Degree Of Cell Death Research Articles

Evaluation of Tolerance and Selection of Heat-Tolerant Woody Plants against Heat Stress

Heat-tolerant species have become increasingly important because of climate change; however, a selection system for woody plants is not well established. This study was conducted to establish a selection system for heat-tolerant woody plant species and to select heat-tolerant species. After selecting heat-tolerant woody plants and applying heat stress to 27 species, the electrolyte leakage index (ELI) was measured. The ELI of the heat-tolerant species was lower than that of the non heat-tolerant species, and they survived well after heat stress treatment. For the preselected species, the degree of cell death was measured by Evans blue staining method, and the heat stress recovery ability was measured by a 3,3′-diaminobenzidine (DAB) staining method. The species showed less cell death even after heat treatment, and oxidative stress was low after recovery from heat stress. Traditional screening methods are mainly performed through field screening; however, this is difficult because it requires many samples and considerable time. The results of this study are relatively rapid, reproducible, and highly sensitive, so it is judged to be a method that can complement the existing traditional method as a heat-tolerant plant selection system. The results of this study can be widely used for the selection and breeding of heat-tolerant plants in preparation for climate change.

A Nondestructive, Real-Time Annexin V Apoptosis Assay.

This chapter describes a simple, nondestructive, annexin V apoptosis detection method that can be employed in real time over a 48-h test exposure. The real-time functionality allows for temporal resolution of apoptotic and cell death responses during the test exposure and obviates the need for onerous sample preparation and time course protocols associated with other annexin V methods. Further, this technique is eminently accessible to a wide range of laboratories because it does not require flow cytometry or other cytometric methods. It was developed for use with a variety of microplate well densities and with standard multimodal plate readers. The central feature of this assay is that it continuously reports the residency status of phosphatidylserine (PS) on the exofacial surface of a cell as it is translocated from the inner membrane leaflet during the apoptotic process. This homogenous, no-wash assay is made possible by two optimized and distinct annexin V fusion proteins which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During an apoptotic event, the luminescent signal arising from an assay well is proportional to the number of cells with PS exposure, and fluorescence intensity correlates with the degree of cell death (secondary necrosis). Conversely, untreated cells contribute negligible luminescent or fluorescent signals throughout the time course. The data collected from these assay measures provide for both standard potency determinations and kinetic characterization of dose- and agent-dependent apoptotic responses, from early through late phases.

6-Gingerol and Semisynthetic 6-Gingerdione Counteract Oxidative Stress Induced by ROS in Zebrafish.

6-Gingerol (1) is one of the major components in ginger and developing new synthetic methodologies could bring semisynthetic analogs with improved therapeutic properties. Towards this, multigram scale isolation of 6-gingerol with excellent purity was optimized using a simple and robust extraction, followed by column purification. Synthesis of 6-gingerdione, 7 from 6-gingerol was then achieved through selective -OTBDMS protection, DMP oxidation and deprotection reaction sequence for the first time. Compounds 1, 7 and 8 (dehydrozingerone) exhibited excellent cell-free antioxidant properties in DPPH, ABTS, superoxide radical scavenging assay and H2 O2 assay at 10-50 μM concentrations. The hemolytic study suggests that up to 50 μM, all three compounds did not exhibit toxicity to human erythrocytes. When H2 O2 treated zebrafish larvae groups (96hpf) were exposed to compounds 1, 7 and 8, it increases the SOD (19, 19.1 and 18.7 U/mg protein), CAT (18.1, 16.5, and 15.8 μmol/mg levels and decreases the lipid peroxidation level (13, 15 and 18 nmol/mg protein), respectively. In vivo ROS levels and degree of cell death were studied using DCFDA and Acridine orange assays. Compounds 1, 7 and 8 decreases the ROS and cell death level significantly. Taken together, compounds 1, 7 and 8 exhibit excellent antioxidant properties, counteract H2 O2 induced oxidative stress, reduces cell death in zebrafish larvae.

294-OR: RIPK3-Mediated Necroptosis Is an Alternative Form of TNFa-Induced ß-Cell Death

