10μM Concentration Research Articles

EXTH-23. NEW DNA-CROSSLINKING CHEMOTHERAPY IS EFFECTIVE AGAINST POST-TEMOZOLOMIDE MISMATCH REPAIR-DEFICIENT PATIENT-DERIVED HYPERMUTANT GLIOMAS

Abstract The DNA mismatch repair (MMR) pathway triggers glioblastoma (GBM) apoptosis when base mismatches induced by the alkylating agent temozolomide (TMZ) cannot be repaired. GBMs often develop hypermutation and resistance to TMZ through acquired inactivation of DNA MMR enzymes (MSH2, MSH6, MLH1, PMS2). A newly developed therapeutic, KL-50, fluoro-ethylates DNA bases, leading to DNA inter-strand cross-links. This form of DNA damage triggers MMR-independent apoptosis. Thus, KL-50 can target MMR-deficient tumor cells. Previous work showed that KL-50 is effective against GBM cell lines that were rendered TMZ-resistant through shRNA knockdown of MMR genes (PMID 35901163). In those studies, KL-50 was most effective when used in combination with an MGMT enzyme inhibitor, or in MGMT-deficient cells. Here, we extend those findings with a preclinical trial of KL-50 in GBM patient-derived xenografts (PDX), including PDX models of post-TMZ GBM that acquired MMR mutations and hypermutation through serial TMZ exposure (PMID 31852831). Among TMZ-naïve PDX, KL-50 extended survival of engrafted mice by 24 days for GBM6 (partially MGMT-deficient), and by 38 days for GBM12 PDX (fully MGMT-deficient) compared to vehicle (p<0.0001, Log-Rank). Combining KL-50 with 4.0 Gy radiation further extended survival of GBM12 mice (9 days, p=0.02), but not GBM6 mice. With engraftments of GBM6R-m185, a post-TMZ, MMR-deficient derivative of GBM6, 14/14 (100%) of KL-50 treated mice are still alive 60 days post-engraftment compared to 0/13 (0%) of vehicle control mice (median survival 37 days, p<0.0001, experiment ongoing). Complementary in vitro studies showed that GBM6R-m185 cells are more sensitive to KL-50 than TMZ at matched concentrations of 50µM (p=0.001, one-way ANOVA) and 100µM (p=0.047). These data demonstrate the potential of KL-50 in treating post-TMZ MMR-deficient recurrent gliomas.

Silicon exhibits dose-dependent impact on barley growth

As population growth accelerates, agriculture’s significance in our lives remains paramount. However, this surge in population has led to land degradation and increased food scarcity. The impact of silicon (Si) on plants has garnered significant attention in agricultural research. This study was aimed at examining the impact of Si on barley (Hordeum vulgare) plants by administering varying concentrations (5µM, 10µM, 20µM, 50µM, 100µM, 200µM, 500µM, 1000µM) and assessing their effects on plant growth parameters, particularly root and shoot lengths, and root fresh weight. Our study employed a controlled experimental setup to observe how barley plants respond to varying concentrations of Si. The data indicates that Si concentrations up to 10µM offer advantageous effects on barley compared to the control group, suggesting its potential in bolstering agricultural productivity. Similarly, concentrations of 20µM, 50µM, and 100µM were identified as safe for plant growth, opening avenues for their agricultural utilization. However, concentrations of 500µM and 1000µM resulted in complete inhibition of plant growth, emphasizing the necessity for cautious Si application in agriculture to prevent adverse impacts on crop yields. These findings underscore the importance of fine-tuning Si concentrations in agricultural practices to optimize benefits while mitigating potential risks to plants.

T-type calcium channel modulation by hydrogen sulfide in neuropathic pain conditions.

Neuropathic pain can appear as a direct or indirect nerve damage lesion or disease that affects the somatosensory nervous system. If the neurons are damaged or indirectly stimulated, immune cells contribute significantly to inflammatory and neuropathic pain. After nerve injury, peripheral macrophages/spinal microglia accumulate around damaged neurons, producing endogenous hydrogen sulfide (H2S) through the cystathionine-γ-lyase (CSE) enzyme. H2S has a pronociceptive modulation on the Cav3.2 subtype, the predominant Cav3 isoform involved in pain processes. The present review provides relevant information about H2S modulation on the Cav3.2 T-type channels in neuropathic pain conditions. We have discussed that the dual effect of H2S on T-type channels is concentration-dependent, that is, an inhibitory effect is seen at low concentrations of 10µM and an augmentation effect on T-current at 100µM. The modulation mechanism of the Cav3.2 channel by H2S involves the direct participation of the redox/Zn2+ affinity site located in the His191 in the extracellular loop of domain I of the channel, involving a group of extracellular cysteines, comprising C114, C123, C128, and C1333, that can modify the local redox environment. The indirect interaction pathways involve the regulation of the Cav3.2 channel through cytokines, kinases, and post-translational regulators of channel expression. The findings conclude that the CSE/H2S/Cav3.2 pathway could be a promising therapeutic target for neuropathic pain disorders.

