ObjectiveCinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of giant toads and is utilized in traditional Chinese medicine (Chan Su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Our lab is familiar with CINO and has shown it to inhibit cytotrophoblast cells function. Recently, it has been shown that CINO also inhibits the lung cancer cell function, and has been further implicated in other disease processes. In the present study, we propose to pursue this potential application of CINO using ovarian tumor cell line SK-OV-3.Study DesignWe evaluated the in-vitro effect of CINO on ovarian cancer cell line SK-OV-3. Cells were treated with 0.1, 1, 5, and 10 µM CINO. Cell proliferation was measured using a CellTiter Assay (Promega), which is a colorimetric method for determining the number of viable cells. Cell migration was measured using a CytoSelect Assay (Cell Biolabs). Cell invasion was measured using a FluoroBlok Assay (BD). Cell viability was measure using a CellTiter Assay (Promega). Cell cycle progression was evaluated by a Cell Cycle Phase Determination Kit (Cayman Chemical) and apoptosis was evaluated by an Apoptotic Blebs Assay Kit (Cayman Chemical). Cell cycle arrest and apoptotic signaling was determined by fluorescence-activated cell sorting (FACS) analysis.ResultsCINO at ≥0.5 µM inhibited SKOV-3 cell proliferation, migration, and invasion (p<0.05). There was a higher (p<0.05) percentage of S phase cells in groups treated with CINO at 0.5 µM. CINO at ≥0.5 µM down regulated expression of PCNA and caused cell death.ConclusionThis data demonstrates that CINO impairs SK-OV-3 cell function via cell cycle arrest and apoptotic signaling. These findings demonstrate the complex nature of this compound. Not only is CINO directly modulating the actions of the Na/K ATPase through classic mechanism of cardiotonic steroids, but is also directly influencing the nuclear expression of proteins involved in cell cycle progression and DNA repair. Additional investigational studies looking into the molecular pathways involved in altering cell cycle and entry into apoptosis are warranted.In conclusion, we have shown CINO to impair SK-OV3 cell function via cell cycle arrest and apoptotic signaling and suggest that CINO might be further investigated as a novel anti-ovarian cancer agent.

ObjectiveCinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Previously we have shown that CINO inhibits the cytotrophoblast cell function. Recently other study has shown that CINO inhibits A549, a lung cancer cell function. In this study, we assessed the effect of CINO on three different ovarian cancer cell lines; SK-OV-3, CRL-1978 and CRL-11731 to confirm whether the effect of CINO is cell specific.Study DesignWe evaluated the effect of CINO on three ovarian cancer cells SK-OV-3, CRL-1978, and CRL-11731 function in vitro. Each Cell lines were treated with different concentrations of CINO (0.1, 1, 5 and 10 µM). For each cell line cell proliferation, migration and invasion were measured by using a CellTiter Assay (Promega), Cytoselect Assay (Cell Biolabs) and by using a FluoroBlock Assay (BD) respectively. Proliferating Cell Nuclear Antigen (PCNA) was also evaluated in cell lysates of CINO treated these 3 ovarian cancer cells by western blot analysis. Cell Cycle arrest and Cell viability were determined by fluorescence-activated cell sorting (FACS) analysis. We also performed Annexin V staining on CINO treated these 3 ovarian cancer cell lines by immunofluorescence to evaluate the pro-apoptotic protein expression. In addition mitochondrial membrane potential has also been measured for all these 3 ovarian cell lines after CINO treatment using MMP kit, by FACS analysis.ResultsConcentration of CINO at 0.5 µM inhibit SK-OV-3, CRL-1978, and CRL-11731 ovarian cancer cells proliferation, migration and invasion without cell death and loss of cell viability but cell viability differs for each cell line. Each cell lines differ in response to CINO doses for PCNA expression as well as Annexin V pro-apoptotic protein expression. CINO decreases mitochondrial membrane potential for SK-OV-3 but for CRL-1978 and CRL-11731 increases in response to CINO treatment.ConclusionCINO is cell specific, as each cancer cell line responds differently. These data demonstrate that the mode of action of CINO is different on these 3 types of ovarian cancer cells.

