AB0068 NOVEL CHONDROPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF THE SELECTIVE HUMAN MELANOCORTIN MC3 RECEPTOR AGONIST PG-990 ON SNAP ACTIVATED CHONDROCYTES

Background:Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4].Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear.Objectives:This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes.Methods:The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M ofn=4 samples repeated in triplicate. #p≤0.05vscontrol or *p≤0.05vsstimulus.Results:Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (#p≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (*p≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (*p≤0.05). GAG content was significantly reduced by SNAP by 29% (#p≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (*p≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (*p≤0.05).Conclusion:The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line.References:[1]Hunter DJ and Bierma-Zeinstra S. (2019).Lancet.393: 1745–59.[2]Can VCet al.(2020).Euro J Pharmacol. doi:https://doi.org/10.1016/j.ejphar.2020.172971.[3]Intekhab-Alam NYet al. (2013).Cell death & disease.4: 1-6.[4]Getting SJet al.(2006).Mol Pharmacol70: 1850-1855.[5]Kaneva MKet al.(2014).Biochem Pharmacol92: 336-47.[6]Greco KVet al.(2011).Biochem Pharmacol82: 1919-29.Disclosure of Interests:None declared

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Pituitary Adenylate Cyclase Activating Polypeptide Elicits Neuroprotection Against Acute Ischemic Neuronal Cell Death Associated with NMDA Receptors

Cellular Physiology and Biochemistry ◽

10.1159/000495722 ◽

2018 ◽

Vol 51 (4) ◽

pp. 1982-1995 ◽

Cited By ~ 8

Author(s):

Yuji Kaneko ◽

Julian P. Tuazon ◽

Xunming Ji ◽

Cesario V. Borlongan

Keyword(s):

Cell Death ◽

Adenylate Cyclase ◽

Protein Expression ◽

Cell Viability ◽

Caspase 3 ◽

High Mobility ◽

High Mobility Group ◽

Expression Levels ◽

Pituitary Adenylate Cyclase ◽

Nmdar Subunits

Background/Aims: The endogenous neurotrophic peptides pituitary adenylate cyclase-activating polypeptides (PACAP-27/38) protect against stroke, but the molecular mechanism remains unknown. Methods: Primary rat neural cells were exposed to PACAP-27 or PACAP-38 before induction of experimental acute ischemic stroke via oxygen-glucose deprivation-reperfusion (OGD/R) injury. To reveal PACAP’s role in neuroprotection, we employed fluorescent live/dead cell viability and caspase 3 assays, optical densitometry of mitochondrial dehydrogenase and cell growth, glutathione disulfide luciferase activity, ELISA for high mobility group box1 extracellular concentration, ATP bioluminescence, Western blot analysis of PACAP, NMDA subunits, apoptosis regulator Bcl-2, social interaction hormone oxytocin, and trophic factor BDNF, and immunocytochemical analysis of PACAP. Results: Both PACAP-27 and PACAP-38 (PACAP-27/38) increased cell viability, decreased oxidative stress-induced cell damage, maintained mitochondrial activity, prevented the release of high mobility group box1, and reduced cytochrome c/caspase 3-induced apoptosis. PACAP-27/38 increased the protein expression levels of BDNF, Bcl-2, oxytocin, and precursor PACAP. N-methyl-D-aspartate receptor (NMDAR)-induced excitotoxicity contributes to the cell death associated with stroke. PACAP-27/38 modulated the protein expression levels of NMDAR subunits. PACAP-27/38 increased the protein expression levels of the GluN1 subunit, and decreased that of the GluN2B and GluN2D subunits. PACAP-27, but not PACAP-38, increased the expression level of the GluN2C subunit. Conclusion: This study provides evidence that PACAP regulated NMDAR subunits, affording neuroprotection after OGD/R injury.

