PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53R175, one of the most frequent ‘hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53R175 mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53R175 mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53R175 mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53R175 and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under  The majority of p53 mutations are missense that are localized to six ‘hotspot' residues. Mutations in p53 result in the loss of the wild-type (WT) activity; however, these mutants exert either a ‘dominant-negative' effect on the p53 WT activity or a ‘gain-of-function' effects.3 Humans with a Li–Fraumeni syndrome, an autosomal-dominant disorder owing to germline mutations in p53 gene, are at an increased risk of tumorigenesis.4 Thus targeting p53 mutant offers a promising approach for cancer chemotherapeutics. However, the role of p53 mutant as a target for dietary-related cancer chemopreventive compounds remained to be investigated.Mutations in the p53 gene occur in a variety of human cancers with remarkably high frequencies . The mechanisms proposed for PEITC include inhibition of cytochrome P450s, induction of phase II detoxifying enzymes, cell cycle arrest and apoptosis.12 PEITC-induced oxidative stress contributes to apoptosis;14 however, the exact mechanism(s) underlying its activity and its molecular target(s) are not well understood. This knowledge is crucial for discovering more effective ITCs for the prevention and treatment of cancer. In this study, we investigated p53 mutant as a new target of PEITC-induced apoptosis and tumor suppression.Phenethyl isothiocyanate (PEITC), abundantly present in watercress and cruciferous vegetables, exerts cancer chemopreventive effects in animal models, and epidemiological studies also support the role of dietary ITCs in protection against cancer in humans.R175 mutant  siRNA or p53 siRNA. The p53 protein was reduced by ≥90% after p53 siRNA transfection , OVCAR3 (p53R248)   or A549 p53R248) . Importa or DMSO , demonstR175 mutant was the most sensitive to PEITC  cells transfected with plasmid pcDNA3, pcDNA3-p53R175, pcDNA3-p53R273 or pcDNA3-wtp53 with PEITC. The H1299-pcDNA3-p53R175 cells displayed maximum sensitivity to PEITC . We usedto PEITC . CollectR175 with enzyme-linked immunosorbent assay (ELISA) using conformation-specific anti-p53 antibodies. Incubation of the recombinant glutathione-S-transferase (GST)–p53R175H with PEITC resulted in a ∼2.8-fold increase in the PAB1620 (WT-specific) fraction, whereas the PAB240 (mutant-specific) fraction was decreased by ∼2.6-fold . Therefore, we examined its effect on the conformation of p532.6-fold . An immu2.6-fold . Similar2.6-fold . Importa SK-BR-3  and HOP9 SK-BR-3  cells trR175 in the chromatin fractions. The chromatin-bound fractions of SK-BR-3 cells treated with PEITC showed a dose-dependent increase in p53R175 (μM) enhanced the expression of canonical p53 target genes, specifically p21, MDM2, PUMA, NOXA, BCL2 and BAX in SK-BR-3 cells (R175 knockdown SK-BR-3 cells treated with PEITC (4 μM), suggesting that the induction of p53 targets by PEITC was p53R175 dependent (μM)-treated SK-BR-3, AU565, HOP92 and (10)3/175 cells, transfected with plasmid encoding WT p53-binding element in the p21 promoter region, showed an ∼2–2.5-fold increase in luciferase activity (μM) induced p21 expression in the SK-BR-3 cells, whereas the DNA-damaging agent etoposide failed to do so  selectively depletes p53 mutant protein, but not the WT p53, although the mechanisms of such depletion are still unknown.15 Previous studies have shown that compounds that reactivate mutant p53 also induce its partial depletion.16 To investigate the molecular mechanisms for p53R175 depletion, we evaluated p53 mutant levels with different doses and times of PEITC treatment. PEITC induced significant decrease of p53R175 levels, at the concentration as low as 4 μM,  and 20 μM MG132 showed a significant accumulation of the p53 in the insoluble fractions as well as in the whole-cell lysate (WCL) as compared with cells treated with PEITC or MG132 alone (μM) and 10 μM Nutlin-3 displayed an increase in p53 in the WCL  of PEITC. Aggregation of p53R175 occurs at higher concentrations (≥8 μM) (R175 undergoes autophagy. SK-BR-3 cells co-treated with 8 μM PEITC and 50 μM chloroquine (CHQ), an inhibitor of autophagy, displayed an increase of p53 in the WCL compared with the cells treated with PEITC alone. No significant difference was observed for cells co-treated with 4 μM PEITC and 50 μM CHQ or PEITC alone, suggesting that autophagy was induced at higher concentrations of PEITC (20 ATG5-knockdown SK-BR-3 cells (R175 at 8 or 16 μM but not at 2 or 4 μM concentrations of PEITC compared with cells transfected with NS siRNA (R175 aggregates.SK-BR-3 cells treated with 8 fraction . The ins (≥8 μM) . As protof PEITC . Autopha-3 cells  showed hNS siRNA . ATG5-knNS siRNA , suggestR175 is incapable of binding to zinc.21 As PEITC restored the ‘WT-like' conformation to p53R175, we assessed the effect of zinc on its antiproliferative activity. Co-treatment of SK-BR-3 cells with PEITC and zinc chloride (ZnCl2) in the optimal concentration range (10–20 μM) enhanced the potency of PEITC by ∼3.3-fold  and ZnCl2 (2.5 μM) resulted in significant increase in the PAB1620 fraction, whereas no significant change was detected in the PAB240 fractions  showed a decrease in the GSH levels as compared with the DMSO control (N-acetylcysteine (NAC) or 500 units PEG-catalase) alleviated the effects of PEITC on proliferation and apoptosis, whereas a catalase-specific inhibitor 3-amino-1,2,4-triazole (ATZ) enhanced it , whereas no differences were detected in A549 cells  pathways. SK-BR-3 cells treated with 4 μM PEITC showed pATM-S1981 and pCHK2/Thr68 compared with the DMSO control (R175H leads to the reactivation of the DNA-damage response. The absence of pATM-S1981 and pCHK2-Thr68 in A549 cells treated with PEITC was consistent with the γ-H2AX foci data (DSB activates the ataxia telangiectasia mutated (ATM) serine/threonine protein kinase, which then phosphorylates downstream signaling targets. The p53th PEITC . Therefo control , suggestoci data . Co-treaoci data , suggestR175 mutant affects cell cycle progression. SK-BR-3 cells treated with PEITC (4 μM) displayed a significant increase in G2/M and S phases at 24 h (μM) showed an increase in the G1 phase at 24 h, and no change at 72 h  was observed in mice on the PEITC diet (5 μmol/g AIN-93M)26 as compared with those on the control diet (27 or by the immune response of the nu/nu mice as immune cells were detected in the hematoxylin and eosin (H&E)-stained sections  analysis. A significant reduction of p53 mutant protein levels was detected in animals on PEITC diet .12 Thus the reduced cell viability and increased apoptosis in both p53+/+ and p53−/−, H1299 cells and MEF, by PEITC is consistent with the notion that pathway(s) independent of p53 mutational status also exists,12 such as depletion of tubulin in both WT and p53 mutant cells.31Previous studies have shown that in the WT p53 cells PEITC induced apoptosis in a p53-dependent manner.R175. Besides the antiapoptotic regulator BCL2, PEITC induced the expression of several pro-apoptotic targets, including the members of BH3-only class. Importantly, BCL2 failed to suppress the cell death induced by PEITC. p53 regulates BCL2 family members by engaging multiple transactivation-dependent and -independent effectors.32 It will be interesting to study the crosstalk between the different genes in PEITC-treated cells that ultimately result in the cell death.PEITC restored the transactivation functions to p532 enhanced the antiproliferative activity of PEITC and also significantly increased the PAB1620 (WT-specific) fractions. Although, our results support that the reactivated p53R175 could bind zinc, thereby allowing its proper folding, further experiments are required to understand the mechanism of zinc binding and the enhanced efficiency of PEITC. A metallochaperone function has been demonstrated for a small molecule that could restore WT-like conformation to the p53R175.30 Further studies are required to discern the role of PEITC as a source of zinc for the reactivated p53R175. If PEITC indeed functions as a metallochaperone, it will be interesting to determine its effects on other zinc-binding mutants of p53, for example, C176F and C242F.ZnCl33). Although induction of ROS had no effect on the restoration of p53R175H conformation, the elevated oxidative stress was responsible for the activation of the restored p53R175 and induction of apoptosis. In support of this, ATZ enhances the antiproliferative ability of PEITC, whereas PEG-Catalase or NAC inhibits it.PEITC induced oxidative stress in SK-BR-3 cells, which may be exacerbated by increased ROS in p53 mutant cells  a peak concentration of 1 μM PEITC can be reached in the plasma.35As a proof-of-principle, we demonstrated that p5330 In this study, the inhibitory effect of PEITC was demonstrated under conditions that mimic cancer ‘chemopreventive settings'. In this bioassay, the animals were fed diets containing PEITC before the injection of the p53 mutant cells and formation of tumors, and mutant cells were then injected to mimic the presence of ‘cancerous' or ‘initiated' cells. Mice fed PEITC displayed a statistically significant decrease in tumor volumes, inhibition of proliferation and depletion of the p53R175Hin vivo. The elevated mRNA of p53 target genes from mice fed PEITC provides evidence for p53R175H reactivation.Reactivation of mutant p53 has been shown previously in xenograft models under ‘chemotherapeutic settings' where tumors are formed before the systemic administration of the drugs.R175. Given that the R175 mutation is the third most common missense mutation among p53 mutants in human cancers, with an estimated 5.1% frequency of occurrence,36 PEITC may be developed as a lead compound for tumors with p53R175. Mutations in the p53 gene may occur at different phases during tumorigenesis, such as in the late stages of pancreatic neoplasia,37 hepatocellular carcinoma,38 prostate cancer39 and so on but in the early phases of ductal carcinoma in situ (DCIS), a precancerous lesion of breast40 and liver cancer.41 The occurrence of p53 mutations before the development of invasive breast cancer, particularly in DCIS, suggests the potential of PEITC in the prevention of breast cancer. Besides p53R175, PEITC inhibited the proliferation and induced apoptosis of cell lines with other hotspot mutants, including DNA contact mutants. More studies are needed to understand the mechanisms of growth inhibition of these mutants by PEITC. Nevertheless, the finding that a dietary-related compound restores the ‘WT-like' conformation and functions to the p53R175 opens up an opportunity for implementing a practical and effective target-based strategy for cancer prevention and treatment.Significant efforts have been made to identify small molecules as therapeutics from chemical libraries aimed at reactivating p53 mutants. However, target-based prevention studies by dietary compounds are scarce. Our study elucidates a novel mechanism for PEITC, by preferential targeting of p53+/+) were cultured in Dulbecco's modified Eagle's medium with 10% FBS. (10)3 (p53−/−) MEFs and (10)3-derived MEFs with p53 mutations ((10)3/175 and (10)3/273) were cultured in Dulbecco's modified Eagle's medium with 10% FBS and 400 μg/ml G418. The MEF (10)3 and its derivative cells with human p53 mutations at residues