myotoxin to apoptosis. The obtained data evidenced the antitumor potential of BthTX-I, leading to the search for other mechanisms of action of this toxin and opening prospects for its biotechnological applications for the production of new anti-cancer drugs

Evaluating the microbicidal, antiparasitic and antitumor effects of CR-LAAO from Calloselasma rhodostoma venom

CR-LAAO is an l-amino acid oxidase from Calloselasma rhodostoma snake venom that has been broadly studied regarding its structural and biochemical characteristics, however, few studies have investigated its pharmacological effects. The present study aimed at the evaluation of the biotechnological potential of CR-LAAO by determining its bactericidal, antifungal, leishmanicidal and trypanocidal activity, as well as its cytotoxicity on human tumor and non-tumor cell lines. After 24h of preincubation, CR-LAAO showed bactericidal effects against both Staphylococcus aureus (MIC 0.78μg/mL) and Escherichia coli (MIC 31.25μg/mL) strains, inducing dismantle of bacterial cell walls. After 6h of preincubation with Candida albicans, CR-LAAO was able to inhibit 80% of the yeast growth, and it also showed cytotoxic activity on Leishmania species and Trypanosoma cruzi. Additionally, CR-LAAO showed high cytotoxicity on HepG2 and HL-60 tumor cells (IC50 10.78 and 1.7μg/mL), with lower effects on human mononuclear cells (PBMC). The cytotoxic effects of CR-LAAO were significantly inhibited in the presence of catalase, which suggests the involvement of hydrogen peroxide in its mechanisms of toxicity. Therefore, CR-LAAO showed promising pharmacological effects, and these results provide important information for the development of therapeutic strategies with directed action, such as more effective antimicrobial agents