Intervertebral disc (IVD) degeneration that accelerates the loss of disc structural and functional integrities is recognized as one of the major factors of chronic back pain. Cardiovascular risk factors, such as deficits of apolipoproteins that elevate the levels of cholesterol and triglycerides, are considered critical for the progress of atherosclerosis; notably in the abdominal aorta and its lumbar branching arteries that supply lumbar vertebrae and IVDs. Obstruction of the lumbar arteries by atherosclerosis is presumed to promote lumbar disc degeneration and low back pain. APOE-knockout rabbits have recently been shown to generate hyperlipidemia with increased levels of cholesterol and triglycerides that mimic the symptoms of atherosclerosis in humans. Here, we analysed IVD degeneration in the lumbar spines of ten homozygous APOE-knockout and four wild-type New Zealand White rabbits of matching age to prove accelerated IVD degeneration in APOE-knockout rabbits, since APOE-knockout rabbits could be a beneficial model for therapeutic approaches of degenerative IVD disorders. Experiments were performed using T1/T2-weighted magnetic resonance imaging, 3--2,5-diphenyltetrazolium bromide assay, glucose-oxidase assay, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot. APOE-knockout lumbar spines showed more advanced IVD degeneration, obstructed lumbar arteries and lower enhancement of contrast agent in IVDs. Moreover, lower concentration of glucose, lower number of viable cells and lower concentrations of aggrecan, collagen II and higher concentration of collagen I were detected in APOE-knockout IVDs (p < 0.0001). APOE-knockout in rabbits could induce structurally deteriorating premature IVD degeneration that mimics the symptoms of accelerated IVD degeneration in humans. APOE-knockout rabbits could be used as beneficial model, as they can bypass the standard surgical interventions that are commonly applied in research animals for the induction of enhanced IVD degeneration. Their parallel use in therapeutic approaches of IVD disorders and atherosclerosis could reduce the number of research animals to be used and contribute to the principles of 3Rs . Back pain is a prevalent disorder that affects the majority of adults at some time in their lives . ChronicApolipoprotein E (APOE) is an important apolipoprotein of chylomicrons , VLDL, intermediate density lipoprotein (IDL) and LDL. It serves as a ligand for their receptors and is vital for the metabolism of triglyceride-rich lipoproteins (TGRL) as well as for the transport of cholesterol and other lipids in the blood stream . Deficiein vivo therapeutic approaches of degenerative IVD disorders. Moreover, APOE-knockout rabbits could circumvent the standard surgical interventions that are frequently applied in research animal models for the induction of enhanced IVD degeneration. In addition, they could be used concurrently for therapeutic researches of IVD disorders and atherosclerosis.Because of their susceptibility to develop atherosclerosis as well as their similarity in lipid metabolism and cardiovascular physiology to that of humans, rabbits have become an increasingly interesting animal model for the study of lipid metabolism and atherosclerosis –35. Rece2, Scanbur, Denmark) with an elevated platform. Housing conditions were maintained at 18°C, 50% relative humidity and 12/12 h light/dark cycle. Specific Pathogen Free (SPF) quality of the animals was monitored and confirmed according to FELASA recommendations  were determined by using enzyme-linked immunosorbent assay (ELISA). The ELISA kits were obtained from R & D Systems (United Kingdom) or Uscn Life Science Inc. (USA) and assays were performed according to the instruction manuals. Aggrecan, collagen II and collagen I as major matrix proteins in NP and AF tissues were analyzed. For each sample three independent assays were performed with two replicates.2). The monolayer expanded NP and AF cells were harvested by trypsinization and directly used for three-dimensional culture (3D). For the 3D culture a bovine collagen I scaffold of 24 well plate format was used (Viscofan Bioengineering). Briefly, a scaffold was placed into each well that was filled with 250 μl PBS (pH 7.3 without Ca2+ / Mg2+) and incubated at room temperature for 20 min. The PBS was removed and the plate was left in the operating laminar flow hood overnight. Equilibration of the scaffold was performed for 10 min by adding 250 μl pre-warmed culture medium into each well. NP and AF cells were 3D cultured for two weeks in 500 μl culture medium  by changing the culture medium every two days. NP and AF cells were then harvested by digestion of the scaffold with 0.04% w/v collagenase II in 250 μl culture medium . Cell suspensions were filtered through sterile 75 gm nylon mesh filter and supernatants were centrifuged . Pellets were washed twice with PBS  and directly used for expressional analysis of APOE in NP and AF cells.NP and AF cells were isolated by successive digestion of NP and AF tissues with pronase and collagenase II as described above. After digestion samples were filtered through sterile 75 gm nylon mesh filters and supernatants were centrifuged . Pellets were resuspended in 0.5 ml culture medium DME/F-12 and cultured for 2 weeks in 24 well plates by changing the culture medium every two days . DNA contamination was removed by DNase 1 (Sigma-Aldrich). The concentration of total RNA was quantified at 260 nm using Biospectrometer (Eppendorf) and equal amounts of RNA were used for reverse transcription (RT). The cDNAs were synthesized using TaqMan Reverse Transcription Reagents (Applied Biosystems) and the mRNA levels of APOE and β-Actine  were determined by qPCR using TaqMan gene expression assay (Life Technologies) and LightCycler 480 (Roche Applied Science). The TaqMan Gene Expression Master Mix (1× master mix) supplemented with 200 nM sense, 200 nM antisense primers of APOE, 250 nM APOE-probe and 2 μl of the template DNA was used for PCR reactions in 20 μl of final volume. The PCR program contained an initial denaturation step at 95°C for 15 min, 40 cycles of denaturation at 95°C for 15 s, an extension at 60°C for 1 min, a melt curve stage  and a final extension at 72°C for 10 min. Standard, negative control and sample were run in three replicates of a 96 well plate. The mRNA expression levels were numerically presented using the delta CT (ΔCT) method.The sequence (5'->3') of the applied primers and probes are listed below:AGGAGCTGACCATGCTGATGAPOE-sense: CTGTTGCACACGTCCTCCATAPOE-antisense: 6FAB-CCATGCTGATGGAGGAGACC-BHQ1APOE-probe: CAGAAGGACAGCTACGTGGGBeta-actin-sense: CATGTCGTCCCAGTTGGTCABeta-actin-antisense: 6FAB-GACCCTGAAGTACCCCATCG-BHQ1Beta-actin-probe: Western blot was used to examine the protein expression levels of APOE in NP and AF cells of wild type and APOE-knockout rabbits. Total protein was extracted from 3D cultured NP and AF cells by using cold RIPA buffer containing inhibitors of protease and phosphatase. Total protein concentration in supernatants was determined by BCA Protein Assay Kit. Equal amounts of samples protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis  and transferred to polyvinylidene fluoride (PVDF) membrane (Merck Millipore). Monoclonal anti-APOE antibody produced in mouse  was used as primary antibody and the interaction between the primary antibody and APOE was detected on the membrane by using horseradish peroxidase-conjugated goat anti-mouse secondary antibody  and Amersham ECL Western Blotting Detection kit .Landis and Koch based interpretations with κ statistics and agreement percentage among two observers were used for reliable MRI evaluations of lumbar IVD degeneration –41. StatThe authors declare that all data supporting the findings of this study are available within the paper.The T2-weighted MRI signal characteristics of the lumbar IVDs revealed accelerated disc degeneration in APOE-knockout rabbits as compared to that in the wild-type rabbits. The interobserver reliability agreement for the rating of IVD degeneration with two observers was performed as described before –41 and tAPOE-knockout induced obstruction of Gadobutrol transport to the lumbar IVDs could also obstruct the nutrient transport, such as glucose. Low concentration of glucose as energy source could badly affect the cellular environment within the discs. Using glucose-oxidase assays the glucose concentration in APOE-knockout and wild-type rabbits were detected. The concentration of glucose was measured in serums [μmol/ml] as well as in NP and AF tissues [nmol/mg] of the rabbits. The recorded mean glucose concentration in serum samples of APOE-knockout rabbits [8.21 ± 0.365 μmol/ml] was 15.3% higher than that of the wild-type rabbits [7.12 ± 0.424 μmol/ml] (p < 0.0001) . In contNP and AF cells were directly isolated from lumbar IVDs of wild-type and APOE-knockout rabbits that were immediately harvested after euthanization and their numbers of viable cells were quantified using MTT assays. Decreased numbers of viable cells per milligram of wet tissue [cell number/milligram tissue] were detected in NP and AF tissues of APOE-knockout rabbits. The recorded mean numbers of viable cells in NP and AF tissues of APOE-knockout rabbits were 10867 ± 753 and 20122 ± 1176 respectively; and in wild-type rabbits the respective mean values amounted to 17834 ± 1262 and 28273 ± 1019 (p < 0.0001). Comparing the mean values in wild-type and APOE-knockout rabbits the numbers of viable cells in NP and AF tissues of APOE-knockout rabbits were reduced by 39.1% and 28.8% respectively. In NP tissues of both wild-type and APOE-knockout rabbits lower numbers of viable cells were detected than in the respective AF tissues .The concentration levels of critical extracellular matrix proteins aggrecan, collagen II and collagen I were quantified using ELISA in the total protein extracts of IVD tissues [ng/μg]. In NP and AF tissues of APOE-knockout rabbits decreased levels of aggrecan and collagen II were detected as compared with that in NP and AF tissues of wild-type rabbits. The calculated mean aggrecan concentration levels in NP and AF tissues of APOE-knockout rabbits amounted to 422 ± 10.7 ng/μg and 25.8 ± 1.43 ng/μg respectively. Compared with the respective mean concentration levels (594 ± 11 ng/μg and 35.1 ± 1.4 ng/μg) in NP and AF tissues of wild-type rabbits, the levels of aggrecan in NP and AF tissues of APOE-knockout rabbits were reduced by 28.9% and 26.4% respectively (p < 0.0001) , Fig 5A.Previously, the expression of APOE mRNA in several lipoprotein- and non-lipoprotein-producing tissues, such as liver, intestine, spleen, kidney, adrenal gland and brain, were demonstrated . We examIntervertebral disc degeneration affecting the majority of adults is one of the most common causes of chronic back pain with disability , 1–10. DT2-weighted MRI analysis of the lumbar spines in APOE-knockout rabbits exhibited more advanced disc degeneration grade (DDG IV) as compared with the disc degeneration grade (DDG II) of the wild-type rabbits . After iObstructing the supply of glucose as an energy source could critically affect cell survival and anabolism of extracellular matrix proteins within the discs. Analysis of glucose concentration in IVD tissues of APOE-knockout and wild-type rabbits revealed lower glucose concentration in NP and AF tissues of APOE-knockout rabbits, although higher glucose concentration was detected in their serum samples. Their mean glucose concentrations were reduced by 48.2% in NP tissues and by 33.5% in AF tissues. Comparing the glucose concentrations of AF tissues with NP tissues in the same rabbit group, averagely 54.0% lower glucose concentration was verified in NP tissues of APOE-knockout rabbits and 36.4% lower glucose concentration in NP tissues of wild-type rabbits , Fig 3. in vivo results, which show a tight correlation between the levels of glucose and the number of viable cells, support the results of the previous in vitro study, which exhibited the critical effect of glucose deprivation on the maintenance of IVD cell viability [in vivo reduction in glucose concentration could occur combined with reductions in oxygen concentration and pH-value, which could apparently worsen the cellular physiology within the discs.The number of viable cells and the concentration of extracellular matrix proteins were analysed in IVD tissues of APOE-knockout and wild-type rabbits, as the levels of glucose concentrations could be critical for survival and anabolic activities of IVD cells. Diminished numbers of viable cells were detected in NP and AF tissues of APOE-knockout rabbits. It was reduced by 39.1% in NP tissues and by 28.8% in AF tissues. In NP tissues of both wild-type and APOE-knockout rabbits reduced numbers of viable cells were detected compared to that in AF tissues of the respective rabbits . The rediability . HoweverAs anabolism of extracellular matrix proteins within IVDs could be affected by the reduction of glucose concentration and disc cell survival, the concentration levels of the main matrix proteins were analyzed in NP and AF tissues of APOE-knockout and wild-type rabbits. Decreased concentration levels of the core matrix proteins aggrecan and collagen II were detected in NP and AF tissues of APOE-knockout rabbits. The mean levels of aggrecan were reduced by 28.9% in NP tissues and by 26.4% in AF tissues. Similarly, the mean levels of collagen II were reduced by 28.3% and 36% respectively. Conversely, the mean level of collagen I was increased by 19.1% in AF tissues of APOE-knockout rabbits , Fig 5. in vivo therapeutic approaches in degenerative IVD disorders. Moreover, they can bypass the standard surgical interventions, which are frequently applied in research animal for the induction of enhanced disc degeneration. Also, their parallel use in therapeutic approaches of IVD disorders together with atherosclerosis could contribute to the three principles of Replacement, Reduction and Refinement (3Rs) in animal research [In conclusion, lumbar IVDs of APOE-knockout rabbits exhibited accelerated degeneration, lower enhancement of Gadobutrol, decreased concentration of glucose, diminished number of viable cells along with altered concentration levels the major matrix proteins aggrecan, collagen II and collagen I. IVD degeneration induced by APOE-knockout in rabbits could be characterized by structural deterioration that mimics the symptoms of advanced grade disc degeneration in humans. Thus, APOE-knockout rabbits could be used as valuable model for research –54.


