Ascorbate Treatment Research Articles

Pharmacological Ascorbate Suppresses Growth of Gastric Cancer Cells with GLUT1 Overexpression and Enhances the Efficacy of Oxaliplatin Through Redox Modulation.

Rationale: The antitumor activity of high-dose ascorbate has been re-evaluated recently, but the mechanism underlying cell-specific sensitivity to ascorbate has not yet been clarified.Methods: The effects of high-dose ascorbate on gastric cancer were assessed using cancer cell lines with high and low expression of GLUT1 via flow cytometry and colony formation assays in vitro and patient-derived xenografts in vivo.Results: In this study, we demonstrated that gastric cancer cells with high GLUT1 expression were more sensitive to ascorbate treatment than cells with low GLUT1 expression. GLUT1 knockdown significantly reversed the therapeutic effects of pharmacological ascorbate, while enforced expression of GLUT1 enhanced the sensitivity to ascorbate treatment. The efficacy of pharmacological ascorbate administration in mice bearing cell line-based and patient-derived xenografts was influenced by GLUT1 protein levels. Mechanistically, ascorbate depleted intracellular glutathione, generated oxidative stress and induced DNA damage. The combination of pharmacological ascorbate with genotoxic agents, including oxaliplatin and irinotecan, synergistically inhibited gastric tumor growth in mouse models.Conclusions: The current study showed that GLUT1 expression was inversely correlated with sensitivity of gastric cancer cells to pharmacological ascorbate and suggested that GLUT1 expression in gastric cancer may serve as a marker for sensitivity to pharmacological ascorbate.

131 - Inhibition of Peroxide Removal Enhances Pharmacological Ascorbate and Ionizing Radiation-Induced Cytotoxicity of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer deaths with a poor 5 year survival rate of only 8.2%. PDA cells produce high levels of reactive oxygen species (ROS) compared to normal cells, thereby increasing their susceptibility to oxidative stress-induced cell death. Therefore, targeting pathways that are essential in peroxide removal, such as the enzymes glutathione reductase and thioredoxin reductase, could be utilized as a tool for cancer therapy to further disrupt a cancer cells ability to overcome excess ROS. Pharmacological ascorbate is a pro-oxidant that has been shown to enhance cytotoxicity and radio-sensitize several cancer types through an H2O2-mediated mechanism. The objective of this study was to examine the effects of pharmacological ascorbate on PDA cells as an adjuvant to radiation therapy in addition to therapies that inhibit peroxide removal including auranofin (inhibits thioredoxin reductase) and 2-deoxyglucose or glucose deprivation (inhibits regeneration of NADPH). Human PDA cell lines AsPC-1 and MiaPaCa-2 were evaluated for clonogenic survival after treatment with either auranofin (500 nM), 2-deoxyglucose (25 mM), or glucose deprivation for 24 hrs, in combination with irradiation (1-2 Gy) and pharmacological ascorbate (1-2 mM) for 1 hr. Auranofin was shown to inhibit thioredoxin reductase activity, sensitize PDA cells to pharmacological ascorbate treatment and significantly reduce clonogenic cell survival by an additional 50% or 36% for MiaPaca-2 cells (P≤ 0.0001) and 29% or 39% for AsPC-1 cells (P≤ 0.01-0.0001) at 1 and 2 Gy, respectively. Inclusion of 2-deoxyglucose reduced clonogenic cell survival by an additional 20% or 9.5% for MiaPaCa-2 cells (P≤ 0.001-0.01) and 23% or 8% for AsPC-1 cells (P≤ 0.01-0.0001) at 1 and 2 Gy, respectively. Glucose deprivation enhanced cytotoxicity of MiaPaca-2 cells by an additional 40% or 29% (P≤ 0.0001) at 1 and 2 Gy, respectively. Inhibition of peroxide removal sensitizes PDA cells to irradiation and pharmacological ascorbate cytotoxicity. Therapies incorporating these strategies have the potential to be effective for the treatment of PDA.

