ß-Escin overcomes trastuzumab resistance in HER2-positive breast cancer by targeting cancer stem-like features.

BACKGROUND: The emergence of de novo or intrinsic trastuzumab resistance is exceedingly high in breast cancer that is HER2 positive and correlates with an abundant cancer stem cell (CSC)-like population. We sought to examine the capacity of ß-escin, an anti-inflammatory drug, to address trastuzumab resistance in HER2-positive breast cancer cells. METHODS: The effect of ß-escin on trastuzumab-resistant and -sensitive cell lines in vitro was evaluated for apoptosis, expression of HER2 family members, and impact on CSC-like properties. An in vivo model of trastuzumab-resistant JIMT-1 was used to examine the efficacy and toxicity of ß-escin. RESULTS: ß-escin induced mitochondrial-mediated apoptosis accompanied by reactive oxygen species (ROS) production and increased active p18Bax fragmentation, leading to caspase-3/-7 activation. Attenuation of CSC-related features by ß-escin challenge was accompanied by marked reductions in CD44high/CD24low stem-like cells and aldehyde dehydrogenase 1 (ALDH1) activity as well as hindrance of mammosphere formation. ß-escin administration also significantly retarded tumor growth and angiogenesis in a trastuzumab-resistant JIMT-1 xenograft model via downregulation of CSC-associated markers and intracellular domain HER2. Importantly, ß-escin selectively inhibited malignant cells and was less toxic to normal mammary cells, and no toxic effects were found in liver and kidney function in animals. CONCLUSIONS: Taken together, our findings highlight ß-escin as a promising candidate for the treatment of trastuzumab-resistant HER2-positive breast cancers.

ß-Escin reduces cancer progression in aggressive MDA-MB-231 cells by inhibiting glutamine metabolism through downregulation of c-myc oncogene.

BACKGROUND: The c-myc oncogene, which causes glutamine dependence in triple negative breast cancers (TNBC), is also the target of one of the signaling pathways affected by ß-Escin. METHODS AND RESULTS: We sought to determine how c-myc protein affects glutamine metabolism and the proteins, glutamine transporter alanine-serine-cysteine 2 (ASCT2) and glutaminase (GLS1), in ß-Escin-treated MDA-MB-231 cells using glutamine uptake and western blot analysis. Cell viability, colony formation, migration and apoptosis were also evaluated in MDA-MB-231 cells in response to ß-Escin treatment using MTS, colony forming, wound healing, and Annexin-V assay. We determined that ß-Escin decreased glutamine uptake and reduced c-myc and GLS1 protein expressions and increased the expression of ASCT2. In addition, this inhibition of glutamine metabolism decreased cell proliferation, colony formation and migration, and induced apoptosis. CONCLUSIONS: In this study, it was suggested that ß-Escin inhibits glutamine metabolism via c-myc in MDA-MB-231 cells, and it is thought that as a result of interrupting the energy supply in these cells via c-myc, it results in a decrease in the carcinogenic properties of the cells. Consequently, ß-Escin may be promising as a therapeutic agent for glutamine-dependent cancers.

The Biosurfactant ß-Aescin: A Review on the Physico-Chemical Properties and Its Interaction with Lipid Model Membranes and Langmuir Monolayers.

This review discusses recent progress in physicochemical understanding of the action of the saponin ß -aescin (also called ß -escin), the biologically active component in the seeds of the horse chestnut tree Aesculus hippocastanum. ß -Aescin is used in pharmacological and cosmetic applications showing strong surface activity. In this review, we outline the most important findings describing the behavior of ß -aescin in solution (e.g., critical micelle concentration ( c m c ) and micelle shape) and special physicochemical properties of adsorbed ß -aescin monolayers at the air-water and oil-water interface. Such monolayers were found to posses very special viscoelastic properties. The presentation of the experimental findings is complemented by discussing recent molecular dynamics simulations. These simulations do not only quantify the predominant interactions in adsorbed monolayers but also highlight the different behavior of neutral and ionized ß -aescin molecules. The review concludes on the interaction of ß -aescin with phospholipid model membranes in the form of bilayers and Langmuir monolayers. The interaction of ß -aescin with lipid bilayers was found to strongly depend on its c m c . At concentrations below the c m c , membrane parameters are modified whereas above the c m c , complete solubilization of the bilayers occurs, depending on lipid phase state and concentration. In the presence of gel-phase phospholipids, discoidal bicelles form; these are tunable in size by composition. The phase behavior of ß -aescin with lipid membranes can also be modified by addition of other molecules such as cholesterol or drug molecules. The lipid phase state also determines the penetration rate of ß -aescin molecules into lipid monolayers. The strongest interaction was always found in the presence of gel-phase phospholipid molecules.

