A novel machine learning-based screening identifies statins as inhibitors of the calcium pump SERCA.

We report a novel small-molecule screening approach that combines data augmentation and machine learning to identify Food and Drug Administration (FDA)-approved drugs interacting with the calcium pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) from skeletal (SERCA1a) and cardiac (SERCA2a) muscle. This approach uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, thus allowing to screen with high precision large databases of small molecules, including approved and investigational drugs. We chose SERCA because it plays a major role in the excitation-contraction-relaxation cycle in muscle and it represents a major target in both skeletal and cardiac muscle. The machine learning model predicted that SERCA1a and SERCA2a are pharmacological targets for seven statins, a group of FDA-approved 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in the clinic as lipid-lowering medications. We validated the machine learning predictions by using in vitro ATPase assays to show that several FDA-approved statins are partial inhibitors of SERCA1a and SERCA2a. Complementary atomistic simulations predict that these drugs bind to two different allosteric sites of the pump. Our findings suggest that SERCA-mediated Ca2+ transport may be targeted by some statins (e.g., atorvastatin), thus providing a molecular pathway to explain statin-associated toxicity reported in the literature. These studies show the applicability of data augmentation and machine learning-based screening as a general platform for the identification of off-target interactions and the applicability of this approach extends to drug discovery.

Synthesis, computational docking and biological evaluation of celastrol derivatives as dual inhibitors of SERCA and P-glycoprotein in cancer therapy.

A series of eleven celastrol derivatives was designed, synthesized, and evaluated for their in vitro cytotoxic activities against six human cancer cell lines (A549, HepG2, HepAD38, PC3, DLD-1 Bax-Bak WT and DKO) and three human normal cells (LO2, BEAS-2B, CCD19Lu). To our knowledge, six derivatives were the first example of dipeptide celastrol derivatives. Among them, compound 3 was the most promising derivative, as it exhibited a remarkable anti-proliferative activity and improved selectivity in liver cancer HepAD38 versus human normal hepatocytes, LO2. Compound 6 showed higher selectivity in liver cancer cells against human normal lung fibroblasts, CCD19Lu cell line. The Ca2+ mobilizations of 3 and 6 were also evaluated in the presence and absence of thapsigargin to demonstrate their inhibitory effects on SERCA. Derivatives 3 and 6 were found to induce apoptosis on LO2, HepG2 and HepAD38 cells. The potential docking poses of all synthesized celastrol dipeptides and other known inhibitors were proposed by molecular docking. Finally, 3 inhibited P-gp-mediated drug efflux with greater efficiency than inhibitor verapamil in A549 lung cancer cells. Therefore, celastrol-dipeptide derivatives are potent drug candidates for the treatment of drug-resistant cancer.

Large Scale Conversion of Trilobolide into the Payload of Mipsagargin: 8-O-(12-Aminododecanoyl)-8-O-Debutanoylthapsigargin.

In spite of the impressing cytotoxicity of thapsigargin (Tg), this compound cannot be used as a chemotherapeutic drug because of general toxicity, causing unacceptable side effects. Instead, a prodrug targeted towards tumors, mipsagargin, was brought into clinical trials. What substantially reduces the clinical potential is the limited access to Tg and its derivatives and cost-inefficient syntheses with unacceptably low yields. Laser trilobum, which contains a structurally related sesquiterpene lactone, trilobolide (Tb), is successfully cultivated. Here, we report scalable isolation of Tb from L. trilobum and a transformation of Tb to 8-O-(12-aminododecanoyl)-8-O-debutanoylthapsigargin in seven steps. The use of cultivated L. trilobum offers an unlimited source of the active principle in mipsagargin.