107 - Ascorbate Decreases Circulating Tumor Cells (CTCs) in Mice with Pancreatic Adenocarcinoma (PDAC)

Circulating tumor cells (CTCs) are intact cells, the closest representation of human tissue compatible with immunohistochemistry and traditional pathology protocols. Importantly, CTCs are thought to play a central role in metastatic disease spread by detaching from primary disease sites, entering circulation, and seeding distant tissues. Pharmacological ascorbate has shown promise as a potential adjuvant for advanced pancreatic cancer (PDAC) treatment through its ability to generate H2O2 leading to selective cancer cytotoxicity. The study presented here utilizes an animal model and newly developed PDAC CTC assay to reliably determine the effect of ascorbate treatment on the concentration of CTCs. MIA PaCA-2 human PDAC cells were transfected with pQClucIN and pMSCV-IRES-GFP plasmids to express luciferase and green fluorescent protein (GFP) and facilitate in vivo bioluminescent imaging. A 20 µL matrigel slurry was prepared to suspend 4 x 105 cells for orthotopic intrapancreatic injection into 16 nude mice under ultrasound guidance. On day 35, 10 control mice began twice daily saline injections (4 g/kg) and 6 treatment mice began BID ascorbate injections (4 g/kg). On day 50, CTCs were analyzed by flow cytometry to detect circulating GFP-expressing cancer cells. Tumor growth over time, as measured by bioluminescent imaging signal strength, increased equally between groups prior to treatment but slowed significantly in mice treated with pharmacological ascorbate compared to control (p

Pharmacologic ascorbate induces neuroblastoma cell death by hydrogen peroxide mediated DNA damage and reduction in cancer cell glycolysis

An ascorbate-mediated production of oxidative stress has been shown to retard tumor growth. Subsequent glycolysis inhibition has been suggested. Here, we further define the mechanisms relevant to this observation. Ascorbate was cytotoxic to human neuroblastoma cells through the production of H2O2, which led to ATP depletion, inhibited GAPDH, and non-apoptotic and non-autophagic cell death. The mechanism of cytotoxicity is different when PARP-dependent DNA repair machinery is active or inhibited. Ascorbate-generated H2O2 damaged DNA, activated PARP, depleted NAD+, and reduced glycolysis flux. NAD+ supplementation prevented ATP depletion and cell death, while treatment with a PARP inhibitor, olaparib, preserved NAD+ and ATP levels but led to increased DNA double-strand breakage and did not prevent ascorbate-induced cell death. These data indicate that in cells with an intact PARP-associated DNA repair system, ascorbate-induced cell death is caused by NAD+ and ATP depletion, while in the absence of PARP activation ascorbate-induced cell death still occurs but is a consequence of ROS-induced DNA damage. In a mouse xenograft model, intraperitoneal ascorbate inhibited neuroblastoma tumor growth and prolonged survival. Collectively, these data suggest that ascorbate could be effective in the treatment of glycolysis-dependent tumors. Also, in cancers that use alternative energy metabolism pathways, combining a PARP inhibitor with ascorbate treatment could be useful.

Abstract 238: S-nitrosoglutathione Reductase (GSNOR) Plays a Critical Role in Placental Vascularization Working Through the VEGF-NO Pathway