Inhibition of Migration and Invasion in Melanoma Cells by ß-Escin via the ERK/NF-κB Signaling Pathway.

ß-Escin, a natural triterpene saponin was extracted from Aesculus hippocastanum seeds, which have been widely used to treat inflammation in traditional medicine. In an effort to study the possible anti-tumor effects of ß-escin, we performed wound healing, invasion, and adhesion assays to examine the effects of ß-escin on cell migration, invasion, and angiogenesis. Our results revealed that ß-escin inhibits cell migration as well as motility in B16F10 and SK-MEL5 cells in a dose-dependent manner. RT-PCR and Western blot analysis showed that ß-escin increased TIMP-1, -2 while significantly downregulated phosphorylated extracellular signal-regulated kinase (p-ERK) expression, and suppressing nuclear factor-kappa B (NF-κB) and inhibitor of nuclear factor-kappa B (IκB) expression. Overall, the data from the current study suggest that ß-escin has the potential for inhibiting both metastatic and angiogenic activities, and are the earliest evidence for the involvement of the NF-κB/IκB signaling in ß-escin-induced anti-tumor effects.

ß-Escin Effectively Modulates HUVECS Proliferation and Tube Formation.

In the present study we evaluated the anti-angiogenic activities of ß-escin (the major active compound of Aesculus hippocastanum L. seeds). Human umbilical-vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying the anti-angiogenic effect of ß-escin. We investigated the in vitro effects on proliferation, migration, and tube formation of HUVECs and in vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) angiogenesis assay. Moreover, the effect on gene expressions was determined by the RT2 ProfilerTM human angiogenesis PCR Array. It was found that ß-escin exerts inhibitory effect on the basic fibroblast growth factor (bFGF)-induced proliferation, migration and tube formation, as well as CAM angiogenesis in vivo. The inhibition of critical steps of angiogenic process observed with ß-escin could be partially explained by suppression of Akt activation in response to bFGF. Moreover, the anti-angiogenic effects of ß-escin could also be mediated via inhibition of EFNB2 and FGF-1 gene expressions in endothelial cells. In conclusion, ß-escin affects endothelial cells as a negative mediator of angiogenesis in vitro and in vivo and may therefore be considered as a promising candidate for further research elucidating its underlying mechanism of action.

ß-escin activates ALDH and prevents cigarette smoke-induced cell death.

BACKGROUND: The tobacco use is one of the biggest public health threats worldwide. Cigarette smoke contains over 7000 chemicals among other aldehydes, regarded as priority toxicants. ß-escin (a mixture of triterpenoid saponins extracted from the Aesculus hippocastanum. L) is a potent activator of aldehyde dehydrogenase (ALDH) - an enzyme catalyzing oxidation of aldehydes to non-toxic carboxylic acids. PURPOSE: The aim of this study was to evaluate the effect of ß-escin on ALDH activity, ALDH isoforms mRNA expression and cytotoxicity in nasal epithelial cells exposed to cigarette smoke extract (CSE). METHODS: Nasal epithelial cells from healthy non-smokers were treated with ß-escin (1 µM) and exposed to 5% CSE. After 6- or 24-hours of stimulation cell viability, DNA damage, ALDH activity and mRNA expression of ALDH isoforms were examined. RESULTS: 24 h ß-escin stimulation revised CSE induced cytotoxicity and DNA damage. Cells cultured with ß-escin or exposed to CSE responded with strong increase in ALDH activity. This effect was more pronounced in cultures treated with combination of ß-escin and CSE. The strongest stimulatory effect on ALDH isoform mRNA expression was observed in cells cultured simultaneously with ß-escin and CSE: at 6 h for ALDH1A1 and ALDH3A1, and at 24 h for ALDH1A3, ALDH3A2, ALDH3B1, and ALDH18A1. Combined ß-escin and CSE treatment prevented the CSE-induced inhibition of ALDH2 expression at 24 h. CONCLUSIONS: ß-escin is an effective ALDH stimulatory and cytoprotective agent and might be useful in the prevention or supportive treatment of tobacco smoke-related diseases.

