Annexin PI Staining Research Articles

Cytotoxic activity of non-specific lipid transfer protein (nsLTP1) from Ajwain (Trachyspermum ammi) seeds

BackgroundTrachyspermum ammi, commonly known as Ajwain, is a member of the Apiaceae family. It is a therapeutic herbal spice with diverse pharmacological properties, used in traditional medicine for various ailments. However, all previous studies were conducted using small molecule extracts, leaving the protein’s bioactivity undiscovered.AimThe current study aimed to demonstrate the cytotoxic activity of Ajwain non-specific lipid transfer protein (nsLTP1) in normal breast (MCF10A), breast cancer (MCF-7), and pancreatic cancer (AsPC-1) cell lines. Also, to evaluate its structural stability in human serum as well as at high temperature conditions.MethodsThe cytotoxic activity of Ajwain nsLTP1 was evaluated in MCF-7 and AsPC-1 cell lines using MTT assay. Annexin V-FITC and PI staining were used to detect the early apoptotic and late apoptotic cells. The role of nsLTP1 in inducing apoptosis was further studied by quantifying Bcl-2, Bax, Caspase-3, Survivin, EGFR, and VEGF genes expression using RT-PCR. CD spectroscopy analyzed the nsLTP1 conformational changes after thermal treatment for structure stability determination. The RP-HPLC was used to analyze the nsLTP1 degradation rate in human serum at different time intervals incubated at 37 °C.ResultsAjwain nsLTP1 showed a potent cytotoxic effect in MCF-7 and AsPC-1. The IC50 value obtained in MCF-7 was 8.21 μM, while for AsPC-1 4.17 μM. The effect of nsLTP1 on stimulating apoptosis revealed that the proportions of apoptotic cells in both cell lines were relatively increased depending on the concentration. The apoptotic cells percentage at 20 μM was in MCF-7 71% (***P < 0.001) and AsPC-1 88% (***P < 0.001). These results indicate that nsLTP1 might efficaciously induce apoptosis in multiple types of cancerous cells. Genes expression in MCF-7 and AsPC-1 showed significant upregulation in Bax and Caspase-3 and downregulation in Bcl-2, Survivin, EGFR, and VEGF protein. The CD analysis of nsLTP1 showed a significant thermostable property. In serum, nsLTP1 showed a slow degradation rate, indicating high stability with a half-life of ~ 8.4 h.ConclusionOur results revealed the potential anticancer activity of Ajwain nsLTP1 and its mechanism in inducing apoptosis. It further exhibited thermostable properties at high temperatures and in human serum, which suggested this protein as a promising anticancer agent.

Abstract P4-02-06: Exploiting ceramide homeostasis to target endocrine therapy-resistant breast cancer

Abstract Estrogen receptor (ER)-positive breast cancer accounts for the majority of breast cancer-related deaths. Current treatment includes endocrine therapy (ET), which most women respond to at first. However, up to 40% of women will eventually experience a relapse. Recurrent tumors tend to retain ER expression but no longer respond to ET. There have been multiple genomic and proteomic profiling studies between ET-sensitive and ET-resistant breast cancer cells, but lipidomic studies have been lacking. We have performed targeted and non-targeted lipidomic profiling of ET-sensitive and two models of ET-resistant MCF-7 breast cancer cells and found differences in their sphingolipid profiles, suggesting our overarching hypothesis that ceramide homeostasis is altered in ET-resistance. The current study is designed to understand the functional role of ceramides and their metabolites in ET-resistant breast cancer, with an overall goal to improve the current treatment regimens and quality of life in breast cancer patients. Detection of ceramides and other sphingolipid levels were analyzed by reverse-phase chromatography and LC-QToF-MS. C1P levels were detected by LC-QqQ-MS. Hoechst-PI staining was performed to measure cell death and Annexin-PI staining to detect apoptosis. All cytometric data analyses were performed using a Celigo cytometer. RT-qPCR was performed to analyze mRNA expression. Fold changes were calculated using the ΔΔCt method with multiple housekeeping controls. While numerous changes were observed in lipid species, the most pronounced changes were seen in the sphingolipidome. More specifically, ET-resistant breast cancer cells had lower levels of ceramides and di-hydroceramides. Along with a depletion of ceramides, we have also observed an increase in ceramide-1-phosphate (C1P) levels in the ET-resistant cells. C1P, which is produced by phosphorylation of ceramides by ceramide kinase (CERK), is known to promote cell survival and proliferation. This observation suggests that ET-resistant cells may rely on a higher degree of ceramide turnover to C1P compared to their ET-sensitive counterpart. To test this hypothesis, CERK was inhibited with a small molecule inhibitor (NVP-231), which resulted in increased apoptosis in ET-resistant cells but not in ET-sensitive cells, suggesting ET-resistant cells are more sensitive to CERK inhibition. Although NVP-231 treatment increased ceramide levels in both ET-sensitive and ET-resistant cells, only ET-resistant cells were dependent on ceramide turnover to C1P. To further understand the molecular underpinnings of the differential sensitivity to CERK inhibition, we have investigated the expression of several BCL2 family members, as evidence from the literature suggests that sphingolipids play a critical role in apoptosis by interacting with several BCL2 family members by mechanisms that are not thoroughly understood. We have observed a high positive correlation between BCL2 expression and NVP-231 sensitivity, as well as an inverse correlation between MCL-1 and NVP-231 sensitivity. This suggests that loss of BCL2 in the ET-resistant cells may be a factor for increased sensitivity to CERK inhibition and elevated MCL-1 fails to compensate for that loss. Together, our findings suggest that ET-resistant breast cancer cells have a unique ceramide homeostasis dependence, i.e., rely on a higher degree of ceramide turnover to C1P. Additionally, BCL2 family proteins may play a critical role in determining the sensitivity to CERK inhibition. The dependence of ET-resistant breast cancer cells on C1P production for survival can therefore offer a unique opportunity to increase the therapeutic efficacy and improve patient outcome for women with ET-resistant breast cancer. Citation Format: Purab Pal, Alec Millner, Ekin Atilla-Gokcumen, Jonna Frasor. Exploiting ceramide homeostasis to target endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-06.