Introduction: Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc), which is required for the release of nitric oxide (NO) from nitrosylated proteins. Mice lacking S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesized that impaired placental vascularization, one of the primary causes of preeclampsia is mediated by alteration in S-nitrosylation of the VEGF-NO pathway, and ascorbate treatment rescues this pathologic phenotype. Methods: Pregnant GSNOR –/– and control (WT) mice (n=5-7) were studied at late pregnancy (day 17.5). Ascorbate was provided in drinking water beginning at day 0.5. Fetoplacental capillary density was determined from isolectin staining and reactive nitrosative stress determined from nitrotyrosine staining in placental sections. S-nitrosylation of VEGF was determined using SNO-Rac and eNOS levels by Western blot analysis. Results: Fetoplacental capillary density was reduced 19% in GSNOR –/– fetuses compared to WT (P<0.001). GSNOR –/– placentas exhibited higher nitrotyrosine staining than WT placentas, indicating the presence of nitrosative stress. These increases were associated with reduced level of eNOS protein (P<0.05) and decreased S-nitrosylation of VEGF (P<0.05) in the GSNOR –/– placentas as compared to WT. Ascorbate treatment decreased nitrotyrosine staining, and increased fetoplacental capillary density ~10% (P<0.001), eNOS protein levels (P<0.05) and S-nitrosylation of VEGF (P<0.05) in the GSNOR –/– placentas as compared to WT. Conclusion: These findings suggest that GSNOR plays an essential role in promoting placental vascularization in part working through the VEGF-NO pathway. Ascorbate treatment rescued the nitrosative stress and improved placental vascularization, suggesting that it can be used therapeutically to treat or prevent preeclampsia.

Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H2O2) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H2O2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H2O2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H2O2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H2O2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H2O2 accumulation and high light-responsive gene expression. This is because the H2O2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H2O2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.

Ascorbate induces apoptosis in melanoma cells by suppressing Clusterin expression

Pharmacological levels of ascorbate have long been suggested as a potential treatment of cancer. However, we observed that EC50 of ascorbate was at a similar level for cultured healthy melanocytes and melanoma cells, suggesting a limit of pharmacological ascorbate in treating cancer. Loss of 5-hydroxymethylcytosine (5 hmC) is an epigenetic hallmark of cancer and ascorbate promotes 5 hmC generation by serving as a cofactor for TET methylcytosine dioxygenases. Our previous work demonstrated that ascorbate treatment at physiological level (100 μM) increased 5 hmC content in melanoma cells toward the level of healthy melanocytes. Here we show that 100 µM of ascorbate induced apoptosis in A2058 melanoma cells. RNA-seq analysis revealed that expression of the Clusterin (CLU) gene, which is related to apoptosis, was downregulated by ascorbate. The suppression of CLU was verified at transcript level in different melanoma cell lines, and at protein level in A2058 cells. The anti-apoptotic cytoplasmic CLU was decreased, while the pro-apoptotic nuclear CLU was largely maintained, after ascorbate treatment. These changes in CLU subcellular localization were also associated with Bax and caspases activation, Bcl-xL sequestration, and cytochrome c release. Taken together, this study establishes an impending therapeutic role of physiological ascorbate to potentiate apoptosis in melanoma.

Vitamin C Transporters in Cancer: Current Understanding and Gaps in Knowledge.

Sufficient uptake and whole body distribution of vitamin C (ascorbate) is essential for many biochemical processes, including some that are vital for tumor growth and spread. Uptake of ascorbate into cancer cells is modulated by availability, tumor blood flow, tissue diffusion parameters, and ascorbate transport proteins. Uptake into cells is mediated by two families of transport proteins, namely, the solute carrier gene family 23, consisting of sodium-dependent vitamin C transporters (SVCTs) 1 and 2, and the SLC2 family of glucose transporters (GLUTs). GLUTs transport the oxidized form of the vitamin, dehydroascorbate (DHA), which is present at negligible to low physiological levels. SVCT1 and 2 are capable of accumulating ascorbate against a concentration gradient from micromolar concentrations outside to millimolar levels inside of cells. Investigating the expression and regulation of SVCTs in cancer has only recently started to be included in studies focused on the role of ascorbate in tumor formation, progression, and response to therapy. This review gives an overview of the current, limited knowledge of ascorbate transport across membranes, as well as tissue distribution, gene expression, and the relevance of SVCTs in cancer. As tumor ascorbate accumulation may play a role in the anticancer activity of high dose ascorbate treatment, further research into ascorbate transport in cancer tissue is vital.

Vitamin C puts the pedal to the metal.

Increased iron in cancer cells drives selective sensitization of tumors to ascorbate treatment to prolong survival.