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MiR-24-3p Attenuates IL-1β-Induced Chondrocyte Injury Associated With Osteoarthritis by Targeting BCL2L12

10.21203/rs.3.rs-76065/v1 ◽

2020 ◽

Author(s):

Jin Xu ◽

Xiaozhong Qian ◽

Ren Ding

Keyword(s):

Cell Viability ◽

Inflammatory Cytokines ◽

Caspase 3 ◽

Degenerative Joint Disease ◽

Joint Disease ◽

Luciferase Reporter ◽

Pcr Analysis ◽

Protective Effects ◽

Tnf Α ◽

Pro Inflammatory Cytokines

Abstract Background: Osteoarthritis (OA) is a chronic and degenerative joint disease prevalent in the elderly. MiR-24-3p has been reported to be involved in an OA-resembling environment. However, the functional role and underlying mechanism of miR-24-3p in chondrocyte injury associated with OA remains unknown. Methods: The expression of miR-24-3p was determined in OA cases and control patients, as well as IL-1β-stimulated chondrocyte cell line CHON-001 using reverse transcription quantitative PCR analysis. Cell viability was analyzed by CCK-8 assay. Apoptosis status was assessed by caspase-3 activity detection. The pro-inflammatory cytokines (TNF-α and IL-18) were determined using ELISA assay. The association between miR-24-3p and BCL2L12 was confirmed by luciferase reporter assay.Results: We first observed that miR-24-3p expression level was lower in the OA cases than in the control patients and IL-1β decreased the expression of miR-24-3p in the chondrocyte CHON-001. Functionally, overexpression of miR-24-3p significantly attenuated IL-1β-induced chondrocyte injury, as reflected by increased cell viability, decreased caspase-3 activity, pro-inflammatory cytokines (TNF-α and IL-18). Western blot analysis showed that overexpression of miR-24-3p weakened IL-1β-induced cartilage degradation, as reflected by reduction of MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-5) protein expression, as well as markedly elevation of COL2A1 (collagen type II). Importantly, BCL2L12 was demonstrated to be a target of miR-24-3p. BCL2L12 knockdown imitated, while overexpression significantly abrogated the protective effects of miR-24-3p against IL-1β-induced chondrocyte injury.Conclusions: In conclusion, our work provides important insight into targeting miR-24-3p/BCL2L12 axis in OA therapy.

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Tabetri™ (Tabebuia avellanedae Ethanol Extract) Ameliorates Osteoarthritis Symptoms Induced by Monoiodoacetate through Its Anti-Inflammatory and Chondroprotective Activities

Mediators of Inflammation ◽

10.1155/2017/3619879 ◽

2017 ◽

Vol 2017 ◽

pp. 1-14 ◽

Cited By ~ 8

Author(s):

Jae Gwang Park ◽

Young-Su Yi ◽

Yo Han Hong ◽

Sulgi Yoo ◽

Sang Yun Han ◽

...

Keyword(s):

Cell Line ◽

Proinflammatory Cytokines ◽

Inflammatory Mediators ◽

Molecular Mechanisms ◽

Ethanol Extract ◽

Degenerative Joint Disease ◽

Joint Disease ◽

Cellular Models ◽

Anti Inflammatory

Although osteoarthritis (OA), a degenerative joint disease characterized by the degradation of joint articular cartilage and subchondral bones, is generally regarded as a degenerative rather than inflammatory disease, recent studies have indicated the involvement of inflammation in OA pathogenesis. Tabebuia avellanedae has long been used to treat various diseases; however, its role in inflammatory response and the underlying molecular mechanisms remain poorly understood. In this study, the pharmacological effects of Tabetri (Tabebuia avellanedae ethanol extract (Ta-EE)) on OA pathogenesis induced by monoiodoacetate (MIA) and the underlying mechanisms were investigated using experiments with a rat model and in vitro cellular models. In the animal model, Ta-EE significantly ameliorated OA symptoms and reduced the serum levels of inflammatory mediators and proinflammatory cytokines without any toxicity. The anti-inflammatory activity of Ta-EE was further confirmed in a macrophage-like cell line (RAW264.7). Ta-EE dramatically suppressed the production and mRNA expressions of inflammatory mediators and proinflammatory cytokines in lipopolysaccharide-stimulated RAW264.7 cells without any cytotoxicity. Finally, the chondroprotective effect of Ta-EE was examined in a chondrosarcoma cell line (SW1353). Ta-EE markedly suppressed the mRNA expression of matrix metalloproteinase genes. The anti-inflammatory and chondroprotective activities of Ta-EE were attributed to the targeting of the nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) signaling pathways in macrophages and chondrocytes.