Extracellular vesicles from serum samples of mycobacteria patients induced cell death of THP-1 monocyte and PBMC

BackgroundExtracellular vesicles (EVs) play a key role in cell communication and the pathogenesis of some diseases. EVs may accelerate cell death during the course of mycobacterial infection and are also considered as a new vaccine design, drug delivery, and biomarker candidates. The current study evaluates the effects of EVs from serum samples of mycobacteria-infected patients on THP-1 monocytes and PBMC cells.MethodEVs were purified from the serum, then cultured separately with THP-1 monocytes and PBMCs. The cell death was determined through annexin V-FITC and PI staining. GW4869, an EVs inhibitor, was used to determine if EVs released from serum could increase THP-1 monocytes cell death.ResultsThe cell death was significantly increased in the presence of 10 µg/ml and 5 µg/ml concentrations of the purified EVs (p < 0.05). Minimal cell death was determined in 2.5 µg/ml and 1.2 µg/ml (p < 0.05). Up to 85% of the cells were viable in the presence of the GW4869 inhibitor (p < 0.05).ConclusionDirect infection of the cells with EVs released from mycobacteria-infected patients samples, the multiplicity of infection with the EVs, and virulent or avirulent mycobacteria may change the status of the cell death. The isolated EVs from serum samples of patients with mycobacterial infection accelerated cell death, which means that they might not be considered as an optimal tool for developing drug delivery and vaccine against tuberculosis.

Kynurenic acid- a key metabolite protecting the heart from an ischemic damage

Abstract Background Myocardial ischemia is a major cause of death in patients with renal dysfunction. In order to identify a key metabolite which may protect cardiac function following renal injury, we have recently performed a metabolomics profiling analysis of LV lysates and plasma samples derived from animals that underwent an acute kidney injury (AKI) 1 or 7 days earlier, versus sham-operated controls. The analysis revealed that the kynurenic acid (kynurenate, KYNA) metabolite levels are highly elevated in all tested experimental samples relative to control. Purpose We wished to analyze whether KYNA may protect cardiomyocytes' survival and cardiac function upon an ischemic event and if so, to characterize whether the protecting effect may be linked to better preservation of the cardiac mitochondria. Methods Cellular viability of H9C2 rat cardiac myoblasts grown under normoxic or anoxic conditions with or without KYNA was determined by flow cytometry following Annexin-PI staining. The mitochondrial structure of the cells was determined by live cell staining with green (FITC) and deep red (Cy5) mito-tracker dyes. The potential effect of the metabolite on cardiac function following acute MI was tested in a murine model by echocardiography followed by histological staining of the cardiac sections with Picro Sirius Red. Results KYNA given at 10 mM concentration hardly affected the viability of H9C2 grown under normoxia, however the metabolite rescued the viability of the anoxic cells by 63% and largely improved their mitochondrial structure. Moreover, KYNA diluted in the drinking water of post-MI animals (250mg/ml), highly enhanced their cardiac recovery compared to untreated-animals as determined by echocardiography and collagen staining. Conclusions 1. KYNA may represent a key metabolite absorbed by the heart following AKI. 2. KYNA can enhance cardiac cell viability following an ischemic event both in vitro and in vivo in a mechanism which is mediated, at least in part, by protection of the cardiac mitochondria. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Weizmann Institute-Tel-Aviv Sourasky Medical Center joint research grant KYNA's protection of cardiac cells