Abstract 12750: Ascorbate Prevents Hypertension, Proteinuria and Concentric Hypertrophy by Balancing the Nitroso-redox System in a Model of Preeclampsia, the S-nitrosoglutathione Reductase (gsnor) Deficient Mice

Introduction: Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc). Mice lacking S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, proteinuria, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesize that the PE-like phenotype is mediated by nitroso-redox imbalance and nitrosative stress and can be rescued by ascorbate treatment. Methods: Pregnant GSNOR –/– and control (WT) mice (n=5-7) were provided drinking water ± Asc beginning on day 0.5 of gestation (E0.5). We determined blood pressure using a Millar catheter, relative wall thickness by echocardiography, and placental vascularization by isolectin staining. Cardiomyocytes (CM) were isolated at late stage pregnancy (E17.5) and levels of ROS (2’7’-dichlorofluorescein), nitric oxide (NO, diaminofluorescin) and peroxynitrite (dihydrorhodamine 123) were determined by fluorescence (ΔF/F 0 ). Results: CMs isolated from pregnant GSNOR –/– hearts, exhibited elevated levels of ROS (2.48±0.39 vs. 1.58±0.18 in WT, P<0.05), free nitric oxide (6.65±0.43 vs. 5.59±0.26 in WT, P<0.05) and peroxynitrite (0.75±0.04 vs. 0.39±0.03 in WT, P<0.05). These increases were prevented with Asc treatment (P<0.01), which completely rescued the PE phenotype in GSNOR –/– mothers, including hypertension (105±2 mmHg vs. 95± mmHg in Asc-treated, P<0.05), proteinuria (P<0.05) and relative wall thickness (0.56±0.04 vs. 0.45±0.03 in Asc-treated (P<0.05). Placental vascularization was also significantly improved with Asc treatment in GSNOR –/– mothers. Asc had no effect on these parameters in WT mice. Conclusions: Our findings indicate that nitroso-redox imbalance and nitrosative stress contribute to PE in mice. Asc treatment balanced the nitroso-redox system and rescued the pathological phenotypes in GSNOR –/– mice, suggesting that it can be used therapeutically to treat or prevent preeclampsia.

Thyroid and reproductive hormones disruption as well as kallikrein-3 level in dimethyl nitrosamine-induced toxicity: Effects of ascorbate treatment in male wistar rats

Information on dimethyl nitrosamine (DMN)-induced toxicity on endocrine functions is still scanty. This study therefore investigated the outcomes of DMN-induced toxicity on endocrine (thyroid and reproductive) functions, as well as kallikrein-3 level, and effects of ascorbate treatments in male wistar rats. Thirty animals divided into six groups of five rats each were used. Group I animals were the normal control, group II animals served as vehicle control and were administered a single intraperitoneal dose of normal saline, groups III and IV were intraperitoneally injected with a single dose of 30 mg/kg DMN for 48 h, but group IV animals were post-treated orally with 5.71 mg/kg body weight (400 mg/70 kg) ascorbate for seven days, group V animals were pre-treated with same dose of ascorbate orally for seven days before intraperitoneal injection of DMN, while group VI animals were orally administered ascorbate only for seven days. Compared with control, DMN administration resulted in significant decrease (p < 0.05) in serum total cholesterol, testosterone (TST), luteinizing hormone (LH), free triiodothyronine (fT3), and kallikrein III (KLK-3) levels, as well as non-significant increase in serum thyroid stimulating hormone (TSH) level. Pre-treatment with ascorbate significantly increase LH and KLK-3 levels, while post-treatment significantly increase fT3 level. Also, pre-treatment with ascorbate significantly reduced TSH level, while there was no significant difference in TST level following ascorbate treatments. From our findings and to some extent, ascorbate demonstrates ameliorative effects against DMN-induced hormonal disruption in male wistar rats, and this may be attributed to its antioxidant property.

Transcriptomic and functional pathways analysis of ascorbate-induced cytotoxicity and resistance of Burkitt lymphoma.