R175 and R273 were previously derived3 and were a gift from Dr. Darren R Carpizo.HOP92, OVCAR3 and SW620 were ordered from NCI DTP, DCDT Tumor Repository, Fredrick, MD, USA. H1299, HT29, A549, MDA-MB-231, AU565, SK-BR-3 and MCF7 were obtained from Tissue Culture Source Resource, Georgetown University, Washington, DC, USA. All the cell lines were negative for mycoplasma and cultured in RPMI 1640 with 10% FBS. Normal colon cells CCD841 purchased from ATCC  were cultured in Eagle's minimal essential medium with 10% FBS. 3T3 Balb/c fibroblasts  yielded a desired concentration of PEITC at 1% DMSO. SK-BR-3 cell cultures containing PEITC were plated onto a 96-well microtiter plate at 4000 cells per well in duplicate. As a control, 4000 cells per well were seeded in medium containing 1% DMSO in duplicate. For background subtraction, wells lacking cells but containing medium were used. Plates were incubated at 37 °C for 3 days, followed by the addition of WST-1 reagent for 2 h. OD450 was measured using a microplate reader . Percentage of cell proliferation was calculated as the ratio of OD450 values obtained for respective cells grown in the presence of PEITC compared with the presence of DMSO. Similar assays were performed to determine the effect of PEITC on proliferation of H1299, HOP92, AU565, OVCAR3, SW620, HT29, A549, MCF7, CCD841 and SK-BR-3 cells transfected with p53 siRNA or NS siRNA, HOP92 cells transfected with p53 siRNA or NS siRNA, A549 cells transfected with p53 siRNA or NS siRNA, AU565 cells transfected with p53 siRNA or NS siRNA, SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA, H1299 transfected with pcDNA3, H1299 transfected with pcDNA3-p53R175, H1299 transfected with pcDNA3-p53R273 and H1299 transfected with pcDNA3-wtp53, 3T3 Balb/c fibroblasts, (10)3 MEFs, (10)3/175 MEFs and (10)3/273 MEFs.The effect of PEITC on SK-BR-3 cell proliferation was determined by using the WST-1 assay  as described previously.To determine the effect of the reducing agents, NAC or PEG-Catalase on PEITC activity, SK-BR-3 cells were treated with the indicated concentrations of PEITC, reducing agent or both. Cell proliferation was then measured by performing WST-1 assays as described previously. Similarly cells were either co-treated with PEITC and 2 mM ATZ or PEITC alone and cell proliferation was determined. To determine the effect of zinc on PEITC activity, SK-BR-3 cells were treated with the indicated concentrations of PEITC, zinc or both. Cell proliferation was then measured by performing WST-1 assays as described previously.μl of Lipofectamine 2000 in 1 ml of Opti-MEM (Invitrogen). The mixture was added to cells that subsequently were incubated for 6 h. After 24 h, a second transfection was performed similarly. Seventy-two hours after the initial transfection, cells were harvested for preparing lysate or were treated with PEITC or DMSO at the indicated concentrations, and cell proliferation was measured using WST-1 reagent (Roche) as described previously for SK-BR-3 cells. For ATG5 knockdown in SK-BR-3 cells, ATG5 siRNA  was transfected as described previously except that a single transfection was carried out for 6 h. After 24 h, transfected cells were harvested for preparing the lysates or were treated with PEITC for 4 h before lysates were prepared.The p53 siRNA was obtained from SMARTpool . The siRNA was transfected using Lipofectamine 2000 following the manufacturer's protocol . Briefly, cells were plated to 50–60% confluence in 10-cm dishes 24 h before transfection. The siRNA (0.430 nmol) was mixed with 43 μg) was mixed with 43 μl of Lipofectamine 2000 in 1 ml of Opti-MEM (Invitrogen). The mixture was added to cells that subsequently were incubated for 6 h. After 24 h, transfected cells were treated with PEITC for WST-1 assay or Annexin-V staining as described. The transfected cells were maintained in RPMI 1640 with 10% FBS and 400 μg/ml G418.The plasmids pcDNA3, pcDNA3-wtp53, pcDNA3-p53R175 and pcDNA3-p53R273 were transfected using Lipofectamine 2000 following the manufacturer's protocol (Invitrogen). Briefly, cells were plated to 50–60% confluence in 10-cm dishes 24 h before transfection. The plasmid (14 Escherichia coli cells (BL21DE3) transformed with pGeX4T1-mutp53R175H vector were grown at 37 °C in LB medium containing 100 μg/ml ampicillin to an optical density at 600 nm of 0.4. The expression of recombinant protein was induced by the addition of 0.5 mM isopropyl-1-thio-β-galactopyranoside for 3 h at the same temperature under vigorous shaking. Bacteria were pelleted and lysed in lysis buffer  and protease inhibitors) by freeze–thaw cycles (three times) followed by probe sonication (three cycles of 1 min each). Sonicate was clarified by centrifugation at 18 500 × g for 30 min at 4 °C. The supernatant was transferred to a fresh tube and saved. The pellet was resuspended in sarcosyl buffer (lysis buffer+2% sarcosyl) followed by probe sonication (three cycles of 1 min each). The supernatant fractions from both the steps were diluted 1 : 1 in 1 × PBS and were incubated with glutathione-Sepharose beads  for 2 h at 4 °C with constant rotation. After several washes in PBS, protein was eluted in elution buffer , 300 mM NaCl, 1 mM DTT and protease inhibitors). Twenty-five nanograms of recombinant GST-mutant p53R175H  was incubated with DMSO or 4 μM PEITC for 1 h on ice. The protein samples were then applied to the ELISA plates and incubated at 4 °C for 2 h. The wells were washed with 1 × PBST (containing 0.05% Tween-20) and blocked by 5% skim milk at 4 °C for 2 h. Wells were rinsed and mouse primary antibody (PAB240 or PAB1620) diluted 1 : 1000 in 1 × PBST was added followed by incubation at 4 °C overnight. Wells were washed with 1 × PBST followed by addition of secondary anti-mouse HRP antibody at 4 °C for 1 h. Wells were rinsed with 1 × PBST and substrate was added  followed by measuring chemiluminescence at 450 nM. As a control, purified GST only and purified WT p53 (Thermo Scientific) were used. To determine the effect of ZnCl2, 25 ng of recombinant GST-mutant p53R175H  was incubated with 2.5 μM ZnCl2 or 4 μM PEITC alone or in combination for 1 h on ice. ELISA assay was then performed as described.μl of the fluorochrome conjugated Annexin-V was added in the residual buffer and cells were incubated at room temperature (RT) in the dark for 15 min followed by the addition of 0.5 ml of Annexin-V-binding buffer and 5 μl of PI staining solution (0.1 μg/ml). Cells were then analyzed by a BD LSRFORTESSA instrument . To determine the effect of Nutlin-3 treatment on apoptotic induction, SK-BR-3 and A549 cells were treated with PEITC, Nutlin-3 or both or DMSO at the indicated concentrations for 24 or 72 h. Cells were then prepared for Annexin-V staining as described previously. To determine the effect of the reducing agents NAC or PEG-Catalase, on PEITC-induced apoptosis, SK-BR-3, NS siRNA-transfected SK-BR-3 or p53 siRNA-transfected SK-BR-3 cells were treated PEITC, reducing agent or oxidizing agent alone or PEITC in combination with reducing or oxidizing agent. Cells were then prepared for Annexin-V staining as described previously.The Annexin-V staining was carried out in accordance with the manufacturer's instructions . In brief, SK-BR-3, A549, MDA-MB-231, OVCAR3, H1299 transfected with pcDNA3, H1299 transfected with pcDNA3- p53R175, H1299 transfected with pcDNA3-p53R273 and H1299 transfected with pcDNA3-wtp53, 3T3 Balb/c, (10)3 (10)3/175 and (10)3/273 cells were treated with PEITC as indicated or DMSO as a control for 3 days. Cells were harvested by scraping, washed once with 1 × PBS and resuspended in 0.5 ml Annexin-V-binding buffer. Cells were collected by centrifugation, 5 SK-BR-3 cells transfected with ATG5 siRNA or NS siRNA were treated with DMSO or PEITC at the indicated concentrations for 72 h. To assay apoptosis, the cytoplasmic histone-associated DNA fragments indicative of ongoing apoptosis were measured quantitatively using the cell death detection ELISA PLUS photometric enzyme assay (Roche).μM) or 1% DMSO as a control for 6 h in slide chambers with four wells (ThermoFisher Scientific). Cells then were washed twice with 1 × PBS and fixed with formaldehyde (3.7%) at RT for 15 min. Fixed cells were washed four times with 1 × PBS and treated with 0.5% Triton X-100 (Sigma) at RT for 5 min. Cells were washed four times with 1 × PBS containing 0.5% Tween-20 and blocked with 10% goat serum (Sigma) overnight at 4 °C. Cells were washed four times with 0.1% Tween-20 and incubated with mouse PAB240  or mouse PAB1620  that recognizes specifically the mutant or p53 WT, respectively, overnight at 4 °C. After four washes with 0.1% Tween-20, cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG  at RT for 2 h. Cells were washed four times with 0.1% Tween-20 and coated with Prolong Gold Anti-Fade reagent (Invitrogen) containing DAPI. Coverslips were placed on the chamber slides, and cells were cured at RT in the dark for 24 h. Immunofluorescence analyses were performed with a Zeiss LSM 510 META NLO inverted Axiovert 200 M laser scan microscope  with a Plan-Apochromat 63 × 1.4 numerical aperture oil immersion objective lens. Images were captured using the Photomultiplier Tube detectors and analyzed using the Image J software . Fluorescent staining intensity was quantified using the Metamorph software .SK-BR-3, A549, H1299 and (10)3/175 cells were treated with PEITC  was used as a primary antibody.To determine the effect of PEITC on γ-H2AX foci formation in SK-BR-3 cells transfected with p53 siRNA or NS siRNA or A549 cell lines, cells were treated with 4 g for 10 min at 4 °C, and the supernatant was collected. The supernatants were diluted in lysis buffer, and 200 μg of the lysate was gently tumbled at 4 °C for 1 h with protein G-agarose beads (Roche). The lysates obtained after preclearing were then gently tumbled at 4 °C for 2 h with mouse PAB240 antibody . Protein G-agarose beads were then added to the suspensions and incubation was performed for 2 h at 4 °C. The beads were washed four times with lysis buffer supplemented with protease inhibitors, and the immunoprecipitates were eluted by boiling in Laemmli buffer and resolved on 4–12% SDS-PAGE. Immunoprecipitated p53 was detected by western blotting using FL393 (Santa Cruz Biotechnology) as a primary antibody. For the secondary antibody, peroxidase-labeled anti-mouse IgG  was used. The blot was developed using the ECL Prime Western Blot Detection Kit according to the manufacturer's protocol . As a control, the blot was stripped and then reprobed with anti-p53 (DO-1) antibody  or anti-GAPDH antibody . The density of the p53 bands in the PEITC treated samples relative to that of DMSO control was determined using the Gene Tools software .SK-BR-3, A549 and HOP92 cells were treated with the indicated concentration of PEITC or 1% DMSO as a control for 6 h. For preparation of cell lysates, cells were harvested and washed once with 1 × PBS, cell pellets were suspended in lysis buffer (20 mM Tris-Cl (pH 8.0), 137 mM sodium chloride, 10% glycerol, 1% NP-40, 2 mM EDTA) and Protease inhibitors  and the cells were incubated on ice for 30 min. The cell suspension was centrifuged at 18 500 × g for 10 min at 4 °C and the supernatant was collected, unless mentioned otherwise. The remaining pellet was defined as insoluble fractions. Insoluble fractions were dissolved in 2% SDS lysis buffer (65 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2% SDS, 50 mM DTT). For WCL fractions, cells were harvested and the pellets were dissolved in 2% SDS lysis buffer as described previously. The