A paraguayan toad Rhinella schneideri preparation based on Mbya tradition increases mitochondrial bioenergetics with migrastatic effects dependent on AMPK in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Paraguay, healers from the Mbya culture treat cancer with a recipe prepared with the native toad Rhinella schneideri. However, the chemical composition and biological effects of the recipe remain unknown. AIM OF THE STUDY: The aim is to determine the composition of the traditional preparation made using the toad R. schneideri and to evaluate its effect on human breast cancer (BC) cells. MATERIALS AND METHODS: The metabolites contained in the preparation were concentrated using XAD-7 resin, and the concentrate was analyzed by HPLC-MS/MS. The effect of the preparation was assessed in normal (MCF10F) and BC cells (MDA-MB-231 and MCF7). The mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) levels, and cell cycle progression were determined by flow cytometry. The oxygen consumption rate (OCR) was measured by Clark electrode, and fibronectin-dependent migration in normoxia and hypoxia-like conditions were evaluated by transwell assay. RESULTS: From the Amberlite-retained extract from the preparation, 24 compounds were identified, including alkaloids, amino acids, bufadienolides, and flavonoids, among others. The crude extract (CE) did not affect cell cycle progression and viability of BC cell lines. Moreover, it did not make cancer cells more sensitive to the cytotoxic effect of the chemotherapeutics doxorubicin and teniposide. On the other hand, the CE reduced the menadione-induced ROS production and increased NADH, Δψm, and the OCR. Respiratory complexes I and III as well as ATP synthase levels were increased in an AMPK-dependent manner. Moreover, the CE inhibited the migration of BC cells in normoxia and a hypoxia-like condition using CoCl2 as a HIF1α-stabilizing agent. This latter effect involved an AMPK-dependent reduction of HIF1α levels. CONCLUSIONS: The Paraguayan toad recipe contains metabolites from the toad ingredient, including alkaloids and bufadienolide derivatives. The CE lacks cytotoxic effects alone or in combination with chemotherapeutics. However, it increases mitochondrial bioenergetics and inhibits the cancer cell migration in an AMPK-dependent manner in BC cells. This is the first report of the in vitro anticancer effect of a traditional Rhinella sp. toad preparation based on Mbya tradition.

Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae).

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a ß-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 µg) and edema-forming (DEM = 26.00 ± 1.15 µg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

Extracellular Matrix Signals as Drivers of Mitochondrial Bioenergetics and Metabolic Plasticity of Cancer Cells During Metastasis.

The role of metabolism in tumor growth and chemoresistance has received considerable attention, however, the contribution of mitochondrial bioenergetics in migration, invasion, and metastasis is recently being understood. Migrating cancer cells adapt their energy needs to fluctuating changes in the microenvironment, exhibiting high metabolic plasticity. This occurs due to dynamic changes in the contributions of metabolic pathways to promote localized ATP production in lamellipodia and control signaling mediated by mitochondrial reactive oxygen species. Recent evidence has shown that metabolic shifts toward a mitochondrial metabolism based on the reductive carboxylation, glutaminolysis, and phosphocreatine-creatine kinase pathways promote resistance to anoikis, migration, and invasion in cancer cells. The PGC1a-driven metabolic adaptations with increased electron transport chain activity and superoxide levels are essential for metastasis in several cancer models. Notably, these metabolic changes can be determined by the composition and density of the extracellular matrix (ECM). ECM stiffness, integrins, and small Rho GTPases promote mitochondrial fragmentation, mitochondrial localization in focal adhesion complexes, and metabolic plasticity, supporting enhanced migration and metastasis. Here, we discuss the role of ECM in regulating mitochondrial metabolism during migration and metastasis, highlighting the therapeutic potential of compounds affecting mitochondrial function and selectively block cancer cell migration.

FRI-1 Is an Anti-Cancer Isoquinolinequinone That Inhibits the Mitochondrial Bioenergetics and Blocks Metabolic Shifts by Redox Disruption in Breast Cancer Cells.

Since breast cancer (BC) cells are dependent on mitochondrial bioenergetics for promoting proliferation, survival, and metastasis, mitochondria highlight as an important target for anticancer drug discovery. FRI-1, methyl 1, 3-dimethyl-5, 8-dioxo-5, 8-dihydro-4-isoquinolinecarboxylate, was previously described as a selective cytotoxic compound on cancer cell lines, however, details on the mechanism of action remain unknown. In this work, we describe that FRI-1 inhibits mitochondrial bioenergetics, producing apoptosis in MCF7 and MDA-MB-231 BC cell lines. FRI-1 decreases the maximal oxygen consumption rate (OCR), Δψm, NADH, and ATP levels, with a notable increase of mitochondrial reactive oxygen species (ROS) production, promoting AMPK activation with pro-survival effects. Moreover, FRI-1 inhibits the metabolic remodeling to glycolysis induced by oligomycin. In isolated tumoral mitochondria, FRI-1 increases Complex I and III-dependent OCR state 2, and this is sensitive to rotenone and antimycin A inhibitor additions, suggesting a redox cycling event. Remarkably, α-ketoglutarate and lipoic acid supplementation reversed and promoted, respectively, the FRI-1-induced apoptosis, suggesting that mitochondrial redox disruption affects 2-oxoglutarate dehydrogenase (OGDH) activity, and this is involved in their anticancer mechanism. Consistent with this, the combination of FRI-1 and CPI-613, a dual inhibitor of redox-sensible tricarboxylic acid (TCA) cycle enzymes PDH and OGDH, produced extensive BC cell death. Taken together, our results suggest that FRI-1 exhibits anticancer effects through inhibition of mitochondrial bioenergetics by redox disruption in BC cells.