Targeted Delivery of Curcumin Rescues Endoplasmic Reticulum-Retained Mutant NOX2 Protein and Avoids Leukocyte Apoptosis.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitors could be used to rescue mutant H338Y-gp91phox protein of a particular type of CGD with a CybbC1024T mutation, leading to endoplasmic reticulum (ER) retention of the mutant protein. In this study, we developed a novel mouse model with the CybbC1024T mutation on a Cybb knockout background and investigated the therapeutic effects of ER-targeted delivery of the SERCA inhibitor, curcumin, with poly(lactic-coglycolic acid) (PLGA) nanoparticles (NPs). We found that PLGA encapsulation improved the efficacy of curcumin as a SERCA inhibitor to induce ER calcium release. ER-targeting curcumin-loaded PLGA NPs reduced and delayed extracellular calcium entry and protected the cells from mitochondrial damage and apoptosis. In vivo studies showed that ER-targeting curcumin-loaded PLGA NPs treatment enhanced neutrophil gp91phox expression, ROS production and peritoneal bacterial clearance ability of the CybbC1024T transgenic Cybb -/- mice. Our findings indicate that ER-targeted delivery of curcumin not only rescues ER-retained H338Y-gp91phox protein, and hence leukocyte function, but also enhances the bioavailability and reduces cytotoxicity. Modulation of ER function by using organelle-targeted NPs may be a promising strategy to improve the therapeutic potential of curcumin as a treatment for CGD.

Content decline of SERCA inhibitors saikosaponin a and d attenuates cardiotoxicity and hepatotoxicity of vinegar-baked Radix bupleuri.

Improper usage of unprocessed Radix bupleuri root (chaihu) may cause cardiotoxicity and liver injury. Baking herb with vinegar is believed to attenuate the adverse responses. However, the chemical and molecular basis involved remained unclear. To this end, we investigated the in vitro toxicity of saikosaponin a, c, d, and their hydrolysates saikosaponin b1 and b2. Results showed that SSa and SSd possessed higher affinity with sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) by molecular docking, and exhibited stronger toxic responses on cardiomyocytes and hepatocytes than the other three saikosaponins in equivalent concentrations. Further, SSa and SSd induced LC3 puncta formation in U2OS-mCherry-EGFP-LC3 cells. Blockage of autophagy by 3-methyladenine did not abrogate the cytotoxicities induced by SSa and SSd. In parallel, none of SSc, SSb1, or SSb2 caused cell injury. Our study reveals how changes in chemical ingredients are connected to the toxicity of Chaihu during vinegar baking process and also provides a guidance for structure optimization to reduce drug induced toxicity.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA.

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA's distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA's ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

Molecular Modeling of Fluorescent SERCA Biosensors.

Molecular modeling and simulation are useful tools in structural biology, allowing the formulation of functional hypotheses and interpretation of spectroscopy experiments. Here, we describe a method to construct in silico models of a fluorescent fusion protein construct, where a cyan fluorescent protein (CFP) is linked to the actuator domain of the Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). This CFP-SERCA construct is a biosensor that can report on structural dynamics in the cytosolic headpiece of SERCA. Molecular modeling and FRET experiments allow us to generate new structural and mechanistic models that better describe the conformational landscape and regulation of SERCA. The methods described here can be applied to the creation of models for any fusion protein constructs and also describe the steps needed to simulate FRET results using molecular models.

Small molecule RL71 targets SERCA2 at a novel site in the treatment of human colorectal cancer.

While targeted agents are an important part of the treatment arsenal for colorectal cancer, there is still a lack of efficient small-molecule targeted agents based on the understanding of pathogenic molecular mechanisms. In this study, curcumin analog RL71 displayed potent cytotoxicity towards human colon cancer cells with an IC50 of 0.8 µM in SW480 cells and inhibited xenotransplanted tumor growth in a dose-dependent manner. Using affinity chromatography, we identified sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 as the binding target of RL71. Most notably, RL71 demonstrated special binding to SERCA2 at a novel site with the lowest estimated free energy -8.89 kcal mol(-1), and the SERCA2 residues critical for RL71 binding were identified. RL71 suppressed the Ca(2+)-ATPase activity of SERCA2 both in vitro and in vivo, accompanied by the induction of endoplasmic reticulum stress leading to apoptosis and G2/M cycle arrest in SW480 cells. In addition, RL71 showed synergistic cytotoxicity with the pan-SERCA inhibitor thapsigargin. These results suggest that RL71 could be a selective small-molecule inhibitor of SERCA2, and that it may serve as a lead compound for the study of targeted colorectal cancer therapy.

Related flavonoids cause cooperative inhibition of the sarcoplasmic reticulum Ca²âº ATPase by multimode mechanisms.