Necroptosis is an inflammatory and immunogenic form of lytic cell death that can occur downstream of TNFα signaling. Receptor interacting protein kinase 3 (RIPK3) mediates TNFα-induced necroptosis when caspase activity is repressed, but this pathway has not been examined in insulin-producing β-cells. As TNFα contributes to β-cell death in the setting of diabetes and since we found RIPK3 is expressed in INS-1 β-cells and mouse islets, we hypothesized that β-cells are susceptible to necroptosis. To test this hypothesis, INS-1 cells were treated with TNFα (40 ng/ml) in the absence or presence of a synthetic pan-caspase inhibitor (zVAD, 50 μM) for 24 h, cell death was monitored in real-time, and caspase activity was determined. TNFα increased INS-1 cell death (1.63 ± 0.13 vs. 2.44 ± 0.13 fold, n=7, p<0.001) and caspase activity, consistent with apoptosis. When caspases were inhibited with zVAD, TNFα no longer increased caspase activity, but induced a similar degree of cell death (1.63 ± .013 vs. 2.82 ± 0.23 fold, n=7, p<0.001), suggesting an alternate mechanism under these conditions. Moreover, when TNFα-induced cell death was amplified by the SMAC/DIABLO mimetic BV6 (5 μM), we again observed similar levels of cell death with or without zVAD. To further investigate the mechanism of cell death under caspase inhibition, we used the RIPK3 inhibitor GSK ‘872 (5 μM). GSK ‘872 had no effect on TNFα+BV6-induced cell death when caspases were activated (3.35 ± 0.61 vs. 3.48 ± 1.21 fold, n=3, p=0.93), but reduced cell death when caspases were inhibited (4.69 ± 0.57 vs. 1.81 ± 0.57 fold, n=3, p=0.04). These data indicate that TNFα induces β-cell death independent of caspase activity level. However, the mechanism by which TNFα elicits β-cell death does depend on caspase activity, with apoptosis occurring when caspases are activated, and RIPK3-mediated necroptosis occurring when caspases are inhibited. Additional studies are required to understand the factors that may promote β-cell necroptosis in the setting of human diabetes. Disclosure C. J. Contreras: None. L. Lin: None. M. F. Hogan: None. A. Oberst: None. S. E. Kahn: Advisory Panel; Self; Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk, Pfizer Inc. A. T. Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659, I01BX001060)

Investigating the Effect of Substrate Stiffness on the Redox State of Cardiac Fibroblasts Using Scanning Electrochemical Microscopy.

Cardiac fibrosis, in which cardiac fibroblasts differentiate into myofibroblasts, leads to oversecretion of the extracellular matrix, results in increased stiffness, and facilitates disequilibrium of cellular redox state, further leading to oxidative stress and various degrees of cell death. However, the relationship between the matrix stiffness and the redox status of cardiac fibroblasts remains unclear. In this work, we constructed an in vitro cardiac fibrosis model by culturing cardiac fibroblasts on polyacrylamide gels with tunable stiffness and characterized the differentiation of cardiac fibroblasts to myofibroblasts by immunofluorescence staining of α-smooth muscle actin. We then applied scanning electrochemical microscopy (SECM) with a depth scan mode to in situ and quantitatively assess the redox status by monitoring the glutathione (GSH) efflux rate (k) through the redox reaction between GSH (a typical indicator of cellular redox level) released from cardiac fibroblasts and SECM probe-oxidized ferrocenecarboxylic acid ([FcCOOH]+). The SECM results demonstrate that the GSH efflux from the cardiac fibroblasts decreased with increasing substrate stiffness (i.e., mimicking the increased fibrosis degree), indicating that a more oxidizing microenvironment facilitates the cell differentiation and GSH may serve as a biomarker to predict the degree of cardiac fibrosis. This work provides an SECM approach to quantify the redox state of cardiac fibroblasts by recording the GSH efflux rate. In addition, the newly established relationship between the redox balance and the substrate stiffness would help to better understand the redox state of cardiac fibroblasts during cardiac fibrosis.

Aberrant expression of cell-free nucleosomes in dermatomyositis/polymyositis.

Nucleosomes are typically located intracellularly, and extracellular levels of nucleosomes indicated the degree of cell death. We postulated aberrant nucleosomes expression in dermatomyositis (DM) and polymyositis (PM). To assess the aberrant expression of circulating nucleosomes in DM and polymyositis. 76 consecutive DM and PM patients from 1 September 2017 to 31 October 2018 were enrolled in this study, in addition to 20 healthy controls. The levels of circulating nucleosomes, with 16 kinds of detectable myositis-specific antibodies (MSAs) were detected in IIMs patients using enzyme-linked immunosorbent assay kit (ELISA). Receiver operating characteristic (ROC) curve analysis was performed for evaluating the ability of this candidate marker for detecting DM/PM. The results showed that the levels of circulating nucleosomes in DM/PM patients were significantly higher than that in normal individuals. Specifically, elevated levels of nucleosomes were associated with MDA5 Ab, ARS, and TIF1γAb. In addition, elevated levels of circulating nucleosomes correlated with skin eruption. This study has evidenced nucleosomes as a potential new biomarker of DM/PM. Aberrant nucleosomes expression occurs in myositis patients and the difference in nucleosomes expression between patients with and without skin eruption is statistically significant. A unique expression profile of elevated serum nucleosomes was detected in DM/PM-MDA5 Ab(+), ASS, and TIF1γAb(+).

