Role of glyoxalase-1 in trabectedin-resistant myxoid liposarcoma.

e23566 Background: Glyoxalase-1 (Glo1) is involved in the detoxification of the endogenous reactive metabolite, methylglyoxal (MG), whose abnormal accumulation increases adduct levels and induce cell apoptosis. Previous studies demonstrated that increased Glo-1 expression was associated with cancer chemotherapy resistance. We performed bioinformatics analyses finding that Glo-1 mRNA over-expression was correlated with worse prognosis in patients affected by Soft Tissue Sarcoma (STS). On the bases of these evidences, we investigated the potential role of Glo1 expression as biomarker of tumor growth and drug resistance in STS. Methods: Trabectedin and MG cytoxicity was evaluated by MTT viability assay measured at spectrofluorometer. Apoptosis was analyzed by flow cytometry using Annexin V antibody and propidium iodide. Glo-1 expression analysis was performed by Western Blot using a mouse monoclonal anti-human Glo-1 antibody (NBP1-19015). Synergy analysis was calculated using Combenefit software and statistical analysis was performed by Student-t test using the program GraphPad-Prism. Results: As STS trabectedin resistant model, we used a myxoid-liposarcoma cell line (402-91 ET cells) that are not responsive to clinical doses of trabectedin contrary to the parental sensitive cell line (402-91 WT cells). Intriguingly, we found higher Glo-1 protein levels in 402-91 ET cells compared to 402-91 WT cells. The treatment of 402-91 ET cells with the specific Glo1 inhibitor S-p-bromobenzylglutathione cyclopentyl diester (BBGC), in combination with trabectedin (PharmaMar), significantly inhibited cell viability and increased apoptosis than trabectedin alone. In particular, the addition of BBGC reduced trabectedin EC50 (half-maximal effective concentration) from 20.4nM to 5.92nM in 402-91 ET cells, similar to that observed in 402-91 WT cells (4.92nM). To investigate if cell death was induced by MG accumulation following Glo1 inhibition, we evaluated 402-91 ET cell viability after treatment with different doses of MG in combination with trabectedin. The addition of MG restored sensitivity to trabectedin in 402-91 ET cells as well as BBGC. Conclusions: Our results highlight a new potential mechanism of trabectedin resistance mediated by Glo-1 over-expression. The use of the specific Glo-1 inhibitor, BBGC, restores trabectedin sensitivity in resistant cells leading to MG accumulation that, in turn, promotes cell death and apoptosis. These data provide a strong rationale to investigate Glo-1 inhibition strategy, in combination with trabectedin, in STS in vivo models.

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Mitochondrion-Dependent Cell Death in TDP-43 Proteinopathies

Biomedicines ◽

10.3390/biomedicines9040376 ◽

2021 ◽

Vol 9 (4) ◽

pp. 376

Author(s):

Chantal B. Lucini ◽

Ralf J. Braun

Keyword(s):

Cell Death ◽

Oxygen Species ◽

In Vivo Models ◽

Dependent Cell ◽

Cell Death Mechanisms ◽

Sting Pathway ◽

Mitochondrial Membrane Permeabilization

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

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Human Drug Efflux Transporter ABCC5 Confers Acquired Resistance to Pemetrexed in Breast Cancer

10.21203/rs.3.rs-105352/v2 ◽

2021 ◽

Author(s):

Jihui Chen ◽

Zhipeng Wang ◽

Shouhong Gao ◽

Kejin Wu ◽

Fang Bai ◽

...

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Cell Lines ◽

Abc Transporters ◽

Control Group ◽

Apoptosis Pathway ◽

Over Expression ◽

Mcf 7

Abstract AimPemetrexed, a new generation antifolate drug, is approved for the treatment for locally advanced or metastatic breast cancer, but factors affecting the efficacy and resistance of it have yet to be fully explicit. ATP-binding cassette (ABC) transporters have been reported as prognostic and adverse effects predictors of many xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and may contribute to treatment regimen optimization for breast cancer.MethodsFirstly, the expression of ABC transporters family members was measured in cell lines, thereafter examined the potential role of ABC transporter in conferring resistance to pemetrexed in primary cancer cell lines isolated from 34 breast cancer patients, and then the role of ABCC5 in mediating transport of pemetrexed and apoptosis pathway in MCF-7 cell line was assessed. Finally, the functions of ABCC5 on therapeutic effect of pemetrexed was evaluated in breast cancer bearing mice.ResultsThe expressions of ABCC2, ABCC4, ABCC5 and ABCG2 were significantly increased in pan-resistance cell line, and the ABCC5, the most obvious one, was 5.21 times higher than that of the control group. The expression of ABCC5 was inversely correlated with sensitivity (IC50) of pemetrexed (r = 0.741; p<0.001) in breast cancer cells from 34 patients. Furthermore, we found that the expression of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cell line, and the IC50 were 0.06 μg/ml and 0.20 μg/ml in ABCC5 knock-down and over-expression cells, respectively. In in vivo study, we found ABCC5 affected the sensitivity of pemetrexed in breast cancer bearing mice, and the tumor volume was much larger in ABCC5 over-expression group than that in control group (2.7 folds vs 1.3 folds).ConclusionsOur results indicated ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and may be a biomarker for regimen optimization of pemetrexed in breast cancer treatment.