IP3R1) promoter in AML patient-derived primary cells. Wogonoside activates IP3R1, in turn, promotes release of Ca2+ from endoplasmic reticulum, and eventually leads to cell differentiation. Our in vivo study further confirms that wogonoside can promote PLSCR1 and IP3R1 expression in primary AML cells and reduce the AML cell counts in engrafted nonobese diabetic/severe combined immunodeficient mice. Taken together, our findings provide new insight into the mechanism of wogonoside-induced differentiation and anti-leukemic effect on primary AML cells, suggesting the therapeutic potential of wogonoside for AML, especially for non-APL AML.Multiple lines of evidence have demonstrated that increased expression of phospholipid scramblase 1 (PLSCR1) is involved in the differentiation of acute myeloid leukemia (AML) cells by several differentiation-inducing agents including ATRA and phorbol 12-myristate 13-acetate. However, none of these agents can achieve nonhomogenous subcellular distribution of PLSCR1. We have demonstrated that wogonoside possesses differentiation and anti-leukemic effects in AML cell lines by promoting PLSCR1 trafficking into nucleus. Here we report that wogonoside promotes the expression of PLSCR1 and enhances its nuclear translocation and binding to the 1, 4, 5-trisphosphate receptor 1 ( Primary AML cells maintain the basic nature and biological activity of AML samples and are more close to clinical practice compare with AML cell lines. These cells exhibit several similarities in terms of morphological structure and functional activity with the organism, and therefore provide a good experimental subject for screening anti-leukemia drugs.Wogonoside, a flavonoid extracted from 2+ level.10 PLSCR1 also acts as a signaling molecule critical to cell signaling, maturation and apoptosis.12 An earlier investigation on protein–protein interaction networks demonstrated that PLSCR1 binds and modulates the activities of several proteins involved in growth factor and cytokine signaling, including epidermal growth factor receptor, c-Src, shc, onzin and the proto-oncogene c-Abl.15 Moreover, the observed induction of PLSCR1 by interferons suggests the possible roles of PLSCR1 in immune/stress responses, cell cycle regulation and apoptosis.17 Overexpression of PLSCR1 is proposed to inhibit tumorigenesis, induce apoptosis and facilitate the differentiation of myeloid cells.19 Recently, potential roles of PLSCR1 in hematopoiesis and leukemogenesis were reported.11 It is also demonstrated that PLSCR1 contributes to leukemia cell differentiation induced by ATRA and/or phorbol 12-myristate 13-acetate (PMA). Conversely, silencing PLSCR1 inhibits ATRA/PMA-induced leukemic cell differentiation.20 In addition, hematopoietic myeloid precursor cells from PLSCR1-/- mice showed defective colony formation and impaired granulocyte terminal differentiation upon stimulation with selective growth factors.19 Further investigation revealed that there is significantly decreased expression of PLSCR1 in AML-M1, -M5a and -M5b compared with that in normal bone marrow (BM) cells, and that higher PLSCR1 mRNA levels are associated with significantly longer overall survival in patients with AML.21 These findings clearly indicate that PLSCR1 contributes to leukemic cell differentiation and the longer survival of AML patients.PLSCR1 is a calcium-binding endofacial plasma membrane protein originally shown to accelerate redistribution of plasma membrane phospholipids (PLs) between the inner and outer leaflets following elevation of intracellular Ca22 Previous research has revealed that PLSCR1 is transported to the nucleus after exposure to cytokine stimulation, where it enhances expression of the inositol 1, 4, 5-triphosphate receptor type 1 (IP3R1) gene by directly binding to the promoter region of IP3R1.23 These data support a mechanism of receptor-mediated nuclear import of PLSCR1 and suggest a potential nuclear function for this plasma membrane protein, which has been a focus of recent research. Inspired by these findings, we investigated the effect of PLSCR1 on differentiation of leukemic cells involving its subcellular localization and nuclear function.Nuclear PLSCR1 could regulate specific protein function via binding directly to some genes.2+ from the endoplasmic reticulum (ER),24 we investigated intracellular Ca2+ fluctuation in wogonoside-treated primary AML cells. Ca2+ acts as a pervasive intracellular second messenger that participates in essential biological processes, including secretion, cell proliferation, differentiation and motility.25 In this study, we showed that wogonoside stimulates the differentiation of primary AML cells via a mechanism involving the PLSCR1-IP3R1-Ca2+ pathway. Our findings further clarify the key effect of nucleus PLSCR1 on leukemia differentiation therapy and suggest the mechanism of wogonoside’s anti-leukemic activity, supporting the potential of developing wogonoside as a novel therapeutic agent for clinical treatment of AML patients.In view of the findings that activated IP3R1 stimulates the release of CaμM) treatment (μM) in primary AML cells (μM) treatment not only increased the expression of PLSCR1 in #2 primary AML cells, but also promoted translocation of PLSCR1 into nucleus.To verify the effects of wogonoside on PLSCR1 expression in primary AML cells, we evaluated the PLSCR1 expression level in primary cells from 23 clinical AML patients after wogonoside  exerted significant effects on the expression of several cycle- and differentiation-related proteins, including upregulation of PLSCR1, p21Cip1 and p27Kip1 and downregulation of c-Myc. Results of western blot analysis of whole-cell lysates showed that the total expression of PLSCR1 was increased in #2 sample cells after treatment with 150 μM wogonoside for 24 h. Following elevation of intracytoplasmic PLSCR1, expression of cyclin p27Kip1 was markedly increased. Furthermore, levels of p21Cip1 and IP3R1 were significantly upregulated and c-Myc markedly inhibited after treatment with 150 μM wogonoside for 48 h (μM) for 0, 12, 24, 48, 72 and 96 h. Reverse transcription-polymerase chain reaction (RT-PCR) results showed that PLSCR1 mRNA expression started to increase at 24 h, and reached a relatively high level after 48 h of wogonoside treatment. The IP3R1 level was increased at the 48-h time point of wogonoside treatment  whose PLSCR1 expression levels were markedly upregulated by wogonoside  were transfected with PLSCR1 small interfering RNA  and the efficacy of transfection monitored using western blotting. Cell differentiation analyses were subsequently performed by using nitroblue tetrazolium (NBT) reduction assay, Giemsa staining and FACS assay. Notably, upon silencing of PLSCR1, wogonoside-induced differentiation effects on #2 and #19 primary AML cells were significantly reduced. For example, the nucleocytoplasmic ratio and the expression of CD11b and CD14 were essentially unchanged, and NBT reduction activity induced by wogonoside was basically abolished . In primrd blood . CD34+ c control . These fCip1 and p27Kip1 upregulation and c-Myc downregulation, which could be reversed by PLSCR1 silencing  was used to analyze the expression of PLSCR1-IP3R1-related cell cycle and differentiation proteins in #2 and #19 primary AML cells. With PLSCR1 silencing, wogonoside-induced upregulation of IP3R1 was reduced . Moreoveilencing . Collectin vivo, we assessed the expression patterns of cycle- and differentiation-related proteins in U937 xenografts of BALB/c nude mice.26 Similar to the in vitro results, levels of PLSCR1, p21Cip1, p27Kip1 and IP3R1, the tissue proteins of U937 xenografts, were all increased after administration of wogonoside. On the other hand, the c-Myc level was decreased with wogonoside treatment  staining for CD45 revealed that engrafted AML cells had located and proliferated in BM of transplanted mice . Wogonoside facilitated recovery and triggered tumor regression, as indicated by the low spleen weights of treated mice.4 The weights of other organs were not markedly changed. In addition, wogonoside noticeably prolonged survival in AML-bearing mice  compared with the control group, and slightly in mice bearing AML cells from sample #1 . Results of hematoxylin and eosin (H&E) staining showed that samples of saline-treated control group display hepatosplenomegaly, accompanied by ballooning degeneration of liver cells and multinucleated giant cell infiltration in spleen. Lungs of mice in the saline-treated control group exhibited hyperemia of alveolar cavities, accompanied by acute inflammatory cell infiltration. In addition, saline-treated control mice had renal tubular degeneration and myocardial hypertrophy. Consistently, treatment with wogonoside relieved these symptoms to a noticeable degree in different organs (In a NOD/SCID mouse model engrafted with primary human AML cells (sample #2) via tail vein, of mice , suggestal blood , indicatted mice . On the tributed . Subsequtributed . The rest organs . Taken t+ cells infiltrated into BM of primary AML cell-bearing NOD/SCID mice by using immunofluorescence confocal microscopy. Our results showed that wogonoside treatment noticeably increased the expression of PLSCR1 and IP3R1 and enhanced the nuclear distribution of PLSCR1  for 0, 12, 24, 48, 72 and 96 h. Upregulation of intracellular Ca2+ level was observed at 48 h. Peak intracellular Ca2+ level was reached at 72 h and remained till 96 h, in the presence of 150 μM wogonoside -N, N, N’, N’-tetraacetic acid (EGTA), an extracellular Ca2+ chelator. In primary AML cells pretreated