AML-381: Isolated Bioactive Lipid from Leishmania donovani Induces Apoptosis in AML Cells via Regulating MAPK Signals

The current therapy for cancer treatment, especially in leukemic survivor emphasis, needs to be evolved more. In the current scenario, the microbial source of different origins, including live, attenuated, or genetically modified microbial species or cellular components in active or inactive form or cellular metabolites could be a very promising therapeutic intervention for leukemic therapy. Leishmania sp. is a protozoal parasite whose bioactive lipid (pLLD) has significant anti-inflammatory and anti-cancer properties. Acute myeloid leukemia shows the highest degrees of malignancies and lethality among the elderly and in children. In addition, the chances of secondary infection in AML are very high. Therefore, it was very interesting to find if pLLD could effectively inhibit proliferation of leukemic cells, including acute promyelocytic leukemia (HL-60), acute lymphoblastic leukemia (MOLT-4), acute myeloid leukemia (U937), and chronic myeloid leukemia (K562). Among the four cell lines, U937 showed most significant death in the presence of pLLD in respect to their morphological changes and DNA fragmentation. As apoptosis is a promising approach for cancer therapy, we also evaluated the effect of pLLD in U937 cells with annexin-PI staining and a cell cycle arrest assay using flow cytometry. In the process of programmed cell death, mitochondria play a major role; we also checked mitochondrial membrane potential and cellular reactive oxygen species of this AML cell line in the presence and absence of pLLD with JC-1 and DCFDA dye and found apoptosis was induced by pLLD in U937 cells in a time-dependent manner. pLLD also regulated the cell signals in U937 cells by altering ERK-JNK-p38 levels of the MAPK pathway and simultaneously maintained pro-/anti-apoptotic protein levels (Bax, Bad, Bcl2, t-Bid) assayed by western blot and flow cytometry analysis. Thus, taken together, it was found that pLLD could induce apoptosis in AML cells through regulating MAPK signaling.

Curcumin-like structure (CCA-1.1) induces permanent mitotic arrest (Senescence) on Triple-negative breast cancer (TNBC) cells, 4T1

Triple-negative breast cancer (TNBC) remains as the deadliest cancer type due to the lack of treatment options. Hence, several attempts have been made to develop new anticancer for TNBC therapy. This study intended to challenge curcumin analog (CCA)-1.1, which is derived from pentagamavunone-1 structure, against the 4T1 cell line and TNBC cell model, covering the cytotoxic activity in correlation with cell cycle progression, apoptosis induction, reactive oxygen species (ROS) generation, and senescence evidence. The cell viability, cell cycle profile, apoptosis induction, intracellular ROS level, and senescence induction were determined in vitro using trypan blue exclusion, propidium iodide (PI) staining, Annexin-PI staining, dichlorofluorescein diacetate staining, and senescence-associated-β-gal method. CCA-1.1 showed cytotoxic activity on 4T1 cells, giving half maximal inhibitory concentration value of 3M, but was less toxic on non-cancerous 3T3-L1 cells. CCA-1.1 induced rapid cell death and inhibited cell cycle progression at the mitotic phase. Instead, of causing apoptosis, CCA-1.1 induced mitotic catastrophe. Furthermore, CCA-1.1 itself increased the intracellular ROS level and induced senescence, possibly through catastrophic cell death. Altogether, our preliminary study strengthens the potency of CCA-1.1 for its anticancer activities against TNBC cells and prospective to be pharmaceutically developed as a novel candidate for cancer therapy.

Curcumin-Galactomannoside Complex inhibits the Proliferation of Human Cervical Cancer Cells: Possible Role in Cell Cycle Arrest and Apoptosis.