fractions were collected by centrifuging the lysate at 18 500 × g for 10 min at 4 °C. Then 30–50 μg of the lysates were loaded on 4–12% SDS/PAGE. Protein was transferred onto a PVDF membrane, and the blots were developed using the ECL Prime Western Blot Detection Kit according to the manufacturer's protocol (Amersham). The antibodies for p21, Bax, ATM, pATM S1981, CHK2, pCHK2 Thr68 and p53 (DO-1) were purchased from Santa Cruz Biotechnology and GAPDH antibody was from Novus Biologicals. The antibody for ATG5  was a gift from Dr. Shivendra V Singh.Different lysis buffers were used to prepare soluble, insoluble and WCL fractions. For lysate (soluble fraction) preparation, cells were harvested and washed twice with 1 × PBS. RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 2 mM EDTA) was added to the cells, and the cells were incubated on ice for 30 min. Then the cell suspension was centrifuged at 18 500 × g for 5 min. Cell pellets were washed once with ice-cold PBS and transferred to 1.5-ml microcentrifuge tubes followed by centrifugation at 500 × g for 2 min. Pellets were stored at −80 °C prior to chromatin fractionation following the manufacturer's instruction  to generate nuclear soluble and chromatin-bound protein fractions. Ten micrograms of protein from the soluble nuclear extract and the chromatin-bound nuclear extract for the samples from DMSO- or PEITC-treated cells were resolved on 4–12% SDS-PAGE and transferred to PVDF membranes. Blots were probed with p53 (DO-1) antibody . Histone H3 and TopoIIB, which served as markers for the chromatin and soluble nuclear fractions, respectively, were detected with rabbit anti-Histone H3 polyclonal (Thermo Scientific) and mouse anti-TopoIIB monoclonal (Santa Cruz Biotechnology) antibodies.SK-BR-3 cells were treated with the indicated concentrations of PEITC or DMSO as a control for 4 h. Cells were trypsinized and harvested by centrifugation at 500 × μM PEITC or DMSO as a control for 4 h. RNA was extracted from the cells using a Qiagen RNeasy Kit , cDNA was synthesized by using High Capacity RNA to cDNA Kit  and the gene expression level was measured by qRT-PCR using TaqMan gene expression assays . The gene expression level is normalized with GAPDH, and the average is presented with S.D. from triplicates of repeated experiments. RNA was extracted from the SK-BR-3 xenograft tissues by using Qiagen RNeasy Kit and was processed further for qRT-PCR as described for the SK-BR-3 cells. The gene expression levels were normalized with GAPDH. Fold changes in the gene expression levels were calculated for each tumor from the treated group relative to the tumors from the control group and the average is presented with S.D.SK-BR-3, SK-BR-3 transfected with p53 siRNA or NS siRNA, H1299 and A549 cells were treated with 4 The levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured using the GSH/GSSG-Glo Glutathione Assay Kit (Promega). Briefly, SK-BR-3 cells were treated with PEITC or DMSO as a control for 4 h. Cells were then processed for the glutathione assay as per the manufacturer's instructions (Promega).43 It was transfected into SK-BR-3, HOP92, AU565, H1299 and MEF ((10)3/175 and (10)/273) cells, followed by treatment with PEITC  for 24 h. The cell lysate was made and the luciferase reporter assay was performed in accordance with the manufacturer's instructions .WWP-Luc (p21/WAF1 promoter) plasmid encoding p53 WT-binding element in the p21 promoter region was a gift from Bert Vogelstein .2+ and Mg2+, trypsinized for 5 min and harvested by centrifugation at 190 × g for 3 min at 4 °C. Cells were washed once with PBS and the pellets resuspended in 1 ml of 70% ethanol and stored at –20 °C overnight. Cells were harvested by centrifugation at 420 × g for 10 min. The cell pellets were washed once with 1 ml cold PBS and resuspended in 1 ml freshly prepared PI staining solution ). The cell suspension was incubated at RT for 30 min in the dark followed by incubation for 30 min at 4 °C. The samples were run on a Becton Dickinson FACS sorter  and the data were analyzed using Mod Fit program .SK-BR-3, SK-BR-3 transfected with p53 siRNA or NS siRNA and A549 cells were treated with PEITC, Nutlin-3 or both or DMSO as a control for 24 or 72 h. Cells then were prepared for flow cytometric analysis. Briefly, cells were washed with PBS free of Cag for 10 min at 4 °C, washed once with PBS and resuspended in RIPA buffer (10 mM sodium phosphate (pH 7.2), 300 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate and 2 mM EDTA) containing a protease and phosphatase inhibitor mixture and were incubated on ice for 30 min and centrifuged at 18 500 × g for 10 min at 4 °C. The supernatant was collected, and 200 μg of the lysate was loaded on 4–12% SDS/PAGE. Following electrophoresis, protein was transferred onto a PVDF membrane, and blot was probed with anti-pATM Ser1981 antibody (1 : 500) (Santa Cruz Biotechnology) or anti-pCHK2 Thr68 antibody (1 : 500) (Santa Cruz Biotechnology). For the secondary antibody, peroxidase-labeled anti-mouse IgG  was used. The blot was developed using the ECL Prime Western Blot Detection Kit following the manufacturer's protocol (Amersham). As a control, the blot was stripped and then reprobed with anti-ATM anti-body  or CHK2 antibody .Cells were treated with either DMSO, ATZ, NAC, PEG-Catalase or PEITC alone or PEITC in combination with ATZ or NAC or PEG-Catalase for 4 h. Cells then were harvested by centrifugation at 1600 × in vivo studies and tumor harvest were performed in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. Mice were weighed and housed under quarantine in polycarbonate cages  for 1 week. Mice were fed an AIN-93M diet and water ad libitum for 1 week of quarantine. After 1 week in quarantine, the mice were placed on either a control AIN-93M diet or an AIN-93M diet supplemented with PEITC (5 μmol PEITC/g diet) (10 mice/group). Both diets were prepared by Research Diets . The number of animals was decided based on previous studies, which used the SK-BR-3 xenograft mouse model to generate statistically significant data, and the concentration of the PEITC in diet was chosen based on our previous bioassay in mice.44 The PEITC concentration in the diet was confirmed to be 4.97±0.16 μmol/g by ethyl acetate extraction followed by cyclocondesation reaction of 1,2-benzenedithiol with the ITCs as described.34 By single-factor ANOVA calculation, there was no statistically significant difference  between the PEITC samples. The food was replenished every alternate day. After 1 week of control and PEITC diets, the mice were injected with exponentially grown 2 × 106 SK-BR-3 cells (suspended in 50 μl of Matrigel) in their left and right mammary fat pads (‘cancer chemoprevention' settings). No mortality or death was observed over the course of the experiment. Tumor formation was assessed externally weekly, and tumor dimensions were measured with Vernier calipers for a 10-week bioassay period. Tumor volumes were calculated with the formula L × W2 × 0.523. The tumors were small in size and were not easily measurable externally. Because of the small size, some of the tumors showed variations in the tumor volume measurements over the weeks. For this reason, only the tumors (n=7) that followed a growth pattern without fluctuations were considered for the final tumor volume calculations. However, no outliers were detected by using the GraphPad software . All the animals were killed, and the tumors were removed and were confirmed by H&E staining as described below. Animal weights (g) were measured weekly. The ITC concentration in the blood of the animals collected at the time of necropsy was evaluated by cyclocondensation reaction of 1,2-benzenedithiol with ITCs as described.34 PEITC concentration was found to be 1.13±0.15 μM (n=3) and 0.37±0.03 μM (n=2) in the blood of the PEITC-treated and control animals, respectively. Blinding to the groups was not possible because of the different diets; experiments were, however, blinded to tumor harvest and histopathological analysis. H&E-stained sections were blindly examined by pathologist for the incidence of tumors.Twenty female athymic nu/nu Balb/c mice  were purchased from Charles River Laboratories . All μm, de-paraffinized with xylenes and rehydrated through a graded alcohol series. Heat-induced epitope retrieval was performed by immersing the tissue sections at 98 °C for 20 min in 10 mM citrate buffer (pH 6.0) with 0.05% Tween. Immunohistochemical staining was performed using a horseradish peroxidase-labeled polymer from Dako   according to the manufacturer's instructions. Briefly, slides were treated with 3% hydrogen peroxide and 10% normal goat serum for 10 min each and exposed to primary antibodies p53 (DO-7)  for 1 h at RT and Ki-67  overnight at 4 °C. Slides were exposed to the appropriate HRP-labeled polymer for 30 min and DAB chromagen (Dako) for 5 min. Slides were counterstained with Hematoxylin , blued in 1% ammonium hydroxide, dehydrated and mounted with Acrymount . Consecutive sections with the primary antibody omitted were used as negative controls. The sections were examined under an Olympus BX61 microscope at × 200 magnification. Representative images were captured from the entire tumor section using a DP70 camera and DP70 software , and images were analyzed using Image J software (NIH). Further, because of the small size of the tumors, four sections per tumors were analyzed for determining the cell number. The sections were stained with different antibodies as mentioned and for each antibody 20 pictures were taken from different areas on slide to count the total cell number. The data presented for each tumor are the average of the total number of cells from different antibody stainings.H&E-stained slides were examined and tissues were confirmed as tumors by histopathological examination. Immunohistochemistry was performed at Histopathology and Tissue Shared Resources, Georgetown University following standard protocols. Briefly, tissues were sectioned at 5 n=12) lysate from each group was prepared by homogenizing the tissue in 20w/v of lysis buffer . Twenty-five micrograms of the lysates were loaded on 4–12% SDS/PAGE, transferred onto a PVDF membrane and the blot was probed with p53 (DO-1) antibody as described previously. To perform qRT-PCR assay with the SK-BR-3 tumor xenograft tissues, RNA was extracted from tumor tissues (n=4) using the Qiagen RNeasy Kit and gene expression level was measured as described previously.As we were limited by the amount of tumor tissue, especially from the mice on the PEITC-supplemented diet, we divided the tumors randomly to perform western blotting and qRT-PCR analyses. For western blotting analysis, the SK-BR-3 tumor xenograft (t-test. Differences were considered statistically significant at P-values of ≤0.05. All statistical tests were two-sided.Statistical differences in tumor volume and biological end points were evaluated with a two-tailed Student's