Flavonoids are group of plant-derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio-accumulate within humans and are considered to have beneficial health effects, including cancer chemoprotection. One mechanism proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca²âº ATPase. An investigation was undertaken with respect to the mechanism of inhibition for three flavonoids: quercetin, galangin and 3,6 dihydroxyflavone (3,6-DHF). Each inhibited the Ca²âº ATPase with K(i) values of 8.7, 10.3 and 5.4 µM, respectively, showing cooperative inhibition with n ~ 2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E1 conformation and reduced [³²P]-ATP binding. However, both galangin and 3,6-DHF increased the affinity of Ca²âº for the ATPase by decreasing the Ca²âº-dissociation rate constant, whereas quercetin had little effect. Ca²âº-induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6-DHF and galangin (but not with quercetin), indicating that Ca²âº-associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP-dependent phosphorylation and dephosphorylation, whereas 3,6-DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites: one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesize that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids.

Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells.

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase pump, leading to autophagy induction through the activation of the Ca(2+)/calmodulin-dependent kinase kinase-AMP-activated protein kinase-mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

Contribution of endoplasmic reticulum Ca2+ regulatory mechanisms to the inflammation-induced increase in the evoked Ca2+ transient in rat cutaneous dorsal root ganglion neurons.

Persistent inflammation results in an increase in the magnitude and duration of high K(+)-evoked Ca(2+) transients in putative nociceptive cutaneous dorsal root ganglion (DRG) neurons. The purpose of the present study was to determine whether recruitment of Ca(2+)-induced Ca(2+) release (CICR) contributes to these inflammation-induced changes. Acutely dissociated, retrogradely labeled cutaneous DRG neurons from naïve and complete Freund's adjuvant inflamed adult male Sprague-Dawley rats were studied with ratiometric microfluorimetry. Ryanodine only attenuated the duration but not magnitude of the high K(+)-evoked Ca(2+) transient in neurons from inflamed rats. However, there was no significant impact of inflammation on the potency or efficacy of ryanodine-induced block of the caffeine-evoked Ca(2+) transient, or the impact of sarco-endoplasmic reticulum ATPase (SERCA) inhibition on the high K(+)-evoked Ca(2+) transient. Furthermore, while there was no change in the magnitude, an inflammation-induced increase in the duration of the caffeine-evoked Ca(2+) transient was only observed with a prolonged caffeine application. In contrast to the high K(+)-evoked Ca(2+) transient, there was no evidence of direct mitochondrial involvement or that of the Ca(2+) extrusion mechanism, the Na(+)/Ca(2+) exchanger, on the caffeine-evoked Ca(2+) transient, and block of SERCA only increased the duration of this transient. These results indicate the presence of Ca(2+) regulatory domains in cutaneous nociceptive DRG neurons within which cytosolic Ca(2+) increased via influx and release are highly segregated. Furthermore, our results suggest that changes in neither CICR machinery nor the coupling between Ca(2+) influx and CICR are primarily responsible for the inflammation-induced changes in the evoked Ca(2+) transient.

Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal.

AIM: To investigate the effects of Lizhong Tang, an herbal product used in traditional Chinese medicine, on mouse small intestine interstitial cells of Cajal (ICCs). METHODS: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. All of the experiments were performed at 30-32 °C. RESULTS: ICCs generated pacemaker potentials, and Lizhong Tang produced membrane depolarization in current-clamp mode. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang. Pretreatment with thapsigargin (a Ca²âº-ATPase inhibitor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang. However, pacemaker potentials were completely abolished in the presence of an external Ca²âº-free solution, and under this condition, Lizhong Tang induced membrane depolarizations. Furthermore, When GDP-ß-S (1 mmol/L) was in the pipette solution, Lizhong Tang still induced membrane depolarizations. In addition, membrane depolarizations were not inhibited by chelerythrine or calphostin C, which are protein kinase C inhibitors, but were inhibited by U-73122, an active phospholipase C inhibitors. CONCLUSION: These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.

Hypoglycemic effect of Bumelia sartorum polyphenolic rich extracts.

