Marinobufagin

Telocinobufagin and Marinobufagin Produce Different Effects in LLC-PK1 Cells: A Case of Functional Selectivity of Bufadienolides.[Pubmed:30223494]

Int J Mol Sci. 2018 Sep 14;19(9). pii: ijms19092769.

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na(+)/K(+)-ATPase inhibition, they activate signal transduction via protein(-)protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and Marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 muM (Marinobufagin) for pig kidney Na(+)/K(+)-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for Marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted Marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3beta inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3beta at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/beta-catenin pathway by acting upstream to beta-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.

Revealing of endogenous Marinobufagin by an ultra-specific and sensitive UHPLC-MS/MS assay in pregnant women.[Pubmed:29853035]

Talanta. 2018 Sep 1;187:193-199.

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the alpha1-isoform of Na(+),K(+)-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100mmx3mm; 3microm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.[Pubmed:29287997]

Toxicol Lett. 2018 Mar 15;285:121-131.

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 72h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC50 values ranging from 0.15 (leukemia) to 7.35 (larynx) muM] 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: 10.88muM] 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: 7.5muM) 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.