Background:Cervical cancer is the most common cancer and has the highest morbidity rate of gynaecological malignancies in women worldwide. So, the development of effective anti-cancer agents to treat this condition is vital. Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade, free-curcuminoids (curcumin-galactomannoside complex; CGM) on cervical cancer cells (HeLa) of human origin. In this study, we examined the anticancer potential of CGM as well as its effects on the cell cycle and the apoptosis of HeLa cancer cell. Methods:Determination of anti-proliferative and apoptosis validation of CGM on HeLa cells was performed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide and annexin-V-fluorescein isothiocyanate assays. Measurement of Reactive Oxygen Species (ROS) production, Caspase activities and protein expression experiments were performed to investigate the potential mechanisms of action in the apoptotic process. Results:The cytotoxic assays revealed that the CGM showed inhibition of cell survival and exhibited high cytotoxic activity against HeLa cells at 25 μg/mL. Further studies on morphological changes were done in CGM-treated cervical cancer cells contributing to apoptosis. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that CGM induced apoptosis in HeLa cell lines at 25 μg/mL. By the supplementation of CGM showed an increase in Bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. Conclusion:The evidence obtained from this study suggests that CGM is a potent and promising natural formulation against cervical cancer cells via induction of apoptosis through ROS mediated mitochondrial damage in HeLa cells. Hence, CGM could be further explored as a potential lead in treating cancer.

Anti-apoptotic effect of Nisin as a prebiotic on human mesenchymal stem cells in harsh condition.

Mesenchymal stem cells (MSCs) are progenitor cells of connective tissue with the ability of proliferation, self-renewal, and multilineage differentiation that make it a promising source with an enormous potential to be utilized for tissue repairing and vehicles of cell-based gene therapy. The low survival rate of MSCs following transplantation is their drawback. Preconditioning with some factors is a novel and effective strategy, improving the survival of the cells by protecting them from harmful conditions and result in the good recovery of injured tissues. Nisin is a prebiotic with antimicrobial activity. This manuscript aimed to evaluate the effect of Nisin preconditioning of MSCs on in vitro cell viability. MSCs were cultured and preconditioned with Nisin in different concentrations. Then, they are separately exposed to H2O2 and serum deprivation. Cell survival and cell apoptosis were evaluated by MTT assay and Real-time PCR, respectively. Furthermore, Annexin-PI staining and caspase activity was performed to visualize apoptotic cells. MSC-Nisin viability and proliferation significantly increased when exposed to H2O2 and serum deprivation, compared to that of MSCs. About 250 and 500IU/mL of Nisin donate a significant anti-apoptotic impact to MSCs. Our data suggest that preconditioning with Nisin has been improved cell viability and the anti-apoptotic capacity of MSCs. However, the mechanism related to the protective properties of preconditioning and using this strategy in stem cell therapy requires more research.

Static magnetic field halves cryoinjuries of vitrified mouse COCs, improves their functions and modulates pluripotency of derived blastocysts

This study was performed with the aim of evaluating the influence of static magnetic field (SMF) of 60 mT on mouse Cumulus Oocytes Complexes (COCs) vitrification. The COCs were vitrified in the presence (Vit_SMF+) and absence of SMF (Vit_SMF−). Along with these groups, non-vitrified or fresh COCS, which exposed (nVit_SMF+) and non-exposed (nVit_SMF−) to magnetic field, were also considered. Survival and viability rates and mitochondrial activity as well as ultrastructure of oocytes were examined by trypan blue Staining (TBS), Annexin-PI Staining, JC1 staining and transition electron microscopy, respectively. Following in vitro fertilization (IVF) and embryo development, gene expression was carried out through qRT-PCR at blastocyst (BL) stage. The survival rate in Vit_SMF+ and Vit_SMF− decreased meaningfully in comparison with nVit_SMF− (P < 0.05), but there was no significant difference between SMF+ and SMF− groups. The mitochondrial activity in Vit_SMF− was significantly reduced compared to the nVit_SMF− group (P < 0.05), however its value in Vit_SMF+ returned to the control level. Ultrastructural study demonstrated that SMF could protect the COCs from cryoinjuries and reduced damaged features in ooplasm of the vitrified oocytes. There was no significant difference in fertilization rate. Although, BL formation was the highest rate in the Vit_SMF+ group, it was just substantially higher than the non-vitrified groups (P < 0.05). The significant changes of Oct4, Cdx2 and Nanog genes expression due to vitrification (Vit_SMF−) or SMF (nVit_SMF+) treatments (P < 0.05) as compared to control (nVit_SMF−), returned to the natural level after using SMF in vitrified derived blastocysts (Vit_SMF+). Totally based on the results, it is clear that static magnetic field improves mitochondrial potential activity and ultrastructure of mouse vitrified COCs. In addition, SMF enhances the embryo cleavage rate to blastocyst stage and modulates pluripotency in blastocyst embryos derived from vitrified COCs.