Ascorbate is a pro-oxidant that generates hydrogen peroxide–dependent cytotoxity in cancer cells without adversely affecting normal cells. To determine the mechanistic basis for this phenotype, we selected Burkitt lymphoma cells resistant to ascorbate (JLPR cells) and their ascorbate-sensitive parental cells (JLPS cells). Compared with JLPS cells, the increased glucose uptake in JLPR cells (with upregulated glucose transporters, increased antioxidant enzyme activity, and altered cell cycling) conferred ascorbate–induced cytotoxicity and resistance. Transcriptomic profiles and function pathway analysis identified differentially expressed gene signatures for JLPR cells and JLPS cells, which differential expression levels of five genes (ATF5, CD79B, MHC, Myosin, and SAP18) in ascorbate-resistant cells were related to phosphoinositide 3 kinase, cdc42, DNA methylation and transcriptional repression, polyamine regulation, and integrin-linked kinase signaling pathways. These results suggested that coordinated changes occurred in JLPR cells to enable their survival when exposed to the cytotoxic pro-oxidant stress elicited by pharmacologic ascorbate treatment.

Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis

Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.

Abstract 66: Ascorbate Prevents Hypertension, Proteinuria and Concentric Hypertrophy by Balancing the Nitroso-redox System in a Model of Preeclampsia, the S-nitrosoglutathione Reductase (gsnor) Deficient Mice

Introduction: Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc). Mice lacking S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, proteinuria, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesize that the PE-like phenotype is mediated by nitroso-redox imbalance and nitrosative stress and can be rescued with ascorbate treatment. Methods: Pregnant GSNOR –/– and control (WT) mice (n=5-7) were provided drinking water ± Asc beginning at day 0.5 of gestation (E0.5). We determined blood pressure using a Millar catheter, relative wall thickness (RWT) by echocardiography, and placental vascularization by isolectin staining. Cardiomyocytes (CM) were isolated at late stage pregnancy (E17.5) and fluorescent dyes used to determine levels of ROS (2’7’-dichlorofluorescein), nitric oxide (NO, diaminofluorescin) and peroxynitrite (dihydrorhodamine 123). Results: Isolated CMs from pregnant GSNOR –/– hearts, exhibited elevated levels of ROS (2.48±0.39 vs. 1.58±0.18 ΔF/F 0 ), free NO (6.65±0.43 vs. 5.59±0.26 ΔF/F 0 ) and peroxynitrite (0.75±0.04 vs. 0.39±0.03 ΔF/F 0 ) compared to WT. These increases were prevented with Asc treatment (P&lt;0.01), which completely rescued the PE phenotype in GSNOR –/– mothers, including hypertension (105±2 mmHg vs. 95± mmHg in Asc-treated, P&lt;0.05), proteinuria (P&lt;0.05) and RWT (0.56±0.04 vs. 0.45±0.03 in Asc-treated (P&lt;0.05). Placental vascularization was also significantly improved with Asc treatment in GSNOR –/– mothers. Asc had no significant effect in WT mice. Conclusion: Our findings indicate that nitroso-redox imbalance and nitrosative stress contributes to PE in mice. Asc treatment balanced the nitroso-redox system and rescued the pathological phenotypes in GSNOR –/– mice, suggesting that it can be used therapeutically to treat or prevent preeclampsia.

Abstract 2995: Mechanisms of high dose ascorbate inhibiting pancreatic cancer growth and metastasis