Here we report that murine CD4 T cells lacking Neuroplastin (Nptn−/−), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn−/− T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn−/− T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn−/− T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.The outcome of T cell activation is determined by mechanisms that balance Ca Cytosolic Ca2+ levels ([Ca2+]i) return to baseline due to both Ca2+ uptake into stores and extrusion across the plasma membrane via PMCAs3. The degree to which the TCR is engaged by ligands on the APC together with extrinsic factors, such as cytokines, determines the amplitude and the spatio-temporal profile of Ca2+ signals4. The Ca2+ signal acts on calcineurin, a phosphatase, which in turn dephosphorylates nuclear factor of activated T cells (NFAT), triggering its nuclear translocation and thus transcription of T cell activation genes6. Products of these genes include cytokines that induce polarization of T cells to T helper 1 (Th1) or T helper 2 (Th2) profiles to direct cellular or humoral responses, respectively7. Th1 differentiation strongly correlates with sustained TCR-induced Ca2+ signaling, while the Th2 profile is associated with weaker and more transient Ca2+ signals10.Activation of the T cell receptor (TCR) by cognate peptide on an antigen-presenting cell (APC) leads to release of Ca13. Loss of PMCA1 in mice is embryonic lethal, while PMCA4 knock out mice are viable but male-sterile11. PMCAs are controlled at the transcriptional, splicing and post-translational level14. In Jurkat T cells, PMCA4 activity is stimulated by Ca2+ 3 but inhibited through spatial confinement in microdomains at the immune synapse, where Ca2+ sequestration by mitochondria or association with membrane proteins STIM1 and POST restricts its local activity at the synapse17. These modifications to PMCA localization ensure that TCR-induced Ca2+ signaling succeeds in activating downstream targets such as NFAT.The PMCA family comprises four members, two of which are expressed in T cells: PMCA1 and PMCA418. CD147 is involved in T cell development and activation20. In contrast, nothing is known about Neuroplastin in T cells. Neuroplastin is expressed as two highly gylcosylated splice variants called Np65 and Np55, with 3 and 2 Ig domains, respectively21. Np65 exhibits trans-homophilic binding22 and is mainly expressed in neurons, while Np55 is more broadly expressed21. Np65 is required for learning and memory23 and for synaptic structure and plasticity25. To date, few non-neuron-specific interaction partners of Np55 have been identified28. We report here that Neuroplastin interacts with PMCA and is required for stabilizing PMCA expression. Nptn−/− T cells show elevated Ca2+ levels and increased nuclear NFAT, and this phenotype was mimicked in PMCA1 heterozygous T cells. Finally, we show that loss of Neuroplastin from T cells results in the production of elevated levels of cytokines and a strong bias towards Th1 polarization.Neuroplastin is a transmembrane protein of the Ig superfamily and a close paralog of CD147 (Basigin/EMMPRIN)21, differential glycosylation of Np55 accounts for the appearance of multiple bands in lymphoid tissues. In extracts from purified CD4 T cells, Neuroplastin was present as a band of 45 kDa that was upregulated in activated CD4 blast cells  mice with 1:1 mixtures of bone marrow cells derived from wt and Nptn−/− donors carrying specific cell surface markers (Ly5.1 versus Ly5.2). When competing with wt cells, Nptn−/− precursor cells within an otherwise normal cellular environment were moderately hindered in colonizing the thymus. However, progression through thymic developmental stages and peripheral T cell homeostasis appeared normal when assessed for relative abundance of Nptn−/− thymocyte and T cell subpopulations . Western blot analysis confirmed that Neuroplastin-immunoprecipitates from wt thymic lysates contained PMCA1 and PMCA4 . We first confirmed that Neuroplastin interacts with PMCA1 and PMCA4 in BMDMs by co-immunoprecipitation  and the rate constant K (0.0078 ± 0.0006 s−1vs 0.0092 ± 0.0003 s−1) as determined by exponential fit were reduced in mutant T cells, whereas the plateau was elevated (0.206 ± 0.004 a.u. vs 0.115 ± 0.002 a.u.)  and rate constant (0.306 ± 0.038 s−1 vs 0.354 ± 0.026 s−1) and by an elevated plateau (0.835 ± 0.026 a.u. vs 0.603 ± 0.016 a.u.) . First we observed that, similar to Neuroplastin, PMCA1 and PMCA4 were upregulated following T cell activation  Neuroplastin physically associates with PMCAs and (ii) that this interaction underlies a previously unnoticed requirement for Neuroplastin in murine T cell activation. We showed that its loss in T cells resulted in a profound reduction of both PMCA isoforms along with reduced Ca2+ clearance following stimulation. Importantly, all aspects of impaired Ca2+ regulation were phenocopied by genetic reduction of PMCA1 levels. Of note, we did not detect SERCAs or mitochondrial Ca2+ uniporters in the here presented screen nor in a series of MS-based analyses of Neuroplastin-specific immunoprecipitates isolated from mouse brain samples under less stringent conditions. To our knowledge, particular roles for PMCA1 and 4 in murine T cells have not been addressed and studies on human T cells have focused on PMCA4. Our study uncovered a previously unreported localization of murine PMCA1 to the immune synapse. The fact that heterozygosity of Pmca1 already resulted in altered Ca2+ homeostasis argues for a pivotal role of this isoform in murine T cells. Along this line, the elevated Ca2+ in both Nptn−/− and Pmca1/−+ T cells was accompanied by increased activation of NFAT and NFAT-dependent cytokine production. Coexpression of PMCAs and Np55 in many cell types suggests that this complex is a universal module in Ca2+ signaling. In fact, we have also noticed reduction of PMCA in Nptn−/− brains, though to a lesser extent than in T cells23. Thus, Neuroplastin regulation of Ca2+ clearance via PMCA may explain some of its neuronal roles in synaptogenesis, long-term potentiation, and memory processes25.Neuroplastin has multiple functions in the nervous systemNptn−/− samples. Susceptability of PMCAs to proteolytic cleavage by calpain or by caspases 1 and 3 is well-established13. However, these proteases affect PMCA isoforms differently and typically produce detectable subfragments, which we never observed. It is rather conceivable that Neuroplastin stabilizes PMCAs co- and/or post-translationally in a chaperone-like manner, i.e. by supporting proper folding and membrane insertion of PMCAs as a prerequisite for further trafficking and surface expression. Loss of Neuroplastin may in turn lead to increased removal of PMCA molecules by co-translational, ER-associated protein degradation (ERAD)31, a mechanism that may even operate on nascent polypeptide chains32. Indeed, this would explain why neither our immunofluorescence labelings nor cellular fractionations pointed to an accumulation of PMCAs within secretory pathway compartments in Nptn−/− cells. Interestingly, valosin-containing protein , a major player in ERAD and quality control of protein folding33, was among the few interaction partners for Neuroplastin that we identified by mass spectrometry when samples were prepared under stringent conditions . Therefore, concomitant reduction in both PMCA isoforms due to loss of Neuroplastin may account for impaired development of common lymphoid precursor subpopulations. Despite the strong reduction of PMCA in Nptn-deficient T cells, the elevated cytosolic Ca2+ levels did not apparently interfere with thymic T cell development. We cannot rule out, however, that a lower signaling threshold during thymocyte selection may have led to alterations in the TCR repertoire, thereby eliminating T cells prone to even more severe Ca2+ phenotypes. Moreover, our data indicate that T cells cope with PMCA reduction, at least in part, by increased uptake of Ca2+ into intracellular stores.We found that in a competitive situation, 2+ clearance, one would expect that normal baseline levels would always be reached in truly resting cells, even when PMCA levels are strongly reduced as in the case of Nptn deficiency. However, Nptn−/− (and to a lesser extent Pmca1/−+) T cells displayed a robust increase in baseline [Ca2+]i. This may reflect ongoing Ca2+ flux during T cell development and naïve T cell homeostasis, with the consequence to persistently challenge the limited capacity of the remaining PMCA molecules. Indeed, while routinely scanning the surface of antigen presenting cells, naïve T cells experience tonic TCR signaling, which has been implicated in the maintenance of T cell responsiveness to antigen35. With PMCAs reduced, activity of SERCA gains impact on Ca2+ clearance, consistent with the observed Ca2+ overload of both the ER and mitochondria36. Notably, preliminary assessment of SERCA protein levels did not reveal up- or down-regulation in the absence of Neuroplastin.While the number and activity of PMCAs determine the kinetics of Ca2+]i was elevated by ~ 65% in Nptn−/− T cells, whereas, consistent with a milder PMCA reduction, it was elevated by only ~30% in Pmca1+/− T cells. Still, nuclear NFAT levels and TCR-induced IFNγ production were similarly increased in cells of either genotype. Also, for both genotypes we observed that the portion of naïve T cells with instantaneous [Ca2+]i values far above mean was much larger than for wt. This observation would be consistent with more frequent fluctuations, possibly oscillations, within individual cells. In fact, it is well-established that frequent Ca2+ oscillations are particularly efficient in maintaining nuclear NFAT37. Moreover, recent studies on non-immune cells demonstrated that PMCAs actively shape SOCE-induced Ca2+ oscillations in an isoform-specific manner38 and silencing of PMCA1 and 4 was found to increase Ca2+ oscillations and nuclear translocation of NFATc1 in osteoclasts39. Thus, while our FACS-based Ca2+ measurements did not resolve oscillations, we assume that reduced PMCA levels in both Nptn−/− and Pmca1+/− T cells exert their effects on NFAT and cytokine production at least in part via increased Ca2+ oscillation.The mean baseline ex, and acquired for 10 min. Ca2+ kinetics were analyzed using FlowJo and Excel. Instantaneous single cell ratios from different experiments were summarized as mean ± SD. Mean baseline ratios were averaged over 30 s and normalized to the average wt ratio for each experiment. Ca2+ decay phases were analyzed by fitting an exponential function to the mean ratios using Prism software. Since exponential decay after TCR-induced Ca2+ peak levels is delayed due to simultaneous influx and efflux, the best fit one-phase exponential regression was performed starting 1 min after the mean maximum ratio. For the rapid Ca2+ decay after thapsigargin-induced SOCE, the best fit one-phase exponential regression was performed for the first 30 s after removal of Ca2+. The fitted curve was calculated as Y = P + (Y0 − P) * EXP(−K * t) with Y0: ratio at t = 0, P: plateau ratio, K: rate constant [1/s], half time: ln(2)/K [s]. The initial rate was calculated at t = 0 from the first derivation of the fitted function with initial rate = −K * (Y0 − P).T cells from different donors were labeled separately with anti-CD4 using donor-specific fluorescent conjugates and mixed for further treatments and Cawt or Nptn−/− CD4 T cells were stimulated on immobilized anti-CD3 (5 μgl/ml) and anti-CD28 (1 μg/ml) for 3 to 5 days. B cells were stimulated overnight with 10 μg/ml LPS (Sigma) and loaded with 50 μM pOva (JPT). T and B cells were mixed 1:1, centrifuged 90 s at 300xg, carefully resuspended in R10 and incubated for 90 min at 37 °C on poly-L-lysine coated cover slips for synapse formation. T-B pairs were fixed with 4% PFA/ 5% sucrose and labeled overnight with anti-Neuroplastin in BD Perm/Wash buffer and subsequently with anti-PMCA1 in 0.05% Triton/ 10% donkey normal serum for 2 h followed by staining with donkey secondary antibodies and DAPI. Immune synapses were imaged on a Leica TCS SP5 confocal microscope.For immune synapse formation, OT-II transgenic wt and Nptn−/− cells were