Effect of Proline on Cell Death, Cell Cycle, and Oxidative Stress in C6 Glioma Cell Line.

Since proline metabolism has been implicated to play an underlying role in apoptotic signaling and cancer, and hyperprolinemic patients present susceptibility to tumors development, this study investigated the effect of proline on cell death, cell cycle, antioxidant enzymes activities, and immunocontent/activity of proteins involved in cell death/survival signaling pathways in C6 glioma cells. C6 cells were incubated with proline (0-5mM) for 1h, 24h, 48h, 72h, or 7days. Proline in high concentrations slightly decreased LDH release, and no cytotoxic effect was seen by Annexin-PI staining. Superoxide dismutase and catalase activities were increased by proline (1mM) after 72h, suggesting an increase in reactive species levels. Acetylcholinesterase activity was inhibited by proline at 1, 3, and 5mM. The cell cycle progression was not altered. Results from Western blot analyses showed that proline at 1mM after 72h increased p-NF-ĸB and decreased acetylcholinesterase immunocontent but did not altered AKT, p-AKT, GSK3β, and p-GSK3β. Taken together, the data suggest that high proline levels seems to favor the signaling pathways towards cell proliferation, since acetylcholinesterase, which may act as tumor suppressor, is inhibited by proline. Also, p-NF-κB is increased by proline treatment and its activation is related to tumor cell proliferation and cellular response to oxidants. Proline also induced oxidative stress, but it appears to be insufficient to induce a significant change in cell cycle progression. These data may be related, at least in part, to the increased susceptibility to tumor development in hyperprolinemic individuals.

S100A8 and S100A9 promote endothelial cell activation through the RAGE‑mediated mammalian target of rapamycin complex 2 pathway.

S100 calcium binding protein A8 (S100A8) and A9 (S100A9) belong to the S100 family of calcium-binding proteins and have important roles in inflammation. They increase endothelial cell proliferation, thereby affecting inflammation, angiogenesis and tumorigenesis. However, the mechanism of action of S100A8/9 in endothelial cells needs further study. Therefore, the present study sought to investigate the effects of S100A8/9 on the proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and their mechanism of action. The viability of HUVECs was determined through a Cell Counting Kit-8 assay. The effect of S100A8/9 on the proliferation of HUVECs was detected by flow cytometry. Migration was evaluated by a Transwell migration assay. Apoptosis was evaluated by Annexin V-FITC and PI staining via flow cytometry. Western blot analysis and reverse transcription-quantitative polymerase chain reaction assays were performed to evaluate the activation of the phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mTOR pathway and mTOR complex 2 (mTORC2). We previously confirmed that S100A8/9 were consistently overexpressed at 1 and 7 days post-surgery in a rabbit vein graft model, which is the period when apoptosis changes to proliferation in neointimal hyperplasia. In the present study, proliferation, viability and migration were increased after treating HUVECs with S100A8/9. S100A8/9 stimulated the PI3K/Akt/mTOR pathway and mTORC2, which was significantly suppressed by a receptor for advanced glycation end products (RAGE)-blocking antibody. Furthermore, depleting expression of RAGE or mTORC2 protein components (rapamycin-insensitive companion of mTOR) by small interfering RNA was found to reduce the cell viability, migration and angiogenesis of S100A8/9-treated HUVECs. The development of neointimal hyperplasia is a complex process initiated by damage to endothelial cells. In conclusion, S100A8/9 has an important role in intimal hyperplasia by promoting cell growth and angiogenesis via RAGE signaling and activation of mTORC2.

New Diterpene Compound from Euphorbia connate Boiss., 3,7,14,15-Tetraacetyl-5-Propanoyl-13(17)-Epoxy-8,10(18)-Myrsinadiene, Inhibits the Growth of Ovarian Cancer Cells by Promoting Mitochondrial-Mediated Apoptosis