The Effect of Lanthanum (III) Nitrate on the Osteogenic Differentiation of Mice Bone Marrow Stromal Cells.

To study the species of lanthanum (III) nitrate (La[NO3]3) dispersed in cell media and the effect on the osteoblast differentiation of bone marrow stroma cells (BMSCs). Different La-containing precipitations were obtained by adding various concentrations of La(NO3)3 solutions to Dulbecco's modified Eagle medium (DMEM) or DMEM with fetal bovine serum (FBS). A series of characterisation methods, including dynamic light scattering, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and protein quantification were employed to clarify the species of the different La-containing precipitations. The primary BMSCs were isolated, and the cell viability, alkaline phosphatase activity, and the formation of a mineralised nodule of BMSCs were tested when treated with different La-containing precipitations. The La(NO3)3 solutions in DMEM could form LaPO4, which exits in the particle formation, while the La(NO3)3 solutions in DMEM with FBS could form a La-PO4-protein compound. When treated with La(NO3)3 solutions in DMEM, the cell viability of the BMSCs was inhibited at the concentrations of 1, 10, and 100μM at 1day and 3days. Meanwhile, the supernatant derived from the La(NO3)3 solutions in DMEM did not affect the cell viability of the BMSCs. In addition, the precipitate derived from the La(NO3)3 solutions in DMEM added to the complete medium inhibited the cell viability of the BMSCs at concentrations of 10μM and 100μM. When treated with La(NO3)3 solutions in DMEM with FBS, the derived precipitate and supernatant did not affect the cell viability of the BMSCs, except for the concentration of 100μM La(NO3)3. The La-PO4-protein formed from the La(NO3)3 solutions in DMEM with FBS inhibited the osteoblast differentiation of BMSCs at the concentration of 1μM La(NO3)3 (P < 0.05) but had no effect on either the osteoblast differentiation at the concentrations of 0.001 and 0.1μM or on the formation of a mineralised nodule at all tested concentrations of La(NO3)3. Overall, La(NO3)3 solutions in different cell culture media could form different La-containing compounds: La-PO4 particles (in DMEM) and a La-PO4-protein compound (in DMEM with FBS). The different La-containing compounds caused different effects on the cell viability, osteoblast differentiation, and the formation of a mineralised nodule of the BMSCs. The La-containing precipitation inhibited the osteoblast differentiation by inhibiting the expression of osteoblast-related genes and proteins, providing a theoretical basis for clinical doctors to apply phosphorus-lowering drugs such as lanthanum carbon.

Effects of fatty acid-ethanol amine (FA-EA) derivatives on lipid accumulation and inflammation.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10μM could significantly decrease mRNA levels of IL-6, IL-1β, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1β in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.

Antisense oligonucleotides (ASO) to increase progranulin production in frontotemporal dementia

Frontotemporal Dementia (FTD) is a debilitating disease that causes the individual to slowly lose their personalities, emotions, and control over social behaviors as disease progresses. FTD is caused due to significant neuronal loss in the frontal and temporal lobes of the brain. Though there is no treatment or cure yet for FTD, research has identified that the protein progranulin may be responsible for this neuronal loss, as a lack of progranulin has been linked to the degradation of neurons in FTD patients. Therefore, an effective treatment for FTD must aim to increase the production of progranulin in the brain. Previous research papers (Jiao J et al, 2010*) found that MicroRNA 29 (miR‐29) was responsible for regulating the translation of hPGRN (human progranulin) RNA through binding to the 3’UTR and preventing translation, suggesting that preventing miR‐29 from binding to the 3’UTR of hPGRN RNA should increase the overall production of progranulin. Antisense oligonucleotides (ASO) achieve this through binding to the hPGRN RNA and disrupting the binding site of miR‐29 and therefore preventing miR‐29 from binding. In our study, neuroblastoma cells were transfected with luciferase DNA as a surrogate measure of progranulin translation and then treated with increasing concentrations of M5 ASO and evaluated for luciferase expression. The results indicated that there was an increase in luciferase activity with the increasing concentrations of ASO, specifically concentrations of 10μM and 20μM which had a significant increase in progranulin production. Further tests were done in an attempt to confirm that the ASOs are working through binding to the 3’UTR and preventing miR‐29 from binding, however these tests came back inconclusive. These findings provide a beneficial insight into further development of these ASOs as possible treatments and therapies for FTD.*Jiao J, Herl LD, Farese RV, Gao FB. MicroRNA‐29b regulates the expression level of human progranulin, a secreted glycoprotein implicated in frontotemporal dementia. PLoS One. 2010 May 10;5(5):e10551

Heparin-induced and caffeine or ouabain supplemented capacitation of frozen-thawed yak (Bos grunniens) spermatozoa.