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Polydatin Alleviates Lipopolysaccharide-Triggered Inflammatory Injury Through Up-Regulating miR-125b in Chondrogenic ATDC5 Cells

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.18:108-114 ◽

2019 ◽

Vol 18 (1) ◽

pp. 108-114

Author(s):

Liu Haifeng ◽

Zhang Fan ◽

Wu Huiming ◽

Wang Qinglai ◽

Zhang Kuixian ◽

...

Keyword(s):

Cell Viability ◽

Caspase 3 ◽

Interleukin 1 ◽

Coiled Coil ◽

Joint Disease ◽

Protective Effects ◽

Down Regulation ◽

Inflammatory Injury ◽

Atdc5 Cell ◽

Anti Inflammatory

Osteoarthritis is a bone-joint disease prevalent in older people characterized by joint inflammation. In traditional Chinese medicine, polydatin plays an important anti-inflammatory role. This study analyzed the potential effects and possible internal mechanisms of polydatin on osteoarthritis. First, lipopolysaccharide-induced osteoarthritis injury was established in chondrogenic ATDC5 cells. Lipopolysaccharides significantly stimulated inflammatory injuries in ATDC5 cells as exemplified by a decrease in cell viability and an increase in inflammatory cytokine secretions including interleukin-6, tumor necrosis factor-a, and interleukin-1. Moreover, lipopolysacchrides also increased Cleaved caspase-3 and Cleaved Poly (ADP-ribose) polymerase to promote cell apoptosis. Second, polydatin showed significant protective effects against lipopolysaccharide-induced inflammatory injury, again exemplified by increased cell viability, decreased inflammatory cytokines, Cleaved caspase-3, and Cleaved poly (ADP-ribose) polymerase. Lastly, miR-125b and its binding target Rho-Associated Coiled-Coil Containing Protein Kinase 1 were closely associated with regulatory effects of polydatin against lipopolysaccharide-stimulated ATDC5 cell inflammatory injuries. Polydatin alleviated lipopolysaccharide-stimulated inflammatory injuries via the down-regulation of miR-125b. The present study concludes that polydatin plays an anti-inflammatory role in lipopolysaccharide-stimulated ATDC5 cell inflammatory injuries via the down-regulation of miR-125b.

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Simultaneous Treatment with P53 Overexpression and Interferon γ Exerts a Dramatic Increase in Apoptosis Induction of U87 Cells

Galen Medical Journal ◽

10.31661/gmj.v10i0.2270 ◽

2021 ◽

Vol 10 ◽

pp. e2270

Author(s):

Zahra Abbasy ◽

Hamid Zaferani Arani ◽

Mahsa Ale-Ebrahim ◽

Vihan Moodi ◽

Javad Nematian ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cell Line ◽

Inflammatory Cytokine ◽

Caspase 3 ◽

Simultaneous Treatment ◽

P53 Overexpression ◽

Study Results ◽

Anti Inflammatory ◽

U87 Cells

Background: Gliomas possess low immunogenicity, which is an inevitable hinder in front of cancer immunotherapy. Different interferons (IFNs) may proceed apoptosis instead in p53-dependent or independent pathways. P53 induces the anti-inflammatory programmed cell death in cancer cells; on the other hand, IFN gamma (IFNγ) is a modulatory/pro-inflammatory cytokine. There are contradictory reports of whether this cytokine can possess an anti- or pro-cancerous impact on tumors. Hence, we aimed to investigate the possible cooperative apoptotic effect of the P53 and IFNγ over expressions on the U87 glioblastoma cell line. Materials and Methods: The P53 expressing vector was amplified by Escherichia coli BL21. This vector was confirmed by the aid of sequencing. At the next step, U87 cells were transfected using lipofectamine. Cells were treated with P53 vector and/or IFNγ. The type of cellular death investigated by flow cytometry and the expression level of cleaved caspase-3 protein was also precisely demonstrated by western blotting. Results: Sequencing results revealed that inserted P53 was identical with human P53. Western blot results revealed that both IFNγ and P53 overexpression could up-regulate cleaved caspase-3 protein expression in this cell line. Interestingly, flow cytometry data determined that concurrent treatment with P53 exogenous overexpression and IFNγ induces about 70% apoptosis in U87; more than the sum of cell death occurs after IFNγ or P53 overexpression alone (~18%+21%=39%). Conclusion: The present study results showed that p53-overexpression and IFNγ could ultimately induce up-regulation of the caspase-3 and ultimately significant apoptosis increasing in the U87 cell line. Although IFNγ is believed to be a pro-inflammatory cytokine and P53 is an anti-inflammatory agent, our results demonstrated that they could act synergistically to induce apoptosis in U87 cells. [GMJ.2021;10:e2270]