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Group III phospholipase A2 downregulation attenuated survival and metastasis in ovarian cancer and promotes chemo-sensitization

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01985-9 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Upasana Ray ◽

Debarshi Roy ◽

Ling Jin ◽

Prabhu Thirusangu ◽

Julie Staub ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Viability ◽

Phospholipase A2 ◽

Mtt Assay ◽

Metabolic Inhibitor ◽

Autophagic Flux ◽

Ciliated Cells ◽

Group Iii

Abstract Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited cancer progression. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

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YAP inhibits autophagy and promotes progression of colorectal cancer via upregulating Bcl-2 expression

Cell Death and Disease ◽

10.1038/s41419-021-03722-8 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Lan Jin ◽

Yunhe Chen ◽

Dan Cheng ◽

Zhikai He ◽

Xinyi Shi ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Death ◽

Transcriptional Coactivator ◽

Inhibitory Protein ◽

Potential Therapeutic Target ◽

Related Cell ◽

Autophagy Inhibition

AbstractColorectal cancer (CRC) is one of the most aggressive and lethal cancers. The role of autophagy in the pathobiology of CRC is intricate, with opposing functions manifested in different cellular contexts. The Yes-associated protein (YAP), a transcriptional coactivator inactivated by the Hippo tumor-suppressor pathway, functions as an oncoprotein in a variety of cancers. In this study, we found that YAP could negatively regulate autophagy in CRC cells, and consequently, promote tumor progression of CRC in vitro and in vivo. Mechanistically, YAP interacts with TEAD forming a complex to upregulate the transcription of the apoptosis-inhibitory protein Bcl-2, which may subsequently facilitate cell survival by suppressing autophagy-related cell death; silencing Bcl-2 expression could alleviate YAP-induced autophagy inhibition without affecting YAP expression. Collectively, our data provide evidence for YAP/Bcl-2 as a potential therapeutic target for drug exploration against CRC.

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Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22010202 ◽

2020 ◽

Vol 22 (1) ◽

pp. 202

Author(s):

Josephin Glück ◽

Julia Waizenegger ◽

Albert Braeuning ◽

Stefanie Hessel-Pras

Keyword(s):

Cell Death ◽

Cell Viability ◽

Pyrrolizidine Alkaloids ◽

Defense Mechanism ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

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Abstract 1118: Engineered cell line panel complemented with in vivo models to enable next-generation target therapy discovery in cancer

10.1158/1538-7445.am2021-1118 ◽

2021 ◽

Author(s):

Zhixiang Zhang ◽

Xiaohe Shi ◽

Xiangyang Zuo ◽

Qingyang Gu ◽

Qunsheng Ji

Keyword(s):

Cell Line ◽

Target Therapy ◽

Next Generation ◽

In Vivo Models ◽

Cell Line Panel

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Apoptosis and in vivo models to study the molecules related to this phenomenon

Einstein (São Paulo) ◽

10.1590/s1679-45082010rb1685 ◽

2010 ◽

Vol 8 (4) ◽

pp. 495-497 ◽

Cited By ~ 1

Author(s):

Adriana Luchs ◽

Claudia Pantaleão

Keyword(s):

Cell Death ◽

B Cell ◽

Ectopic Expression ◽

B Cell Lymphoma ◽

Murine Models ◽

In Vivo Models ◽

Large B Cell Lymphoma ◽

Pathological Conditions ◽

Pharmacological Targets

ABSTRACT Apoptosis or programmed cell death is a physiological process, essential for eliminating cells in excess or that are no longer necessary to the organism, acting on tissue homeostasis, although the phenomenon is also involved in pathological conditions. Apoptosis promotes activation of biochemical pathways inside cells called caspase pathway, of the proteins responsible for the cleavage of several cell substrates, leading to cell death. Antiapoptotic members of the Bcl-2 family (B cell CLL/lymphoma 2), that belong to the intrinsic route of the activation of caspases, such as Bcl-xL (extra-large B-cell lymphoma) and Bcl-w (Bcl-2-like 2), act predominantly to prevent that pro-apoptotic members, such as Bax (Bcl-2-associated X protein) and Bak (Bcl-2 relative bak) lead to cell death. Antiapoptotic molecules are considered potentially oncogenic. Murine models are known to be valuable systems for the experimental analysis of oncogenes in vivo, and for the identification of pharmacological targets for cancer and to assess antitumor therapies. Given the importance of tumorigenesis studies on the immune responses to cancer and the possibility of investigating the participation of antiapoptotic molecules in tumor progression in vivo, the development of new models may be platforms for studies on tumorigenesis, immune antitumor responses, investigation of the ectopic expression of antiapoptotic molecules and immunotherapies for tumors.

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Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-137477 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Sara El Hoss ◽

Sylvie Cochet ◽

Auria Godard ◽

Hongxia Yan ◽

Michaël Dussiot ◽

...

Keyword(s):

Cell Death ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Ineffective Erythropoiesis ◽

Hypoxic Conditions ◽

Cd34 Cells ◽

F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

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