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

Author(s)

Cássio Prinholato da Silva

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2012-10-19

Examining board members

Suely Vilela; Lusania Maria Greggi Antunes; Jose Roberto Giglio

• Advisor: Suely Vilela

Abstract

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. (AU)

• FAPESP's process: 10/03243-3 - Evaluation of damage pathway signs and cytokines inflammatory induction in the presence of BthTX-I toxin isolated from Bothrops jararacussu venom.
• Grantee: Cássio Prinholato da Silva
• Support Opportunities: Scholarships in Brazil - Master