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Effects of Glucosamine and Chondroitin Sulfate on Cartilage Metabolism in OA: Outlook on Other Nutrient Partners Especially Omega-3 Fatty Acids

International Journal of Rheumatology ◽

10.1155/2011/969012 ◽

2011 ◽

Vol 2011 ◽

pp. 1-17 ◽

Cited By ~ 67

Author(s):

Jörg Jerosch

Keyword(s):

Fatty Acids ◽

Chondroitin Sulfate ◽

Degenerative Joint Disease ◽

Joint Disease ◽

Omega 3 Fatty Acids ◽

Omega 3 ◽

Collagen Hydrolysate ◽

Cartilage Metabolism ◽

Promising Therapeutic Approach ◽

Anti Inflammatory

Osteoarthritis (OA) is a degenerative joint disease that is characterized by increasing loss of cartilage, remodeling of the periarticular bone, and inflammation of the synovial membrane. Besides the common OA therapy with nonsteroidal anti-inflammatory drugs (NSAIDs), the treatment with chondroprotectives, such as glucosamine sulfate, chondroitin sulfate, hyaluronic acid, collagen hydrolysate, or nutrients, such as antioxidants and omega-3 fatty acids is a promising therapeutic approach. Numerous clinical studies have demonstrated that the targeted administration of selected micronutrients leads to a more effective reduction of OA symptoms, with less adverse events. Their chondroprotective action can be explained by a dual mechanism: (1) as basic components of cartilage and synovial fluid, they stimulate the anabolic process of the cartilage metabolism; (2) their anti-inflammatory action can delay many inflammation-induced catabolic processes in the cartilage. These two mechanisms are able to slow the progression of cartilage destruction and may help to regenerate the joint structure, leading to reduced pain and increased mobility of the affected joint.

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MO1048PHYTOTHERAPY ON RENAL HYPOXIA: THE PREVENTIVE EFFECTS OF HYDROXYTYROSOL - PRELIMINARY IN VITRO RESULTS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab113.004 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Terézia Kamasová ◽

Ana Sofia Abreu ◽

Fátima Paiva-Martins ◽

Luís Belo ◽

Alice Santos-Silva ◽

...

Keyword(s):