Our goal was to investigate heparin-induced capacitation of frozen-thawed yak sperm and to assess the effects of caffeine or ouabain supplementation with heparin on sperm capacitation. Sperm were incubated with varying heparin concentrations, namely 0, 12.5, 25, 50 and 100μg/ml, for 0, 15, 30 and 60min. In every treatment, sperm capacitation was assessed using microscopic examination of the sperm acrosomal status and western blot analysis of the levels of tyrosine phosphorylation (Tyr-P). Based on our results, the optimal condition for frozen-thawed yak sperm capacitation was a 30-min exposure to 50-μg/ml heparin. Next, we incubated frozen-thawed yak sperm with 50-μg/ml heparin, along with varying concentrations of caffeine supplementation, namely 0, 2.5, 5 and 10mM for 30min. Interestingly, caffeine significantly increased yak sperm acrosome reaction (AR) and Tyr-P (p<.05). The optimal caffeine concentration was 5mM, followed by 2.5 and 10mM, with the lowest AR and Tyr-P found in sperm cells that did not receive any caffeine. To examine the effects of ouabain on sperm capacitation, we next incubated frozen-thawed yak sperm with 50-μg/ml heparin, along with varying concentrations of ouabain, namely 0, 25, 50 and 100µM for 30min. We demonstrated that ouabain supplementation did not alter yak sperm AR or Tyr-P in sperm cells, relative to the control (p>.05). In summary, our findings suggested that caffeine acts synergistically with heparin to increase yak sperm capacitation, but ouabain does not synergize with heparin to promote yak sperm capacitation.

The modulating effect of methoxy-derivatives of 2'-hydroxychalcones on the release of IL-8, MIF, VCAM-1 and ICAM-1 by colon cancer cells.

Colon cancer is one of the leading causes of death in the world. The search for effective and minimally invasive methods of treating colon cancer is the aim of modern medicine. Chalcones and their derivatives have shown an anticancer activity. The aim of the study was to evaluate the effect of methoxy-derivatives of 2'-hydroxychalcones: 2'-hydroxy-3"-methoxychalcone (TJ3), 2'-hydroxy-2"-methoxychalcone (TJ6) and 2'-hydroxy-4"-metoxychalcone (TJ7) at the concentrations of 10µM and 25µM on the release of IL-8, MIF, VCAM-1, ICAM-1 by colon cancer SW480 and SW620 cell lines. The cytokines and adhesion molecules were detected using the Bio-Plex Magnetic Luminex Assay and the Bio-Plex Suspension Array System. Our results showed that all tested methoxy-derivatives of 2'-hydroxychalcone compounds significantly reduced ICAM-1 released by SW480 cancer cells. The tested compounds at both concentrations did not significantly affect VCAM-1 released by SW480 and SW620 cancer cell lines. All methoxy-derivatives significantly reduced the concentration of MIF in dose dependent manner on SW480 cells. The TJ3 at the concentration of 25µM significantly decreased IL-8 secreted by SW480 and SW620 cancer cells. Our results demonstrated that tested methoxy-derivatives of 2'-hydroxychalcones showed modulating effect on colon cancer cells.

Cell death induction of dibutyl phthalate (DBP) on primary brain cells from adult zebrafish

The presence of phthalates as plasticizer on pharmaceutical products as well as in aquatic environments requires serious attention. Phthalates are compounds widely used in the manufacturing industry and has been associate with multiple adverse health effects to human. Among the family of phthalates Dibutyl phthalate (DBP) is well used for pharmaceutical products. Limited information however has been generated on the effect of DBP on the brain. The objective of this investigation was to characterize potential neurotoxicity resulting from short term exposure to DBP. Considering the efficacy and sensitivity of the zebrafish model as an early stage- screening tool, and their neurological and biochemical similarities to those of human, this study evaluates the effect of DBP on primary brain cells of the adult zebrafish. This well optimized and implemented methodology can be consider a guide for future studies using primary cells of adult zebrafish given the reduction on reagents, and the number of cells used for treatment. For indirect confirmation of extracted nervous tissue, the Acetylcholinesterase activity (AChEs) assay kit was implemented. In our study primary cells from adult zebrafish brain were exposed to DBP for 24 hours at aerobic environment at concentrations from 3uM to 100μM. The effect on cell viability as well as cell death mechanisms was determined. Results indicate that adult primary brain cells viability is compromised as low as 8uM of DBP. In terms of mechanism, cells undergo apoptosis and autophagy at concentrations of 8μM and 100μM. This study confirms that low concentrations of DBP over a 24-hour period induces apoptosis and autophagy, potentially activating degradation of brain cells and altering biochemical processes of the nervous system. This research presents a novel approach for future molecular studies using brain primary cells as model to screen pharmaceuticals and environmental agents for risk assessment.