A ruthenium(II)-curcumin compound modulates NRF2 expression balancing the cancer cell death/survival outcome according to p53 status

BackgroundTumor progression and tumor response to anticancer therapies may be affected by activation of oncogenic pathways such as the antioxidant one induced by NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor and the pathways modified by deregulation of oncosuppressor p53. Often, oncogenic pathways may crosstalk between them increasing tumor progression and resistance to anticancer therapies. Therefore, understanding that interplay is critical to improve cancer cell response to therapies. In this study we aimed at evaluating NRF2 and p53 in several cancer cell lines carrying different endogenous p53 status, using a novel curcumin compound since curcumin has been shown to target both NRF2 and p53 and have anti-tumor activity.MethodsWe performed biochemical and molecular studies by using pharmacologic of genetic inhibition of NRF2 to evaluate the effect of curcumin compound in cancer cell lines of different tumor types bearing wild-type (wt) p53, mutant (mut) p53 or p53 null status.ResultsWe found that the curcumin compound induced a certain degree of cell death in all tested cancer cell lines, independently of the p53 status. At molecular level, the curcumin compound induced NRF2 activation, mutp53 degradation and/or wtp53 activation. Pharmacologic or genetic NRF2 inhibition further increased the curcumin-induced cell death in both mutp53- and wtp53-carrying cancer cell lines while it did not increase cell death in p53 null cells, suggesting a cytoprotective role for NRF2 and a critical role for functional p53 to achieve an efficient cancer cell response to therapy.ConclusionsThese findings underline the prosurvival role of curcumin-induced NRF2 expression in cancer cells even when cells underwent mutp53 downregulation and/or wtp53 activation. Thus, NRF2 inhibition increased cell demise particularly in cancer cells carrying p53 either wild-type or mutant suggesting that p53 is crucial for efficient cancer cell death. These results may represent a paradigm for better understanding the cancer cell response to therapies in order to design more efficient combined anticancer therapies targeting both NRF2 and p53.

Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication

Cisplatin is a very effective chemotherapeutic, but severe and permanent hearing loss remains a prevalent side effect. The processes underpinning cisplatin-induced ototoxicity are not well understood. Gap junction channels composed of connexin (Cx) subunits allow for the passage of small molecules and ions between contacting neighboring cells. These specialized channels have been postulated to enhance cisplatin-induced cell death by spreading “death signals” throughout the supporting cells of the organ of Corti. This study sought to investigate the role of Cx43 in cisplatin-induced ototoxicity using organotypic cochlear cultures from control and two Cx43-mutant mouse strains harboring either a moderate (Cx43I130T/+) or severe (Cx43G60S/+) reduction of Cx43 function. Cochlear cultures from Cx43-mutant mice with a severe reduction in Cx43-based gap junctional intercellular communication (GJIC) had an enhanced number of hair cells that were positive for cleaved caspase 3, a marker of active apoptosis, after cisplatin treatment. In cisplatin-treated organotypic cochlear cultures, there was a decrease in the co-localization of Cx26 and Cx30 compared with untreated cultures, suggesting that cisplatin causes reorganization of connexin composition in supporting cells. Both Cx26 and Cx30 protein expression as well as GJIC were decreased in organotypic cochlear cultures treated with the gap-junction blocker carbenoxolone. When cisplatin and carbenoxolone were co-administered, there were no differences in hair cell loss compared with cisplatin treatment alone. Using cisplatin-treated control and Cx43-ablated organ of Corti derived HEI-OC1 mouse cells, we found that greatly reducing GJIC led to preferential induction of an ER stress pathway. Taken together, this study strongly suggests that inhibition of GJIC in organ of Corti cells does not lead to differential susceptibility to cisplatin-induced ototoxicity. Although cisplatin causes the same degree of cell death in gap junction competent and incompetent cochlear cells, the engagement of the mitochondrial dysregulation and ER stress differs.

Association of the Polymorphism rs13259960 in SLEAR With Predisposition to Systemic Lupus Erythematosus.

Genome-wide association studies have identified many susceptibility loci for systemic lupus erythematosus (SLE). However, most of these loci are located in noncoding regions of the genome. Long noncoding RNAs (lncRNAs) are pervasively expressed and have been reported to be involved in various diseases. This study aimed to explore the genetic significance of lncRNAs in SLE. A genome-wide survey of SLE risk variants in lncRNA gene loci was performed in Han Chinese subjects (4,556 with SLE and 9,451 healthy controls). The functional relevance of an SLE risk variant in one of the lncRNA genes was explored using biochemical and molecular cell biology analyses. In vitro loss-of-function and gain-of-function strategies were used to clarify the functional and phenotypic relevance of this SLE susceptibility lncRNA. Moreover, correlation of this lncRNA with the degree of apoptosis in the peripheral blood of SLE patients was evaluated. A novel SLE susceptibility locus in a lncRNA gene, designated SLEAR (for SLE-associated RNA), was identified at the single-nucleotide polymorphism rs13259960 (odds ratio 1.35, Pcombined = 1.03 × 10-11 ). The A>G variation at rs13259960, located in an intronic enhancer, was found to impair STAT1 recruitment to the enhancer that loops to the SLEAR promoter, resulting in decreased SLEAR production in peripheral blood mononuclear cells from patients with SLE (3 with the G/G genotype, 22 with A/G, and 103 with A/A at rs13259960; P = 0.0241). Moreover, SLEAR interacted with the RNA binding proteins interleukin enhancer binding factor 2, heterogeneous nuclear RNP F, and TATA-binding protein-associated factor 15, to form a complex for transcriptional activation of the downstream antiapoptotic genes. In addition, SLEAR regulated apoptosis of Jurkat cells in vitro, and its expression level was correlated with the degree of cell death in the peripheral blood of patients with SLE (r = 0.824, P = 2.15 × 10-8 ; n = 30). These findings suggest a mechanism by which the risk variant at rs13259960 modulates SLEAR expression and confers a predisposition to SLE. Taken together, these results may give insights into the etiology of SLE.