with EGTA, wogonoside treatment increased intracellular Ca2+ level compared with cells only pretreated with EGTA  in primary AML cells derived from 13 samples  in all 23 peripheral blood samples of AML patients. In sample #2, upregulation of PLSCR1 was observed either outside or inside nuclei after wogonoside treatment, and it was also detected in peripheral blood cells of AML-bearing NOD/SCID mice. It is known that PLSCR1 is imported into the nucleus where it binds genomic DNA.21 Indeed, nuclear translocation of PLSCR1 was observed after treatment with wogonoside for 48 h in primary AML cells when expression of PLSCR1 was significantly upregulated, indicating that expression upregulation and nuclear translocation of PLSCR1 may be the common cause of differentiation induction. The nuclear-localized PLSCR1 specifically binds to a segment of the 5′-promoter of IP3R1 in a nucleotide sequence-specific manner, enhancing transcription of this gene.23 IP3R1 is known to have a central role in IP3-mediated mobilization of intracellular Ca2+ stores from the ER of diverse cells and is required for cell growth, maturation and differentiation.29 In our study, IP3R1 was detected in CD45+ cells, which were sporadically distributed in BM of wogonoside-treated AML-bearing NOD/SCID mice, suggesting it may be associated with wogonoside’s anti-leukemia effects. Moreover, our in vitro study showed that wogonoside promoted the nuclear import of PLSCR1 and facilitated its binding to the IP3R1 promoter sequence, transcriptionally activated IP3R1 expression, and triggered the release of Ca2+ from ER in primary AML cells. Further investigation showed that Ca2+, downstream of IP3R1, is significantly upregulated starting at 48 h, and reaches a peak plateau at 72 h in the presence of 150 μM wogonoside. Silencing PLSCR1 by siRNA reversed the elevation of Ca2+ level induced by wogonoside, and Ca2+ level was also inhibited by 2-APB, an inhibitor of IP3R1, and underwent a sharp decline at 48 h. However, we observed a rebound in Ca2+ level at 96 h, suggesting the involvement of exogenous Ca2+. 2-APB has been reported to elicit both stimulatory and inhibitory effects on Ca2+ influx through CRAC channels.31 So EGTA was used to eliminate the influence of exogenous Ca2+ and involvement of CRAC channels. When extracellular Ca2+ was removed by EGTA, wogonoside still increased intracellular Ca2+ level and this effect was inhibited by 2-APB. Wogonoside-induced differentiation was almost abrogated after incubation with 2-APB for 96 h, suggesting that elevated intracellular Ca2+ level has a key role in the differentiation process of leukemic cells. Removing extracellular Ca2+ by EGTA could not eliminate the differentiation effects as 2-APB did, indicating that wogonoside-induced differentiation effects was dependent on intracellular Ca2+ level but not extracellular Ca2+ influx. Based on these findings, we speculate that the PLSCR1-pathway is responsible for wogonoside-induced primary AML cell differentiation.It has been well known that either ATRA or PMA-induced differentiation of AML cells is attributed to the upregulation of PLSCR1. However, the inducible PLSCR1 is predominantly localized outside nuclei with little distribution in the nucleus.2+, our study demonstrates that PLSCR1-related molecular events induced by wogonoside in either primary AML cells or U937 xenografts are parallel to those observed in AML cell lines. Previous studies have demonstrated that p27Kip1 and p21Cip1 are required for leukemic cell differentiation.34 Moreover, PLSCR1 induction significantly elevated p27Kip1 protein by inhibiting its degradation and increases p21Cip1 by increased transcription and reduced degradation, when c-Myc protein level can be decreased.12 Consistently, during wogonoside-induced myeloid differentiation of primary AML cells, significant upregulation of p21Cip1 and p27Kip1 expression was observed, and the c-Myc level was significantly decreased. PLSCR1 silencing by siRNA in primary AML cells led to almost complete abrogation of wogonoside-induced myeloid differentiation, p27Kip1/p21Cip1 upregulation and c-Myc downregulation, indicating that wogonoside-induced effects on p27Kip1, p21Cip1 and c-Myc probably are mediated by PLSCR1. Our results showed that p27Kip1 level started to increase when extra-nuclear PLSCR1 was upregulated (12 h), the time point earlier than either p21Cip1 upregulation (72 h) or c-Myc downregulation (48 h) happened, suggesting different regulatory ways. We speculate that the expression of p27Kip1 would be affected by cytoplasmic PLSCR1, and p21Cip1 and c-Myc would more likely be regulated by nuclear PLSCR1. The exact mechanism by which PLSCR1 regulates expression of these downstream proteins in wogonoside-treated primary AML cells requires further investigation.Except the effect on regulation of IP3R1 and CaFor these experiments, primary patient-derived AML cells were selected because of their similarity to the physiological state of AML patients. The findings in our study are in good agreement with clinical observations from individuals with AML theoretically. We assessed the differentiation induction of primary cells from samples that showed different responses to wogonoside in terms of PLSCR1 expression. Wogonoside enhanced the expression of PLSCR1 and showed the highest anti-leukemia activity in samples  with low background PLSCR1 expression. We further observed that wogonoside-induced differentiation of sample (#3) with high background PLSCR1 expression although its expression level was barely affected. However, wogonoside-induced differentiation was not observed on non-responsive sample (#1) with low background PLSCR1 expression . These f2+ pathway leading to the differentiation of primary AML cells  was dissolved in dimethylsulfoxide (DMSO) as a stock solution at 0.5 M. Stock solution was stored at −20 °C, and freshly diluted with medium to the final concentration (150 μM) before each experiment. The final DMSO concentration did not exceed 0.1%. Cells treated with the highest concentration of DMSO were used as control in the corresponding experiments. For in vivo analyses, wogonoside (4 mg/ml) was made into a freeze–dried power formulation by Dr. Xue Ke from College of Pharmacy, China Pharmaceutical University, and mice were injected intraperitoneally (i.p.) with or without wogonoside (80 mg/kg). ATRA was dissolved in DMSO as a stock solution at 0.01 M and used as positive control in the corresponding experiments.For 35 Fluo-3AM was purchased from Beyotime . RPMI-1640 medium and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corporation .NBT, EGTA and 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich . 2-APB and PLSCR1 siRNA (#1) were purchased from Santa Cruz Biotechnology , PLSCR1 siRNA #2 was purchased from ThermoFisher Scientific , and transfection was performed using Lipofectamine 2000 reagent , according to the manufacturer’s instructions.Cip1, p27Kip1, IP3R1, c-Myc and β-actin were obtained from Santa Cruz Biotechnology; antibodies against PLSCR1 was obtained from Abnova . IRDye 800-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from Rockland . Alexa Fluor 488 donkey anti-goat IgG (H+L) antibody was purchased from Life Technologies .Fluorescein isothiocyanate (FITC) anti-human CD14 and phycoerythrin (PE) and FITC anti-human CD45 antibodies were obtained from Miltenyi Biotec Inc. . PE anti-human CD11b antibodies were obtained from eBioscience . Primary antibodies against p21et al.36 Primary leukemic cells were cultured in RPMI-1640 medium, supplemented with 10% FBS, 100 U/ml of benzyl penicillin and 100 μg/ml of streptomycin in a humidified environment with 5% CO2 at 37 °C.Primary leukemic cells from newly diagnosed AML patients without prior therapy  were collected using lymphocyte–monocyte separation medium . The protocol of collection of cells from patients complied with guidelines in the Declaration of Helsinki, and was approved by the First Affiliated Hospital of Nanjing Medical University’s institutional review board and the appropriate ethics committees. A signed informed consent was obtained from each patient. Primary leukemia cells isolation was performed as described previously in Hussong 26 Animals were subcutaneously injected with 2 × 106 U937 cells in 0.1 ml matrigel . When tumors were already palpable (50-100 mm3), the mice were divided randomly into two groups (n=5 per group), a control group  and a wogonoside-treated group (80 mg/kg). The treatment was carried out by i.p. injection every other day for 14 days. The dose was determined based on our preliminary studies (data not shown). At the end of the experiment, animals were killed and tumors were prepared for western blot.Female BALB/c nude mice   were used for the transplantation of U937 cells.6 cells per mouse, n=6 per group) via tail vein in 24 h following the radiation treatment. Animals in the control group were injected with physiological saline to evaluate the effects of injection on survival. Seven days later, the mice were injected i.p. with or without wogonoside (80 mg/kg) every other day for 30 days.27 Next, the animals were inspected daily for 30 days. Finally, peripheral blood were prepared for flow cytometry after the human leukemia cells labeled with huCD45 and the BM were used to perform IHC and immunofluorescent staining. Besides, heart, liver, spleen, lung and kidney were collected for H&E staining.Female NOD/SCID immunodeficient mice (6–9 weeks old)  were sublethally irradiated (2.4 Gy), and were engrafted with primary human AML cells  on Animal Care.Animals were maintained in an air-conditioned and pathogen-free environment  under controlled lighting (12 h light/day) and supplied with standard laboratory food and water 37 Three hundred cells were counted from three different fields for each data point.38 Cells were stained using Giemsa stain for morphologic assessment of differentiation. Fluorescence intensity of CD11b and CD14 was analyzed with a FACS Calibur flow cytometer .39 Data were based on the examination of 10 000 cells per sample selected randomly from 5 × 105 cells.Cell differentiation was assessed by NBT reduction as previously reported.40 Then equal amounts of extracts (50 μg) were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto the PVDF membranes . The blots were incubated with specific antibodies overnight at 4 °C followed by IRDyeTM800-conjugated secondary antibody for 1 h at 37 °C. Detection was performed using the Odyssey Infrared Imaging System .Preparation of whole-cell lysates was performed as described previously.