Abstract 1289: Conjugation of indomethacin with novel oleanolic acid oximes increases its Nrf2 and NF-κB signaling pathways modulating effect in pancreatic cancer cells

Abstract Chronic inflammation is a key factor in the etiology of neoplastic diseases, including pancreatic cancer. Nonsteroidal anti-inflammatory drugs (NSAIDs) were approved for the chemoprevention of colon tumors and suggested for pancreas cancers prophylaxis. Anti-inflammatory and anti-tumorigenic activities are also exerted by naturally occurring and synthetic triterpenoids. Coupling of triterpenoid analogues with NSAIDs may enhance this effect and prevent the unfavorable side effects related to NSAIDs long-term use. In this study novel oleanolic acid oxime (OAO) derivatives conjugated with indomethacin (IND) differing in substitution group at position C-17 were evaluated in the context of their possible modulating effect of Nrf2-ARE and NF-κB signaling pathways in pancreatic cancer cells.PSN-1 cells were incubated for 24h with IND and OAO-IND derivatives at the concentrations of 10µM and 20µM selected based on the results of the MTT assay. The activation of Nrf2 and NF-κB was assessed by the evaluation of its translocation into the nucleus and binding to specific DNA sequences by the ELISA assay. Expression of Nrf2, SOD-1, NF-κBp50, NF-κBp65 and COX-2 was evaluated by RT-PCR and Western blot methods.Cell viability was affected mostly by 3-indomethacinoxyiminoolean-12-en-28-oic acid morpholide and 3-indomethacinoxyiminoolean-en-28-oic acid benzyl ester. The level of Nrf2 in the nucleus and binding to ARE sequence was decreased in PSN-1 cells, similarly as was NF-κBp50 and NF-κBp65 binding and nuclear accumulation. As result of diminished activation of these transcription factors decreased expression of SOD-1 and COX-2 i.e. mRNA and protein levels were found. These results indicate that the OAO-IND derivatives, particularly morpholide and benzyl ester conjugates, are more potent suppressors of NF-κB signaling pathway than IND alone. Moreover, conjugation of IND with novel OAO may protect cancer cells against chemoresistance through inhibition of the Nrf2-ARE pathway. Those novel compounds might be considered the potential modulators of hepatocellular carcinoma therapy and chemopreventive agents. Funding: This work was supported by Polish National Science Centre, grant 2016/21/B/NZ7/01758. Citation Format: Wanda Baer-Dubowska, Maria Narozna, Violetta Krajka-Kuźniak, Barbara Bednarczyk-Cwynar. Conjugation of indomethacin with novel oleanolic acid oximes increases its Nrf2 and NF-κB signaling pathways modulating effect in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1289.

Influence of Arbuscular Mycorrhizae on Callusing and root colonization of Tea (Camellia sinensis) Clones in Kenya

Mycorrhizal fungi are a major component of the soil micro flora in many ecosystems, but usually have limited saprophytic abilities. Arbuscular mycorrhizal fungi (AMF) are an important component of soil life and soil chemistry. In soil, phosphorus may be present in relatively large amounts, but much of it is poorly available because of the very low solubility of phosphates by formation of complexes with iron, aluminum, and calcium, leading to soil solution concentrations of 10μm or less and very low mobility. Tea is a major income earner in the country, but yields are declining since high yielding tea varieties have a major problem with rooting and take so long in the nursery. The current study was initiated to investigate the role between Mycorrhizae and plants to explain rooting and growth rates during early stages of tea establishment. It was conducted at James Finlay in Kericho County, Kenya. The experiment was laid out in a Randomized Complete Block Design (RCBD) with factorial arrangements. Phosphorus treatments consisted of a standard rate of 107.66kg ha -1, two clones of the tea (S15/10 and SC 12/28) and two mycorrhizal strains (Glomus mosseae and Glomus intraradices) plus one control without mycorrhizae. Data was collected on rate of callusing, chlorophyll content and rate of root infection by mycorrhizal fungus. Application of 50kg Mycorrhizae ha-1 exhibited the highest callusing rate on clone SC 15/10 with significant differences (P≤0.05) observed on the chlorophyll content from week 1 to week 30 where the standard application of phosphorus plus 50kg Mycorrhizae ha-1 on clone S 15/10 had the highest content consistently throughout the trial. The highest frequency of mycorrhizae colonization in the rhizosphere was observed when 70kg ha-1 was added under clone SC 12/28. AMF strains are recommended for use on tea propagation in improving callusing rate and the chlorophyll content at a rate of 50kg Mycorrhizae ha-1.