Different gene expression profiles in iPSC-derived motor neurons from ALS8 patients with variable clinical courses suggest mitigating pathways for neurodegeneration.

Amyotrophic lateral sclerosis type 8 (ALS8) is an autosomal dominant form of ALS, which is caused by pathogenic variants in the VAPB gene. Here we investigated five ALS8 patients, classified as 'severe' and 'mild' from a gigantic Brazilian kindred, carrying the same VAPB mutation but displaying different clinical courses. Copy number variation and whole exome sequencing analyses in such individuals ruled out previously described genetic modifiers of pathogenicity. After deriving induced pluripotent stem cells (iPSCs) for each patient (N = 5) and controls (N = 3), motor neurons were differentiated, and high-throughput RNA-Seq gene expression measurements were performed. Functional cell death and oxidative metabolism assays were also carried out in patients' iPSC-derived motor neurons. The degree of cell death and mitochondrial oxidative metabolism were similar in iPSC-derived motor neurons from mild patients and controls and were distinct from those of severe patients. Similar findings were obtained when RNA-Seq from such cells was performed. Overall, 43 genes were upregulated and 66 downregulated in the two mild ALS8 patients when compared with severe ALS8 individuals and controls. Interestingly, significantly enriched pathways found among differentially expressed genes, such as protein translation and protein targeting to the endoplasmic reticulum (ER), are known to be associated with neurodegenerative processes. Taken together, the mitigating mechanisms here presented appear to maintain motor neuron survival by keeping translational activity and protein targeting to the ER in such cells. As ALS8 physiopathology has been associated with proteostasis mechanisms in ER-mitochondria contact sites, such differentially expressed genes appear to relate to the bypass of VAPB deficiency.

Amifostine Prophylaxis in Irradiated Breast Reconstruction: A Study of Oncologic Safety In Vitro.

Indications for adjuvant radiation therapy (XRT) in breast cancer have expanded. Although highly effective, XRT damages surrounding tissues and vasculature, often resulting in delayed or compromised breast reconstruction. Thus, effective yet safe methods of radiation injury prophylaxis would be desirable. Amifostine is a Food and Drug Administration-approved radioprotectant; however, concerns about its potential to also protect cancer remain. The purpose of this study was to evaluate the oncologic safety of amifostine (AMF) in vitro and determine its effect on human breast cancer cells in the setting of XRT. One ER+/PR+/Her2- (MCF-7) and two ER-/PR-Her2- (MDA-MB-231, MDA-MB-468) breast cancer cell lines were investigated. Female fibroblasts were used as controls. Cells were treated with WR-1065, the active metabolite of AMF, 20 minutes before 0Gy, 10Gy, or 20Gy XRT. Live and dead cells were quantified; percent cell death was calculated. WR-1065 treatment significantly preserved viability and reduced healthy female fibroblasts death after XRT compared with untreated controls. All three breast cancer cells lines exhibited radiosensitivity with substantial cell death. Cancer cells retained their radiosensitivity despite WR-1065 pretreatment, achieving the same degree of cell death as untreated controls. This study demonstrated the proficiency of AMF to selectively protect healthy cells from XRT while breast cancer cells remained radiosensitive. These results support the oncologic safety of AMF in breast cancer in vitro. Further investigation is now warranted in vivo to ascertain the translational potential of using AMF as a radioprotectant to improve breast reconstruction after radiation treatment.

Abstract GMM-051: A CLIA-CERTIFIED HIGH-THROUGHPUT DRUG SCREENING PLATFORM FOR OVARIAN CANCER TO INFORM PERSONALIZED CANCER CARE AND DISCOVER NOVEL COMBINATIONS