μg/ml) for 20 min at room temperature. The coverslips were inverted onto slides and immersed in a mounting medium. The images were captured with a confocal microscope at × 1000 magnification .Cells were collected onto the coverslips and fixed in ice-cold methanol for 10 min. Then, coverslips were permeabilized in 0.2% (v/v) Triton X-100 for 20 min and blocked with BSA buffer (PBS containing 3% BSA) for 1 h at room temperature. Then, cells were incubated with primary anti-PLSCR1 antibody (1:10) at 37 °C for 1 h and then 4 °C overnight, followed by incubation with Alexa Fluor 488 donkey anti-goat IgG (H+L) antibody (1:500) for 1 h at 37 °C. The coverslips were washed and counterstained with DAPI working solution (100 μg/ml) for 20 min at room temperature. The slides were covered by coverslips and immersed in a mounting medium. The images were captured with a confocal microscope at × 1000 magnification .The BM slides were permeabilized in 0.2% (v/v) Triton X-100 for 20 min and blocked with BSA buffer (PBS containing 3% BSA) for 1 h at room temperature. Then, the BM slides were incubated with primary anti-IP3R1 antibody (1:50) or anti-PLSCR1 antibody (1:10) at 37 °C for 1 h and then 4 °C overnight, followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) antibody (1:500) or Alexa Fluor 488 donkey anti-goat IgG (H+L) antibody (1:500) for 1 h at 37 °C. Next, the BM slides were incubated with CD45-FITC or CD45-PE antibody (1:10) at 37 °C for 1 h. Then slides were washed and counterstained with DAPI working solution , was used to evaluate the specificity of PLSCR1-binding site in the IP3R1 gene promoter region (5′-CTTAAAGTGCAGTAACCATGTGGATGTGCTGCT-3′), together with the complementary oligonucleotide (5′-AGCAGCACATCCACATGGTTACTGCACTTTAAG-3′) double-stranded probes. The anti-PLSCR1 antibody was used for supershift experiments. The results were photographed using a Bio-Rad phosphorimager and analyzed with Image Lab Software, Version 3.0 .EMSA assay was performed according to the modified method as described previously.41 The primer sequences were as follows:RT-PCR was performed according to the manufacturer’s instructions.human PLSCR1-sense (5′-CTGACTTCTGAGAAGGTTGC-3′);human PLSCR1-antisense (5′-GAATGCTGTCGGTGGATACTG-3′);human IP3R1-sense (5′-TGACGAGAACCTGCCCTAT-3′);human IP3R1-antisense (5′-TCCTTTCGCCATCTTGCT-3′);human GAPDH-sense (5′-TCGTGGAAGGACTCATGACC-3′);human GAPDH-antisense (5′-TCCACCACCCTGTTGCTGTA-3′).35Cells were plated in six-well plates with fresh RPMI-1640 medium. The siRNA transfection was performed using Lipofectamine 2000 reagent according to the manufacturer’s instructions.μM Fluo-3AM, which combined with Ca2+ and produced strong fluorescence. After incubating for 60 min at 37 °C in the dark, the cells were resuspended with 500 μl PBS and the fluorescence intensity were measured by FACS Calibur flow cytometry  at Ex./Em. –488/525 nm.Cells were collected and loaded with 1 g for 5 min at 4 °C was performed to collect total cells, followed by resuspension in 500 μl PBS and detection using FACS Calibur flow cytometry. In this model, CD45+ cells appeared to be human leukemia cells.Whole blood of NOD/SCID mice killed via eyeball extirpation was depleted of red blood cells using red blood cell lysis buffer (eBioscience). For FACS analysis, cells were stained with anti-human CD45 antibodies at 4 °C for 30 min. Centrifugation at 350 To detect PLSCR1 expression, NOD/SCID mouse blood cells were stained with CD45 antibodies at 4 °C for 1 h and washed with PBS containing 0.5% BSA. After incubation with 1 × fixation/permeabilization buffer (eBioscience) for 30 min and 1 × permeabilization buffer (eBioscience) washes, cells were incubated with PLSCR1 antibody at 4 °C for 1 h and washed three times with PBS/BSA (0.1% BSA), followed by incubation with donkey anti-goat (FITC) secondary antibody at 4 °C for 1 h in the dark. Experiments were performed on an FACS Calibur flow cytometer.To characterize the histological alterations, organs and BM from six NOD/SCID mice in each experimental group were immersed in 10% formaldehyde (pH 7.4) fixative for 24 h, embedded in paraffin, cut into sections 4 mm thick, and stained with H&E using standard histological techniques. IHC against CD45 was performed with standard techniques.post-hoc test. Comparisons between two groups were analyzed using two-tailed Student's t-tests. A P-value <0.05 was considered statistically significant.All data were expressed as mean±S.D. from at least three independent experiments performed in a parallel manner. Statistical analysis of multiple group comparisons was performed by one-way analysis of variance (ANOVA) followed by the Bonferroni


Acute myeloid leukemia (AML) is a complex disease with a variety of presentations. A large pericardial effusion is rare, occurring in less than 0.5% of all patients with AML prior to treatment. A 34-year-old male presented with dyspnea, malaise, and weight loss. On physical exam, he was noted to be hypoxic, tachypneic, tachycardic, and hypotensive. He had cervical lymphadenopathy and jugular venous distention. His WBC count was 110 bil/L with 33% blasts. Bone marrow biopsy confirmed AML with 60% blasts. Leukemic cells were also seen in the cerebrospinal fluid on lumbar puncture. An echocardiogram revealed a large pericardial effusion causing tamponade. He underwent emergent pericardiocentesis, and malignant cells were present in the pericardial fluid. Induction therapy with standard dose cytarabine and daunorubicin was initiated, and bone marrow biopsy 14 days later showed no residual AML. This case demonstrates the importance of a thorough evaluation of each organ system when caring for a patient with AML. Acute myeloid leukemia (AML) is a complex disease with a variety of presentations. Majority of patients present with symptoms related to bone marrow failure such as fatigue, bruising or bleeding, fever, and infection . PericarA 34-year-old male without any significant past medical history presented to the hospital with shortness of breath, malaise, and unintentional weight loss. On physical exam, he was noted to be hypoxic, tachypneic, tachycardic, and hypotensive. He had palpable cervical lymphadenopathy and jugular venous distention. Complete blood count showed hyperleukocytosis (white blood cell count (WBC) of 110 bil/L with 33% blasts) and severe anemia (hemoglobin of 4.0 g/dL). Peripheral blood smear demonstrated increased blasts with promonocytes consistent with AML . His EKGGiven his dyspnea, hemodynamic abnormalities, and jugular venous distension, an emergent echocardiogram was obtained which revealed a large pericardial effusion with right atrial and ventricular collapse consistent with cardiac tamponade . PericarHis dyspnea, tachypnea, tachycardia, hypotension, and hypoxia resolved following pericardiocentesis. Given his hemodynamic instability, treatment with standard dose 7 + 3 cytarabine with daunorubicin was preferred over high-dose cytarabine with daunorubicin.The pericardial effusion did not reaccumulate, and he did not require a repeat pericardiocentesis. Bone marrow biopsy performed on day 14 of treatment did not show any residual AML. His blood counts recovered to normal, and he was started on intrathecal methotrexate. He is currently undergoing bone marrow transplant evaluation.Pericardial tamponade as an initial presentation of AML is a rare entity. A thorough literature review identified only three prior reported such cases in adults –5. The cApproximately half of patients with symptomatic pericardial disease and an underlying cancer may have nonmalignant pericardial disease including radiation pericarditis, drug-induced pericarditis, infection, hypothyroidism, autoimmune disorders, and idiopathic pericarditis . In our Although poorly understood, the pathophysiology behind the relationship between leukemia and acute pericardial disease is thought to be related to multiple factors including hemorrhage caused by concurrent thrombocytopenia, infections due to underlying immune deficiency, and direct malignant cell infiltration , 8. WhilOur case also highlights the importance of evaluating for CNS involvement in patients with AML. Central nervous system (CNS) involvement occurs in around 0.6% of adult patients with AML at initial presentation . These pIn conclusion, our case demonstrates the importance of a thorough evaluation of each organ system when caring for a patient with AML. Pericardial effusion causing cardiac tamponade can be one of the initial presentations of AML and requires early recognition and prompt intervention to improve outcome.


