Pomiferin targets SERCA, mTOR, and P-gp to induce autophagic cell death in apoptosis-resistant cancer cells, and reverses the MDR phenotype in cisplatin-resistant tumors in vivo.

Drug resistance in cancer has been classified as innate resistance or acquired resistance, which were characterized by apoptotic defects and ABC transporters overexpression respectively. Therefore, to preclude or reverse these resistance mechanisms could be a promising strategy to improve chemotherapeutic outcomes. In this study, a natural product from Osage Orange, pomiferin, was identified as a novel autophagy activator that circumvents innate resistance by triggering autophagic cell death via SERCA inhibition and activation of the CaMKKß-AMPK-mTOR signaling cascade. In addition, pomiferin also directly inhibited the P-gp (MDR1/ABCB1) efflux and reversed acquired resistance by potentiating the accumulation and efficacy of the chemotherapeutic agent, cisplatin. In vivo study demonstrated that pomiferin triggered calcium-mediated tumor suppression and exhibited an anti-metastatic effect in the LLC-1 lung cancer-bearing mouse model. Moreover, as an adjuvant, pomiferin potentiated the anti-tumor effect of the chemotherapeutic agent, cisplatin, in RM-1 drug-resistant prostate cancer-bearing mouse model by specially attenuating ABCB1-mediated drug efflux, but not ABCC5, thereby promoting the accumulation of cisplatin in tumors. Collectively, pomiferin may serve as a novel effective agent for circumventing drug resistance in clinical applications.

Mechanisms of cell death induced by hexabromocyclododecane (HBCD) involves apoptosis, autophagy, and ER stress.

Hexabromocyclododecane (HBCD), was a widely utilized brominated flame retardant, commonly found in a wide range of household products. The pervasiveness of HBCD has identified the presence of this chemical in foods and in human tissues. Therefore, HBCD has been identified as a chemical of concern. The aim was to investigate the degree of cytotoxicity of HBCD in a range of cell lines derived from different tissues, (including hematopoietic, nerve, liver, and kidney-derived cells) with a view of determining any differential cell type effects. In addition, this study also investigated the mechanism(s) by which HBCD could cause cell death. The results showed that HCBD was considerably more toxic to leukocyte-derived (RBL2H3) and neuronal-derived (SHSY-5Y) cells with LC50 values of 1.5 and 6.1 µM, respectively, compared to cells derived from liver (HepG2) and kidney (Cos-7), which had LC50 values of 28.5 and 17.5 µM, respectively. A detailed investigation of the mechanism(s) of cell death showed that HBCD caused, at least in part, Ca2+ -dependent cell death, caspase-activated apoptosis, and autophagy, but there was little evidence for either necrosis or necroptosis occurring. Furthermore, it was shown that HBCD can also induce the ER stress response which is a known trigger of both apoptosis and autophagy and therefore this could be one of the crucial events by which cell death is initiated. As each of these cell death mechanisms was investigated in at least two different cell lines and no differences were identified, it is likely that the mode of action is not cell-type specific.

Tspan18 is a novel regulator of the Ca2+ channel Orai1 and von Willebrand factor release in endothelial cells.

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

Regulation and roles of Ca2+ stores in human sperm.

[Ca(2)(+)]i signalling is a key regulatory mechanism in sperm function. In mammalian sperm the Ca(2)(+)-permeable plasma membrane ion channel CatSper is central to [Ca(2)(+)]i signalling, but there is good evidence that Ca(2)(+) stored in intracellular organelles is also functionally important. Here we briefly review the current understanding of the diversity of Ca(2)(+) stores and the mechanisms for the regulation of their activity. We then consider the evidence for the involvement of these stores in [Ca(2)(+)]i signalling in mammalian (primarily human) sperm, the agonists that may activate these stores and their role in control of sperm function. Finally we consider the evidence that membrane Ca(2)(+) channels and stored Ca(2)(+) may play discrete roles in the regulation of sperm activities and propose a mechanism by which these different components of the sperm Ca(2)(+)-signalling apparatus may interact to generate complex and spatially diverse [Ca(2)(+)]i signals.

Systems Biology Approach Reveals a Calcium-Dependent Mechanism for Basal Toxicity in Daphnia magna.