Abstract BACKGROUND: Metastatic disease in ovarian cancer is difficult to treat and patients often exhaust standard-of-care regimens. To gain a better understanding of potential treatments, genomic data can be used. However, the interpretation of the genomic data for ovarian cancer is challenging given complex mutation patterns and the absence of recurrent druggable alterations beyond DNA repair deficiency. Here we present the first CLIA certified high-throughput functional assay employing organoid cultures derived from primary patient specimens to directly aid oncologists for personalized treatment selection in combination with genomic data (P.A.R.I.S® Assay, SEngine Precision Medicine, Seattle, WA). EXPERIMENTAL PROCEDURES: Organoids are exposed to a library of 123 clinically relevant drugs. The library was developed to include chemotherapies and FDA approved drugs currently used in the clinic, as well as promising drugs undergoing clinical trials. Compounds are evaluated at a multi-dose response curve and ranked by SPM™ score, which weighs both the sensitivity (degree of cell death) and specificity, which compares the response of the patient's tumor cells to the drug relative to all prior patients. In addition to single agent screens, optimal combinations can also be tested. The results are further integrated with genomic data when available, and drug-responses are reported to the clinician. SEngine has performed >200 drug screens across >20 different tumor types and established high reproducibility of the high-throughput platform. Ovarian samples were derived from either ascites or biopsies. RESULTS: Of the 7 samples directly derived from patients, 1 was from ascites, 4 were from biopsies or surgical excisions, and 2 had both. In addition, 13 cell lines were screened with the SEngine drug library. While every patient exhibited a unique pattern of response consistent with the heterogeneity of the complex genetic landscape of ovarian cancer, sensitivities for certain drugs such as HDAC, PI3K and tyrosine-kinase inhibitors were frequently found. Several n=1 cases will be presented to highlight correlations with retrospective clinical responses as well as with genomic alterations. These results highlight how genomic data and functional testing can be combined to optimize personalized cancer care. To identify optimal drug combinations with PARP inhibitors, we have performed an unbiased screen where patient derived ovarian cancer cell lines were challenged with SEngine drug library in the presence of rucaparib. The screen indicated multiple drugs, such as bromodomain, BCL2 and cyclin dependent kinase (CDK) inhibitors as well as dasatinib as potential sensitizers to rucaparib. Confirmation of these results in additional patient derived organoids and PDX models are in progress. IMPACT: We developed a robust ex vivo screening platform to objectively quantify patient specific sensitivity to a panel of more than 123 oncology drugs and potential novel combinations. SEngine is compiling a registry capturing clinical data, outcome following the P.A.R.I.S® test, and genomic data. Combining the power of high-throughput technology and organoid isolation with genomic data will enable the rapid selection of optimal individualized therapies as single agents or in combination and guide design of future clinical trials. Citation Format: Franz X Schaub, Sabrina Maisel, Reid Shaw, Rachele Rosati, Caroline Bridgwater, Michael Churchill, Robert Diaz, Stephanie Tatem Murphy, Shalini Pereira, Christopher Kemp, VK Gadi, and Carla Grandori. A CLIA-CERTIFIED HIGH-THROUGHPUT DRUG SCREENING PLATFORM FOR OVARIAN CANCER TO INFORM PERSONALIZED CANCER CARE AND DISCOVER NOVEL COMBINATIONS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-051.

The effects of oxygen concentration on cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in precision-cut lung slices

Although animal models are often used in drug research, alternative experimental models are becoming more popular as they reduce animal use and suffering. Of particular interest are precision-cut lung slices, which refer to explants – with a reproducible thickness and diameter – that can be cultured ex vivo. Because lung slices (partially) reflect functional and structural features of whole tissue, they are often applied in the field of immunology, pharmacology, toxicology, and virology. Nevertheless, previous research failed to adequately address concerns with respect to the viability of lung slices. For instance, the effect of oxygen concentration on lung slice viability has never been thoroughly investigated. Therefore, the main goal of this study was to investigate the effect of oxygen concentration (20 vs. 80% O2) on the degree of cell death, anti-oxidant transcription, acute inflammation, and cell proliferation in lung slices. According to the results, slices incubated at 20% O2 displayed less cell death, anti-oxidant transcription, and acute inflammation, as well as more cell proliferation, demonstrating that these slices were considerably more viable than slices cultured at 80% O2. These findings expand our knowledge on lung slices and their use as an alternative experimental model in drug research.

ABCB7 is a gatekeeper of both programmed cell death, apoptotic and non-apoptotic cell death

Abstract Background The ATP-binding cassette (ABC) transporters are superfamily proteins of cellular translocation machinery. Many ABC transporters release drugs to give multidrug resistance. However, ABC transporters existing in intracellular compartments give drug resistance by regulating biological processes, rather than direct release of drugs. The current study aimed to investigate the molecular mechanism that causes the drug resistance of the mitochondrial ABC transporters in cancer cells. Methods RNA-sequencing and bioinformatics analyses were performed to identify key mitochondrial ABC transporters and ABC transporter subfamily B member 7 (ABCB7)-mediated transcriptomic alterations. We investigated role of ABCB7 in iron homeostasis by examining iron concentration and reactive oxygen species (ROS) generation. Drug resistance and cellular survival mechanisms were understood by comparing the type and degree of cell death according to the ABCB7 expression. Results ABCB7 is highly expressed in patients with glioblastoma and has a negative correlation with prognosis. The increase in ABCB7 gives resistance to drug and the loss of ABCB7 leads to cell death. ABCB7 increases the expression of hypoxia-inducible factor 1 alpha (HIF1α) and activates HIF1α signaling by regulating iron concentration. When ABCB7 expression is maintained high in cancer cells, hypoxia-independent activation of HIF1α inhibits cell death by suppressing leucine zipper downregulated in cancer 1 (LDOC1). However, when ABCB7 is decreased, non-apoptotic cell death is induced by the change of iron concentration, and apoptosis which is inhibited by HIF1α signal is also induced. Additionally, because ABCB7 is essential for survival of universal cancer cells, it is presented as a pan-cancer core fitness gene. Conclusions Our findings demonstrate the ABCB7 inhibits both apoptotic and non-apoptotic cell death via maintenance of iron homeostasis and hypoxia-independent HIF1α signaling. Editorial acknowledgement Cancer Growth Regulation Laboratory for their discussion and technical assistance. Legal entity responsible for the study The authors. Funding National Research Foundation of Korea (NRF) [grant numbers: 2015R1A5A1009024 and 2017R1E1A1A01074205], the School of Life Sciences and Biotechnology for BK21 Plus, Korea University and Institute of Animal Molecular Biotechnology Korea University. Disclosure All authors have declared no conflicts of interest.