Ovarian cancer due to the difficulties in early clinical diagnosis and absence of successfull treatment has poor prognosis and high mortality among all gynecological malignancies. Many evidence has revealed that plants of the Euphorbiaceae family are precious sources of novel bioactive compounds with anti-tumor activities. 3,7,14,15-tetraacetyl-5-propanoyl-13(17)-epoxy-8,10(18)-myrsinadiene (TPEM) is a new myrsinane-type diterpene isolated recently by our group from aerial parts of Euphorbia Connata and the present study was aimed to explore its inhibitory effects on growth of two human ovarian cancer cells, OVCAR-3, and Caov-4. The obtained results indicated that growth of OVCAR-3 and Caov-4 cells was significantly inhibited by TPEM in a dose-dependent manner, with the IC50 values of 41.27 ± 1.52 and 36.44 ± 2.41 µM, respectively. Furthermore, using Annexin V-FITC and PI staining it was confirmed that the induced cell death was mainly mediated through apoptotic pathway. Further observations such as decrease in the mitochondrial membrane potential (ΔΨm), increase in the activity of caspase-3 and elevation of the Bax/Bcl-2 ratio suggested the role of mitochondria in the induction of apoptosis by TPEM. ROS level was also remarkably increased in OVCAR-3 and Caov-4 cells in response to TPEM treatment. In conclusion, these findings provide first evidences about potential anticancer properties of TPEM.

Rutin loaded liquid crystalline nanoparticles inhibit lipopolysaccharide induced oxidative stress and apoptosis in bronchial epithelial cells in vitro

Airway inflammation and infections are the primary causes of damage in the airway epithelium, that lead to hypersecretion of mucus and airway hyper-responsiveness. The role of reactive oxygen species (ROS) and their components in the pathophysiological mechanisms of airway inflammation have been well-studied and emphasized for the past several decades. Rutin, a potent bioflavonoid, is well-known for its antioxidant, anti-inflammatory, especially in bronchial inflammation. However, poor solubility and rapid metabolism have led to its low bioavailability in biological systems, and hence limit its application. The present study aims to investigate the beneficial effects of rutin-loaded liquid crystalline nanoparticles (LCNs) against lipopolysaccharide (LPS) induced oxidative damage in human bronchial epithelial cell line (BEAS-2-B) cells in vitro. LPS was used to stimulate BEAS-2-B cells, causing the generation of nitric oxide (NO) and other reactive oxygen species (ROS) that had led to cellular apoptosis. The levels of NO and ROS were detected by, Griess reagent kit and dichlorodihydrofluorescein diacetate (DCFH-DA) respectively, whereas, cell apoptosis was studied by Annexin V-FITC and PI staining. The findings revealed that rutin-loaded LCNs significantly reduced NO, ROS levels and prevented apoptosis in BEAS-2B cells. The observations and findings provide a mechanistic understanding of the effectiveness of rutin-loaded LCNs in protecting the bronchial cells against airway inflammation, thus possessing a promising therapeutic option for the management of airway diseases.

MiR-483-3p promotes cell proliferation and suppresses apoptosis in rheumatoid arthritis fibroblast-like synoviocytes by targeting IGF-1

Accumulating evidence suggests that miR-483−3p is implicated in maintaining biological properties in human cancers. However, its biological roles in rheumatoid arthritis (RA) remain unknown. miR-483−3p levels in synovial tissue samples and fibroblast-like synoviocytes (FLSs) were determined using quantitative real-time PCR. The CCK-8 assay and EdU staining were performed to assess cell proliferation in RA FLSs after transfection with miR-483−3p mimics or inhibitor. Flow cytometry with Annexin V-FITC staining or PI staining was performed to assess apoptosis or cell cycle progression in RA FLSs, respectively. miR-483−3p was upregulated in RA, which markedly promoted cell proliferation, induced the G0/G1-to-S phase transition, and suppressed apoptosis in RA FLSs, whereas miR-483−3p silencing yielded opposite results. Moreover, insulin growth factor 1 (IGF-1) was detected as a direct miR-483−3p target. IGF-1 silencing partially restored cell proliferation, the G0/G1-to-S phase transition, and apoptosis suppression in RA FLSs via miR-483−3p inhibition. Our results showed that miR-483−3p promotes RA FLSs proliferation by targeting IGF-1, suggesting a potential strategy for diagnostic and treatment strategy for RA.

Dextran stabilized fullerene soot induced toxicity on alveolar epithelial cells (A549 cells)