Cell Viability ◽

Cell Line ◽

Therapeutic Potential ◽

Quantitative Polymerase Chain Reaction ◽

Oxidative Status ◽

Hypoxic Conditions ◽

Thiazolyl Blue Tetrazolium Bromide ◽

Anti Inflammatory ◽

Preventive Effects

Abstract Background and Aims Renal hypoxia plays a key role in the pathophysiology of acute kidney injury and in the progression of chronic kidney disease, potentiating other important risk factors for renal disease, such as oxidative stress, renal fibrosis, and inflammation. Hydroxytyrosol (HT) is a phenolic compound extracted from olives and olive-derived products, that has been shown to detain potent in vitro antioxidant and anti-inflammatory activity. The aim of this study was to evaluate the preventive therapeutic potential of HT on a cellular model of renal hypoxia. Method A cell line of normal adult proximal tubular epithelium (HK-2 cell line) was used to determine the effects of the chemical induction of hypoxia with cobalt chloride (CoCl2), as well as the preventive potential of HT on the elicited effects. For this purpose, HK-2 cells were exposed for 24 h to 254 µM CoCl2, to mimic the hypoxic conditions, or pre-incubated for 1 h with 5 µM HT and further exposed to the CoCl2 for 24 h more. Cell viability was assessed by the thiazolyl blue tetrazolium bromide reduction assay. Oxidative status was evaluated by the measurement of reactive oxygen and nitrogen species (ROS and RNS) and reduced glutathione (GSH) levels, by using standardized fluorometric and colorimetric assays. The expression of several genes related to the hypoxic, inflammatory, and fibrotic responses was determined by quantitative polymerase chain reaction (PCR). Results CoCl2-exposed HK-2 cells (hypoxic conditions) showed a significant decrease in cell viability (p < 0.0001 vs. control), and a disruption of the oxidative status, characterized by an increase of ROS and RNS production of about 6-fold over control cells (p < 0.0001) and a decrease in GSH intracellular levels of nearly 50 % (p < 0.05). Although the pre-exposure to HT showed no significant effects on the loss of cell viability elicited by CoCl2, the presence of HT prior to induction of hypoxia reduced the generation of ROS and RNS (p < 0.05 for HT + CoCl2 vs. CoCl2) and prevented the GSH depletion (GSH levels for HT + CoCl2 were similar to those of control) elicited by CoCl2. When compared to control cells, CoCl2-exposed HK-2 cells also showed increased expression of genes related to hypoxia (HIF1A, p < 0.05; GAPDH, p < 0.0001), as well as of modulators of inflammation (IL6, p < 0.0001) and fibrosis (TGFB1, p < 0.05). Importantly, the expression of these genes was partially or even totally suppressed by the pre-exposure of cells to HT (GAPDH, p < 0.01 for HT + CoCl2 vs. CoCl2; expression of HIF1A, IL6 and TGFB1 for HT + CoCl2 was similar to that of control). Conclusion Our data supports the potential for a multiplicity of preventive effects of HT, providing antioxidant, anti-inflammatory and anti-fibrotic defenses to renal cells under hypoxic conditions. Importantly, the development of safe and effective therapeutic approaches based on phytochemicals such as HT, may present substantial advantages for renal patients over synthetic drugs, including fewer side effects, significantly lower price, and ease of administration in the form of dietary supplements. Acknowledgments This work was supported by Applied Molecular Biosciences Unit (UCIBIO), financed by national funds from FCT/MCTES (UIDB/04378/2020), by North Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024), and co-financed by FCT/MCTES (PTDC/OCE-ETA/32492/2017) and FEDER/COMPETE 2020 (POCI-01-0145-FEDER-032492).

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Re-Wiring BCL-2 Family Anti-Apoptotic Protein Dependencies through Modulating Phosphorylation to Re-Sensitize Venetoclax-Resistant Lymphoid Malignancies to BCL-2 Inhibition

Blood ◽

10.1182/blood-2020-140455 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-36

Author(s):

Stephen Jun Fei Chong ◽

Mary C Collins ◽

Liam Hackett ◽

Matthew S. Davids

Keyword(s):