Bumelia sartorum (Sapotaceae) is used ethnomedicinally for treatment of several diseases, including diabetes mellitus. The aim of this work was to investigate the hypoglycemic effect of B. sartorum extracts, rich in polyphenolic compounds, and the possible mechanisms of action. Assessment of B. sartorum hypoglycemic activity was performed from the blood glucose level in normoglycemic mice after administration of the extract by oral gavage. The hypothesis that sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition could prolong the increase in cytoplasmic Ca2+ concentration, thus leading to an increase of insulin release was evaluated. The enzyme inhibition was measured by ATP hydrolysis using SERCA1 isolated from rabbit skeletal muscle. The total content of phenolic compounds was determined by the Folin-Ciocalteau method. The ethyl acetate (EtOAc) partition and F5 fraction obtained from B. sartorum, both of them rich in polyphenolics, were shown to have a hypoglycemic effect on normoglycemic mice, more significant than that of the known antidiabetic drug, glibenclamide used as a standard comparable compound. Both samples significantly inhibited SERCA activity. Different extracts of B. sartorum, rich in polyphenolic compounds, were able to reduce blood glucose in normoglycemic mice and inhibit SERCA activity. SERCA inhibition may be one of the possible mechanisms involved in glucose decrease.

Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer.

Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. Sarco/endoplasmic reticulum calcium ATPase (SERCA) channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies "credential" SERCA as a therapeutic target in cancers associated with NOTCH1 mutations.

The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway.

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442.

Triggered firing and atrial fibrillation in transgenic mice with selective atrial fibrosis induced by overexpression of TGF-ß1.

BACKGROUND: Calcium transient triggered firing (CTTF) is induced by large intracellular calcium (Ca(i)) transient and short action potential duration (APD). We hypothesized that CTTF underlies the mechanisms of early afterdepolarization (EAD) and spontaneous recurrent atrial fibrillation (AF) in transgenic (Tx) mice with overexpression of transforming growth factor ß1 (TGF-ß1). METHODS AND RESULTS: MHC-TGFcys(33)ser Tx mice develop atrial fibrosis because of elevated levels of TGF-ß1. We studied membrane potential and Ca(i)transients of isolated superfused atria from Tx and wild-type (Wt) littermates. Short APD and persistently elevated Ca(i) transients promoted spontaneous repetitive EADs, triggered activity and spontaneous AF after cessation of burst pacing in Tx but not Wt atria (39% vs. 0%, P=0.008). We were able to map optically 4 episodes of spontaneous AF re-initiation. All first and second beats of spontaneous AF originated from the right atrium (4/4, 100%), which is more severely fibrotic than the left atrium. Ryanodine and thapsigargin inhibited spontaneous re-initiation of AF in all 7 Tx atria tested. Western blotting showed no significant changes of calsequestrin or sarco/endoplasmic reticulum Ca(2+)-ATPase 2a. CONCLUSIONS: Spontaneous AF may occur in the Tx atrium because of CTTF, characterized by APD shortening, prolonged Ca(i) transient, EAD and triggered activity. Inhibition of Ca(2+) release from the sarcoplasmic reticulum suppressed spontaneous AF. Our results indicate that CTTF is an important arrhythmogenic mechanism in TGF-ß1 Tx atria.

In vitro effect of valepotriates isolated from Valeriana glechomifolia on rat P-type ATPases.

Valepotriates are iridoids found in variable amounts in Valerianaceae and might be among the bioactive compounds which confer anxiolytic properties to the Valeriana species. On the other hand, unspecific cytotoxicity has also been described. Presently, however, no particular molecular target has been defined for these compounds. Here we studied the effect of valtrate, acevaltrate, and 1- ß-acevaltrate isolated from Valeriana glechomifolia on the enzymatic activity of rat P-type ATPases. Valepotriates did not affect rat skeletal muscle sarco/endoplasmic reticulum Ca²âº-ATPase (SERCA) activity at the highest concentration used (100 µM). In contrast, the same concentration inhibited roughly half of the total H⁺/K⁺-ATPase activity from rat gastric epithelium (valtrate 54.6 ± 3.2 %, acevaltrate 60.7 ± 7.3 %, 1- ß-acevaltrate 50.2 ± 3.1 %; mean ± SEM, n = 3-5). Finally, these substances showed the highest inhibitory potency toward Na⁺/K⁺-ATPase, and the inhibition curves obtained provided a similar IC50 (in µM) for rat kidney α1 isoform (valtrate 21.2, acevaltrate 22.8, 1- ß-acevaltrate 24.4) and brain hemispheres α2/ α3 isoforms (valtrate 19.4, acevaltrate 42.3, 1- ß-acevaltrate 38.3). Our results suggest that P-type ATPases are differentially inhibited by valepotriates and that Na⁺/K⁺-ATPase might be one of their molecular targets in vivo.