first separately labeled with anti-CD4 and mixed as described above. Confocal images with maximal DAPI signal were captured to determine ROIs for nuclear NFAT quantification using ImageJ. Nuclear NFAT intensities were normalized to the mean of wt cells for each experiment.For immunofluorescent NFATc2 and DAPI staining of CD4 T cells, we used the Fixation/Permeabilization Kit (ebioscience). For optimized comparability, 2, 750 U/ml Benzonase (Sigma), containing protease inhibitors). After centrifugation at 15000xg for 20 min, supernatants were used for analysis.For Western blots cell pellets were homogenized by incubation at 4 °C for 30 min in Triton-homogenization buffer  were homogenized in saccharose HEPES buffer . The homogenate was centrifuged at 1000xg for 10 min to remove nuclei and debris. Supernatant was centrifuged for 1 h at 100000xg. Pellets were washed in homogenization buffer  and centrifuged as above. The resulting pellet was then rehomogenized in the same buffer containing 0.5% Triton X-100. After incubation at 4 °C for 1 h the material was centrifuged at 20800xg for 1 h.Total brains from adult mice were homogenized in homogenization buffer, centrifuged at 100000xg for 1 h and washed once. The pellet was then rehomogenized in buffer containing 0.5% Triton X-100. After 1 h incubation samples were spun again as above.Triton X-100 solubilized membrane proteins from spleen, thymus and total brain were ethanol precipitated, and deglycosylation was then performed using the Glyco Profile IV chemical deglycosylation kit (Sigma). Deglycosylated protein was purified by dialysis .2 and Benzonase (Sigma), containing protease inhibitors) incubated at 4 °C for 30 min and spun at 15000xg for 20 min. The resulting supernatant was precleared by 30 min incubation with ProteinG SepharoseTM 4 Fast Flow . The lysate was then incubated with Neuroplastin antibody overnight. Protein G Sepharose beads were added for 2 h at 4 °C. Beads were washed three times with washing buffer  followed by a short rinse in 20 mM Tris/150 mM NaCl. For SDS-PAGE, bound proteins were eluted with 1x Rotiload (Roth). For mass spectrometry, beads were washed three times with PBS and finally resuspended in 50 mM ammonium bicarbonate.Cell pellets were homogenized by carefull pipetting in Digitonin homogenization buffer  kit according to the manufacturer’s instructions (Thermo Fisher Pierce). Samples were solubilized in sample loading buffer  at concentrations of approximately 1.5 μg/μl and run on 4–20% SDS-Polyacrylamide gels. For immunodetection proteins were transfered onto nitrocellulose and incubated with the primary antibodies as indicated. Immunoreactivity was detected according to standard protocols using an ECL Imager  or ECL-films. Images below saturation or films with the shortest exposure time still showing all expected bands were used for quantification by Image-J. For each blot, band intensities were normalized relative to the respective loading control and statistically analyzed using Prism software.Neuroplastin immunoprecipitate beads were washed with PBS and resuspended in 50 mM ammonium bicarbonate. Cysteins were reduced with 2 mM dithiothreitol for 30 min at room temperature and subsequently ß-methylthiolated by addition of 10 mM methyl methanethiosulfonate. Digestion was performed by addition of 0.5 µg trypsin (Promega) and incubation overnight at 37 °C. Peptides were extracted by pooling the primary supernatant and the supernatant of a subsequent washing step using 0.1% (v/v) trifluoroacetic acid (TFA).Peptides were purified with reversed-phase C18 ZipTip nano-columns (Millipore), eluted with 0.1% TFA/ 70% ACN, and dried. Protein identification was performed by high-resolution mass spectrometry on a hybrid dual-pressure linear ion trap/orbitrap mass spectrometer  equipped with an EASY-nLC Ultra HPLC (Thermo Scientific). For analysis, peptide samples were adjusted to 10 µl 0.1% TFA/ 1% ACN and fractionated on a 75 µm (ID), 25 cm PepMap C18-column, packed with 2 µm resin (Dionex/Thermo Scientific). Separation was achieved through applying a gradient from 2 to 35% ACN in 0.1% formic acid over 150 min at a flow rate of 300 nL/ min. An Orbitrap full MS scan was followed by up to 15 LTQ MS/MS runs using collision-induced dissociation (CID) fragmentation of the most abundantly detected peptide ions. Essential MS settings were as follows: full MS ; MS/MS . Normalized collision energy was set to 35%, and activation time to 10 ms. Raw data processing and protein identification were performed using PEAKS Studio V.8.0 (Bioinformatics Solutions). False discovery rate was set to <1%.All datasets generated and analyzed during the current study are available from the corresponding authors on reasonable request.Supplementary Information


Finally, we performed chemoprotection studies in an in vivo mouse model. Of 35 patients, 23 (65%) expressed CCR2 by FCM in PB. Two cell lines expressed high levels of CCR2 (THP-1 and murine AML). RT-PCR and WB confirmed CCR2 production. CCL2 solid phase ELISA showed significantly lower levels of CCL2 in PB and BM compared to normal controls. Chemotaxis experiments confirmed a dose-dependent migration in AML primary cells expressing CCR2 and THP-1 cells. A significant inhibition of transmigration was seen after CCL2/CCR2 blockade. Proliferation of CCR2+ AML cell lines was slightly increased (1.4-fold) after axis stimulation. We observed a non-significant increase in phase S THP-1 cells exposed to CCL2 and a concomitant decrease of cells in G1. The chemotherapy studies did not show a protective effect of CCL2 on cytarabine-induced apoptosis or synergy with chemotherapy after CCL2/CCR2 blockade both in vitro and in vivo. In conclusion, CCL2/CCR2 axis is expressed in the majority of monocytoid AML blasts. The axis is involved in cell trafficking and proliferation but no in vitro and in vivo chemotherapy protective effect was seen.Acute myeloid leukemia (AML) has a high mortality rate despite chemotherapy and transplantation. Both CXCR4/SDF-1 and VLA-4/VCAM1 axes are involved in leukemia protection but little is known about the role of CCL2/CCR2 in AML biology and protection against chemotherapy. We measured CCR2 expression in AML cell lines and primary AML cells by flow cytometry (FCM), real time PCR (RT-PCR) and western blot (WB). CCL2 production was quantified by solid phase ELISA in peripheral blood (PB) and bone marrow (BM) serum. We measured chemotaxis in a transwell system with different concentrations of CCL2/CCR2 blockers; cell cycle with BrDU and propidium iodide and proliferation with yellow tetrazolium MTT. We determined synergy in  Acute myeloid leukemia (AML) is a complex disease with an elevated mortality rate despite high intensity therapies . The mecCCL2 belongs to the family of ß-chemokines . Its genin vivo solid tumor models , it was shown that CCL2/CCR2 axis mediated the migration of MSC into the tumor and also showed evidence of CCL2-mediated protumor effect. CCR2 -/- mice had attenuated tumor growth compared to wild-type mice [The role of CCL2/CCR2 axis in cancer is largely unknown. In ype mice . In humaype mice . Interesype mice . In anotype mice . In FIP1ype mice .In this study we show in a series of experiments with both AML cell lines and primary AML cells an important role of CCL2/CCR2 axis in AML cell trafficking and proliferation but not in protection against chemotherapy.PR/+ strain has been previously described and was maintained on a C57BL/6 × 129/SvJ F1 background [C57BL/6J and 129Sv/J mice were obtained from the Jackson Laboratory . The mCGckground . Hybrid 6 cells/mouse) were injected intravenously via the tail vein into genetically compatible B6129F1 recipients, without pre-treatment with any radiation or chemotherapy conditioning.Acute promyelocytic leukemia cells (APL) from the spleens of mCG-PML-RAR knock in mice (B6129F1) were harvested and cryopreserved . APL celPlerixafor (AMD3100)  was supplied as a sterile isotonic aqueous solution at 20 mg/ml and was administered at a dose of 5 mg/Kg as a subcutaneous injection. The CCR2 antagonist, SC202525  was supplied as a sterile lyophilized powder, soluble in DMSO (100 mM). SC202525 was administered at a dose of 2 mg/Kg as a subcutaneous injection.Colony-forming cell (CFC) or colony-forming unit (CFU) assays were performed by plating blood in methylcellulose medium for mouse cells supplemented with recombinant cytokines  according to standard techniques . The assTo ensure leukemia development and engraftment, peripheral blood (PB) samples were taken from the tail of mice for complete blood counts using an automated cell counter  and flow cytometry (FCM) . The spleens from dead or euthanized animals were analyzed for evidence of AML. Single-cell suspensions from PB samples were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD117, phycoerythrin (PE)-conjugated anti-mouse CD34 and allophycocyanin (APC)-conjugated anti-mouse Ly-6G and Ly-6C  . A minimum of 10,000 events were acquired for each sample by FCM and data analyzed using FACSDiva software .Cytarabine (Ara-C) was purchased from Pfizer . Recombinant human CCL2, CCL8 and CCL7 were purchased from R&D Systems .The murine leukemia APL cells (myeloid origin) were kindly provided by Dr. John DiPersio . The AML cell lines U-937 (monocytoid), THP-1 (monocytoid) and Kasumi (M2), were purchased from ATCC . All cell lines (murine and human) were cultured in RPMI 1640 , supplemented with 10% (v/v) FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin, non-essential amino acids and 2 mM L-glutamine in a humidified incubator at 37°C with 5% carbon dioxide.de novo AML after written informed consent in accordance with institutional guidelines set forth by the Catholic University of Chile and before chemotherapy treatment. This study was reviewed and approved by the Catholic University of Chile School of Medicine Institutional Ethics Committee before samples were taken. BM aspirates collected during standard diagnostic procedures were obtained from the posterior iliac crest and collected in a heparinized syringe. Mononuclear cells were purified by Ficoll-Hypaque density-gradient centrifugation as described by the manufacturers . Blast purity exceeded 90%. Primary cells were resuspended in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin, non-essential amino acids and 2 mM L-glutamine in a humidified incubator at 37°C with 5% carbon dioxide. Table 1shows the general diagnostic laboratory data for the 35 AML patients in the study. The percentage of leukemia blasts corresponds to the amount of blasts present in whole BM or PB samples, before gradient separation.Primary AML samples from PB or bone marrow (BM) were obtained from patients with newly diagnosed For CCR2 expression studies, AML cell lines, BM or PB samples were stained at room temperature with saturating concentrations of phycoerythrin-conjugated anti-CCR2, fluorescein isothiocyanate-conjugated anti-CD14, peridinin chlorophyll–conjugated CD45 and allophycocyanin-conjugated anti-CD34/Kit monoclonal antibodies for 1 hour. Samples were washed once with 5ml BD FACS lysing solution  and washed again with 5ml phosphate buffered saline and then analyzed by FCM. We considered positive for CCR2 expression all AML samples with FCM readings above 2% as determined by data from all screened patients and respective isotype controls.THP-1 cells were seeded on 96-well plates and treated with various concentrations of recombinant human CCL2  for 24 hours before cultures were harvested and analyzed by FCM. The amount of cells in each sample was