Abstract 127: Oleanolic acid oxime derivatives modulate NF-κB signaling pathway leading to cell cycle arrest in pancreatic cancer cells

Abstract Chronic inflammation is considered an important risk factor in the etiology of neoplastic diseases, including pancreatic cancer (PC). Naturally occurring triterpeinoid - oleanolic acid (OA) was shown to possess anti-inflammatory and anti-tumorigenic activities in the in vitro as well as in vivo models. The limited bioavailability of OA justifies searching for its more available derivatives. In this study novel oleanolic acid oxime derivatives (OAO) differing in substitution group at position C-17 were evaluated in the context of their possible modulating effect of NF-κB signaling pathway and subsequently cell cycle arrest and proliferation of pancreatic cancer cells - line PSN-1. PSN-1 cells were incubated with OA and OAO derivatives at the concentrations of 10µM, 20µM and 30 µM selected basing on the results of MTT assay for 24h. The activation of NF-κB was assessed by measuring the level of binding of its active subunits p65 and p50 to specific DNA sequence using the ELISA assay, and their translocation from cytosol to nucleus by Western blot. For genes expression evaluation (NF-κB, COX-2, ERK1/2 and JNK) the RT-PCR (mRNA) and bead-based sandwich type immunoassay on the Luminex®MAGPIX instrument (protein) was applied. Muse®Cell Analyzer was used to flow cytometric assessment of cells proliferation based on Ki67 expression and the cell cycle progression was assessed by propidium iodide staining. The OAO-morpholide derivative (18) and OAO-benzyl ester derivative (14) reduced the most efficiently the NF-κB activation and expression resulting in a diminished level of COX-2 protein in the cytosolic fraction. The expression of JNK and ERK1/2, the elements of MAPK signaling cascade, was not significantly affected by any of the tested OAO derivatives. Treatment with OAO-morpholide derivative (18) also induced the cell cycle arrest leading to accumulation of the cells in S/G2 phase. None of the tested OAO derivatives affected significantly the cells proliferation measured in term of Ki67 expression. These results indicate that the OAO derivatives, particularly morpholide conjugates are more potent suppressors of NF-κB signaling pathway than parent OA. The specificity of OAO structure substitution seems to be crucial for the NF-κB modulation and subsequent possible anti-inflammatory effect. Downregulation of NF-κB by these compounds does not seem to be connected with the suppression of MAPK signaling pathways. Funding: This work was supported by Polish National Science Centre, grant 2016/21/B/NZ7/01758. Citation Format: Maria Narożna, Violetta Krajak-Kuźniak, Robert Kleszcz, Wanda Maria Baer-Dubowska. Oleanolic acid oxime derivatives modulate NF-κB signaling pathway leading to cell cycle arrest in pancreatic cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 127.

New benzimidazole-aldehyde hybrids as neuroprotectors with hypochlorite and superoxide radical-scavenging activity.

Many neurodegenerative disorders include oxidative stress-mediated pathology. Melatonin and its metabolites act as endogenous reactive oxygen species (ROS) scavengers and antioxidants. N,N'-Disubstituted benzimidazole-2-thiones with extended side chains could exert antioxidant and neuroprotective properties due to structural similarities to melatonin. The toxicological potential of the compounds was evaluated by monitoring the synaptosomal viability and the levels of reduced glutathione (GSH) in isolated rat brain synaptosomes. The neuroprotective effects were assessed in vitro in a model of 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. The capability to decrease superoxide anion radical and hypochlorite was evaluated by luminol-dependent chemiluminescent assays. Compounds 5-7 containing residues of veratraldehyde, vanillin, and syringaldehyde at concentration 250μM, preserved at the highest degree the synaptosomal viability and GSH levels. Further screening of compounds 5-7 at lower concentrations of 100μM, 10μM, and 1μM, respectively, demonstrated that 6 and 7 do not show any toxicity within this concentration range. In the model of 6-OHDA-induced oxidative stress, 6 revealed concentration-dependent, neuroprotective, and antioxidant activities similar to melatonin. All the three compounds demonstrated ability to decrease the chemiluminescent scavenging index (CL-SI) in the hypochlorite containing system. In the superoxide system, the hydrazones exhibited different effects on the signal. Our studies suggest that the benzimidazole-aldehyde hybrids act as direct ROS scavengers and membranes' stabilizers against free radicals. Thus, they play a role in the antioxidative defense system and have a promising potential as therapeutic neuroprotective agents for the treatment of neurodegenerative disorders.

Dose-Dependent Iron Chelating Effects of Eltrombopag on in Vitro Human Megakaryopoiesis