A rapid method for total Β-escin analysis in dry, hydroalcoholic and hydroglycolic extracts of Aesculus hippocastanum L. by capillary zone electrophoresis.

INTRODUCTION: Seeds of Aesculus hippocastanum L. are used in European phytotherapy to treat inflammatory and vascular problems, and also to help in the regulation of the microcirculation. Thus, the quality control of herbal medicines using this species is important. OBJECTIVE: To develop and to optimise a capillary zone electrophoresis method to determine total ß-escin in different extracts of A. hippocastanum L. METHODS: The optimal condition found through chemometric approach was: 25 mmol/L of bicarbonate-carbonate buffer, pH 10.3; +20 kV of voltage; 20°C of cartridge temperature; direct ultraviolet detection at 226 nm; 13 mbar injection for 5 s and analysis time within 6 min. RESULTS: Repeatability, coefficient of variation (CV; %) = 3.19, 3.07 and 1.89 (n = 12), and intermediate precision, CV (%) = 3.05, 3.53 and 2.99 (n = 24) for dry, hydroalcoholic and hydroglycolic extracts, respectively were achieved. The accuracy was evaluated through recovery tests in concentration levels of 100, 150 and 200 g/L, ranging from 98.17 to 104.68%. The proposed method exhibited linearity (r = 0.9983) in the concentration range from 101.4 to 907.2 g/L and limits of detection and quantification equal to 11.63 and 38.76 g/L respectively. CONCLUSION: A fast and reliable methodology for determination of total ß-escin was successfully validated and applied on extracts of A. hippocastanum L. demonstrating its usefulness to quality control of medicines containing this plant species.

Effect of Escin Alone or in Combination with Antifungal Agents on Resistant Candida glabrata Biofilms: Mechanisms of Action.

Nowadays, the increase in antimicrobial-resistant fungi (AMR) is certainly a major health concern, and the development of alternative therapeutic strategies has become crucial. Natural products have been used to treat various infections, and their chemical properties contribute to the performance of their biological activities, such as antifungal action. The various virulence factors and mechanisms of resistance to antifungals contribute to making Candida glabrata one of the most frequent agents of candidiasis. Here we investigate the in vitro and in vivo activity of ß-escin against Candida glabrata. The ß-escin MICs were determined for a reference strain and two clinical isolates of C. glabrata. Furthermore, growth kinetics assays and biofilm inhibition/eradication assays (crystal violet) were performed. The differences in the expression of some anti-biofilm-associated genes were analyzed during biofilm inhibition treatment so that reactive oxygen species could be detected. The efficacy of ß-escin was evaluated in combination with fluconazole, ketoconazole, and itraconazole. In addition, a Galleria mellonella infection model was used for in vivo treatment assays. Results have shown that ß-escin had no toxicity in vitro or in vivo and was able to inhibit or destroy biofilm formation by downregulating some important genes, inducing ROS activity and affecting the membrane integrity of C. glabrata cells. Furthermore, our study suggests that the combination with azoles can have synergistic effects against C. glabrata biofilm. In summary, the discovery of new antifungal drugs against these resistant fungi is crucial and could potentially lead to the development of future treatment strategies.

Beneficial effects of ß-escin on muscle regeneration in rat model of skeletal muscle injury.

