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

Abstract

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.

Keywords: Hypoxia; Migrastatics; Mitochondrial bioenergetics; Paraguayan toad recipe; Proliferation; Rhinella schneideri.