Eltrombopag (ELT), a small molecular thrombopoietin (TPO) mimetic approved for children and adults, could offer a therapeutic alternative to selected neonates and young children with chronic thrombocytopenias. ELT has also been proposed as a potential anti-cancer drug due to its anti-proliferative effects in tumor cells, which are mediated by its strong iron chelating properties. This raises the potential concern that rapidly proliferating normal cells, like bone marrow cells in neonates or young infants, could also be susceptible to the anti-proliferative effects of ELT. In this study, we first assessed the responses of neonatal (cord blood, CB) and adult (peripheral blood, PB) megakaryocyte (MK) progenitors to increasing concentrations of ELT, TPO and Romiplostim (ROM) in vitro. Consistent with the previously described pattern of neonatal megakaryopoiesis, CB progenitors generated 10-times more MKs, which were of lower ploidy but had higher CD42 surface expression levels than PB-MKs. Unlike TPO or ROM, ELT exhibited dose-dependent opposing effects on in vitro megakaryopoiesis: Low concentrations (≤6µM) stimulated megakaryopoiesis, but high concentrations (30µM) suppressed MK differentiation and proliferation, as evidenced by a severe reduction in the percentage and absolute number of CD41+ cells after 7 days of culture. The toxic effects of high ELT concentrations were not abrogated by the addition of TPO at concentrations achieved in hyporegenerative thrombocytopenias (3 ng/mL), and were reproduced by the addition of the iron chelators deferoxamine (DFO) or deferiprone (DFP) at concentrations of 100µM to MK cultures, suggesting iron deficiency as a potential mechanism. To further assess the iron chelating effects of ELT in human MKs, we used the calcein assay. In K562 cells, as well as in CB MKs, ELT concentrations &gt;10µM reduced the labile intracellular iron pool (LIP) to lower levels than the iron chelator DFP at a concentration of 200 µM, indicating the strength of ELT's iron chelating properties. These concentrations of ELT also resulted in a severe reduction of mitochondrial transmembrane potential, detected by the JC-1 assay, decreased proliferation (EdU click assay) and apoptosis (Annexin-V binding). During MK differentiation, committed MK progenitors (CD34+/CD41+ cells) were the cell population most sensitive to the antiproliferative and apoptotic effects of high-dose ELT. Next, we evaluated whether the iron status of differentiated MK progenitors would influence their responses to ELT. To test this, we generated iron-depleted and iron-repleted CB-MK progenitors by culturing CD34+ cells for 7 days in the presence of apo-transferrin (iron-free) or holo-transferrin (iron-saturated), followed by a 72 hour culture with different concentrations of ELT. These studies revealed a strong iron status/ELT dose interaction (interaction p&lt;0.0001); In iron-depleted cultures, 30 µM ELT and 100 µM DFO similarly impaired MK expansion and induced MK apoptosis, but these effects were attenuated in iron-repleted cultures. Interestingly, iron-depleted adult PB progenitors did not show reduced MK expansion or increased apoptosis in response to 30 µM ELT, suggesting a lower susceptibility of adult megakaryopoiesis to the iron chelating effects of ELT. Taken together, these findings suggest that iron status is an important variable affecting the response to ELT, particularly in neonates and children. Disclosures Cooper: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Porter:Celgene: Consultancy, Honoraria; Protagonism: Honoraria; Agios: Consultancy, Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria; Bluebird bio: Consultancy, Honoraria. Bussel:Regeneron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; RallyBio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kezar Life Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Physician Education Resource: Speakers Bureau; 3S Bio: Speakers Bureau; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Tranquil: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Momenta Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; UCB: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Sola-Visner:Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.

Is 1,8-Cineole-Rich Extract of Small Cardamom Seeds More Effective in Preventing Alzheimer's Disease than 1,8-Cineole Alone?

The present study demonstrates the efficacies of synthetic 1,8-cineole and an 1,8-cineole-rich supercritical carbon dioxide (SC-CO2) extract of small cardamom seeds in preventing oligomerization of amyloid beta peptide (Aβ42) and inhibiting iron-dependent oxyradical production in vitro. The oligomerization of Aβ42 was monitored by thioflavin T assay and MALDI-TOF analysis of the oligomers. The iron-dependent production of oxygen free radicals was detected by fluorometric benzoate hydroxylation assay. We observed that both pure 1,8-cineole and 1,8-cineole-rich extract of small cardamom seeds at concentrations of 50µM and 100µM prevented the production of reactive hydroxyl radicals from a mixture of Fe2+ and ascorbate. However, the 1,8-cineole-rich extract of small cardamom seeds prevented in vitro Aβ42 oligomerization more effectively vis-à-vis the synthetic (99% pure) 1,8-cineole. Additional study on SHSY5Y cells indicated that both pure 1,8-cineole and 1,8-cineole-rich SC-CO2 extract of small cardamom seeds prevented iron-dependent cell death. Since oxidative damage, Aβ42 aggregation and loss of cell viability (iron-induced) are characteristics of onset of Alzheimer's disease pathology, our results suggest a putative therapeutic role of 1,8-cineole-rich extract of small cardamom seeds over pure 1,8-cineole in preventing this neurodegenerative disease.

LHRH Targeted Chonderosomes of Mitomycin C in Breast Cancer: An In Vitro/ In Vivo Study.

