A New Approach to Inhibiting Triple-Negative Breast Cancer: In Vitro, Ex Vivo and In Vivo Antiangiogenic Effect of BthTx-II, a PLA2-Asp-49 from Bothrops jararacussu Venom.

Phospholipases A2 (PLA2) represent a superfamily of enzymes widely distributed in living organisms, with a broad spectrum of pharmacological activities and therapeutic potential. Anti-angiogenic strategies have become one of the main tools in fighting cancer. In this sense, the present work reports the inhibition of tumor angiogenesis induced by Asp-49 BthTX-II using in vitro, ex vivo and in vivo approaches. We demonstrate that BthTx-II inhibited cell adhesion, proliferation, and migration of human umbilical vein endothelial cells (HUVEC), as well as caused a reduction in the levels of endothelial growth factor (VEGF) during in vitro angiogenesis assays. BthTx-II was also able to inhibit the sprouting angiogenic process, by the ex vivo germination assay of the aortic ring; in addition, this toxin inhibited the migration and proliferation of HUVEC in co-culture with triple-negative breast cancer cells (e.g., MDA-MB-231 cells). Finally, in vivo tumor suppression and anti-angiogenic activities were analyzed using MDA-MB-231 cells with Matrigel injected into the chorioallantoic membrane of chicken embryo (CAM) for 7 days treatment with BthTx-II, showing a considerable reduction in vessel caliber, on the size and weight of tumors. Together, these results suggest an important antiangiogenic and antitumor role for BthTx-II, as a potential prototype for the development of new tools and antitumor drugs in cancer therapy.

BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: An example of snake venom phospholipases A2 versatility.

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.

BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: an example of snake venom phospholipases A2 versatility

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.

Antitumor and antimetastatic effects of PLA2-BthTX-II from Bothrops jararacussu venom on human breast cancer cells.

This work shows the antitumor and antimetastatic effects of BthTX-II, an Asp-49 PLA2 from Bothrops jararacussu venom, on MDA-MB-231 human triple negative breast cancer cells. BthTX-II caused a dose-dependent cell death of MDA-MB-231 cells when compared with the non-tumorigenic breast cells by inducing apoptosis and autophagy. BthTX-II was also able to decrease the proliferation and to inhibit cell cycle progression. We also observed an upregulation of the ATM gene, which is responsible for cell-cycle arrest and DNA repair such as CCND1, CCNE1, CDC25A, E2F1, AKT1 and AKT3. Interestingly, BthTX-II inhibited invasion, migration and 3D cell growth of MDA-MB-231 cells, as well as inhibited the epithelial-mesenchymal transition (EMT) of this cell by increasing E-cadherin (CDH-1) and decreasing TWIST1, CTNNB1, vimentin and cytokeratin-5 expression. In conclusion, these results showed that BthTX-II displays antitumor and antimetastatic effects on MDA-MB-231 cells and may be useful for the development of new approaches and therapeutic strategies to manage triple negative breast cancer.