The canonical SOCE is defined as the Ca2+ entry that occurs in response to near-maximal depletion of Ca2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca2+ entry pathway in cancer cell migration has not been adequately assessed.Store-operated Ca2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays.The present study investigated the involvement of AA-induced Ca2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca2+ entry.We showed that both the store-dependent and AA-induced Ca2+ entry pathway is an important signal for GEPNET cell migration.Taken together, these data revealed that an alternative Orai3-dependent CaThe online version of this article (10.1186/s12935-018-0529-8) contains supplementary material, which is available to authorized users. Following restoration of 2.2 mM Ca2+ to the bath solution, a second, larger transient elevation in [Ca2+]cyt was observed indicative of SOCE. As shown in Fig. 2+ release. While, the average amplitude of the CPA-mediated release in control cells was 0.13 ± 0.02 ratio units, treatment with SKF and 2-APB resulted in Ca2+ release with the average amplitudes of 0.14 ± 0.02 and 0.13 ± 0.01 ratio units, respectively.An initial set of experiments was performed to pharmacologically characterize store-operated and arachidonic acid (AA)-activated forms of Ca2+ entry pathway. Application of exogenous 1–30 µM AA induced elevations in [Ca2+]cyt in a concentration-dependent manner . In contrast, the AA-induced rise in [Ca2+]cyt was not diminished by treatment with 50 μM 2-APB and was 2.21 ± 0.12 ratio units (n = 3), demonstrating that the SOCE and the AA-induced Ca2+ entry pathways were pharmacologically distinguishable.We next tested whether addition of AA activated a distinct Ca2+ buffering, clearance or release from internal stores. The rates of the Mn2+-induced quench of the fluorescence response following activation or inhibition of SOCE and the AA-induced responses was determined and compared against the rate of quenching in unstimulated cells. As shown in Fig. 2+ entry channel, treatment with AA resulted in rapid quenching of the fura-2 fluorescence signal. In this case the rate of quenching was 14.36 ± 0.59 FU/s (n = 4), indicating that the rate of entry was approximately 2.5 fold faster than the CPA-evoked rate of entry, and 14 times faster than the unstimulated rate of entry. In contrast to the CPA-treated cells, 2-APB treatment had no significant effect on the rate of AA-induced entry, and the rate of quenching was 24.62 ± 5.26 FU/s (n = 4). Treatment with SKF however, resulted in significant diminishment of entry. The normalized rate of AA-induced quenching was reduced to 1.45 ± 0.12 FU/s (n = 4) by treatment with SKF.We next performed a series of manganese quench assays to measure the rates of quenching and follow divalent ion entry independent of potential contribution by Ca2+]cyt in the absence of extracellular Ca2+. Therefore, we asked if the rise in [Ca2+]cyt resulted from ER Ca2+ release and whether this release was sufficient to depleted the [Ca2+]ER and trigger SOCE. To determine whether AA-induced Ca2+ release from the ER, we pre-treated the cells in nominal Ca2+ containing saline with thapsigargin (TSG), an irreversible inhibitor of the SERCA pump, and then applied AA. As shown in Fig. 2+ from ER stores. Subsequent application of AA did not induce significant additional release. The mean amplitude of this signal was only 0.007 ± 0.001 ratio units (n = 4). In contrast, in time-matched control experiments (lower panel), where TSG treatment was omitted, AA evoked Ca2+ release with mean amplitude of 0.24 ± 0.05 ratio units (n = 4), a value similar in magnitude to TSG-evoked release. These results indicated that AA mobilized Ca2+ release from a TSG- or CPA-sensitive intracellular store of Ca2+. Thus, to test whether AA treatment depleted [Ca2+]ER, a signal that is necessary to trigger canonical SOCE, we reversed the sequence of treatment as shown in Fig. 2+]cyt gradually increased to a new steady state about 200 s. Subsequent addition of TSG resulted in an additional release of Ca2+ with mean amplitude of 0.08 ± 0.01 ratio units (n = 3). However, this Ca2+ release signal was significantly smaller than that evoked by TSG alone (on average 0.19 ± 0.03 ratio units (n = 3) as shown in a time-matched control experiment   AA-induced mobilization of [Ca2+]ER did not activate Ca2+ entry and (2) that continuous presence of AA was needed to sustain AA-induced Ca2+ entry.As shown in Fig. nits n = . Howevernits n = . Howevernits n = . Howevernits n = . However2+ entry pathways could be distinguished functionally in BON cells, we investigated the requirements of Orai1 and Orai3 channel subunits for these modes of Ca2+ entry. Specific channel subunits in BON cells were selectively knocked down by transfecting with plasmids that expressed short hairpin RNAs (shRNAs) for Orai1, Orai3 and scrambled sequences for either genes of interest as controls. Since, previous work indicated that BON cells express low levels of Orai2 mRNA, the role of this homolog in mediating Ca2+ entry was not assessed in the current study [Because SOCE and the AA-induced Cant study . Western2+ entry were investigated using live cell Ca2+ imaging methods. As shown in Fig. 2+ release signal. The average amplitude of the CPA-evoked release signal in cells that were transfected with scrambled shRNA was 0.16 ± 0.02 ratio units and that for cells transfected with Orai1 and Orai3 shRNAs were 0.15 ± 0.01 and 0.12 ± 0.01 ratio units, respectively.The functional consequences of knockdowns of Orai homologs on Ca2+ entry in cells transfected with scrambled shRNA was 1.68 ± 0.32 ratio units (n = 5), cells that were transfected with Orai1 or Orai3 shRNAs had significantly reduced peak amplitudes of 0.29 ± 0.09 (n = 6) and 0.29 ± 0.10 (n = 6) ratio units, respectively. These results indicated that both Orai1 as well as Orai3 were required for mediating Ca2+ entry in response to AA and that channels that contained predominantly Orai1 were required for SOCE.In contrast to the SOCE responses, knockdown of either Orai1 or Orai3 significantly decreased the amplitude of the AA-induced responses compared to cells transfected with scrambled shRNA. While the mean amplitude of the AA-induced Ca2+ entry to assess their relative effectiveness to induce migration in BON cells. We first tested whether the induced cell migration required extracellular Ca2+. As shown in Fig. 2+ had minimal effect on the basal migration compared to SFM alone. For example, the percentage of cells migrated in SFM was 3.89 ± 0.40% (n = 4) while the percentage of cells migrated in a reduced Ca2+-containing SFM was 1.96 ± 0.40% (n = 3). Moreover, direct activation of SOCE by treatment of BON cells with either 1 μM TSG or 30 µM CPA failed to enhance cell migration significantly above basal levels with only 3.54 ± 0.47% cells migrated (n = 3). In contrast, migration of BON cells was enhanced approximately threefold in response to treatment with 6 μM AA where 14.43 ± 1.61% of cells migrated (n = 4). Reducing extracellular Ca2+ abrogated the AA-induced enhancement of cell migration  consistent with a requirement for Ca2+ entry. To further assess the relative contributions of store-operated- and AA-induced Ca2+ entry in BON cell migration, the pharmacological inhibitors of SOCE, SKF and 2-APB, were tested on AA-induced BON cell migration. Treatment with SKF significantly reduced the AA-induced cell migration. In these experiments only 2.82 ± 0.58% cells on average migrated (n = 4). In contrast, 2-APB had no significant effect on the AA-induced enhancement of cell migration when compared to AA alone. In these experiments 12.76 ± 0.53% cells on average migrated (n = 4). It was interesting to note that in contrast to the acute effects of AA-treatment where Ca2+ entry was enhanced in the presence of 2-APB, inclusion of 2-APB did not enhance AA-induced cell migration. This most likely resulted from a difference in the time courses of treatment for Ca2+ imaging and Boyden chamber experiments. Taken together, activation of AA-induced Ca2+ entry, but not SOCE, could mobilize a sub-population of BON cells to migrate in a Boyden chamber.We next selectively activated store-operated or AA-induced Ca2+ entry via Orai1/3 channels [However, because AA can be readily metabolized into other bioactive classes of compounds such as leukotrienes and prostaglandins, we assessed whether AA-metabolites contributed in BON cell migration. Moreover reports by Mohamed Trebak’s group demonstrated that leukotriene-C4 could mediate a store-independent Cachannels , 24. To 2+ entry is thought to be mediated by a channel that contains both Orai1 and Orai3, we investigated the role of these homologs in AA-induced cell migration. As shown in Fig. 2+ entry is the dominant Ca2+ entry pathway regulating BON cell migration in vitro.Because AA-induced CaTransformed cells can exhibit a migratory phenotype that correlates with increased metastatic potential. In BON cells that express both epithelial and neuroendocrine features, this can be achieved via neuroendocrine-to-mesenchymal transition (NMT). This transition is characterized by a reduction in neuroendocrine markers and altered expression of mesenchymal proteins –34. ThusIn these experiments, cells were plated onto coverslips or applied to transwell inserts, serum starved for 4 h, incubated overnight or for up to 48 h in growth medium supplemented with 6 µM arachidonic acid or 0.1% DMSO as vehicle control and subsequently assessed by immunofluorescence. Morphological changes induced by AA treatment were visualized by F-actin staining using fluorescently labeled phalloidin. The majority of control cells appeared clustered and were rounded or oblong in shape ~ 97%), whereas the majority of AA treated cells ~ 93%) exhibited an elongated, flattened shape that was often decorated with multiple cellular processes. Additional images of these morphologies are shown in Additional file % exhibit%, whereaOne hallmark of NMT is a reduction in the neuroendocrine secretory granule protein CgA. Under our experimental conditions, approximately 34% of cells in control groups exhibited perinuclear and subplasmamembrane distribution of CgA signal. Following overnight AA treatment, less than 15% of cells showed labeling. Figure Furthermore, as shown in Fig. In contrast, we did not observe changes in expression levels of the mesenchymal proteins α-SMA and Snail1 following AA treatment (data not shown). The lack of an effect may be in part be explained the surprising expression of these markers in BON cells prior to AA treatment.2+ signals and cell migration. These findings support the hypothesis that AA-induced Ca2+ entry through an Orai1/Orai3 containing channel was important for migration of a sub-population of BON cells.Previous work from our laboratory provided molecular and functional characterization of the SOCE pathway  in a varRecent work from several groups has demonstrated a requirement of Orai1-containing channels for SOCE and cell migration in some tumor cell lines. Although knockdown of Orai1 suppressed BON cell migration, it was unlikely that the suppression was mediated by inhibition of a canonical store-operated channel. This idea was supported by the observation that direct activation of SOCE by TSG-induced store depletion was ineffective at stimulating migration. Although this finding is tempered by the caveat that prolonged treatment with TSG can induce ER stress and apoptosis.2+ entry channel that contained Orai3 as well as Orai1 subunits. Consistent with this proposal, we found that treatment with AA resulted in Ca2+ influx that was distinct from canonical SOCE. Moreover, activation of this pathway stimulated migration of BON cells, and that when inhibited, abrogated the enhancement in migration. These data showed that Orai1 was necessary for mediating SOCE, whereas AA-induced Ca2+ entry as well as BON cell migration required both Orai1 and Orai3.Our contention is that it was more likely that the knockdown of Orai1 inhibited an AA-induced Ca2+ entry channel characterized here is reminiscent of the store-independent ARC channel described by Shuttleworth’s group [2+ entry in vascular smooth muscle cells in response to thrombin stimulation [2+ entry or cell migration in our system, pharmacological inhibition of AA-metabolism did not suppress AA-induced cell migration. This indicated that the enhancement of cell migration observed was largely due to AA itself and not its metabolite.The AA-induced Ca’s group , 36. Mormulation . Althoug2+ entry or AA-evoked BON cell migration, consistent with previous work that showed 2-APB does not block, and may even enhance, conductance in Orai3-containing channels. In contrast, the broader spectrum channel blocker SKF effectively blocked Ca2+ entry through Orai1- and Orai3-containing channels. These findings indicated a novel mechanism by which Orai-mediated Ca2+ entry may contribute to migration in this biologically idiosyncratic group of cancers.Assessment of migration using pharmacological tools was also consistent with the involvement of an Orai3-containing channel. Typically, 2-APB has been used as a tool to block SOCE and has been shown in some studies to inhibit cell migration. For example, 2-APB was shown to block EGF-induced cell migration in nasopharyngeal carcinoma  and woun2+ entry has been observed in other cell types [In this context, it should be noted that a mutual antagonism between SOCE- and AA-evoked Call types –39 and t2+ channel has relevance for GEPNET pathophysiology. For example, these cancers are thought to arise from neurosecretory cells in the gastrointestinal tract and commonly metastasize to the liver. Both the small intestine and liver exhibit elevated concentrations of AA [2-driven synthesis of AA in hippocampal neurons [Based on our findings it is tempting to speculate that the AA-induced Cans of AA , 41 comp neurons . Bradyki neurons . Moreove neurons . Althoug2+ entry through an Orai1/Orai3 heteromeric ion channel and that this mode of Ca2+ entry is important for migration in a GEPNET cell-line. The findings suggest that AA-induced Ca2+ entry may help set the migratory potential of these tumor cells and identify Ca2+ entry through the Orai3-containing channel as a novel signal for BON cell migration that may be exploited for therapeutic prevention of recurring GEPNET metastasis.The current manuscript demonstrates that treatment with AA induced CaAdditional file 1: Figure S1. Calcium entry amplitude and dynamics evoked by AA treatments. (A) Bar graph showing the peak amplitude increases in cytosolic Ca2+ in fura-2 loaded BON cells induced by application of different concentrations of arachidonic acid. Peak ratio changes for 1, 3, 6, 10 and 30 µM AA applications was 0.45 ± 0.19, 0.69 ± 0.24, 1.48 ± 0.18, 2.10 ± 0.27 and 4.71 ± 0.72, respectively (n = 3). B. Examples of typical Ca2+ dynamics induced by AA treatment. On restoration of extracellular Ca2+ we observed two typical responses: a more common, complex waveform (black) and a slower, gradual and sustained rise (red). Scale bars = 100 s and 0.500 ratio units.Additional file 2: Figure S2. Selective knockdown of Orai channel subunit proteins. Western blot data demonstrating that silencing of one Orai channel paralog does not induce changes in expression of the other paralog. shRNAs are indicated as scrambled (Scr) or selective for Orai 1 (O1) or Orai 3 (O3). Actin expression was used as a loading control.Additional file 3: Figure S3. Morphological changes induced in BON cells by overnight treatment with 6 µM AA. Cell shapes were visualized by treatment with phallotoxin fluorescently-labeled with Alexa Fluor 546 (red) or 633 (cyan). Confocal images of cells in culture are shown following treatment with vehicle contro (DMSO) or AA as indicated.