Fullerene comprises the major allotrope of carbon holding several fruitful potentials to be applied in various industrial and biomedical scenarios. Scientists have acquired large number of data on fullerene research using its derivatives like C60, C70 etc. Nevertheless, a precise focus on fullerene soot nanopaticles and its toxic impacts in living tissue is still behind mainstay even if it represents the crude parent form of all other derivatives. Present study addresses an acute toxicity profiling of fullerene soot nanoparticles in alveolar epithelial cells (A549) as a paradigm of pulmonary exposure. Surface functionalization was given for fullerene soot nanoparticles using dextran polymer as a mean to establish a stable homogenous dispersion (denoted as dFSNPs hereafter). Following functionalization, dFSNPs were characterized for various parameters including size, surface charge, morphology and functional groups using DLS, Zeta potential analysis, TEM and FT-IR measurements respectively. Effective dextran functionalization was evident from the characteristic peaks in FTIR spectra. Cell viability assessed using MTT and NRU assays; both of which showed a dose dependent cytotoxic response. Thymidine incorporation also confirmed similar trend in viability rate. In accordance with literatures, DCFHDA assay confirmed free radical scavenging activity of fullerene nanoparticles. An altered cellular morphology was observed under fluorescent microscope. Sub-cellular functionalities including lysosomal integrity and mitochondrial stability were found to be compromised at highest tested concentration of dFSNPs (160 μg/ml) without any genotoxic impacts within nuclear premises. FACS analysis following Annexin-PI staining confirmed apoptotic cell death. Hence the overall study substantiated dose dependent toxicity of dFSNPs which is likely to occur during pulmonary exposure.

LncRNA RMST-mediated miR-107 transcription promotes OGD-induced neuronal apoptosis via interacting with hnRNPK

The long noncoding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (RMST) silencing has been demonstrated to protect against ischemic brain injury in vivo and neuron injury in vitro. However, its underlying mechanisms in the progression of ischemic stroke have not been well explored. The expression of RMST in oxygen-glucose deprivation (OGD)-treated HT-22 hippocampal neuron cell line was examined using quantitative Real-Time PCR (qRT-PCR). CCK-8 cell viability and apoptotic cell detection using Annexin V-FITC and PI staining coupled with flow cytometry were performed to determine the pro-apoptotic role of RMST in HT-22 hippocampal neuron cell line. Furthermore, RNA pull-down, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) and dual-Luciferase reporter assays were performed to determine the mechanism of RMST in OGD-induced HT-22 cell apoptosis. In the results, RMST was highly expressed in OGD-treated HT-22 cells. Altered RMST expression led to marked changes in HT-22 cell proliferation and apoptosis. Mechanistically, RMST indirectly activated p53/miR-107 signaling pathway via interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and fulfilled its pro-apoptotic function in HT-22 cells. In conclusion, our data indicated that the RMST/hnRNPK/p53/miR-107/Bcl2l2 axis plays an important role in regulating neuronal apoptosis.

Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

76P - Proton-sensitizing effect of small molecule inhibitor of P300 histone acetyltransferase C646 in human pancreatic cancer cells

BackgroundHistone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Recent studies have demonstrated that several chromatin-regulating proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. Even though histone acetylation is one of the key mechanisms of epigenetic regulation, relatively little is known about the cancer therapeutic potential of HAT inhibitors. This is in stark contrast to the well-studied effects of HDAC inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase in human pancreatic cancer cells. MethodsCell viability assay (CCK-8; Dojindo), Clonogenic survival assay, Cell cycle analysis (PI staining). Annexin V-FITC and PI staining, γ-H2AX foci, Western blot, BxPC-3 Xenograft tumor model, TUNEL assay. ResultsAsPC-1, BxPC-3 and Mia-paca2 cells exhibited increase in radiosensitivity when exposed to C646 at all doses of proton beam tested. C646 pre-treatment induce led to increase of sub-G1 population and abolishment of G2/M arrest. Flow cytometry analysis with annexin V staining and Western blot analysis showed that The pre-treatment with C646 significantly enhanced proton-induced apoptotic cell death through the down-regulation of anti-apoptotic molecules. Our in vivo results demonstrate synergistic effects of combination therapy with C646 and proton beam. In BxPC-3 xenograft tumor model, C646 pre-treatment increased proton-induced tumor growth inhibition and apoptotic cell death in tumor tissues. In addition, the combination treatment of C646 with proton beam increased DNA damage and decreased activation of DNA repair pathway compared with proton alone. ConclusionsOur results suggest that the C646 may increase proton- induced apoptosis through the modulation of various pro- and anti- apoptotic molecules in human pancreatic cancer cells. These results provide proof of principle that the inhibition of histone acetylation by HATis can be exploited to develop new proton-sensitizer. Legal entity responsible for the studyThe authors. FundingNational Research Foundation of Korea (NRF). DisclosureAll authors have declared no conflicts of interest.