Mitomycin C (MMC) is an anti-cancer drug used for the treatment of breast cancer with limited therapeutic index, extreme gastric adverse effects and bone marrow suppression. The purpose of the present study was the preparation of a dual-targeted delivery system of MMC for targeting CD44 and LHRH overexpressed receptors of breast cancer. MMC loaded LHRH targeted chonderosome was prepared by precipitation method and was characterized for their physicochemical properties. Cell cycle arrest and cytotoxicity tests were studied on cell lines of MCF-7, MDA-MB231 and 4T1 (as CD44 and LHRH positive cells) and BT-474 cell line (as CD44 negative receptor cells). The in vivo histopathology and antitumor activity of MMC-loaded chonderosomes were compared with free MMC in 4T1 cells inducing breast cancer in Balb-c mice. MMC loaded LHRH targeted chonderosomes caused 3.3 and 5.5 fold more cytotoxicity on MCF-7 and 4T1 cells than free MMC at concentrations of 100μM and 10μM, respectively. However, on BT-474 cells the difference was insignificant. The cell cycle test showed no change for MMC mechanism of action when it was loaded in chonderosomes compared to free MMC. The in vivo antitumor studies showed that MMC loaded LHRH targeted chonderosomes were 6.5 fold more effective in the reduction of tumor volume than free MMC with the most severe necrosis compared to non-targeted chonderosomes in pathological studies on harvested tumors. The developed MMC loaded LHRH targeted chonderosomes were more effective in tumor growth suppression and may be promising for targeted delivery of MMC in breast cancer.

Abstract 3867: Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer

Abstract Introduction and Objective: Prostaglandin E2 (PGE2) is a lipid-derived signaling agent that promotes tumorigenesis via increasing stemness, proliferation, and angiogenesis. PGE2 levels are elevated in multiple cancers, including bladder (BCa) where they may play a pathological role. PGE2 is generated in part by cyclooxygenase enzymes (COX-1/COX-2). Clinical trials using the COX-2 specific inhibitor celecoxib to prevent progression and recurrence of non-muscle invasive BCa report hazard ratio reductions; however, celecoxib fails to significantly outperform standard of care. These studies are dose-limited by cardiotoxicity present with COX inhibition, but have suggested higher doses of celecoxib may yield significant results. Microsomal PGE2 synthase-1 (mPGES-1) is one of two terminal enzymes (downstream of COX-1/2) associated with PGE2 production. Inhibition of mPGES-1 does not affect synthesis of the prostaglandins responsible for cardiotoxicity but still reduces PGE2 levels. Using a model of carcinogen induced BCa, we have previously established mPGES1-/- have significantly smaller bladder tumors by weight, reduced pathological stage, reduced PGE-2 metabolites, and reductions in percent tumor involvement in the bladder compared to WT mice. The purpose of this study was to further understand mPGES mediated tumorigenesis using in vitro cellular models. We hypothesized pharmacological inhibition with the mPGES-1 specific inhibitor MF63 would reduce PGE2 levels leading to reduced cellular proliferation and reduced PGE2 release in vitro. Methods: mPGES1 protein and RNA levels were measured in benign (UROtsa) or malignant BCa cell lines using western blotting and qPCR respectively. PGE2 levels were assessed by ELISA. Cell proliferation was assessed using the hexosaminidase assay and colony formation assays. Cell death was assessed using lactate dehydrogenase (LDH) release. Results: In vitro, BCa cell lines HT-1376, RT-4 and HTB-9 significantly upregulate mPGES1 as compared to a benign urothelial cell line (UROtsa). To assess the capacity of MF63 to effect PGE2 production, we treated HT-1376 and HTB-9 cells with MF63 at increasing concentrations and assessed PGE2 levels. MF63 significantly reduced PGE2 production after 24h in both cell lines. Furthermore, pharmacological inhibition of mPGES1 with MF63 at concentrations of 10µM and above significantly reduced cellular proliferation after 48 or 72 hours using both colony formation and the hexosaminidase assay to assess proliferation. Finally, concentrations of 50µM MF63 robustly induced cell death as measured by LDH release. Conclusions: These data show that inhibition of mPGES1 in vitro with the inhibitor MF63 results in decreases in PGE2 levels and reduced cellular proliferation, likely due in part to cell death induction. mPGES1 inhibitors are expected to have improved safety profiles and given our results, may have therapeutic potential in BCa. Citation Format: Benjamin Leeland Woolbright, Jordan Barney, Van Schloegel Schloegel, Erika Abbott, John Arthur Taylor. Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3867.

Hydrolysis of the non-canonical cyclic nucleotide cUMP by PDE9A: kinetics and binding mode.