TNF and lower levels of XIAP in diagnostic samples, as well as with NPM1 mutation. In a large set of primary AML samples, these data provide novel insights into factors regulating Smac mimetic response in AML and have important implications for the development of Smac mimetic-based therapies and related diagnostics in AML.Apoptosis is deregulated in most, if not all, cancers, including hematological malignancies. Smac mimetics that antagonize Inhibitor of Apoptosis (IAP) proteins have so far largely been investigated in acute myeloid leukemia (AML) cell lines; however, little is yet known on the therapeutic potential of Smac mimetics in primary AML samples. In this study, we therefore investigated the antileukemic activity of the Smac mimetic BV6 in diagnostic samples of 67 adult AML patients and correlated the response to clinical, cytogenetic and molecular markers and gene expression profiles. Treatment with cytarabine (ara-C) was used as a standard chemotherapeutic agent. Interestingly, about half (51%) of primary AML samples are sensitive to BV6 and 21% intermediate responsive, while 28% are resistant. Notably, 69% of ara-C-resistant samples show a good to fair response to BV6. Furthermore, combination treatment with ara-C and BV6 exerts additive effects in most samples. Whole-genome gene expression profiling identifies cell death, TNFR1 and NF-κB signaling among the top pathways that are activated by BV6 in BV6-sensitive, but not in BV6-resistant cases. Furthermore, sensitivity of primary AML blasts to BV6 correlates with significantly elevated expression levels of  CEBPA and NPM1 are considered as provisional AML entities in the WHO classification  from adult AML patients at diagnosis were provided by the German-Austrian AML Study Group (AMLSG) with patient-informed consent obtained in accordance with the Declaration of Helsinki and institutional review board approval from all participating centers. Mononuclear cells were Ficoll gradient purified, and the percentage of leukemic cells/blasts was at least 80% following enrichment. Patient age at the time of diagnosis ranged from 20.3 to 81.7 years (median 57.0 years). Clinical characteristics at the time of diagnosis are detailed in Samples . CEL files  were normalized and filtered using BRB Array Tools Version 3.7.2 by applying the JustRMA algorithm and previously reported filtering criteria [Using GeneChip Human Genome U133 Plus 2.0 Arrays  gene expression was profiled in 24 AML samples [12 untreated, diagnostic samples of BV6-resistant  or GraphPad Prism 4 .Microarray data were analyzed using BRB Array Tools Version 3.7.2 as previously described . Group-w


Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, and generally considered to be caused by environment and genetic factors. In this study, we combined a genome-wide haplotype association study (GWHAS) and gene prioritization strategy to mine AML-related genetic affect factors and understand its pathogenesis. A total of 175 AML patients were downloaded from the public GEO database (GSE32462) and 218 matched Caucasian controls were from the HapMap Project. We first identified the linkage disequilibrium (LD) blocks and performed a GWHAS to scan AML-related haplotypes. Then we mapped these haplotypes to the corresponding genes as candidate. And finally, we prioritized all the AML candidate genes based on the similarity with 38 known AML susceptibility genes. The results showed that 1754 haplotypes were significant associated with AML (P<1E-5) and mapped to 591 candidate genes. After prioritizing all 591 AML candidate genes, we obtained four genes ranking at the front as AML risk genes: RUNX1, JAK1, PDGFRA, and FGFR2. Among them, RUNX1, JAK1 and PDGFRA had been confirmed as AML risk genes. In particular, we found that the gene FGFR2 was a novel AML susceptibility gene with a haplotype TT (rs7090018 and rs2912759) showed significant association with AML . In a word, our findings might provide a new perspective to understand the pathogenesis of AML. Acute myeloid leukemia (AML) is one of the cancer in myeloid line of blood cells, characterized by clonal expansion of myeloid progenitors in the bone marrow and peripheral blood . AML wasKühn MW, et al shared a genome-wide CBF-AML SNP-array data set with pediatric and adult CBF-AML patients GSE32462). Based on the mutation analyses and CNAs analyses, they identified multiple activating mutations in KIT, FLT3, JAK2, NRAS, and KRAS, and revealed that in some cases, more than one gene within the RAS/Kinase signaling pathway were activated 62. Based. Thus weIn this study, a total of 739,981 autosomal SNPs were included, which shared in both GPL6801 platform and the HapMap database. After the quality control (detail see methods), our study obtained a total of 734,624 eligible autosomal SNPs. Then a genome-wide haplotype association study was performed, which contained 393 samples . We identified 118,057 haplotype blocks, which contained total 519,865 haplotypes. For each haplotype, we carried out a chi-square test to assess the relationship with AML risk. In the end, 1,754 haplotypes located on 1,089 block regions showed significant correlation with AML (P< 1E-5) .According to the chromosome location information, 1,754 significant haplotypes were mapped to 591 genes, and regarded them as AML-related candidate genes. Meanwhile, we retrieved 38 known AML susceptibility genes from OMIM and GAD database, which had been confirmed to associate with AML risk. After that, we analyzed the similarity on the structure and function between two gene sets, and total 42 features were included, such as Ensembl Est, Gene Ontology, KEGG, Blast, HPRD, and so on (See methods for more detailed). We performed all above analyses by the Endeavour software. At last, four genes, RUNX1, JAK1, PDGFRA, and FGFR2, had lower P-value (P<0.05), and regarded as high-risk AML genes (Table The runt related transcription factor 1 (RUNX1) gene, also named as AML1, PEBP2, located on chromosome 21q22.3, and the physical location was: 36,160,098bp (start) – 36,421,595bp (end). The RUNX1 gene ranked the first, and also was the only gene existed in both training and candidate gene sets. From the detail information on Figure The janus kinase 1 (JAK1) gene located from 65,298,906bp (start) to 65,432,187bp (end) on chromosome 1p32.3-p31.3. The detailed information could be seen on the Figure From our GWHAS, a LD block was found on the platelet derived growth factor receptor alpha (PDGFRA) gene with a physical location from 55,063,358bp to 55,102,425bp. Among six haplotypes, two haplotypes GGGCTC and GGGCTT showed significant association with AML   gene located on chromosome 10q26 with the physical location: 123,237,844bp (start) – 123,357,972bp (end). From Figure Acute myeloid leukemia was recognized as a complex disease of hematopoietic stem cell disorders. It was classified into several categories based on underlying genetic alterations to facilitate diagnosis and prognosis by the World Health Organization (WHO). Among them, CBF-AML was characterized by the presence of distinct cytogenetic abnormalities . The genRecent years, the development of high-resolution genome-wide scanning technologies had given us abundant genetic variation resources for mining disease pathogenesis. Although still a lot of work need to be done, we believe that our work is effective and feasible. The study about conserved linkage disequilibrium chromosome regions can provide a new perspective for subsequent understanding of the pathogenesis about AML.ftp://ftp.ncbi.nih.gov/hapmap) [In this study, we downloaded all the original genotypes data of Caucasian CBF-AML patients from GEO database (accession GSE32462) under the chip platform GPL6801 (Affymetrix SNP 6.0 array), which contained 930,662 SNP markers. The purpose of our study was to identify the genetic risk factors of acute myeloid leukemia and avoid the effect of acquired somatic mutation. Therefore we selected the germline DNA of 175 pediatric and adult CBF-AML patients obtained from bone marrow or blood at remission. Considering the International HapMap Project could provide a variety of geographically neighboring reference populations, we compared the similarity of allele frequencies between HapMap Caucasian populations and GSE32462, and selected the highest correlation the HapMap CEU and TSI populations as a reference. Finally, 218 unrelated Caucasian control individuals were downloaded from phase II+III of the International HapMap Project  , 35. We On the basis of the autosomal SNPs data of 393 case-control samples, we performed a GWHAS to identify AML-related haplotypes. First, we used the Four Gamete Tests (FGT) method to assess the linkage disequilibrium (LD) blocks . Then thWe mapped all the AML-related candidate haplotypes to genes based on the chromosome location information. All the genes location information was queried from the NCBI gene database. If a gene shared the same chromosome fragment with at least one AML candidate haplotype, it would be regarded as an AML candidate gene.http://www.bioapp.org/research/AMLhaplotype). Then up to 42 available data sources were used to train features and construct models, which generally contained the following categories: gene and protein function, chemical information, bio-molecular pathways, phenotypic information, interaction networks, expression profiles, expression ontologies, sequence features [https://endeavour.esat.kuleuven.be/Default.aspx) [In some ways, the genes involved in the defined biological processes or diseases often shared some related characteristics, such as the similarity for sequence, functional annotation, expression and regulation information , 38. Thefeatures . The glofeatures . In this