The expanding diversity and ever increasing amounts of man-made chemicals discharged to the environment pose largely unknown hazards to ecosystem and human health. The concept of adverse outcome pathways (AOPs) emerged as a comprehensive framework for risk assessment. However, the limited mechanistic information available for most chemicals and a lack of biological pathway annotation in many species represent significant challenges to effective implementation of this approach. Here, a systems level, multistep modeling strategy demonstrates how to integrate information on chemical structure with mechanistic insight from genomic studies, and phenotypic effects to define a putative adverse outcome pathway. Results indicated that transcriptional changes indicative of intracellular calcium mobilization were significantly overrepresented in Daphnia magna (DM) exposed to sublethal doses of presumed narcotic chemicals with log Kow ≥ 1.8. Treatment of DM with a calcium ATPase pump inhibitor substantially recapitulated the common transcriptional changes. We hypothesize that calcium mobilization is a potential key molecular initiating event in DM basal (narcosis) toxicity. Heart beat rate analysis and metabolome analysis indicated sublethal effects consistent with perturbations of calcium preceding overt acute toxicity. Together, the results indicate that altered calcium homeostasis may be a key early event in basal toxicity or narcosis induced by lipophilic compounds.

Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 µm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

Exogenous heat shock proteins HSPA1A and HSPB1 regulate TNF-α, IL-1ß and IL-10 secretion from monocytic cells.

Endogenous molecules, such as heat shock proteins (HSP), can function as danger signals when released into the extracellular environment in response to cell stress, where they elicit an immune response such as cytokine secretion. There has also been some suggestion that contamination of exogenous HSPs with lipopolysaccharide (LPS) may be responsible for these effects. This study investigates the effects of exogenous HSPA1A and HSPB1 on the activation of immune cells and the resulting secretion of cytokines, which are involved in inflammatory responses. To address whether exogenous HSPs can directly activate cytokine secretion, naïve U937 cells, differentiated U937 cells and peripheral blood mononuclear cells (PBMCs) were treated with either exogenously applied HSPA1A or HSPB1 and then secreted IL-1ß, TNF-α and IL-10 were measured by ELISA. Both HSPs were able to induce a dose-dependent increase in IL-10 secretion from naïve U937 cells and dose-dependent IL-1ß, TNF-α and IL-10 secretion were also observed in differentiated U937 cells and PBMCs. We also observed that CD14 affects the secretion levels of IL-1ß, TNF-α and IL-10 from cells in response to exogenous HSP treatment. In addition, HSPA1A and HSPB1 were shown to interact with CD14, CD36 and CD11b extracellular receptor proteins. Several approaches used in this study indicate that HSP-induced cytokine secretion is largely independent of any contaminating LPS in the samples.

Bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)-methane (bis-phenol) is a potent and selective inhibitor of the secretory pathway Ca²âº ATPase (SPCA1).

The secretory pathway Ca(2+) ATPase (SPCA) provides the Golgi apparatus with a Ca(2+) supply essential for Ca(2+)-dependent enzymes involved in the post-translational modification of proteins in transit through the secretory pathway. Ca(2+) in the Golgi apparatus is also agonist-releasable and plays a role in hormone-induced Ca(2+) transients. Although the Ca(2+) ATPase inhibitors thapsigargin is more selective for the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA) than for SPCA, no inhibitor has been characterised that selectively inhibits SPCA. A number of inhibitors were assessed for their selectivity to the human SPCA1d compared to the more ubiquitous human SERCA2b. Each isoform was over-expressed in COS-7 cells and the Ca(2+)-dependent ATPase activity measured in their microsomal membranes. Both bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane(bis-phenol) and 2-aminoethoxydiphenylborate (2-APB) selectively inhibited SPCA1d (with IC(50) values of 0.13 µM and 0.18 mM, respectively), which were of 62- and 8.3-fold greater potency than the values for hSERCA2b (IC(50) values; 8.1 µM and 1.5mM, respectively). Other inhibitors tested such as bis-phenol-A, tetrabromobisphenol-A and trifluoperazine inhibited both Ca(2+) ATPases similarly. Furthermore, bis-phenol was able to mobilize Ca(2+) in cells that had been pre-treated with thapsigargin. Therefore we conclude that given the potency and selectivity of bis-phenol it may prove a valuable tool in further understanding the role of SPCA in cellular processes.

A plethora of interacting organellar Ca2+ stores.

The endoplasmic reticulum is not the only major agonist-releasable Ca2+ store within cells; it is now clear that virtually all organelles so far studied have the ability to act as mobilizable Ca2+ stores. From recent findings with regard to Ca2+ transportation and Ca2+ homeostasis within a variety of cell organelles such as the mitochondria, nucleus, Golgi and lysosomes, it emerges that many of these organellar Ca2+ stores appear to interact with each other, adding a further level of complexity to Ca2+ signalling events.

Regucalcin ameliorates doxorubicin-induced cytotoxicity in Cos-7 kidney cells and translocates from the nucleus to the mitochondria.