counted for 50 seconds.THP-1 cells cultured in RPMI 1640 were incubated with recombinant human CCL2 (10 ng/ml) or a mixture of recombinant human CCL8 (50 mg/ml), CCL7 (50 mg/ml) and CCL2 (10 mg/ml) for at least 2 hours before treatment with Ara-C . AML cells were exposed to Ara-C for 24 hours before analysis of cell viability or apoptosis.5 cells/well), U-937 (3x 105 cells/well) or primary AML cells (8x 105 cells/well) grown in RPMI supplemented with 2% FBS, were placed in the upper chamber (500 μl) exposed to different doses of recombinant chemokine CCL2 (optimum dose 10 ng/ml) placed in the lower chamber. Cell cultures were incubated and allowed to migrate overnight (15 hours) at 37°C with 5% carbon dioxide and subsequently cells from both chambers were harvested and counted by FCM analysis for 50 seconds/sample and also counted using a Neubauer hemocytometer to determine transmigration. Results are expressed as the percentage migration relative to control (spontaneous) migration. THP-1 and U-937 migration to cytokine SDF-1 was included as a positive control. SDF-1 is the ligand for CXCR4, a pivotal receptor involved in AML cell trafficking and mobilization. For migration-inhibition experiments, THP-1 or primary AML cells were pre-incubated for 30 minutes with a blocking human CCR2 monoclonal antibody (10 μg/ml) (R&D systems) or CCR2 receptor antagonist, sc202525 (5 nM). In some experiments, cell migration was assayed in the presence of neutralizing monoclonal antibody against CCL2 (5 μg/ml) (R&D systems). Isotype-matched antibodies were used as negative controls.Migration of THP-1 and primary AML cells from 3 patients with the highest CCR2 expression by flow cytometry was assayed using the Transwell system  as previously described . THP-1 -2,5-diphenyl tetrazolium bromide) assay . Two hours before ending the 24-hour treatment, 10 μl of MTT  was added to each well, and the samples were incubated for 2 hours at 37°C. Cells were lysed and MTT crystals solubilized by the addition of 100 μl of 0.02 N HCl in isopropanol. The absorbance of each well was determined at 590 nm using a BioTek microplate reader . Cell viability (%) was calculated relative to the control.THP-1 cell apoptosis was assessed by FCM using the annexin V-FITC apoptosis detection kit as described by the manufacturers . In short, THP-1 cells were grown in 24-well plates before treatment with Ara-C for 24 hours. Cultures were harvested and washed once in phosphate-buffered saline (PBS) and resuspended in 1X binding buffer in PBS with 1% BSA and incubated with Annexin V-FITC and APC anti-mouse Ly-6G and Ly-6C (Gr-1) (BD Pharmingen) for 30 minutes. Cells were then washed once with PBS and resuspended in 1X binding buffer in PBS supplemented with PI. All data was acquired on a BD FACSCanto II cytometer and analyzed using FACSDiva software (BD Biosciences)).THP-1 cells grown in 24-well plates with or without recombinant human CCL2 (50 mg/ml) for 24 hours were incubated with bromodeoxyuridine at various time-points as described by the manufacturers . THP1 cells were harvested, fixed and labelled with anti-BrdU fluorescein isothiocyanate antibody and 7-AAD to analyze cell cycle status by FCM.® ECL Western blotting substrate (Thermo Scientific). The following anti-human antibodies were used for immunoblotting: CCR2 and β-actin .Cell pellets were washed twice in ice cold PBS and lysed with lysis buffer and protease inhibitors (20 mM Tris (pH 7.5), 1% triton, 10% glycerol, 137 mM NaCl and 2 mM EDTA, 250 μM PMSF, 5μg/ml leupeptin) for 20 mins, followed by centrifugation at 10,000 rpm for 10 min at 4°C. Protein concentration of the supernatant was measured using Bio-Rad protein assay dye reagent . Fifty micrograms of proteins were equally loaded to a 10% SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membrane (PVDF) . Membranes were developed using Pierce® green master mix  according to the manufacturer’s instructions. qRT-PCR was carried out with a StepOne Applied Biosystems thermal cycler. All the reagents used were from Applied Biosystems. Reactions were run in triplicate and the genes of interest were normalized to glyceraldehyde-3P-dehydrogenase (GAPDH). The primers sequences were as follows: CCR2 forward primer: TGACAGGCACAGATGAATGG; CCR2 reverse primer: ATCATCTCCTGGCTGAATGC; GAPDH forward primer: CACCCAGAAGACTGTGGATGG; GAPDH reverse primer: CCACCAC-CCTGTTGCTGTAG.Total RNA was extracted using Trizol (Invitrogen) as described by the manufacturer. Complementary DNA was subsequently synthesized from total cellular RNA using MMLV reverse transcriptase . Real-time PCR (qRT-PCR) was performed using fast SYBRBM and PB samples from AML patients and healthy donors were centrifuged for 5 mins at 2,000 rpm room temperature to obtain the blood plasma. Plasma samples were processed to quantify the amount of autocrine CCL2 (ligand for CCR2 receptor) production by solid phase enzyme-linked immunosorbent assay (ELISA) using the human CCL2 quantikine ELISA kit (R&D Systems). The assay was followed according to the manufacturer’s instructions.*P < 0.05.All data is given as means ± S.D. of at least three independent experiments. Statistical analysis of data was made using one-way analysis of variance (ANOVA) followed by Bonferroni`s least significant difference post hoc test. Statistical analysis was performed using GraphPad Prism 5 statistical package. The significance level chosen for the statistical analysis was To evaluate the role of CCL2 /CCR2 in AML biology, we first determined CCR2 expression in human AML cell lines  and the murine AML cell line (APL) by flow cytometry (FCM), western blot (WB) and real time PCR (qPCR). FCM analysis with CCR2 MoAb demonstrated high surface expression of CCR2 in THP-1 and APL cells lines, whereas low CCR2 expression was observed with human AML cell lines U-937 and Kasumi . Similarde novo AML patients  and 6 healthy donors (with informed consent). We found 23/35 AML patients (65%) had CCR2 expression compared to their isotype controls. Mean CCR2 expression was 19% (range 3.5–90%) in PB and 20% (range 3–90%) in BM. We processed 35 newly diagnosed To further confirm our findings on CCR2 expression in these AML patient samples, we detected CCR2 receptor expression by WB for patients in which we had enough material for protein purification. To explore the possible role of CCL2/CCR2 axis on AML cell trafficking, we subsequently determined whether the incubation of THP-1 and primary AML cells with human recombinant CCL2 could stimulate AML cell migration in vitro. Migration of THP-1 and primary AML cells was determined as described in Materials and Methods. We used a transwell system with permeable supports with 3μm pores. We first exposed THP-1 cells placed on the upper chamber to various concentrations of CCL2 placed in the lower compartments of the migration system. As shown in We then performed migration studies with patient AML samples. We also investigated whether the CCL2/CCR2 axis shows an effect on AML cell proliferation and cell cycle when exposed to CCL2. First, the THP-1 cells were incubated with various concentrations of CCL2  for 24 hours and the data was analyzed by FCM. As observed in We have also optimized the bromo-deoxyuridine (BrdU) labeling technique to investigate THP-1 cell cycle analysis. THP-1 cells were pre-incubated for 4 hours with CCL2 (50 ng/ml) before staining with BrdU. Cells were allowed to proliferate overnight before FCM analysis. Interestingly, data showed a slight increase in S phase (DNA synthesis) of THP-1 cells with CCL2 treatment (50ng/ml) compared to THP-1 control . FurtherWe also performed both proliferation and migration studies with the APL cells. Migration studies showed significantly lower APL migration than THP-1 cells (data not shown). Proliferation studies with CCR2+ cell lines THP-1 and APL, and CCR2- cell line U937 showed that both CCR2+ cell lines proliferated significantly more after exposure to CCL2 compared to CCR2- cell line U937 (data not shown).We previously published that the BM stromal cells secrete soluble factors that protect human AML cells from Ara-C induced apoptosis . We explWe performed a solid phase ELISA with PB and BM blood plasma samples from 18 recruited patient samples  to quantify the amount of autocrine CCL2 production. As observed in We previously demonstrated in vivo that the BM stroma supports and protects AML cells as a mechanism from chemotherapy resistance . It is wWe then investigated whether the CCL2/CCR2 axis plays a role in vivo in AML mobilization and homing. For these in vivo studies, healthy B6 x 129 F1 mice (n = 16) were used to explore WBC mobilization using the CCR2 antagonist, sc-202525 (2 mg/Kg). Healthy mice were subcutaneously injected with the CXCR4 inhibitor, AMD3100  (n = 4), sc-202525 (2 mg/Kg) (n = 4) or with DMSO only (vehicle control group) (n = 4). PB samples were taken at 30 min, 1, 2, 4, 8 hours post-treatment. At each time point, we performed WBC counts and CFU assays to study the mobilization of WBCs and HPCs from the BM to the PB. As observed in 6 APL cells. To ensure a minimum percentage of circulating APL cells, eight days after APL injection, mice were screened for leukemia engraftment and trafficking by FCM and WBC count. All mice presented comparable numbers of circulating WBCs and APL blasts ) in PB and mice were separated into 2 groups. The following day, mice were treated with a subcutaneous injection of sc-202525 (2 mg/Kg) (n = 5) or DMSO treatment . PB samples were taken at 0, 1, 2, 4 and 7 hours post-treatment for WBC count and FACS analysis. All mice in the sc-202525 treatment group showed an increase in WBCs  were intravenously injected with 2x10 in WBCs . However in WBCs . Due to Despite steady improvements in AML therapy over recent years, AML is still a highly deadly disease, with only 25% of adults surviving 5 years post-diagnosis . Most AMGreat progress in cellular and molecular biology has allowed us to better understand the process of AML development and evolution , 23. Howin vivo experiments and human trials, CXCR4 blockade has demonstrated survival improvement of AML mice models [Surface molecules are potential targets for novel therapies against AML. For example, a clear role in AML biology of CXCR4 receptor has been shown. In e models , 24 and e models .in vitro experiments that upon co-culture of mesenchymal stromal cells (MSC) with AML cells, there was a significant increase of MSC-derived CCL2 secretion [Little is known about CCL2/CCR2 axis in AML biology. Recently it has been demonstrated in a series of ecretion .In our series, high expression of CCR2 was observed in AML cell lines and 65% of human AML samples (exclusively monocytoid AML) detected by FCM, WB and qPCR. Several series using human AML samples have shown similar results in CCR2 expression, mostly detected by FCM , 27. QuaOverall, we observed that our patients had significantly lower CCL2 plasma levels than normal controls. In contrast to our data, Manzur et al  showed iin vivo model [To determine CCR2 functionality, we performed a number of experiments to evaluate AML cell trafficking. We showed that under the exposure to CCL2 (CCR2 ligand), a significant dose-dependent transwell migration was seen in cells expressing CCR2, but not in those not expressing the CCR2 receptor. Furthermore, after blockade with a synthetic CCR2 inhibitor (SC202525) as well as monoclonal antibodies against CCL2 and CCR2, a significant inhibition in transwell migration was also observed. As expected, none of the cells lacking CCR2 expression showed transmigration