Here we demonstrate that loss of Bax, but not Bcl-2 or Bak, inhibits this sustained Ca2+ elevation. What is more, in the absence of Bax, thapsigargin-elicited responses were decreased; and in two-photon-permeabilised bax−/− cells, Ca2+ loss from the ER was reduced compared to WT cells. The Ca2+-like peptides, CALP-1 and CALP-3, which activate EF hand motifs of Ca2+-binding proteins, significantly reduced excessive Ca2+ signals and necrosis caused by two BH3 mimetics: BH3I-2′ and gossypol. In the presence of CALP-1, cell death was shifted from necrotic towards apoptotic, whereas CALP-3 increased the proportion of live cells. Importantly, neither of the CALPs markedly affected physiological Ca2+ signals elicited by ACh, or cholecystokinin. In conclusion, the reduction in passive ER Ca2+ leak in bax−/− cells as well as the fact that BH3 mimetics trigger substantial Ca2+ signals by liberating Bax, indicate that Bax may regulate Ca2+ leak channels in the ER. This study also demonstrates proof-of-principle that pre-activation of EF hand Ca2+-binding sites by CALPs can be used to ameliorate excessive Ca2+ signals caused by BH3 mimetics and shift necrotic death towards apoptosis.BH3 mimetics are small-molecule inhibitors of B-cell lymphoma-2 (Bcl-2) and Bcl-xL, which disrupt the heterodimerisation of anti- and pro-apoptotic Bcl-2 family members sensitising cells to apoptotic death. These compounds have been developed as anti-cancer agents to counteract increased levels of Bcl-2 proteins often present in cancer cells. Application of a chemotherapeutic drug supported with a BH3 mimetic has the potential to overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the success rate of the treatment. We have previously shown that the BH3 mimetics, BH3I-2′ and HA14-1, induce Ca HA14-1 was the first BH3 mimetic obtained by molecular modelling that had the ability to displace Bax from Bcl-2, followed by the induction of cell death.7 Later, a family of seven members of BH3 mimetics, called BH3 inhibitors (BH3Is), was developed and shown to displace Bak peptide from Bcl-xL, trigger apoptosis, cytochrome c release and caspase activation.9 A natural BH3 mimetic gossypol, isolated from the cotton plant (Gossypium), is also capable of inhibiting Bcl-2, Bcl-xL and Mcl-1.11BH3 mimetics are small-molecule synthetic inhibitors of Bcl-2 and Bcl-xL, specifically developed as anti-cancer agents.in vitro to TRAIL-induced apoptosis.12 Gossypol (AT-101) was found to increase radiation efficacy in head and neck cancer cell lines in vitro;13 and recently underwent phase II clinical trials in combination with docetaxel14 and with androgen deprivation therapy.15 A particularly interesting approach is to develop chemically tailored BH3 mimetics that interact specifically with the anti-apoptotic proteins overexpressed in a target cancer type.8Simultaneous application of a chemotherapeutic agent and a BH3 mimetic can potentially overcome drug resistance in cancers overexpressing anti-apoptotic Bcl-2 proteins and thus increase the treatment success rate. BH3I-2′ and HA14-1 were shown to sensitise leukaemic cells 2+ homoeostasis. Previously we have reported that HA14-1 and BH3I-2′ deplete the ER Ca2+ store causing a sustained elevated cytosolic Ca2+ concentration in pancreatic acinar cells (PACs).16 Others have demonstrated that HA14-1 causes Ca2+ deregulation in platelets, HeLa and HEK-293T cells.17 This immediately triggers questions about safe and specific use of BH3 mimetics, especially given the crucial role of Ca2+ in the regulation of a wide variety of intracellular process including muscle contraction,18 enzyme secretion,19 fertilisation,21 cell proliferation22 and death.23 This is particularly important in the pancreas, as physiological Ca2+ oscillations control enzyme secretion in PACs, whereas abnormal Ca2+ signals are the hallmark of the initial stages of a severe necrotising disease of the pancreas – acute pancreatitis.26 This study aims to assess the effects of BH3 mimetics on Ca2+ handling in relation to activation of pro-apoptotic Bax in PACs. Also, we draw conclusions about the involvement of Bax in intracellular Ca2+ signalling and propose means to reduce the excessive Ca2+ signals enabling modulation of the cell death mechanisms.As exciting as the prospect of Bcl-2 inhibition might seem, accumulating evidence shows that BH3 mimetics often affect intracellular Ca2+ release from the intracellular stores.16 Pharmacological inhibition of inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) led to a decreased Ca2+ release from the ER of permeabilised PACs but did not completely block it. This indicates that IP3Rs and RyRs were not the primary source of BH3 mimetic-elicited Ca2+ release but merely amplified it.16 Here PACs, isolated from wild-type (WT) mice as well as animals with a loss-of-function mutation in one of the following genes: bcl-2, bak or bax were treated with 5 μM BH3I-2′ , whereas in bax−/− cells the Ca2+ signal generation was largely abolished (2+]i (P<0.001). The responses to BH3I-2′ or HA14-1 in bax−/− cells, if present at all, were mainly short-lasting Ca2+ transients, whereas sustained elevation was mostly inhibited  cells was higher than for WT cells ,27 did not significantly affect the responses to BH3I-2′ in bax−/− cells ; the latter was completely inhibited by BAPTA  and RyRs (by 10 μM ruthenium red).28 Average traces . Inhibition of Ca2+ extrusion by 1 mM La3+ preserved the difference in size of the thapsigargin-induced Ca2+ release between WT and bax−/− cells  resulted in only very slightly diminished responses in bax−/− cells in the first 100 s (62.7±3.7 a.u.) compared to WT cells  and bax−/− cells  are short peptides designed by inversion of the hydrophobic pattern of EF hand Ca2+ store with thapsigargin leads to opening of store-operated Ca2+ entry (SOCE) channels in the plasma membrane and influx of Ca2+ into the cytosol.33 As seen in 2+]i which, in the absence of external Ca2+, is transient due to the extrusion of Ca2+ by the plasma membrane Ca2+ pumps. When Ca2+ is added to the external solution, [Ca2+]i increases again, due to SOCE (2+ induced similar [Ca2+]i elevations (μM) of CALP-1  compared to the control , whereas apoptosis markedly increased (P<0.05) compared to BH3I-2′ alone. In contrast, CALP-3 did not affect apoptosis, but caused a reduction in necrotic cells (P<0.05) accompanied by an increase in the proportion of live cells (P<0.05).Excessive cytosolic Ca BH3I-2′ . The con pattern . In the 2+.38 Similarly to BH3I-2′ and HA14-1, in the absence of extracellular Ca2+, bax−/− cells did not respond to gossypol ; CALP-1 markedly decreased the proportion of cells that responded at all (P<0.05) and the proportion of apoptotic cells was increased (P<0.05). CALP-3 reduced gossypol-induced necrosis (P<0.05) and increased the fraction of live cells (P<0.05).It has been reported that gossypol is also capable of mobilising intracellular Ca plateau . Preincugossypol ; P<0.001d at all . The effd at all  were verd at all . In the 2+ signal generation is potentially very useful but given the crucial role of Ca2+ in the regulation of a wide variety of intracellular processes, it was important to test whether CALPs also affect physiological Ca2+ signalling. Nanomolar doses of ACh trigger enzyme secretion in PACs, an event controlled by Ca2+ signals.24 Here, 50 nM ACh induced repetitive [Ca2+]i oscillations (μM CALP-1 (39 CCK (5 pM), induced oscillatory Ca2+ responses in acinar cells  in order to induce Ca2+-dependent cell death of similar proportions of apoptosis and necrosis  while increasing slightly the proportions of both live and apoptotic cells; whereas 100 μM CALP-3 substantially reduced necrosis (P<0.05), leading to an increase in the number of live cells (P<0.05). The apoptotic fraction was essentially unaffected by CALP-3.As the primary mechanism by which CALPs exert their effects is the pre-activation of EF hand Ca2+ entry , it is u43 An increasing number of reports indicates that Bcl-2 itself may either directly or indirectly modulate intracellular Ca2+ fluxes such as: (1) Ca2+ release from the ER by binding to IP3Rs or RyRs;46 (2) Ca2+ reuptake into the ER by SERCA;47 (3) cellular Ca2+ extrusion by PMCA (plasma membrane Ca2+ ATPase);48 (4) and mitochondrial Ca2+ load.49It is now commonly accepted that Bcl-2 proteins are distributed in different cell compartments, not only at the outer mitochondrial membrane, but also in the cytosol, at the nuclear envelope and the ER.2+ release from the intracellular stores in mouse PACs and in the rat pancreatic cancer cell line AR42J.16 This Ca2+ release was not completely blocked by inhibition of IP3Rs and RyRs indicating involvement of other Ca2+ channels. We have also shown that BH3I-2′ and HA14-1 displace Bax from Bcl-2 and Bcl-xL.16 Here we provide new insights into the mechanism of this phenomenon by demonstrating that loss of Bax, but not Bak or Bcl-2, substantially inhibited Ca2+ responses to BH3I-2′  Ca2+ chelation by BAPTA completely abolished both apoptosis and necrosis induced by BH3I-2′ and (2) reduction of Ca2+ signals elicited by CALP-1 or CALP-3  or a significant shift in cell death towards apoptosis (CALP-1).Although it can be triggered by sustained elevations of [Ca68 However, development of synthetic non-peptide compounds activating EF hand Ca2+-binding motifs could potentially overcome these limitations, providing a useful pharmacological switch of cell death mode, particularly in cancer therapies.Our results indicate that application of CALPs, especially CALP-1, could improve the outcome of BH3 mimetic-based therapies. Although the pharmacokinetic properties of CALPs have not yet been studied in detail, it is likely that CALPs share characteristics of other short peptides, such as limited cell permeability and metabolic instability.2 1, glucose 10; pH 7.2. KHEPES (intracellular solution) consisted of (mM): KCl 130, NaCl 18, MgCl2 1, HEPES 10, ATP 3, EGTA 0.1, CaCl2 0.05; pH 7.2.The main reagents for cell isolation and imaging include: Fluo-4 AM, Fura-2 AM, Fluo-5N AM and BAPTA AM ; collagenase ; inorganic salts . Other reagents: HA14-1 ; BH3I-2′, gossypol ; caffeine and thapsigargin ; CALP-1, CALP-3, ruthenium red and CDN1163 . NaHEPES buffer was prepared as follows (mM): NaCl 140, KCl 4.7, HEPES 10, MgClbcl-2−/− , bax−/− (B6.129X1-Baxtm1Sjk/J) were obtained from The Jackson Laboratories ; bak−/− (B6.129-Bak1tm1Thsn/J) colony was a gift of Professor David Mark Pritchard (University of Liverpool); bax−/− strain extensively crossed into the CD1 background was also obtained from Professor Alun Davies (Cardiff University).69 Transgenic mice were bred in house either from null (bak−/−) or heterozygous  parents; the genotype of each mouse was confirmed by PCR reaction with primers suggested by the supplier. All animals were housed in the institutional animal unit, maintained on a 12 h light cycle on a standard rodent chow diet with free access to water. The mice were killed according to Schedule 1 of Animals (Scientific Procedures) Act 1986, dissected and the pancreatic tissue was removed for further experimental procedures.All procedures involving animals were performed in accordance with the UK Home Office regulations. C57BL/6J mice  were supplied by Charles River Laboratories ; transgenic mice 2+. Freshly isolated pancreas was washed twice in NaHEPES, injected with collagenase  and subsequently incubated at 37 °C for 15 min in the collagenase solution to allow digestion of the tissue. After incubation, the pancreas was broken down by pipetting, suspended in NaHEPES, spun , resuspended in NaHEPES and spun again. Finally, isolated PACs were suspended in NaHEPES and loaded with