The non-canonical cyclic nucleotide cUMP and the phosphodiesterase PDE9A both occur in neuronal cells. Using HPLC-coupled tandem mass spectrometry, we characterized the kinetics of PDE9A-mediated cUMP hydrolysis. PDE9A is a low-affinity and high-velocity enzyme for cUMP (Vmax = ~ 6μmol/min/mg; Km = ~ 401μM). The PDE9 inhibitor BAY 73-6691 inhibited PDE9A-catalyzed cUMP hydrolysis (Ki = 590nM). Docking studies indicate two H-bonds between the cUMP uridine moiety and Gln453/Asn405 of PDE9A. By contrast, the guanosine moiety of cGMP forms three H-bonds with Gln453. cCMP is not hydrolyzed at a concentration of 3μM, but inhibits the PDE9A-catalyzed cUMP hydrolysis at concentrations of 100μM or more. The probable main reason is that the cytosine moiety cannot act as H-bond acceptor for Gln453. A comparison of PDE9A with PDE7A suggests that the preference of the former for cGMP and cUMP and of the latter for cAMP and cCMP is due to stabilized alternative conformations of the side chain amide of Gln453 and Gln413, respectively. This so-called glutamine switch is known to be involved in the regulation of cAMP/cGMP selectivity of some PDEs.

Abstract 2505: Baicalein reverses chronic stress-induced ovarian cancer promotion through GABA receptors

Abstract Studies suggest that chronic stress accelerate the progression of ovarian cancer, and the promotion effects can be abrogated by β-blockers. However, β-blockers should be assessed and tailored to each cancer patient as they will exert toxic effects in overdose. Baicalein, a widely used Chinese herbal medicine, identified as a new type of anticancer drug with an obvious function of anti-anxiety in animal experiments and whose mechanism may be mediated by GABA receptors. The focus of the current study was to determine whether baicalein could block the effects of chronic stress on tumor growth and investigate the underlying mechanism that relates to GABA receptors. Two kinds of ovarian cancer cell lines (OVCAR-8 and CAOV-3) were selected to be investigated in the inhibitory effect of baicalein on cancer cells. Cell CCK-8 assay, wound healing assay, transwell assay and flow cytometry were used for in vitro experiments. Chronic stress was induced by a restrained stress model in nude mice carrying xenografts in the presence and absence of treatment with baicalein. Xenograft sizes and weight were measured after 4 weeks. Norepinephrine was measured by HPLC and GABA was measured by immunoassays in blood and tumor tissues. Expression of GABA receptors in the xenografts was analyzed by real-time quantitative PCR, western blotting and immunohistochemistry (IHC). Our results showed that ovarian cancer cell lines exhibited significant promotion in the presence of NE concentrations of 10Μm and baicalein concentrations of 10μM could reverse its effects by Cell CCK-8 assay and wound healing assay. Daily restraint stress resulted in significantly increased tumor growth in tumor model of ovarian cancer in nude mice. And this increase was completely blocked with daily baicalein treatment. Chronic stress attenuated expression of GABA receptors in vivo and in vitro; conversely, baicalein increased expression of GABA receptors. Overall, our data suggest that baicalein stimulated GABA receptors activation to reduce tumor growth in ovarian cancer. Considering the stimulatory effects of chronic stress on cancer growth, our study implicates baicalein as a potential medicine for blocking the deleterious effects of chronic stress. Citation Format: Ke Li, Yu Kang, Yinhua Yu. Baicalein reverses chronic stress-induced ovarian cancer promotion through GABA receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2505.

Selective inhibition of endogenous antioxidants with Auranofin causes mitochondrial oxidative stress which can be countered by selenium supplementation

Auranofin is a thiol-reactive gold (I)-containing compound with potential asa chemotherapeutic. Auranofin has the capacity to selectively inhibit endogenous antioxidant enzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx), resulting in oxidative stress and the initiation of a pro-apoptotic cascade. The effect of Auranofin exposure on TrxR and GPx, and the potential for cellular protection through selenium supplementation was examined in the non-cancerous human cell line Swan-71. Auranofin exposure resulted in a concentration dependent differential inhibition of selenoprotein antioxidants. Significant inhibition of TrxR was observed at 20nM Auranofin with inhibition of GPx from 10µM. Significant increases in reactive oxygen species (ROS) were associated with antioxidant inhibition at Auranofin concentrations of 100nM (TrxR inhibition) and 10µM (TrxR and GPx inhibition), respectively. Evaluation of mitochondrial respiration demonstrated significant reductions in routine and maximal respiration at both 100nM and 10μM Auranofin. Auranofin treatment at concentrations of 10μM and higher concentrations resulted in a ∼68% decrease in cellular viability and was associated with elevations in pro-apoptotic markers cytochrome c flux control factor (FCFc) at concentration of 100nM and mitochondrial Bax at 10μM. The supplementation of selenium (100nM) prior to treatment had a generalized protective affect through the restoration of antioxidant activity with a significant increase in TrxR and GPx activity, a significant reduction in ROS and associated improvement in mitochondrial respiration and cellular viability (10µM ∼48% increase). Selenium supplementation reduced the FCFc at low doses of Auranofin (<10μM) however no effect was noted on either FCFc or Bax at concentrations above 10μM. The inhibition of antioxidant systems in non-cancerous cells by Auranofin is strongly dose dependent, and this inhibition can be altered by selenium exposure. Therefore, Auranofin dose and the selenium status of patients are important considerations in the therapeutic use of Auranofin as an agent of chemosensitization.