BACKGROUND: Recent advancements in understanding ß-escin action provide basis for new therapeutic claims for the drug. ß-escin-evoked attenuation of NF-κB-dependent signaling, increase in MMP-14 and decrease in COUP-TFII content and a rise in cholesterol biosynthesis could be beneficial in alleviating muscle-damaging processes. PURPOSE: The aim of this study was to investigate the effect of ß-escin on skeletal muscle regeneration. METHODS: Rat model of cardiotoxin-induced injury of fast-twich extensor digitorum longus (EDL) and slow-twich soleus (SOL) muscles and C2C12 myoblast cells were used in the study. We evaluated muscles obtained on day 3 and 14 post-injury by histological analyses of muscle fibers, connective tissue, and mononuclear infiltrate, by immunolocalization of macrophages and by qPCR to quantify the expression of muscle regeneration-related genes. Mechanism of drug action was investigated in vitro by assessing cell viability, NF-κB activation, MMP-2 and MMP-9 secretion, and ALDH activity. RESULTS: In rat model, ß-escin rescues regenerating muscles from atrophy. The drug reduces inflammatory infiltration, increases the number of muscle fibers and decreases fibrosis. ß-escin reduces macrophage infiltration into injured muscles and promotes their M2 polarization. It also alters transcription of muscle regeneration-related genes: Myf5, Myh2, Myh3, Myh8, Myod1, Pax3 and Pax7, and Pcna. In C2C12 myoblasts in vitro, ß-escin inhibits TNF-α-induced activation of NF-κB, reduces secretion of MMP-9 and increases ALDH activity. CONCLUSIONS: The data reveal beneficial role of ß-escin in muscle regeneration, particularly in poorly regenerating slow-twitch muscles. The findings provide rationale for further studies on ß-escin repositioning into conditions associated with muscle damage such as strenuous exercise, drug-induced myotoxicity or age-related disuse atrophy.

The Natural Product ß-Escin Targets Cancer and Stromal Cells of the Tumor Microenvironment to Inhibit Ovarian Cancer Metastasis.

The high mortality of OvCa is caused by the wide dissemination of cancer within the abdominal cavity. OvCa cells metastasize to the peritoneum, which is covered by mesothelial cells, and invade into the underlying stroma, composed of extracellular matrices (ECM) and stromal cells. In a study using a three-dimensional quantitative high-throughput screening platform (3D-qHTS), we found that ß-escin, a component of horse chestnut seed extract, inhibited OvCa adhesion/invasion. Here, we determine whether ß-escin and structurally similar compounds have a therapeutic potential against OvCa metastasis. Different sources of ß-escin and horse chestnut seed extract inhibited OvCa cell adhesion/invasion, both in vitro and in vivo. From a collection of 160 structurally similar compounds to ß-escin, we found that cardiac glycosides inhibited OvCa cell adhesion/invasion and proliferation in vitro, and inhibited adhesion/invasion and metastasis in vivo. Mechanistically, ß-escin and the cardiac glycosides inhibited ECM production in mesothelial cells and fibroblasts. The oral administration of ß-escin inhibited metastasis in both OvCa prevention and intervention mouse models. Specifically, ß-escin inhibited ECM production in the omental tumors. Additionally, the production of HIF1α-targeted proteins, lactate dehydrogenase A, and hexokinase 2 in omental tumors was blocked by ß-escin. This study reveals that the natural compound ß-escin has a therapeutic potential because of its ability to prevent OvCa dissemination by targeting both cancer and stromal cells in the OvCa tumor microenvironment.

Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach.

Ca2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the 'added buffer' approach [1] with perforated patch-clamp recordings [2]. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

Perforated Whole-Cell Recordings in Automated Patch Clamp Electrophysiology.

The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca2+), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.

Influence of Acetylated Annealed Starch on the Release of ß-Escin from the Anionic and Non-Ionic Hydrophilic Gels.

Naturally sourced products introduced to human nutrition and rediscovered for therapy include polysaccharides from potatoes. The starch may obtain unique properties via acetylation with acetic anhydride at 13 cm3/100 g of starch as the basic dose of reagent used in industrial conditions. The hydrogel formulation was applied as a carrier for escin included in the dry extract of Aesculus hippocastanum. Six hydrogels were evaluated (methylcellulose, polyacrylic acid-Carbopol 980 NF and polyacrylate crosspolymer 11-Aristoflex Velvet) with various concentrations of the modified starch. The kinetic studies of in vitro ß-escin release were carried out in purified water at 37 ± 0.5 °C using a paddle apparatus at 50 rpm and a time period of 7 h. The criterion for the most suitable model was based on a high correlation coefficient of evaluated release profiles. The addition of modified annealed acetylated potato starch resulted in prolongation of ß-escin release.

ß-Escin alleviates cobalt chloride-induced hypoxia-mediated apoptotic resistance and invasion via ROS-dependent HIF-1α/TGF-ß/MMPs in A549 cells.