Reactive oxygen species are involved in cell death in wheat roots against powdery mildew

Inoculation of wheat (Triticum aestivum L.) leaves with wheat powdery mildew fungus (Blumeria graminis f. sp. tritici) induces the cell death in adventitious roots. Reactive oxygen species (ROS) play a key role in respond to biotic stress in plants. To study the involvement of ROS and the degree of cell death in the wheat roots following inoculation, ROS levels and microstructure of root cells were analyzed in two wheat cultivars that are susceptible (Huamai 8) and resistant (Shenmai 8) to powdery mildew fungus. At 18 d after powdery mildew fungus inoculation, only Huamai 8 displayed the leaf lesions, while root cell death occurred in both varieties. Huamai 8 had a high level of ROS accumulation, which is associated with increased root cell degradation, while in Shenmai 8, there was little ROS accumulation correlating with slight root cell degradation. The molecular study about the expression levels of ROS scavenging genes (MnSOD and CAT) in wheat roots showed that these genes expression decreased after the leaves of wheat was inoculated. The difference between Huamai 8 and Shenmai 8 on subcellular localization of H2O2 and O2– was corresponded with the different down-regulation of the genes encoding for superoxide dismutase and catalase in two wheat cultivars. These results suggested that ROS were involved in the process by which powdery mildew fungus induced cell death in wheat roots.

Commonly Used Chemotherapy Drugs Differentially Determine Microenvironment-Mediated Protection, Via Mitochondrial Transfer, to B-Precursor Acute Lymphoblastic Leukaemia Cells

Murine models of Acute Lymphoblastic Leukaemia (ALL) suggest relapse arises not from intrinsic chemoresistance by genetically distinct cells, but from a subset of cells protected within a specific niche.To confirm the existence of such a niche in patients with ALL and elucidate the mechanism by which stromal cells (MSC) protect ALL cells we isolated MSC from bone marrow of 70 B-ALL patients enrolled on the UKALL14 trial. Immunostaining for f-actin, gene expression profiling and cytokine/chemokine quantification showed that a significant proportion of bone marrow specimens, especially after treatment with a cytarabine (AraC)-containing block, contained MSC with an activated phenotype, analagous to cancer associated fibroblasts (ALL-CAF).We demonstrated that primary ALL cells, ALL cell lines, AraC and daunorubicin (but not vincristine (VCR) or dexamethasone (dex)) generated ALL-CAF de novo from both healthy donor MSC and the MSC cell line HS27a. Notably, the chemotherapy drugs induced distinct morphological changes and differential alpha-smooth muscle expression (figure 1a).Control of oxidative stress via modification of reactive oxygen species (ROS) presented a potential unifying explanation for ALL-CAF generation by both ALL cells and chemotherapy. Using flow cytometry we demonstrated that AraC significantly increased ROS in the B-ALL cell line, SEM, in monoculture but in co-culture with HS27a, ROS was significantly lowered and was not impacted by AraC. The MSC co-culture-mediated reduction in ROS in co-culture corresponded to a significant reduction in cell death (10.5% vs 36%, p = 0.0001). By contrast, VCR did not impact ROS significantly and Dex reduced it. Both were significantly more effective than AraC at inducing SEM cell death in co-culture (VCR 20.2% vs AraC 10.5%, p = 0.0003; Dex 39.1% vs AraC 10.5%, p = 0.0007), despite inducing the same degree of cell death in monoculture.We hypothesised that mitochondrial transfer between ALL-CAF and B-ALL cells could provide a generalised mechanism to overcome the deleterious impact of cell-intrinsic and chemotherapy-driven ROS in B-ALL cells. A ‘mitotracker’ flow cytometry assay showed differential mitochondrial transfer from HS27a to B-ALL cells, in proportion to the baseline ROS levels. We confirmed that mitochondria could also be transferred from healthy donor MSC co-cultured with primary patient ALL cells. Furthermore, AraC, but not VCR or Dex, significantly enhanced mitochondrial transfer, and did so in a dose-dependent manner. To rule out passive transfer of dye, we used the murine stromal line MS5 as an alternative mitochondria donor to SEM cells. Murine mitochondrial, but not nuclear, DNA was clearly seen in flow-sorted SEM cells after co-culture with MS5, at baseline and at higher levels after AraC therapy. We also directly visualised the transfer of mitochondria along tunnelling nanotubes (TNT) by time-lapse confocal imaging (figure 1b). To confirm that mitochondrial transfer was essential in MSC ‘rescue’ of ALL cells, we generated HS27a cells deficient in mitochondria following prolonged culture with low dose ethidium bromide. The mitochondrially-deficient cells retained viability as well as the ability to become ALL-CAF but were clearly defective in their ability to rescue SEM ALL cells from AraC induced cell death.To confirm the functional impact of mitochondrial transfer via TNT, we used actin inhibitor latrunculin B (LatB) and the microtubule damaging agent nocodazole which both significantly blocked the phenomenon. Both LatB and nocodazole significantly restored AraC-related cytotoxicity. Colchicine, another microtubule damaging agent had a similar impact to nocodazole. VCR completely overcame the protective impact of HS27a on AraC cytotoxicity and was additive with AraC in the co-culture system.We have shown that CAF-like MSC provide support to ALL cells under oxidative stress by mitochondrial transfer via TNT. This is disrupted by microtubule damaging agents and conditions provoking their formation are mitigated by Dex, both mainstays of ALL therapy. Our data may explain the ineffectiveness of ROS-inducing chemotherapy at eradicating disease at the niche and provides an explanation of why low dose, microtubule damaging agents such as VCR used in maintenance therapy are effective in ALL. Our findings have immediate implications for the design and scheduling of current combination chemotherapies for ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