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis-like factor, marinobufagenin (MBG) has been implicated as a causative factor in preE. We demonstrated that MBG inhibits the proliferation, migration, and invasion of cytotrophoblast (CTB) cells. We have identified a novel human monoclonal antibody with higher specificity than Digibind for MBG. We assessed the attenuation of MBG-induced CTBs dysfunction by three anti-MBG antibodies: 206–208, H1L2, and 3e9.MethodsA panel of anti-MBG antibodies with potential as human therapeutic agents was developed by Panorama Research, Inc.; Sunnyvale, CA (206–208, H1L2). H1L2 was a humanized version of previously described anti-MBG murine antibody 3e9. 206–208 was identified in a human phage antibody library. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG for 48 h. Some cells were pretreated with 206–208, H1L2, or 3e9 for 2h, while others were co-treated with these antibodies prior to MBG treatment. Culture media were collected for analysis of pro-angiogenic and anti-angiogenic factors. Cell viability was measured using a CellTiter Assay kit. Cell lysates were utilized to evaluate the expression of proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.ResultsMBG down-regulated PCNA and up-regulated p38 phosphorylation in CTBs treated with ≥1 nM MBG compared to basal (DMSO treatment) (*p<0.05 for each). Secretion of sFlt-1 and sEng were increased, while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG (*p<0.05 for each). Both anti-MBG antibodies pretreatment and co-treatment significantly up-regulated PCNA and down-regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p<0.05 for each). The anti-proliferative and anti-angiogenic effects of MBG were not due to a cytotoxic effect, as evaluated by a cell viability assay. MBG at 0.1 nM had no effect.ConclusionsWe found that all 3 anti-MBG antibodies attenuate MBG-induced anti-proliferative and anti-angiogenic milieu in cultured CTBs. Here we describe for the first time two fully human anti-MBG antibodies, which can be targeted as therapeutic agents for the development of innovative treatment strategies for preE and potentially other disorders involving aberrant MBG signaling.

ObjectivePreeclampsia (preE), a syndrome of hypertension and proteinuria. Most recently it was demonstrated that high circulating levels of soluble (pro) renin receptor s(P)RR at delivery were associated with preE. In this study the placental expression of (P)RR were evaluated in preE patients and in a rat model of preE as well as in nonhuman primates. We also evaluated the circulatory levels of s(P)RR.Study Design(1) Placental samples were collected from 20 NP and 20 preE consenting patients in an IRB approved prospective study. (2) An established rat model of preE and NP rats (n=10 each) were used. (3) The placental samples from squirrel monkey (NP; n=10) and owl monkey (both early and term, NP, n=1) were collected. The (P)RR expression were measured both by western blotting (WB) and Immunohistochemistry (IHC) using anti-ATP6IP2. The levels of serum s(P)RR were measured by ELISA.ResultsThe placental expression of (P)RR were higher (p<0.05) in preE compared to NP both in patients and rat model. The s(P)RR levels were higher in preE (preE patients: 29.2±4.5; PDS rats: 16.9±1.9 ng/mL) compared to NP (NP human: 19.3±4.2; NP rats: 10.4±3.7 ng/mL). The early placenta of owl monkey expressed higher (P)RR compared to term and were expressed in squirrel monkey placentas.ConclusionsThese data suggest that increased expression of (P)RR in the placenta are related to the occurrence of preE in both patients and rat models. These data also reconfirmed that the high level of circulatory s(P)RR is associated with preE. The higher expression of (P)RR in early owl monkey in compare to term placenta suggests that the (P)RR is important for normal placental development. The expression of (P)RR in nonhuman primates reveals the approach of future studies on owl monkey and squirrel monkey preE models.

ObjectiveDespite growing knowledge of the pathophysiology leading to the development of preeclampsia (PreE) and diabetes mellitus (DM), the interaction between the two disease processes needs to be further examined. This study compared normal pregnancies to those complicated with preE, gestational diabetes mellitus (GDM), and/or pre-existing diabetes in order to assess the effects of elevated glucose on placental development and its potential role in the pathogenesis of preE.MethodsThe chart review was performed in an IRB approved retrospective cross-sectional study of live born singleton deliveries. A total 621 subjects were randomly selected from deliveries occurring between 2008 to 2011 at Baylor Scott & White Memorial hospital. Statistical analysis was performed using Duncan's post-hoc test and ANOVA.ResultsPatients who developed preE had higher systolic and diastolic blood pressures than those who did not develop preE (p<0.05). Patients with either pre-existing diabetes or GDM were older. There was no difference among groups for gravidity (p=0.21) with an average gravidity of 2.7 (1.8SD) for 621 subjects and a range of 1 to 14 pregnancies. Patients with preE delivered earlier in pregnancy than those without preE regardless of diabetes status. However, those with preE and pre-existing diabetes delivered significantly earlier at 35.0+/−0.4 than the other two preE groups (*p<0.05 for each), suggesting more severe condition. Additionally, patients with pre-existing diabetes who developed preE delivered smaller babies than those with pre-existing diabetes without preE (1.00±0.03, p<0.05 for each). However, the development of GDM did not result in smaller babies for those pregnancies with preE.ConclusionsThe development of preE in those with pre-existing diabetes led to growth restriction and more severe disease as evidenced by lower birth weights and earlier gestational ages at delivery. These differences were not seen in GDM pregnancies. This supports the concept that elevated glucose levels during placental development in the first trimester may alter the placenta and lead to restriction later in pregnancy when a second stimulus triggers preE.