Abstract Purpose: High-dose intravenous ascorbate (IVC) bypasses bioavailability barriers of oral ingestion and provides pharmacologic concentrations in tissues, and exhibits pro-oxidant anti-cancer activities. In pancreatic cancer pre-clinical models, ascorbate has been shown to sensitize the tumor to the 1st line chemotherapeutic drug gemcitabine. Here, we investigated the possible mechanisms of actions of ascorbate on pancreatic cancer growth and metastasis. Methods Orthotopic pancreatic cancer mouse model was utilized for evaluation of the effects of ascorbate in inhibiting tumor growth and metastasis. Molecular biological approaches were used in investigation the mechanisms of these effects. Results In the orthotopic mouse tumor model, ascorbate treatment alone decreased pancreatic cancer growth and metastasis. Ascorbate treatment enhanced expressions of epithelial markers and suppressed expression of mesenchymal markers in pancreatic cancer cells. MMP-2 expression and activity were decreased dose-dependently. Tumor tissues from ascorbate-treated mice had markedly increased collagen and desmoplasia in the extracellular matrix. Such fibrosis was not seen in normal tissues from the same mouse. In addition, ascorbate decreased the protein level of deacetylase HDAC6, and inhibited activity of the deacetylase Sirt-2 through depletion of NAD+. As a result, ascorbate robustly increased α-tubulin acetylation in pancreatic cancer cells. Acetylation promoted tubulin polymerization and stabilization, mimicking the cellular outcome of paclitaxel, resulted in inhibition of cell motility and mitosis. Conclusion Pharmacological concentrations of ascorbate affected pancreatic tumor cells and the tumor microenvironment, led to inhibition of tumor growth and metastasis. Citation Format: Qi Chen, Kishore Polireddy, Ruochen Dong, Ping Chen. Mechanisms of high dose ascorbate inhibiting pancreatic cancer growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2995.

Proteomic Analysis in Esophageal Eosinophilia Reveals Differential Galectin-3 Expression and S-Nitrosylation

Background Aims: Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. Methods: Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. Results: Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. Conclusion: Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.

Pharmacologic Ascorbate Treatment of Influenza A In Vitro and In Vivo in Mice

Pharmacologic Ascorbate Treatment of Influenza A In Vitro and In Vivo in Mice

Synergistic effects of ascorbate and sorafenib in hepatocellular carcinoma: New insights into ascorbate cytotoxicity

We investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacologic dose (5–20mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. This was further supported by the finding that ascorbate at a non-cytotoxic dose (1mM) enhanced cell killing caused by glucose oxidase. Consistent with this conclusion, ascorbate treatment caused deregulation of cellular calcium homeostasis, resulting in massive mitochondrial calcium accumulation. Ascorbate acted synergistically with the chemotherapeutic sorafenib in killing Hep G2 cells, but not primary hepatocytes, suggesting adjuvant ascorbate treatment can broaden sorafenib’s therapeutic range. Sorafenib caused mitochondrial depolarization and prevented mitochondrial calcium sequestration. Subsequent ascorbate addition further deregulated cellular calcium homeostasis promoting cell death. Additionally, we present the case of a patient with hepatocellular carcinoma (HCC) who had prolonged regression of a rib metastasis upon combination treatment with ascorbate and sorafenib, indicating that these studies have direct clinical relevance.

Abstract 19282: Transient Bone Morphogenic Protein Antagonism Directs Differentiation of Ipscs Into the Cardiac Neural Crest and Ckit+ Myocardial Progenitor Lineages

Introduction: The signaling pathways that govern cKit+ cardiac progenitor cell (CPC) differentiation into cardiomyocytes (CMs) are unknown. Some studies suggest an essential role in cardiomyogenesis, others suggest a minimal CPC contribution. We studied if it is a non-permissive cardiac milieu that minimizes the generation of CMs from CPCs. Hypothesis: Transient BMP antagonism directs the generation of cardiomyocytes from CPCs. Methods: We lineage-traced CPCs using a novel dual-recombinase responsive indicator mice (cKitCreERT2;Wnt1::Flpe;RC::Fela) and iPSCs derived from cKitCreERT2;IRG (iPSCKit) mice. Results: Intersectional genetic fate-mapping of cKitCreERT2;Wnt1:: Flpe;RC::Fela embryos supported that cKit marks Wnt1-expressing cardiac neural crest (CNC) progenitors, emerging at ~E9.5 and contributing a limited number of cardiomyocytes (n=3). We lineage-traced CPCs during stage-specific cardiogenic differentiation of iPSCKit. Ascorbate treatment promoted differentiation of iPSCKit-derived embryoid bodies (EBs) into Nkx2.5+ myocardium, 45.5%±6.7% of which co-expressed the Cre-reporter EGFP (n=154 EBs; 12 preps), suggesting that CPCs encompass fully competent cardiomyogenic progenitors. Noggin (or Dorsomorphin), a BMP antagonist transiently expressed in the heart at E7.5-E8.5 but not during CNC invasion, directed the differentiation of iPSCkit-EBs into Mesp1+/Isl1+/Nkx2.5+ cardiac mesoderm progenitors (p≤0.0001). The same signaling pathway subsequently directed EBs into the cKit+/Wnt1+/Pax3+/Mitf-H+/Isl1+/Nkx2.5+ CNC lineage (p≤0.0001), while suppressing the generation of WT1+/Tbx18+ epicardium (p&lt;0.05). Stage-specific induction of Cre-recombination delineated that iPSCkit-derived CPCs encompass Mesp1–/cKit+/Nkx2.5+ CNC progenitors, which contributed EGFP+ CNC derivatives, including Nkx2-5+ cardiomyocytes, to 60.7%±7.3% of spontaneously beating EBs (n=147 EBs; 12 preps). Conclusions: Our data show that CPCkit are fully competent CNC-derived cardiomyogenic progenitors, whose differentiation to cardiomyocytes is minimized by a latent Noggin-mediated signaling pathway. Therapeutically exploiting CPCkit, provides an important strategy for maximizing myocardial regeneration.