196 - Metabolic stress promotes sustained activation of protein phosphatase 2A (PP2A) in the islet beta-cell: novel regulation by alpha4, a non-canonical subunit of PP2A

Purpose Despite emerging evidence suggesting requisite roles for protein kinases in islet beta-cell function, protein phosphatases remain an under studied area in islet biology. The PP2A, which accounts for 80% of total serine/threonine phosphatases, has been implicated in many facets of cell regulation including proliferation, survival, and apoptosis. We recently reported hyperactivation of PP2A in islet beta-cells following exposure to high glucose (metabolic stress) conditions, and proposed that hyperactivation of PP2A could lead to dephosphorylation of key proteins necessary for islet function. Herein, we studied putative regulatory roles for alpha4, a non-canonical adaptor regulatory subunit of PP2A, in the sustained activation of PP2A under metabolic stress. Methods Pancreatic islets were isolated by the collagenase digestion method. Human islets were from Prodo Labs. INS-1 832/13 cells, rat and human islets were cultured under basal (2.5 mM glucose) or metabolic stress (20-30 mM glucose) conditions for 24-48 hrs. PP2A activity was determined by immunoprecipitation phosphatase assay. Degree of cell death was detected by Annexin V/Propidium fluorescence staining. Results Metabolic stress significantly increased PP2A activity in INS-1 832/13 cells (~ 3 fold), rodent islets (~ 1.8 fold) and human islets (~ 2.2 fold). Sustained activation of PP2A was also seen in islets derived from pre-diabetic (7 weeks; ~1.4 fold) and diabetic (13 weeks; ~ 2 fold) ZDF rats. Western blot analysis indicated that alpha4 is expressed in INS-1 832/13 cells, rat islets and human islets. Metabolic stress also increased the expression of alpha4 in INS-1 cells and human islets. siRNA-mediated suppression of alpha4 expression markedly (-60%) inhibited high glucose-induced PP2A activity and cell death in INS-1 832/13 cells. Conclusions Alpha4 promotes PP2A activation and dysfunction of pancreatic beta-cells under high glucose-exposure conditions. We conclude that alpha4 could represent a novel target to impede PP2A-derived signaling events leading to beta-cell demise under metabolic stress.

Abstract 1619: Personalized medicine: A CLIA-certified high-throughput drug screening platform for ovarian cancer

