Toxic profile of marinobufagin from poisonous Amazon toads and antitumoral effects on human colorectal carcinomas.

ETHNOPHARMACOLOGICAL RELEVANCE: South Americans natives have extensively used the toad "kururu" to reduce/treat skin infections, cutaneous lesions and sores. They release secretions rich in bufadienolides, polyhydroxy steroids with well-documented cardiotonic and antiproliferative actions, but in vivo antitumoral evaluations in mammals are rare, and toxicological safety has been left in second place. AIMS OF THE STUDY: This investigation used in silico, in vitro and in vivo tools to evaluate acute and subacute toxic effects of marinobufagin and the anticancer action in tumor-bearing mice models. MATERIALS AND METHODS: Initially, in silico toxic predictions were performed, followed by in vitro assays using human and murine normal and tumor lines. Next, acute and subacute studies on mice investigated the behavior, hematological and intestinal transit profile and antitumoral activity of marinobufagin in sarcoma 180- and HCT-116 colorectal carcinoma-transplanted mice for 7 and 15 days, respectively. Ex vivo and in vivo cytogenetic assays in Sarcoma 180 and bone marrow cells and histopathological examinations were also executed. RESULTS: In silico studies revealed ecotoxicological effects on crustaceans (Daphnia sp.), fishes (Pimephales promelas and Oryzias latipes), and algae. A 24-h marinobufagin-induced acute toxicity included signals of central activity, mainly (vocal frenzy, absence of body tonus, increased ventilation, ataxia, and equilibrium loss), and convulsions and death at 10 mg/kg. The bufadienolide presented effective in vitro cytotoxic action on human lines of colorectal carcinomas in a similar way to ouabain and tumor reduction in marinobufagin-treated SCID-bearing HCT-116 heterotopic xenografts. Animals under subacute nonlethal doses exhibited a decrease in creatinine clearance with normal levels of blood urea, probably as a result of a marinobufagin-induced renal perfusion fall. Nevertheless, only minor morphological side effects were identified in kidneys, livers, hearts and lungs. CONCLUSIONS: Marinobufagin has in vitro and in vivo anticancer action on colorectal carcinoma and mild and reversible alterations in key metabolic organs without direct chemotherapy-induced gastrointestinal effects at subacute exposure, but it causes acute ataxia, equilibrium loss, convulsions and death at higher acute exposure.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) µM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC50: 10.88 µM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC50: 7.5 µM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer.

Bufadienolides from amphibians: A promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na+/K+-ATPase inhibition.

Amphibians present pharmacologically active aliphatic, aromatic and heterocyclic molecules in their skin as defense against microorganisms, predators and infections, such as steroids, alkaloids, biogenic amines, guanidine derivatives, proteins and peptides. Based on the discovered bioactive potential of bufadienolides, this work reviewed the contribution of amphibians, especially from members of Bufonidae family, as source of new cytotoxic and antitumor molecules, highlighting the mechanisms responsible for such amazing biological potentialities. Bufonidae species produce bufadienolides related to cholesterol through the mevalonate-independent and acidic bile acid pathways as polyhydroxy steroids with 24 carbons. In vitro antitumor studies performed with skin secretions and its isolated components (specially marinobufagin, telocinobufagin, bufalin and cinobufagin) from Rhinella, Bufo and Rhaebo species have shown remarkable biological action on hematological, solid, sensitive and/or resistant human tumor cell lines. Some compounds revealed higher selectivity against neoplastic lines when compared to dividing normal cells and some molecules may biochemically associate with Na+/K+-ATPase and there is structural similarity to the digoxin- and ouabain-Na+/K+-ATPase complexs, implying a similar mechanism of the Na+/K+-ATPase inhibition by cardenolides and bufadienolides. Some bufadienolides also reduce levels of antiapoptotic proteins and DNA synthesis, cause morphological changes (chromatin condensation, nuclear fragmentation, cytoplasm shrinkage, cytoplasmic vacuoles, stickiness reduction and apoptotic bodies), cell cycle arrest in G2/M or S phases, mitochondrial depolarization, PARP [poly (ADPribose) polymerase] and Bid cleavages, cytochrome c release, activation of Bax and caspases (-3, -9, -8 and -10), increased expression of the Fas-Associated protein with Death Domain (FADD), induce topoisomerase II inhibition, DNA fragmentation, cell differentiation, angiogenesis inhibition, multidrug resistance reversion, and also regulate immune responses. Then, bufadienolides isolated from amphibians, some of them at risk of extinction, emerge as a natural class of incredible chemical biodiversity, has moderate selectivity against human tumor cells and weak activity on murine cells, probably due to structural differences between subunits of human and mice Na+/K+-ATPases.