or inhibition of transmigration. Similar results were observed with cell lines and primary AML cells. These results suggest that CCL2/CCR2 axis presents functional activity in AML blast migration in vitro. A recent analysis also confirmed that CCL2/CCR2 axis is relevant in AML blast migration using a human AML cell line (THP-1) and an vo model .in vitro, CCL2 stimulates CCR2+ cells to proliferate. However, the numbers were very small and its relevance is unclear. Bruserud et al. [in vitro chemoprotective effect of CCL2 and other MCPs after exposing AML cells to cytarabine. It has not been previously published if CCR2-CCL2 axis is involved in the microenvironment protection against chemotherapy, as seen with other axis like CXCR4/SDF-1 or VLA4/VCAM1. One hypothesis is that CCL2/CCR2 is only involved in migration as it occurs with normal leukocyte biology, since different concentrations and durations of co-culture of CCL2 did not induce chemotherapy protection. Also, the addition of other MCPs (MCP-2 and MCP-3) did not induce chemotherapy protection. Our hypothesis was that CCL2/CCR2 axis was related to chemotherapy protection as observed with other axis. However, our studies failed to demonstrate any sign of chemoprotection. Our hypothesis was that cell cycle modification was also involved in chemotherapy protection after inducing cell cycle arrest, but this too was not observed. Actually, we observed a significant increase in S phase and proliferation rate. Other models have shown that this axis is involved in proliferation and tumor progression [in vitro possibly relates to the fact that CCL2/CCR2 axis has to be connected in vivo to other axis like CXCR4/SDF-1 or VLA-4/VCAM1.We also sought to analyze if CCL2/CCR2 axis was involved in AML proliferation. Previous literature has shown that CCL2 is involved in cell proliferation of different cell lines –33. In rd et al.  showed td et al. , 34. In gression –37. We hin vivo model if after CCR2 blockade, CCL2/CCR2 axis was involved in chemotherapy protection. In order to show this, we reproduced a previous model of AML mobilization to expose AML cells from their niche and make them sensitive to chemotherapy [To show this, first we sought to study in an otherapy . OverallRecent evidence suggests that CCL2/CCR2 axis may possibly be involved in AML resistance to chemotherapy, not related to its role in AML migration and proliferation kinetics but related to subsets of immunosuppresive macrophages into the bone, which could be blocked with CCL2/CCR2 inhibitors and that way improve the response to chemotherapy . Also, iFinally, our results suggest that CCL2/CCR2 axis may have a role in a subset of AML patients, especially in monocytoid AML, which represent the leukemias with higher CCR2 expression. In normal monocytes its role is mostly related to cell migration. In our experiments its main role was related to migration and possibly to proliferation but not chemotherapy protection as we initially hypothesized, at least in the way the experiments were designed. It appears that other mechanisms for chemotherapy resistance may be involved including normal cells, like macrophages, as shown by recent evidence. However, multiple mechanisms involving not only the CCL2/CCR2 axis but as Brenner et al suggested , the che


We detected the presence of cyanidin in the intracellular space of β-cells. Cyanidin stimulated insulin secretion and increased intracellular Ca2+ signals in a concentration-dependent manner. The Ca2+ signals were abolished by nimodipine, an l-type voltage-dependent Ca2+ channel (VDCC) blocker or under extracellular Ca2+ free conditions. Stimulation of cells with cyanidin activated currents typical for VDCCs and up-regulated the expression of glucose transporter 2 (GLUT2), Kir6.2, and Cav1.2 genes. Our findings indicate that cyanidin diffuses across the plasma membrane, leading to activation of l-type VDCCs. The increase in intracellular Ca2+ stimulated insulin secretion and the expression of genes involved in this process. These findings suggest that cyanidin could be used as a promising agent to stimulate insulin secretion.Cyanidin is a natural anthocyanidin present in fruits and vegetables with anti-diabetic properties including stimulation of insulin secretion. However, its mechanism of action remains unknown. In this study, we elucidated the mechanisms of cyanidin for stimulatory insulin secretion from pancreatic β-cells. Rat pancreatic β-cells INS-1 were used to investigate the effects of cyanidin on insulin secretion, intracellular Ca This increases intracellular Ca2+ concentration and stimulates insulin secretion ) were: CACCCAAGTCCCGTCGTGAAGT/GATCCACAATGCCACGCTTCTG (Ins); GAAATTCAAGAAGCGAAAAG/CCTGCTGTCACTCTGGTAGTAG  from the three independent experiments (n = 3). Patch-clamp recordings were expressed as representative current–voltage relationship (I/V) for VDCC channels from 24 to 31 cells from each group. The average current amplitude for cyanidin and control and the average traces for cyanidin and diazoxide were compared using unpaired Student’s t-test. Multiple group comparisons for cyanidin and control experiments including insulin secretion, intracellular Ca2+ signal and real-time PCR were carried out using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test . p < 0.05 was considered to be statistically significant.The results of insulin secretion, intracellular CaTo determine whether cyanidin could diffuse across the plasma membrane, Naturstoff reagent A was used. We detected the presence of cyanidin in the intracellular space 10 min after stimulation of cells and treatment with Naturstoff reagent A, but not after cyanidin or reagent alone . The pre50 for cyanidin was 80.08 ± 2.11 µM. The maximum secretion was achieved with 100 µM cyanidin that caused a 2.5-fold increase over basal, whereas 20 mM KCl resulted in a 3-fold increase.Next, we stimulated cells with increasing concentrations of cyanidin to examine whether it could stimulate insulin secretion. The results showed that cyanidin (1–300 µM) markedly increased insulin release from INS-1 cells . The cal2+ are a requirement for insulin secretion, we examined whether cyanidin would induce Ca2+ signals. Stimulation of cells with cyanidin (80 to 300 µM) increased intracellular Ca2+ in a concentration-dependent manner . Removal of extracellular Ca2+ abolished the response to 100 µM cyanidin, but not after store depletion with thapsigarin, a Ca2+ ATPase pump inhibitor in the ER. Cyanidin also failed to increase intracellular Ca2+ under both Ca2+ free and store depletion conditions  that stimulate insulin secretion using Ca2+ signaling [2+ can be due to influx across the plasma membrane and/or release from the ER [2+ was due to influx, as store depletion did not alter the cyanidin response. Furthermore, the l-type VDCC blocker nimodipine abolished the Ca2+ signals to indicate the pathway for Ca2+ influx. In support of this finding, the patch-clamp data confirmed the activation of channels. The opening of l-type Ca2+ channels represents the final common pathway for insulin secretion in pancreatic β-cells. l-type Ca2+ channels comprise five subunits, including α1, α2, β, γ, and δ subunits [l-type Ca2+ channel α1 subunits with high affinity [l-type VDCCs [l-type Ca2+ channel at a different site from that of Bay K 8644, an l-type Ca2+ channel agonist [l-type Ca2+ channel. Other phytochemical compounds such a p-methoxycinnamic acid [2+ influx through VDCCs. This mechanism now appears to be universal among the flavonoids. In our study, we also tested whether cyanidin functions as sulfonylureas, which induces the closure of KATP channels leading to membrane depolarization, Ca2+ influx, and insulin secretion [ATP channel opener, did not impact the effect of cyanidin on Ca2+ signals which is in agreement with genistein [p-methoxycinnamic acid [The insulin secreting activity of cyanidin has been demonstrated in pancreatic β-cells . Our finignaling ,20. Incrm the ER . Our ressubunits . It has affinity . Our finpe VDCCs ,23. Howe agonist . Additiomic acid  stimulatecretion . We founenistein  and p-memic acid . Other dmic acid . It suggl-type Ca2+ channel. Our gene expression analysis during cyanidin stimulation reports a down-regulation of genes associated with insulin secretion in diabetic animal models [6.2 and Cav1.2. The expression of Cav1.2 (a subunit of l-type VDCC) is crucial for insulin secretion because it promotes Ca2+ influx [2+ influx, insulin secretion, and the expression of the Cav1.2 gene. The KATP channel comprises two subunits: the K+ inwardly rectifying (Kir6.2) and sulfonylurea receptor (SUR1). Hyperglycemia decreases Kir6.2 mRNA levels in rat pancreatic islets as well as in INS-1 cells [2+ influx and insulin secretion from HIT-T15 cells [ATP dependent pathway, it might enhance glucose-induced insulin secretion by increasing Kir6.2 expression. Therefore, the up-regulation of genes controlled by cyanidin might be important to increase insulin sensitivity and insulin secretion. Further experiments are required to clarify the effect of cyanidin on these genes expression related to glucose-stimulated insulin secretion (GSIS) under dysfunction of pancreatic β-cells. Finally, there is accumulating evidence that cyanidin can stimulate insulin secretion [In regard to its intracellular localization, flavonoids are absorbed into cells by passive diffusion and membrane transport . We detel models ,33,34. Il models . Adminisl models ,37. In rl models . In addi+ influx , whereas+ influx . Our fin-1 cells  and reduecretion , and inhecretion . Our stuecretion ,43,44. Hecretion . At presl-type VDCCs. This effect promotes Ca2+ influx which stimulates insulin secretion from pancreatic β-cells (6.2 and Cav1.2 genes that could have potential implications on glucose-induced insulin secretion, glucose homeostasis, and diabetes.The results from our study revealed that cyanidin diffuses across the plasma membrane, leading to the activation of  β-cells . Cyanidi