Abstract Background: Metastatic disease in ovarian cancer is difficult to treat and patients often exhaust standard-of-care regimens. To get a better understanding of potential treatments, genomic data is used in some cases. However, this only points to therapeutic options in a minority of cases highlighting an urgent need to develop assays to identify potential therapies. Current functional tests are limited in the number of tested drugs. Here we present the first CLIA certified high-throughput functional assay employing organoid cultures derived from primary patient specimens to directly aid oncologists for personalized treatment selection (P.A.R.I.S. Assay, SEngine Precision Medicine, Seattle, WA). Experimental Procedures: Organoids are treated for 6 days with a library of 123 clinically relevant drugs. Compounds are evaluated at a multi-dose response curve and ranked by SPM score, which weights both the sensitivity (degree of cell death) and specificity, which compares the patient's tumor cells response to the drug relative to all prior patients. The results are further integrated with genomic data and reported to the clinician to highlight treatment options. SEngine has performed >150 drug screens and established high reproducibility including multiple ovarian cancer cases from either ascites, surgical or core biopsies. Results: Here we present two n-of-one patient studies. Patient 1 is a 48 year old woman with positive family history who was diagnosed with late stage serous ovarian cancer. SEngine generated cancer organoids and performed high-throughput screening with a panel of 123 drugs. Sensitivities to PARP inhibitors prompted SEngine to advise for germline testing which revealed a BRCA1 and TP53 mutation. The cells were also uniquely sensitive to paclitaxel and the combination with carboplatin resulted in remission indicating concordance with clinical data. In addition, highlighting the importance of functional screening, a unique response to a sub-group of EGFR inhibitors was identified, of potential consideration in case of recurrence. The second case also has a family history of ovarian cancer, but no BRCA mutations were detected. Genomic data indicated FGF6 and FGF23 amplification which directly corresponded to a unique sensitivity to one FGFR inhibitor (AZD-4547). The organoids were also resistant to PARP inhibitors, consistent with the absence of BRCA mutations. Impact: We developed a robust ex vivo screening platform to objectively quantify patient specific sensitivity to a panel of 123 oncology drugs. SEngine is compiling a registry capturing clinical data, outcome following the P.A.R.I.S. test as well as genomic data. The power of high throughput technology and organoid isolation will enable the rapid selection of optimal individualized therapies as single agents or in combination. Citation Format: Hallie A. Swan, Rachele Rosati, Caroline Bridgwater, Michael J. Churchill, Roland M. Watt, Reid C. Shaw, Stephanie A. Murphy, Robert L. Diaz, Shalini C. Pereira, Franz X. Schaub, Carla Grandori. Personalized medicine: A CLIA-certified high-throughput drug screening platform for ovarian cancer [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 1619.

Cold Atmospheric Plasma Selectively Induces Apoptosis In Renal Adenocarcinoma

The application of cold atmospheric plasma (CAP) in the selective treatment of cancer has drawn increasing attention. However, very few studies have focused on the selective effect of CAP towards cancer compared to the corresponding normal cells, among which the results have been mixed. Guerrero-Preston has shown that CAP selectively kills head and neck squamous cell carcinoma. While the normal oral cavity epithelial cells were killed to some degree, the mechanism of selectivity was not reported [1]. Breast cancer has been shown to be selectively killed relative to mesenchymal stem cells (MSCs) [2], however, no selectivity was found when compared to normal breast epithelial cells [3]. CAP induces apoptosis of lung cancer without affecting bronchial epithelial cells, but no apoptotic pathways were identified or proposed [4]. Ishaq et al. proposed NADPH oxidase (Nox2) as the endogenous ROS source which mediates the ROS level in different types of colon cancer and further induces various degrees of cell death, but this proposed pathway was not verified in normal colon cells [5]. We study the effects of helium cold atmospheric plasma using the Canady Helios Cold Plasma Scalpel on established renal adenocarcinoma and renal epithelial cell lines. Our data demonstrates that kidney adenocarcinoma (769-P) is selectively killed compared to renal epithelial cells (HPRCEC). DR4 (TRAIL-R1) expression was found to be significantly increased following CAP treatment, and may contribute to the selective induction of apoptosis. Differences in DR4 expression following CAP treatment may explain the inconsistent responses of various cell lines to CAP.

Aralia elata inhibits neurodegeneration by downregulating O-GlcNAcylation of NF-κB in diabetic mice.

To investigate the role of O-GlcNAcylation of nuclear factor-kappa B (NF-κB) in retinal ganglion cell (RGC) death and analysedthe effect of Aralia elata (AE) on neurodegeneration in diabetic mice. C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control (CTL) diet at the onset of diabetes mellitus (DM). Two months after injection of streptozotocin or saline, the degree of cell death and the expression of O-GlcNAc transferase (OGT), N-acetyl-b-D-glucosaminidase (OGA), O-GlcNAcylated proteins, and O-GlcNAcylation of NF-κB were examined. AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness (P<0.001 vs CTL, P<0.01 vs DM) and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (P<0.001 vs CTL, P<0.0001 vs DM), glial activation, and active caspase-3 (P<0.0001 vs CTL, P<0.0001 vs DM) were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GlcNAcylation and OGT were increased in diabetic retinas (P<0.0001 vs CTL), and the level of O-GlcNAcylation of the NF-κB p65 subunit was higher in diabetic retinas than in controls (P<0.0001 vs CTL). AE extract downregulated O-GlcNAcylation of NF-κB and prevented neurodegeneration induced by hyperglycemia (P<0.0001 vs DM). O-GlcNAcylation of NF-κB is concerned in neuronal degeneration and that AE prevents diabetes-induced RGC apoptosis via downregulation of NF-κB O-GlcNAcylation. Hence, O-GlcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic possibility to prevent diabetes-induced neurodegeneration.