Doxorubicin (DOX) is a potent anticancer drug, which can have unwanted side-effects such as cardiac and kidney toxicity. A detailed investigation was undertaken of the acute cytotoxic mechanisms of DOX on kidney cells, using Cos-7 cells as kidney cell model. Cos-7 cells were exposed to DOX for a period of 24 h over a range of concentrations, and the LC50 was determined to be 7 µM. Further investigations showed that cell death was mainly via apoptosis involving Ca2+ and caspase 9, in addition to autophagy. Regucalcin (RGN), a cytoprotective protein found mainly in liver and kidney tissues, was overexpressed in Cos-7 cells and shown to protect against DOX-induced cell death. Subcellular localization studies in Cos-7 cells showed RGN to be strongly correlated with the nucleus. However, upon treatment with DOX for 4 h, which induced membrane blebbing in some cells, the localization appeared to be correlated more with the mitochondria in these cells. It is yet to be determined whether this translocation is part of the cytoprotective mechanism or a consequence of chemically induced cell stress.

Recent advances in Membrane Biochemistry.

This Biochemical Society Annual Symposium on Recent Advances in Membrane Biochemistry was organized to bring together experts from across the spectrum of biomembrane disciplines from the biological to the biophysical/structural, with the intention of promoting interactions and collaborations across the field. We were keen that the potential for improving human health that stems from a deeper understanding of membrane structure/function should be acknowledged, especially in the light of the increasing numbers of membrane protein structures that continue to be made available to the biomembrane community. This foreword provides an idea of what was communicated in the various sessions and, we hope, gives an impression of the excitement generated by the speakers and delegates at this over-subscribed Symposium.

A diversity of SERCA Ca2+ pump inhibitors.

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.

Occurrence of chemical pollutants in major e-waste sites in West Africa and usefulness of cytotoxicity and induction of ethoxyresorufin-O-deethylase (EROD) in determining the effects of some detected brominated flame retardants and e-waste soil-derived extracts.

We investigated the occurrence of chemical pollutants in major e-waste sites in West Africa and usefulness of cytotoxicity and induction of ethoxyresorufin-O-deethylase (EROD) in determining the effects of some detected brominated flame retardants (BFRs) and e-waste soil-derived extracts. Analysis of the e-waste site samples using AAS and GC-MS techniques revealed the presence of a range of toxic metals as well as persistent and toxic organic pollutants, respectively, in the vicinity of the e-waste sites. As expected, the occurrence (%) of all the detected chemical pollutants in experimental soils significantly (P < 0.05) differs from occurrence (%) in control soil. The calculated LC50 values on RBL-2H3 cells of the detected tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) were 3.75 µM and 4.2 µM, respectively. Tribromophenol (TBP), dibromobiphenyl (DBB), and decabromodiphenyl ether (DBDE) were remarkably less toxic on RBL-2H3 cells compared with TBBPA and HBCD as they did not reduce RBL-2H3 cell viability below 50% in the tested concentration range (0-20 µM). The study revealed that TBBPA and HBCD could induce significant RBL-2H3 cell death through caspase-dependent apoptosis. The study further shows that the cytotoxicity of some of these BFRs could increase synergistically when in mixtures and potentially activate inflammation through the stimulation of mast cell degranulation. The e-waste soil-derived extracts induced a concentration-dependent increase in EROD activity in the exposed RTG-W1 cells. Ultimately, nonpolar extracts had higher EROD-inducing potency compared with polar extracts and hence suggesting the presence in higher amounts of AhR agonists in nonpolar e-waste soil-derived extracts than polar extracts. Overall, there is urgent need for actions in order to improve the environmental quality of the e-waste sites.

Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria.

HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

Ca2+-stores in sperm: their identities and functions.

Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+](i) and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles that serve as Ca2+ stores in somatic cells. Here, we review i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the probable identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally, we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm.

In vitro cyto-toxic assessment of heavy metals and their binary mixtures on mast cell-like, rat basophilic leukemia (RBL-2H3) cells.