Abstract 2052: A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines

Abstract Introduction: The third leading cause of cancer-related deaths in the United States is pancreatic cancer, &amp;gt;95% of which is Pancreatic Ductal Adenocarcinoma or PDAC. MUC1 is a transmembrane glycoprotein expressed on normal glandular epithelial cells. In PDAC, MUC1 is overexpressed and aberrantly glycosylated and is designated tumor associated MUC1 (tMUC1). Over 80% of human PDAC expresses tMUC1. In a NCI initiated study, out of 75 tumor antigens, MUC1 was ranked the second most targetable antigen. Furthermore, the 72 amino acid cytoplasmic tail of MUC1 (MUC1 CT) is reported to aid in oncogenic signaling leading to tumor progression and metastasis by blocking apoptosis. A novel monoclonal antibody, TAB004, has been developed specifically against tMUC1. TAB004 detects tMUC1 with a high rate of specificity and sensitivity and spares recognition of normal epithelial MUC1. HYPOTHESIS: Treatment with TAB004 curbs PDAC cell survival by blocking MUC1 CT associated oncogenic signaling and renders the cells more susceptible to standard chemotherapy drugs. Methods: Several human PDAC cell lines based on their tMUC1 expression were grown in media with heat-inactivated serum to ensure that it is devoid of complement proteins. Cells were treated with various concentrations of TAB004 antibody, control IgG, 5-FU, paclitaxel, or gemcitabine and the IC50 was determined using the MTT or SRB cell survival assays. Once IC50 was determined, cells were treated with combinations of TAB004 and the chemotherapy drugs. To determine apoptosis, treated cells were stained with Annexin V-FITC and PI, and analyzed by flow cytometer. tMUC1 expression and localization was determined by staining cells with TAB004.Cy5.5 and imaged by confocal microscopy. Data analysis was performed using GraphPad Prism 7.0 and a p value of &amp;lt;0.05 was considered significant. Results: By itself, TAB004 treatment had minimal effect on most of the cell lines tested except for Capan2. The IC50 of TAB004 was ~400ugs/ml for Capan2. However, when combined with Gemcitabine, PTX, or 5-FU, TAB004 significantly increased anti-tumor efficacy of the drugs, especially 5-FU in Capan2, and 5-FU and gemcitabine, and PTX in BxPC3.MUC1. Annexin V-FITC and PI staining confirmed the MTT data in Capan2 cells. Fluorescent images showed that TAB004 internalizes in MUC1-positive PDAC cell lines but not in MUC1-negative cells. Conclusions: TAB004 has a dose-dependent, complement-independent growth-inhibitory effect on Capan2 cells (IC50 is ~400ug/ml). TAB 004 internalizes and activates apoptosis in Capan2 cells. The combination of TAB004 with chemotherapy drugs increases the efficacy of drug-induced killing of PDAC cells. Future studies will determine the mechanism of TAB004 induced apoptosis and sensitization of PDAC cells to drug-induced killing. Citation Format: Mukulika Bose, Pinku Mukherjee. A novel antibody blocks anti-apoptotic activity of MUC1 in pancreatic cancer cell lines [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 2052.

Protective effect of gallic acid on apoptosis of sperm and in vitro fertilization in adult male mice treated with cyclophosphamide.

Alteration of free radicals (reactive oxygen species) causes mammals' sperm damage. Gallic acid (GA) is known as an antioxidant which is effective against oxidative stress. The purpose of this study was to evaluate the antioxidant effects of GA on the sperm apoptosis and in vitro fertilization (IVF) in adult male mice treated with cyclophosphamide (CP). Following a pilot study to find the dose responses of GA, 40 adult male naval medical research institute (NMRI) mice (32 ± 3 g) were divided into five groups (n = 8): control, sham (normal saline, NS: 0.2 mL per day), CP (15 mg kg-1 per week; intraperitoneal, IP), GA (12.5 mg kg -1 per day; IP), and GA+CP. After the treatment, sperm parameters were analyzed. The apoptosis of sperm was measured by Annexin-PI staining method followed by flow cytometry detection. Fertility was assessed by IVF method among the groups. The difference in sperm parameter and fertility rate between the control (% 80.05 ± 6.53) and cyclophosphomide groups (% 51.82 ± 10.78) was significant (P < .001) but GA plus CP (% 78.16 ± 5.71) restored the fertilization rate (P < .001). Also, a remarkable increase was noted regarding apoptotic sperm in CP group vs the control group. The comparison in the five groups shows that GA cotreatment was significantly effective in reducing the apoptosis rate caused by cyclophosphamide (P < .05). It was ultimately attained that GA has a potent antioxidant effect which could inhibit the detrimental effect of CP on the apoptosis and fertility rate of sperm in the mouse.