We also showed that inhibiting the intracellular Ca2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca2+ signals play an essential role in the active cell migration during gastrulation.During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca Xenopus laevis, the gastrulation movements begin with vegetal rotation of the mesodermal sheet1. Subsequently, the leading edge mesoderm (LEM), the most vegetal mesoderm region, touches the inner side of the blastocoel roof (BCR). The LEM then shows directional migration toward the anterior end on the fibronectin-rich BCR5. During this process, axial mesoderm following the LEM undergoes convergent extension, in which cell movements elongate the embryo proper along the anterior-posterior axis and narrow the tissue mediolaterally, eventually forming the rod-shaped notochord6. On the other hand, the prechordal mesoderm that follows the LEM cells plays essential roles in head formation8.Gastrulation is one of the most important processes in the early development of a variety of animals. In vertebrates, this dynamic remodelling process is achieved by the coordinated movements of three germ layers, which contribute to the development of various organs in their proper positions in the body. In the experimental vertebrate model spina bifida10. This anterior tissue migration is thought to be regulated by chemoattractants and/or by cell responses to mechanical signals. As chemoattractants for the directed tissue migration, PDGF7 and SDF-111 secreted from the ectoderm have been implicated, and the LEM cells are thought to receive these chemokine signals via their respective receptors12. On the other hand, the LEM cells’ sensing of local mechanical interactions between the leader cells and follower cells has been reported to determine the asymmetric formation of cell protrusions and the oriented movement of LEM cells13. However, how such mechanical signals are interpreted by the cells remains unknown.The LEM has an indispensable role in the directional migration of the mesodermal sheet, and disrupting this migration causes severe morphological defects in the embryo, such as abnormal notochord formation and 2+ signalling regulates a variety of physiological events, including cell proliferation, apoptosis, differentiation, and cell migration14. In cell migration, Ca2+ signalling regulates the effectors required for cytoskeletal remodelling and the establishment of focal adhesions18. Ca2+ signalling is essential both for the homeostasis of adult animals and for developmental processes such as organogenesis20. It is generally thought that intracellular Ca2+ signalling is regulated by fluctuations in the intracellular Ca2+ concentration, recognized as Ca2+ transients. There are two essential sources of intracellular Ca2+: influx from the extracellular space and release from the endoplasmic reticulum (ER). The influx of extracellular Ca2+ directly depends on cell membrane Ca2+ channels, while the release from the ER’s Ca2+ source depends on Ca2+ channels on the ER membrane14. The roles of these two modes of Ca2+ elevation in Xenopus development have been extensively studied. The Ca2+ transient and wave-like propagation of Ca2+ triggered by fertilization have been well characterized21, and this Ca2+ elevation is known to induce re-entry into the meiotic cell cycle22. In the gastrula stage, Ca2+ transients have been observed in the ectoderm and axial mesoderm24, suggesting that Ca2+ plays important roles in those tissues. Recent reports further indicate that Ca2+ signalling has critical roles in tissue morphogenesis29. Here we sought to clarify the intracellular Ca2+ dynamics and how they contribute to gastrulation cell movements. We first examined the Ca2+ dynamics of the migrating embryonic cells and the function of Ca2+ signals in the Xenopus LEM. We found that Ca2+ transients occur preferentially in the LEM cells during migration and are confined to the front rows of the LEM. Functional analyses in which the intracellular Ca2+ level was depleted by drug treatment and elevated with a Ca2+ ionophore demonstrated that the Ca2+ signal is necessary and sufficient for LEM migration. Finally, we found that Ca2+ transients are required for the polarized lamellipodia formation that accelerates LEM migration. Taken together, these results suggest that local Ca2+ signals in LEM cells contribute to the gastrulation cell movements of vertebrates.Intracellular Ca2+ dynamics in LEM cells during gastrulation, we tested several variants of a FRET-based Ca2+ indicator yellow cameleon (YC)-Nano. We found that YC-Nano3GS30 had the most suitable dynamic range, enabling us to detect basal as well as transient increases in the intracellular Ca2+ of LEM tissue. To express the Ca2+ indicator and to label the cell membrane to visualize cell shape, we injected mRNAs for YC-Nano3GS and membrane-targeting RFP (mRFP), respectively, into the two dorsal blastomeres of 4-cell-stage embryos. However, there are well known technical limitations to observing cellular events in the gastrulating mesoderm, which is underneath the pigmented ectoderm. Therefore, to observe the cells undergoing gastrulation more clearly, we prepared “cap-less” embryos, as previously described  in the LEM displayed wave-like diffusions at the subcellular level . Most transients lasted from 180–210 sec, although the duration ranged from 60 sec to several minutes  and finally ceased migrating soon after the open space was filled with cells, as previously reported. Using the Calls Fig. . To charvel Fig. . These Ctes Fig. . Thus, o2+ transients frequently occurred in the first row of LEM cells  of cell migration, while Ca2+ transients were hardly detected after mantle closure . Therefore, we next used dorsal marginal zone (DMZ) explants , and observed the Ca2+ dynamics and the cell migration by time-lapse imaging of the leading region of the DMZ. BAPTA-AM-treated explants showed greatly reduced Ca2+ transients compared with DMSO-treated control explants , representing a single transient, or for 2 hours, representing continuous and repeated Ca2+ transients, were subjected to the pull-down assay  reduced the migratory activity of the LEM. Conversely, Ca2+ induction by a Ca2+ ionophore (Ionomycin) accelerated the migratory activity of the LEM. Furthermore, we found that Ca2+ elevation enhanced the Rac1 activity. Taken together, these results suggested that Ca2+ signalling at the edge of the LEM regulates the migration activity of the tissue and therefore controls gastrulation cell movements.In this study, we used 2+, we used a FRET-based Ca2+ indicator YC-nano 3GS, and observed the LEM by spinning disc confocal microscopy. This indicator is suitable for measuring the steady-state concentrations30 of intracellular Ca2+and enabled us to observe and characterize the Ca2+ transients in detail under various experimental conditions. We found that almost all of the Ca2+ transients occurred in single cells, and only occasionally did we observe wave-like propagations over multiple cells in the leading mesodermal cells (data not shown) as previously described28. In the leading edge, most of the Ca2+ transients had a duration of 180 to 210 sec. These rather long durations differ from those reported for Xenopus mesodermal cells (90 sec)28 and neural plate (less than 40 sec)27. At the subcellular level, temporal analysis of the Ca2+ dynamics revealed that almost all of the Ca2+ transients showed a wave-like pattern . However, this profile is distinct from calcium transients we observed which are restricted to few cells. Because our explants and cap-less embryo were observed 60 min after manipulation, it is unlikely that the Ca2+ transient is a direct wound response.To visualize Caern Fig. . Ca2+ tr36, and are commonly stimulated by the calcium influx from efflux from ER store; therefore, one of our speculations is that RyR receptor or IP3 receptor are involved in triggering the Ca2+ transients in the LEM. Other sources include extracellular space37, mitochondoria38 and lysosomal calcium efflux in which acidic calcium store of lysosome regulates calcium transient like ER store39.However, we were not able to address what actually triggers the calcium transients in the LEM. Notably, these wave-like patterns are reported in various cells2+ transients reduced the cellular protrusions of the leader cells  was not sufficient to activate Rac1 activity, but continuous Ca2+ stimulation did activate it . The other is an “accumulation model,” which suggests that repeated Ca2+ transients induce cellular protrusion activity when the accumulated signal reaches a certain threshold.Regarding how Ca it Fig. . These rt period  minutes 2+ transients during gastrulation , and purified with a NICK column (Pharmacia). The YC-nano30 and membrane-tagged RFP (mRFP) constructs were previously reported43.For 45.For RT-PCR with drug-treated DMZ explant, 5 explants were dissociated at St. 10.5 and incubated with various drug for 3 hours. The following primers were used: ODC, Cerberus, Xbra and XnotXenopus laevis were induced to ovulate by injecting human gonadotropin. The collected eggs were fertilized in vitro, treated with 3% cysteine for dejellying, and reared in water. For microinjection, embryos were placed in a solution of 0.3% Ficoll in 1x MMR. mRNAs and NPE-caged IP3 were injected into the dorsal region at the four-cell stage. Injected embryos were reared in the same buffer until the appropriate stages.Female adult To treat whole embryos, Ionomycin  in DMSO was added to the medium at st. 11. To treat explants, Ionomycin in DMSO was added to the medium by pipet during live imaging. BAPTA-AM  in DMSO was added to the medium 30 minutes before starting the live imaging.3. The cap-less embryo was plated on a fibronectin (FN)-coated glass-bottom dish. For DMZ explants with LEM, at the early gastrula stage (st. 11–11.5), a 60–80° dorsal marginal zone (DMZ) explant including the LEM was dissected by tungsten needle and immediately mounted onto a FN-coated glass-bottom dish. These mounted explants were incubated for at least 1 hour before microscopic observation. All explants were cut and incubated in Danilchik’s for Amy (DFA) plus bovine serum albumin (BSA) medium46.To prepare cap-less explants, ectoderm was removed from the embryo at the late-gastrula stage (st. 12–12.5), as previously describedTo measure Rac1 activity, we used the Rac1 Pulldown Activation Assay Biochem Kit . DMZ explants expressing myc-tagged xlRac1 were treated with Ionomycin for 3 minutes or 2 hours. To detect Rac1 activity, after Ionomycin or DMSO treatment in DFA medium, the DMZ explants were washed with 1 ml of Steinberg’s solution. The collected explants were lysed in lysis buffer  with a cocktail of protease inhibitors. The supernatant of the lysed sample was collected and used for a pull-down assay with GST-tagged PAK-PBD beads, which bind to active Rac1. The samples were denatured by adding an equivalent volume of 2x SDS sample buffer . After boiling for 5 min, the samples were subjected to SDS-PAGE and blotted onto PVDF membranes (Bio- Rad). For detection, an anti-myc antibody , HRP-conjugated secondly antibody, and ECL kit  were used.To analyse the calcium dynamics, 500 μg of YC-nano 3GS mRNA was dorsally injected into 4-cell-stage embryos. All calcium imaging was done using an inverted microscope  equipped with a spinning disk confocal unit , EMCCD camera , and 445-, 488-, and 561-nm lasers (Andor). All of the images were obtained with 100–500-ms scanning every 15–120 seconds for 0.5–3 hours at 20 degrees Celsius with a 10x  or 20x  objective lens.To provide the UV illumination for uncaging experiments, a mercury lamp-based light source with a band-pass filter  was installed on the spinning disk confocal system, with a shutter controlled by iQ2 software (Andor). To uncage the NPE-Caged IP3, the explants were illuminated for 3 seconds with UV light.Images were analysed using Image J software. The ratio between the YFP and CFP of YC-nano was calculated after background subtraction. We used the average intensity of an empty sample region as the background. A calcium transient was defined as an increase in the ratiometric value greater than the moving median image of the ratiometric value Suppl. .For cell migration analysis, we measured three different ordinates  in the leader cells at three time points . We measured the migration distance for 180 min at the three different ordinates and calculated the average of the three distances as the migration activity.For the measurement migration velocity, we monitored the displacement of cell position from the start (0 min) to the end points (90 min) and velocity was calculated by dividing the distance by 90 min.To quantitate celllular protrusion stability, we measured membranous protrusion areas over 30 min and calculate the average protrusion area per minute as the protrusion stability.All our data in this article will be provided upon request.Supplementary figures and figure legendsSupplementary movie 1Supplementary movie 2Supplementary movie 3Supplementary movie 4Supplementary movie 5Supplementary movie 6Supplementary movie 7