Abstract 394: Transient Bone Morphogenic Protein Antagonism Directs Differentiation of iPSCs into the Cardiac Neural Crest and cKit+ Myocardial Progenitor Lineages

Introduction: The capability of cKit+ cardiac progenitor cells (CPCs) to participate in cardiomyocyte regeneration remains controversial, despite basic and clinical studies supporting such a role. Hypothesis: A non-permissive cardiac milieu minimizes the generation of cardiomyocytes from CPCs. Methods: We lineage-traced CPCs using novel dual-recombinase responsive indicator mice (cKitCreERT2;Wnt1::Flpe;RC::Fela) and iPSCs derived from cKitCreERT2;IRG (iPSCKit) mice. Results: Intersectional genetic fate-mapping of cKitCreERT2;Wnt1:: Flpe;RC::Fela embryos supported that cKit marks Wnt1-expressing cardiac neural crest (CNC) progenitors, emerging at ~E9.5 and contributing a limited number of cardiomyocytes. To decipher the mechanisms underlying cardiomyocyte differentiation of CPCs, we lineage-traced CPCs during stage-specific cardiogenic differentiation of iPSCKit. Ascorbate treatment promoted differentiation of cKit+ iPSC-derived embryoid bodies (EBs) into Nkx2.5+ myocardium, 45.5%±6.7% of which co-expressed the Cre-reporter EGFP (n=154 EBs; 12 preparations), suggesting that CPCs encompass fully competent cardiomyogenic progenitors. Noggin (or Dorsomorphin), a BMP antagonist transiently expressed in the heart at E7.5-E8.5 but not during CNC invasion, directed the differentiation of iPSCkit-EBs into Mesp1+/Isl1+/Nkx2.5+ cardiac mesoderm progenitors (p≤0.0001). Remarkably, the same signaling pathway subsequently directed EBs into the cKit+/Wnt1+/Pax3+/Mitf-H+/Isl1+/Nkx2.5+ CNC lineage (p≤0.0001), while suppressing the generation of WT1+/Tbx18+ epicardium (p&lt;0.05). Stage-specific induction of Cre-recombination delineated that iPSCkit-derived CPCs encompass Mesp1–/cKit+/Nkx2.5+ CNC progenitors which contributed EGFP+ CNC derivatives, including Nkx2-5+ cardiomyocytes, to 60.7%±7.3% of spontaneously beating EBs (n=147 EBs; 12 preparations). Conclusions: Collectively, our data show that CPCkit are fully competent CNC-derived cardiomyogenic progenitors, whose differentiation to cardiomyocytes is minimized by a latent Noggin-mediated signaling pathway. Therefore exploiting CPCkit therapeutically, provides an important strategy for maximizing myocardial regeneration.