We investigated the cytotoxicity and mechanisms of cell death induced by salts of Cadmium (Cd2+), Lead (Pb2+), Arsenic (AsO43-) and Chromium (Cr+6) on RBL-2H3 cells (a model mast cell line). In addition, cyto-toxic effect on cell viability was assessed to reveal their nature of interaction in binary mixture. The individual cytotoxic characteristics of these metals on RBL-2H3 cell viability showed a concentration-dependent reduction of cell viability. We observed that concentration-dependent cytotoxic potency on RBL-2H3 cells of these metals range in the following order Cd2+>Cr+6>As O43- > Pb2+ with LC50 values of 0.11 µM, 93.58 µM, 397.9 µM and 485.3 µM respectively. Additive effects were observed with Pb2+ + Cd2+, Pb2+ + AsO43-, Pb2+ + Cr+6 and AsO43- + Cr+6. The study revealed that Pb2+, Cd2+, AsO43- and Cr+6 could induce significant (P < 0.01) cell death by apoptosis in RBL-2H3. Highly significant necrotic cell death was observed with Pb2+ and Cr+6 (P < 0.01) than Cd2+ and AsO43- (P < 0.05). Overall, it can be deduced that several cell death executing pathways may be concomitantly activated on exposure to heavy metals and the predominance of one over others might depend on the type of heavy metal, concentration and the metabolic state of the cell. Eventually, binary mixtures of some of these metals showed less cytotoxicity than would be expected from their individual actions and may depend on the co-exposure of the metal ions and their modes of action.

Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism.

Resistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells via new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from Nelumbo nucifera, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells, via calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.

Ca2+ signalling plays a role in celastrol-mediated suppression of synovial fibroblasts of rheumatoid arthritis patients and experimental arthritis in rats.

BACKGROUND AND PURPOSE: Celastrol exhibits anti-arthritic effects in rheumatoid arthritis (RA), but the role of celastrol-mediated Ca2+ mobilization in treatment of RA remains undefined. Here, we describe a regulatory role for celastrol-induced Ca2+ signalling in synovial fibroblasts of RA patients and adjuvant-induced arthritis (AIA) in rats. EXPERIMENTAL APPROACH: We used computational docking, Ca2+ dynamics and functional assays to study the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump (SERCA). In rheumatoid arthritis synovial fibroblasts (RASFs)/rheumatoid arthritis fibroblast-like synoviocytes (RAFLS), mechanisms of Ca2+ -mediated autophagy were analysed by histological, immunohistochemical and flow cytometric techniques. Anti-arthritic effects of celastrol, autophagy induction, and growth rate of synovial fibroblasts in AIA rats were monitored by microCT and immunofluorescence staining. mRNA from joint tissues of AIA rats was isolated for transcriptional analysis of inflammatory genes, using siRNA methods to study calmodulin, calpains, and calcineurin. KEY RESULTS: Celastrol inhibited SERCA to induce autophagy-dependent cytotoxicity in RASFs/RAFLS via Ca2+ /calmodulin-dependent kinase kinase-ß-AMP-activated protein kinase-mTOR pathway and repressed arthritis symptoms in AIA rats. BAPTA/AM hampered the in vitro and in vivo effectiveness of celastrol. Inflammatory- and autoimmunity-associated genes down-regulated by celastrol in joint tissues of AIA rat were restored by BAPTA/AM. Knockdown of calmodulin, calpains, and calcineurin in RAFLS confirmed the role of Ca2+ in celastrol-regulated gene expression. CONCLUSION AND IMPLICATIONS: Celastrol triggered Ca2+ signalling to induce autophagic cell death in RASFs/RAFLS and ameliorated arthritis in AIA rats mediated by calcium-dependent/-binding proteins facilitating the exploitation of anti-arthritic drugs based on manipulation of Ca2+ signalling.

The interaction of the brominated flame retardant: tetrabromobisphenol A with phospholipid membranes.

Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy [partition coefficient (Log K(p))=5.7+/-0.7]. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 microM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.

Inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by flavonoids: a quantitative structure-activity relationship study.

Flavonoids are commonly found in fruit and vegetables and have been shown to reach concentrations of several micromolars in human blood plasma. Flavonoids are also believed to have cancer chemoprotective properties. One hypothesis is that flavonoids are able to initiate apoptosis, especially in cancer cells, via a Ca(2+)-dependent mitochondrial pathway. This pathway can be activated through an exaggerated elevation of cytosolic [Ca(2+)], and sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA) play an essential role in ameliorating such changes. In this study, we demonstrate that flavonoids (especially flavones) can inhibit the activity of Ca(2+)-ATPases isoforms SERCA1A and SERCA2B in the micromolar concentration range. Of the 25 flavonoids tested, 3,6-dihydroxyflavone (IC(50), 4.6 microM) and 3,3',4',5,7-pentahydroxyflavone (quercetin) (IC(50), 8.9 microM) were the most potent inhibitors. We show that polyhydroxylation of the flavones are important for inhibition, with hydroxylation at position 3 (for SERCA1A) and position 6 (for SERCA2B) being particularly relevant.