Hypoxia is contributed in various pathophysiological conditions including obesity, cardiovascular diseases, and cancer. In cancer, hypoxia is a salient phenomenon and has been correlated with tumor progression, metastasis, and provoke resistance to therapies in cancer patients, which exert with stabilization of main effector, hypoxia inducible factor-1 alpha (HIF-1α). Therefore, therapeutic targeting of hypoxic responses in cancer is the potential approach to improve the better treatment efficacy. In the present study, we evaluated the effect of ß-Escin (ß-Es) on hypoxia-induced resistance to apoptosis and metastasis in human non-small-cell lung cancer cells. The MTT assay revealed that ß-Es treatment decreased the A549 cells viability under cobalt chloride-induced hypoxia. Apoptotic proteins were analyzed by western blot that showed cancer cells treated with ß-Es induced cell death in hypoxia condition as proteins compared with normoxia. Moreover, we observed that cobalt chloride induced hypoxia through the generation of intracellular reactive oxygen species and stabilized the transcriptional factor HIF-1α, which leads to cancer metastasis. This notion was supported by the migration, invasion, and adhesion assays. Furthermore, hypoxia increased the expression of transforming growth factor-ß, and the activation of matrix metalloproteinases were suppressed by the treatment of ß-Es as well as pretreatment with N-acetylcysteine (NAC). Therefore, we demonstrate that a concurrent activation of HIF-1α, transforming growth factor-ß, and matrix metalloproteinases participate in hypoxia-induced metastasis and that ß-Es prevent A549 cells metastasis by inhibition of reactive oxygen species.

Activation of p53 Gene Expression and Synergistic Antiproliferative Effects of 5-Fluorouracil and ß-escin on MCF7 Cells.

One of the most common malignancies in women is breast cancer. ß-escin has pharmacological anticancer effects. 5-fluorouracil (5-FU) has antimetabolite and antiproliferative properties. The purpose of this study was to investigate the combined effects of 5-FU and ß-escin on apoptosis, colony formation, Bcl-2 signaling protein, and p53 gene expression in MCF7 breast cancer cell line. The cytotoxic effects, the number of colonies, apoptosis, p53 gene expression, and Bcl-2 signaling protein of the combined 5-FU and ß-escin on MCF7 cells were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clonogenic assay, flow cytometry, real-time quantitative polymerase chain reaction, and western blotting methods, respectively. Half-maximal inhibitory concentration values of ß-escin and 5-FU were 80 µg/ml and 2 µM, respectively. The combination of 5-FU and ß-escin on MCF7 cell viability showed a combination index equal to 0.5. The expression of p53 and apoptosis increased in the combination of 5-FU and ß-escin on MCF7 cells compared to that of control group (P < 0.05). In addition, the number of colonies and Bcl-2 signaling protein in combination of 5-FU and ß-escin decreased with respect to untreated control cells or single treatment of 5-FU and ß-escin. The combination of 5-FU and ß-escin not only has synergistic effects by increasing cell apoptosis and p53 gene expression but also decreases Bcl-2 signaling protein in MCF7 cell lines.

Aesculus hippocastanum L. seed extract shows virucidal and antiviral activities against respiratory syncytial virus (RSV) and reduces lung inflammation in vivo.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease and bronchiolitis in children worldwide. No vaccine or specific, effective treatment is currently available. ß-escin is one of the main bioactive constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), and both ß-escin and AH have demonstrated a beneficial role in clinical therapy because of their anti-edematous, anti-inflammatory and antioxidative effects. Besides, we have reported that ß-escin and AH show virucidal, antiviral and immunomodulatory activities against the enveloped viruses HSV-1, VSV and Dengue virus in vitro. In this study, we demonstrate that ß-escin and AH have virucidal and antiviral activities against RSV, as well as NF-κB, AP-1 and cytokine modulating activities in RSV infected epithelial and macrophage cell lines in vitro. Besides, in a murine model of pulmonary RSV infection, AH treatment improves the course of acute disease, evidenced by decreased weight loss, reduced RSV lung titers, and attenuated airway inflammation. In contrast, even though ß-escin showed, similarly to AH, antiviral and immunomodulatory properties in vitro, it neither reduces viral titers nor attenuates lung injury in vivo. Thus, our data demonstrate that AH restrains RSV disease through antiviral and immunomodulatory effect.

Virucidal, antiviral and immunomodulatory activities of ß-escin and Aesculus hippocastanum extract.