The effects of wild ginger (Costus speciosus (Koen) Smith) rhizome extract and diosgenin on rat uterine contractions.

The aim of this study was to investigate the effects of wild ginger (Costus speciosus (Koen) Smith, Costaceae) rhizome extract on uterine contractility. We particularly examined the effects on spontaneous phasic contractions and the mechanisms whereby it exerts its effects. Wild ginger rhizomes were ethanolic extracted and their constituents analyzed. Isometric force was measured in strips of longitudinal myometrium and the effects of the extract studied. The extract (10 mg/100 mL) increased spontaneous contractions. The amplitude and frequency of the phasic contraction were significantly increased along with basal tension. Force produced in the presence of the extract was abolished by inhibition of l-type calcium channels or myosin light chain kinase (MLCK). The actions of the extract were not blocked by the estrogen receptor blocker, fulvestrant. Although significant amounts of diosgenin were present in the extract, we found that, depending upon its concentration, diosgenin had either no effect or was inhibitory on force. Interestingly, the extract induced significant amounts of force in the absence of extracellular calcium, which could be blocked by inhibition of the sarcoplasmic reticulum calcium-ATPase (SERCA), but not fulvestrant. We conclude that wild ginger rhizome extract stimulates phasic activity in rat uterus. Our data suggest that the uterotonic effect is due to nonestrogenic effects and not those of diosgenin. Wild ginger was able to increase contraction via calcium entry on l-type calcium channels and sarcoplasmic reticulum (SR) calcium release. We suggest that wild ginger rhizome extract may be a useful uterine stimulant.

Targeting sarcoplasmic/endoplasmic reticulum Ca²+-ATPase 2 by curcumin induces ER stress-associated apoptosis for treating human liposarcoma.

Human liposarcoma is the most common soft tissue sarcoma. There is no effective therapy so far except for surgery. In this study, we report for the first time that curcumin induces endoplasmic reticulum (ER) stress in human liposarcoma cells via interacting with sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2). Curcumin dose-dependently inhibited the cell survival of human liposarcoma cell line SW872 cells, but did not affect that of human normal adipose-derived cells. Curcumin-mediated ER stress via inhibiting the activity of SERCA2 caused increasing expressions of CHOP and its transcription target death receptor 5 (TRAIL-R2), leading to a caspase-3 and caspase-8 cascade-dependent apoptosis in SW872 cells in vitro and in vivo. Moreover, 70% of human liposarcoma tissues showed an elevated SERCA2 expression compared with normal adipose tissues. Curcumin dose-dependently inhibited the activity of SERCA2, and the interaction of molecular docking and colocalization in ER of curcumin with SERCA2 were further observed. These findings suggest that curcumin may serve as a potent agent for curing human liposarcoma via targeting SERCA2.

Effect of artemisinin on oocyst wall formation and sporulation during Eimeria tenella infection.

The anticoccidial effect of a product extracted from the natural herb Artemisia annua, artemisinin, which has a potential use as a dietary supplement, has been studied. Commercial artemisinin was administered at 10 and 17 ppm in food and tested against infection with Eimeria tenella. A battery trial to quantify the effect of artemisinin on the reproductive and infective capabilities of E. tenella was carried out. For that purpose flow cytometry was combined with electron microscopy and immunofluorescence techniques in order to study the effect of artemisinin on E. tenella gametogenesis. Significantly reduced oocyst output and lesion scores were found in chickens treated with artemisinin. In addition, evidence to support a lower oocyst sporulation rate was obtained. Though the ultrastructural studies showed normal development of gametogenesis in artemisinin-treated chickens, the oocyst wall formation was significantly altered. This resulted in both death of developing oocysts and reduced sporulation rate. Immunofluorescent studies provided evidence that treatment with artemisinin inhibited sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) expression in macrogametes. According to these findings, artemisinin has a deleterious effect on fertilized macrogametes (early zygotes) by inhibiting SERCA. The altered secretion of the wall-forming bodies may be the result of Ca(2+)-dependent ATPase impaired activity which, in turn, is the result of SERCA inhibition.