a Ca2+ sensitive dye as described below.PAC isolation and most of the experimental work was carried out in NaHEPES buffer. Unless otherwise stated, NaHEPES was supplemented with 1 mM Ca2+ indicators: 5 μM Fluo-4 AM for 30 min or 10 μM Fura-2 AM for 1 h. After the incubation the cells were resuspended in fresh NaHEPES and used for experiments at room temperature in a flow chamber perfused with NaHEPES-based extracellular solution. Experiments with Fluo-4 AM were performed using the Leica confocal microscope TCS SPE : × 63 oil objective, excitation 488 nm, emission 500–600 nm. Static images were taken at 512 × 512 pixel resolution and series of images were recorded at 256 × 256 pixel resolution, two consecutive frames were averaged. Fluorescence signals were plotted as F/F0, where F0 was an averaged signal from the first ten baseline images. Experiments with Fura-2 AM were performed using the Nikon Diaphot 200 imaging system : excitation at 365 and 385 nm, emission at 510 nm. The signals were plotted as 365/385 nm ratio or the ratio normalized to baseline values (0R/R).Isolated PACs were loaded at room temperature with one of the CaμM Fluo-5N AM in NaHEPES for 45 min at 37 °C. After the incubation, the cells were resuspended in fresh NaHEPES and used for experiments at room temperature in a flow chamber perfused with KHEPES-based intracellular solution. The cells were permeabilised with two-photon laser beam (720–750 nm),70 which was applied at a small area of the plasma membrane of a PAC. The experiments were performed using the Leica two-photon confocal microscope TCS SP5 with the same settings as for Fluo-4 AM (see above).PACs were loaded with 5 μM CALP-1 or 100 μM CALP-3 for 15 min at room temperature; the remaining two were incubated without pretreatment. Then cell death was induced for 30 min in both samples containing CALPs and in one untreated sample. Depending on the experiment, either one of BH3 mimetics was used (5 μM BH3I-2′ or 5 μM gossypol) or 5 μM menadione together with 100 nM ACh. Analogical experiments were performed with 15 min pretreatment with BAPTA instead of CALPs. The control samples were left untreated. Fifteen  minutes before the end of the incubation, annexin V-FITC and propidium iodide were added to all samples. The cells were visualised with the Leica confocal microscope TCS SPE. Annexin V-FITC specifically stained apoptotic cells , whereas propidium iodide was used for detection of necrotic cells . Multiple pictures (20–35) per treatment group were taken; live, apoptotic and necrotic cells were counted in each treatment group.Cell death assay was performed using Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) according to a modified manufacturer′s protocol. PACs were isolated as described above and divided equally into four experimental groups. Two samples were pretreated with 100 2+ responses, areas under individual traces were calculated according to the formula: F is the recorded fluorescence (or ratio for Fura-2), F0 is the baseline fluorescence (or ratio) and Δt – time interval. Obtained values were then averaged and presented as bar charts with S.E.M. The Student′s t-test was applied for statistical comparison. The significance threshold was set at 0.05 and the range was indicated by asterisks . Where applicable, N indicates the number of individual experiments, whereas n – individual cells.For quantitative analysis of CaU-test with the significance threshold set at 0.05.For cell death assays, three independent experiments were performed for each treatment group; average values and S.E.M. were calculated and the results presented as bar charts. Statistical analysis was performed using the non-parametric Mann–Whitney


HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal CaHIGHLIGHTS- Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.2+-sensitivity, essential for normal responses to adrenaline.- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity.- We have identified a new class of drugs that are capable of both reducing Ca- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers. Troponin, together with tropomyosin and actin constitutes the thin filaments of muscle. In unstimulated muscle contraction is inhibited by the C-terminus of the troponin I subunit binding to actin-tropomyosin and blocking the interaction with myosin crossbridges. Upon stimulation intracellular Ca2+ rises and Ca2+ binds to the troponin C subunit (TnC), the C-terminus of TnI is released from its site on actin and crossbridge cycling can proceed  leading to increased rate and magnitude of contraction. Lusitropy, an increased rate of relaxation allowing for shorter twitch duration, is a key component of adrenergic activation and involves TnI. Phosphorylation of ser22 and 23 of TnI by PKA leads to a 2-fold decrease in Ca2+-sensitivity and a corresponding increase in the rate of Ca2+ release from TnC and is essential for the lusitropic response  and hypertrophic cardiomyopathy (HCM) are often due to single mutations in a protein of the sarcomere and have been extensively studied 2+-sensitivity that is considered to be sufficient to induce the symptoms of HCM. The effect of DCM mutations in contractile proteins on Ca2+-sensitivity is variable with a trend toward deceased Ca2+-sensitivity , compared with similar inactive molecules can provide considerable insight into the mechanism of re-coupling and may lead to the discovery of more potent re-coupling (lead) compounds with therapeutic potential.EGCG and its analogs have been widely studied as Hsp90 inhibitors, so we began our investigations using analogs of EGCG and of Silybin, a natural product with structural similarity, originally studied as Hsp90 inhibitors . To reduce the phosphorylation level (<0.3 mol Pi/mol) troponin was treated with shrimp alkaline phosphatase  and mutant E180G tropomyosin were expressed in a baculovirus/Sf9 system  , tropomyosin (40–60 nM), and troponin (60 nM) to study Ca2+ concentrations to generate Ca2+-activation curves. The data were fitted to the 4-variable Hill equation to yield a value for EC50 and nH. Most assays of the effect of compounds are single measurements. When appropriate EC50 values from replicate experiments were analyzed by paired t-test since the distribution of EC50 has been shown to be normal. Error bars and n values are given in tables and figures.The fraction motile was measured over a range of Ca2+. Most assays were single measurements.For drug screening, motility was measured with paired phosphorylated and unphosphorylated tropomyosin E180G mutant-containing thin filaments at a fixed 0.037 μM Ca2+ activation of contractility using an in vitro motility assay that measures the movement of reconstituted thin filaments over a bed of immobilized myosin heads. A series of measurements, some of which have been previously published . Thus, the effect of EGCG is independent of the mutation that induced uncoupling; in addition, both in man and cat, HCM cases were found where no mutation in sarcomeric genes was detected yet the thin filaments were uncoupled and they could also be re-coupled by EGCG  there were four compounds that were both Hsp90 inhibitors and re-couplers, four that were re-couplers only, four that were Hsp90 inhibitors only and seven that were inactive in both assays  is an example of a compound that is a pure desensitizer as illustrated in Figures r Figure .2+-sensitivity of native thin filaments in either the phosphorylated or unphosphorylated state, in contrast to EGCG  and compound 7 that has a reverse effect differ only by the number and positions of O-methyl groups in the D ring whilst other substitutions in the D ring (compounds 8 and 16) render the compound inactive. In addition, a number of substitutions in the A ring are compatible with recoupling when combined with an appropriate substitution in the D ring   and meta positions.A group of compounds with substitutions containing the core rings A, C, and D of EGCG shift function from good recoupling to no effect and even to reverse re-coupling. The structural differences are small, for instance compound  Figures . Methyla2+-sensitivity. In addition, we have identified a molecular level dysfunction common to both HCM and DCM-causing mutations    Figure . This in2+-sensitivity and unphosphorylated form with high and low Ca2+ sensitivity . Mutations that produce uncoupling can only undergo transition from the unphosphorylated high Ca2+ sensitivity to phosphorylated high Ca2+ sensitivity state and the further transition to low Ca2+ sensitivity is blocked. Dvornikov et al. have proposed structural evidence that a normal phosphorylation-dependent structural transition is blocked in the TnI R145W HCM mutant troponin  could be accounted for if they bound equally to the phosphorylated and unphosphorylated low affinity states. The reverse re-coupler, compound #2+-sensitivity of TnC compatible with our four state model  to troponin subunits have been made, most studies have involved binary complexes (TnI and TnC) and partial sequences  or chromanone (in Silybin) are fundamental. The way in which the substitutions define the structure, charge distribution, hydrogen bonding potential or acidity of the A ring, apparently involved in its intercalation between TnI and TnC, is likely to be critical for the compound effect. In the EGCG analogs it seems that either the B ring  or the D ring  are sufficient to confer re-coupling activity, so one ring may be able to assume the configuration of the other when one of them is absent. In Silybin and Dehydrosilybin, the lignan part is not fully needed for function  and only the B isomers are recouplers, whilst the A stereoisomers of Silybin and Dehydrosilybin are inactive as recouplers. In this case it seems likely that the A isomers have an unfavorable conformation that prevents the A and D rings binding in the same way as the B isoform. It is noteworthy that the B isomer is more active than the A isomer is several different bio-assays (Kren and Walterová, 2+ sensitivity by TnI phosphorylation is the loss of the lusitropic response to beta adrenergic stimulation, which is usually accompanied by blunting of the whole physiological adrenergic response. This is demonstrated most directly by a lack of response to dobutamine stimulation observed at the whole animal, papillary muscle and single cardiomyocyte levels (Song et al., The main effect of uncoupling of the modulation of CaAS, AM, MP, AC performed the experiments reported here; BB, AK, VK, DB prepared and supplied EGCG and silybin analogs; SM obtained funding, directed research and wrote the paper. All the authors contributed to editing and completing the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.