OBJECTIVES: ß-Escin, one of the constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), inhibits NF-κB activation, which plays an important role in HSV-1 replication. The aim was to examine the antiherpetic activity of ß-escin and AH, as well as their effect on the activation of NF-κB and AP-1 and cytokine secretion in epithelial cells and macrophages. METHODS: Cell viability was evaluated using MTT assay, and antiviral and virucidal activity was determined by plaque assay. The effect on NF-κB and AP-1 signalling pathways activation was determined by a luciferase reporter assay, and cytokine production was measured by ELISA. KEY FINDINGS: ß-Escin and AH had virucidal and anti-HSV-1 activities, and the antiviral activity was discovered for other enveloped viruses (VSV and Dengue). Moreover, ß-escin and AH significantly reduced NF-κB and AP-1 activation and cytokine production in macrophages stimulated with HSV-1 and TLRs ligands. However, an enhanced activation of these pathways and an increase in the levels of pro-inflammatory cytokines in ß-escin and AH-treated HSV-1-infected epithelial cells were found. CONCLUSIONS: This study demonstrates virucidal and broad-spectrum antiviral activities for ß escin and AH. Besides, ß-escin and AH modulate cytokine production depending on the stimuli (viral or non-viral) and the cell type under study.

Regulation of RhoA/ROCK and sustained arterial contraction by low cytosolic Ca2+ levels during prolonged depolarization of arterial smooth muscle.

The role of L-type Ca2+ channels (LTCCs) and RhoA/Rho kinase (ROCK) on depolarization-induced sustained arterial contraction lasting several minutes is already known. However, in vivo, vascular smooth muscle cells can be depolarized for longer periods, inducing substantial inactivation of LTCCs and markedly reducing Ca2+ influx into the myocytes. We have examined, in femoral arterial rings, the role of LTCCs and RhoA/ROCK during long-lasting depolarization. Our results reveal a new vasoreactive response after 20-30min of depolarization in 2.5mM external Ca2+ that has not been identified previously with shorter stimuli. Prolonged depolarization-induced arterial contraction was permanently abolished when arterial rings were treated with 100nM external Ca2+ or 20nM nifedipine. However, when Ca2+ influx was restricted, applying ~7µM external Ca2+ solution or 3nM nifedipine, vasorelaxation was transient, and isometric force slowly increased after 30min and maintained its level until the end of the stimulus. Under these conditions, arterial contraction showed the same temporal course of RhoA activity and was sensitive to fasudil, nifedipine and cyclopiazonic acid. Ca2+-response curve in ß-escin permeabilized arteries was also sensitive to ROCK inhibitors. Thus, although long-lasting depolarization inactivates LTCCs, the reduced Ca2+ entry can induce a detectable arterial contraction via RhoA/ROCK activation.

ß-escin reverses multidrug resistance through inhibition of the GSK3ß/ß-catenin pathway in cholangiocarcinoma.

AIM: To develop a safe and effective agent for cholangiocarcinoma (CCA) chemotherapy. METHODS: A drug combination experiment was conducted to determine the effects of ß-escin in combination with chemotherapy on CCA cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of ß-escin and common chemotherapeutics on the proliferation of human CCA cells (QBC939, Sk-ChA-1, and MZ-ChA-1). Immunocytochemistry was used to detect the expression of P-glycoprotein (P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/ß-catenin pathway. The protein levels of P-gp, pS9-GSK3ß, pT216-GSK3ß, GSK3ß, ß-catenin, and p-ß-catenin were further confirmed by western blotting. RESULTS: The drug sensitivity of QBC939 and QBC939/5-fluorouracil (5-FU) cells to 5-FU, vincristine sulfate (VCR), or mitomycin C was significantly enhanced by ß-escin compared with either agent alone (P<0.05). In addition, the combination of ß-escin (20 µmol/L) with 5-FU and VCR was synergic with a combination index<1. Further investigation found that the mRNA and protein expression of P-gp was down-regulated by ß-escin. Moreover, ß-escin induced GSK3ß phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of ß-catenin. Interestingly, activation of the GSK3ß/ß-catenin pathway induced by Wnt3a resulted in up-regulation of P-gp, which was effectively abolished by ß-escin, indicating that ß-escin down-regulated P-gp expression in a GSK3ß-dependent manner. CONCLUSION: ß-escin was a potent reverser of P-gp-dependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3ß/ß-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.