A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids.

We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbent assay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a polyclonal antibody raised against a novel MBG-bovine serum albumin conjugate, this assay achieved an MBG detection limit of less than 9 pg/mL. MBG levels in various rat urine and serum samples were effectively determined using this methodology. Interassay variability averaged 9.8%, while intra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respectively. Recovery of exogenously added MBG averaged 106%, and parallelism data further established the accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerful tool for the possible diagnosis, prevention and management of human hypertensive states, particularly preeclampsia.

Resibufogenin corrects hypertension in a rat model of human preeclampsia.

The study of the pathogenesis of preeclampsia has been hampered by a relative dearth of animal models. We developed a rat model of preeclampsia in which the excretion of a circulating inhibitor of Na/K ATPase, marinobufagenin (MBG), is elevated. These animals develop hypertension, proteinuria, and intrauterine growth restriction. The administration of a congener of MBG, resibufogenin (RBG), reduces blood pressure to normal in these animals, as is the case when given to pregnant animals rendered hypertensive by the administration of MBG. Studies of Na/K ATPase inhibition by MBG and RBG reveal that these agents are equally effective as inhibitors of the enzyme.

Cephalostatin 1 selectively triggers the release of Smac/DIABLO and subsequent apoptosis that is characterized by an increased density of the mitochondrial matrix.

Cephalostatin 1 is a bis-steroidal marine natural product with a unique cytotoxicity profile in the in vitro screen system of the National Cancer Institute, suggesting that it may affect novel molecular target(s). Here we show that cephalostatin 1 induces a novel pathway of receptor-independent apoptosis that selectively uses Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) as a mitochondrial signaling molecule. At nanomolar concentrations, cephalostatin 1 triggers dose- and time-dependent DNA fragmentation in leukemia Jurkat T cells. Apoptosis was found to be dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone blocks cephalostatin 1-mediated DNA fragmentation. The CD95 death receptor as well as other caspase-8-requiring death receptors were not involved because Jurkat T cells lacking the CD95 receptor or caspase-8 and control cells responded equally to cephalostatin 1. Although cephalostatin 1 affects mitochondria by dissipating the mitochondrial membrane potential, neither cytochrome c nor apoptosis-inducing factor is released, as shown by Western blot analysis. Interestingly, cephalostatin 1 selectively triggers the mitochondrial release of the inhibitor of apoptosis antagonist Smac/DIABLO. Overexpression of the antiapoptotic protein Bcl-x(L) delayed both Smac/DIABLO release and onset of apoptosis, suggesting that Smac/DIABLO is required for cephalostatin 1-induced apoptosis. This new mitochondrial pathway is accompanied by marked structural changes of mitochondria as shown by transmission electron microscopy.

QSAR evaluation of the Ch'an Su and related bufadienolides against the colchicine-resistant primary liver carcinoma cell line PLC/PRF/5(1).

QSAR analysis has been used to identify the essential structural requirements for increasing the inhibitory activities of selected bufadienolides from the Chinese drug Ch'an Su (and other sources) against the primary liver carcinoma cell line PLC/PRF/5 (PLC) and the derived colchicine-resistant line (COL). The variable substituent domain of the proposed pharmacophore of the bufadienolides was investigated using a Comparative Molecular Field Analysis (CoMFA) approach. A model with considerable predictive ability was obtained. In addition, the CoMFA results agreed well with the pharmacophore bufadienolide model for the parent PLC line proposed earlier.

Biological activity of a novel nonpeptide antagonist to the interleukin-6 receptor 20S,21-epoxy-resibufogenin-3-formate.

Interleukin (IL)-6 is a key mediator in the regulation and coordination of the immune response and participates in pathogenesis of cancer cachexia, autoimmune disease, and postmenopausal osteoporosis. In the course of a screening program aimed at IL-6 inhibitor from natural products, we isolated 20S,21-epoxy-resibufogenin-3-formate (ERBF) from bufadienolide and examined the effect of ERBF on activities of various cytokines. ERBF dose dependently suppressed IL-6 activity and caused a parallel rightward shift of dose-response curves to IL-6 at concentrations of 0.03 to 10 ng/ml. Analysis of data yields a pA(2) of 5.12 and a slope of 0.99. Selectivity of ERBF on activity of cytokines was examined using cytokine-dependent cell lines. ERBF did not affect IL-2-dependent growth of CTLL-2 cells, IL-3-dependent growth of Baf3 cells, or tumor necrosis factor (TNF)alpha-induced growth suppression in TNFalpha-sensitive L929 cells. ERBF also did not affect IL-4-stimulated expression of FcepsilonR II receptor (CD23) in U-937 cells, the IL-8-induced chemotaxis of human neutrophils, or nerve growth factor-stimulated neuronal differentiation in PC-12 cells. In contrast, ERBF dose dependently suppressed IL-6-induced neuronal differentiation in PC-12 cells. Furthermore, ERBF suppressed only IL-6-induced osteoclast formation without affecting osteoclast formation induced by IL-11, leukemia inhibitory factor, and 1alpha,25-dihydroxyvitamin D(3). In receptor binding assay, unbound (free) IL-6 was increased in a dose-dependent manner by pretreatment with ERBF on IL-6 receptor (IL-6R), suggesting that ERBF suppresses binding of IL-6 to IL-6R. These results clearly indicate that ERBF is a novel specific small molecule to show IL-6 receptor antagonist activity.

Isolation and structure of a 20,21-epoxybufenolide series from "Ch'an Su".

Steroid bufenolides resulting from epoxidation of the 17beta-2-pyrone ring of bufadienolides are rare. Five 20,21-epoxybufenolides, namely, 20S,21-epoxyresibufogenin (1), 20R,21-epoxyresibufogenin (2), 3-O-formyl-20S,21-epoxyresibufogenin (3), 3-O-formyl-20R,21-epoxyresibufogenin (4), and 3-oxo-20S,21-epoxyresibufogenin (5), were isolated from the Chinese toad skin extract drug Ch'an Su. The structures were elucidated by spectroscopic and chemical methods. The configuration at C-20 was assigned by the analysis of difference NOE spectra. The cancer cell (KB and MH-60) growth inhibition by the new 20,21-epoxybufenolides was examined, and 20,21-epoxides 1, 2, and 5 were found to significantly inhibit the leukemia MH-60 cell line.