Cell Death ◽

Protein Expression ◽

Cell Line ◽

Research Funding ◽

Family Members ◽

B Cell Lymphoma ◽

Prolonged Treatment ◽

Lymphoid Malignancies ◽

Phosphatase Inhibitor ◽

Apoptotic Protein

Introduction Resistance to apoptosis is a hallmark of cancer, and modulation of BCL-2 family proteins is an important mediator of such resistance in hematologic malignancies. Despite the clinical efficacy of the BCL-2 inhibitor venetoclax (VEN), prolonged treatment may lead to resistance, such as the BCL2 G101V mutation (Blombery et al, Blood, 2020); however, over half of VEN resistant cases are not explained by known genetic mechanisms. Phosphorylation of BCL-2 at serine-70 (S70pBCL2) or of MCL-1 at threonine-163 (T163pMCL1) have been shown to increase sequestration of the pro-apoptotic protein BAX (Deng et al, JNCI, 2000) and stabilize the level of anti-apoptotic protein MCL-1 (Wang et al, Mol Cancer, 2014), respectively. We hypothesized that the increase in post-translational modifications of BCL-2 family members, in particular S70pBCL2 and T163pMCL1, are novel mechanisms of functional VEN resistance in lymphoid malignancies. We further hypothesized that the FDA-approved phosphatase activator drug FTY720 (fingolimod) would de-phosphorylate these BCL-2 family members and thereby re-sensitize malignant lymphoid cells to VEN-induced apoptosis. Methods A VEN resistant diffuse large B-cell lymphoma cell line (OCI-Ly1-R) as well as peripheral blood mononuclear cells from 12 previously untreated CLL patients co-cultured with human stromal NK-Tert cells were treated ex vivo with VEN +/- FTY720. A VEN sensitive cell line (OCI-Ly1-S) was treated with VEN +/- a phosphatase inhibitor okadaic acid (OA). Western blot was used to evaluate changes in S70pBCL2 and T163pMCL1 protein levels. BH3 profiling via flow cytometry was performed to determine the survival dependence on anti-apoptotic BCL-2 family members via cytochrome c release in response to specific BH3-only peptides and drugs such as VEN applied directly to mitochondria (Ryan et al, Biol Chem, 2016). Cell viability assays (CellTiter-Glo, Trypan Blue and Annexin/Hoechst) were employed to investigate the effects of FTY720 on OCI-Ly1-R resistance to VEN. The BCL-2-BAX interaction was investigated using co-immunoprecipitation in VEN resistant and sensitive cells following treatment with VEN +/- FTY720. T-test, ANOVA and multiple comparison with a statistical significance set at 2-tailed p ≤ 0.05 were used. Results OCI-Ly1-R cells displayed higher S70pBCL2, T163pMCL1 and MCL-1 expression compared to OCI-Ly1-S cells. Notably, the increase in S70pBCL2 was associated with reduced response of VEN-mediated BCL-2-BAX dissociation, while the increase in T163pMCL1 was accompanied by enhanced MCL-1 protein expression. Using BH3 profiling, we found that the increase in S70pBCL2, T163pMCL1 and MCL-1 expression were functionally associated with a decrease in BCL-2 survival dependence (-79.1%, 1μM VEN, P < 0.0001) and an increase in MCL-1 dependence (+52%, 10μM MS1, P < 0.0001) (Fig. A). The addition of FTY720 reversed these observations in OCI-Ly1-R cells, where we observed a decrease in S70pBCL2, T163pMCL1 and MCL-1 protein expression, BCL-2 and BAX interaction, as well as a "re-wired" functional dependence toward BCL-2 (-21.6% 10μM MS1, +27.9% 1μM VEN, P < 0.0001) (Fig. B). Importantly, pre-treatment with FTY720 re-sensitized OCI-Ly1-R cells to VEN-induced cell death (+56.1%, P = 0.0001) (Fig. C). Conversely, treatment with a phosphatase inhibitor, OA, led to an increase in S70pBCL2, T163pMCL1 and MCL-1 expression as well as reduced late death of OCI-Ly1-S cells (-60%, P = 0.0018). We validated our cell line results in primary CLL cells, and again the combination of FTY720 and VEN similarly reduced S70pBCL2, T163pMCL1 and MCL-1 expression, increased BCL-2 dependence, and enhanced VEN-induced cell death (+23.6%, P < 0.0001). Conclusion Increased S70pBCL2 and T163pMCL1 are associated with VEN resistance, in part by inhibiting VEN-induced BCL-2-BAX dissociation and switching the functional survival dependence from BCL-2 to MCL-1. FTY720 re-sensitizes VEN resistant cells by reducing S70pBCL2, T163pMCL1 and MCL-1 expression, dissociating BAX from BCL-2 and "re-wiring" the survival dependence to BCL-2. These preclinical findings support the exploration of this strategy clinically in patients with VEN resistant lymphoid malignancies. Disclosures Davids: Sunesis: Consultancy; AbbVie: Consultancy; Surface Oncology: Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Zentalis: Consultancy; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; MEI Pharma: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Merck: Consultancy; Research to Practice: Honoraria.

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The Combinatorial Activity of Eftozanermin (ABBV-621), a Novel and Potent TRAIL Receptor Agonist Fusion Protein, in Pre-Clinical Models of Hematologic Malignancies

Blood ◽

10.1182/blood-2020-136155 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 41-41

Author(s):

Morey L Smith ◽

Sha Jin ◽

Dong Chen ◽

Haichao Zhang ◽

Jason Huska ◽

...

Keyword(s):

Cell Death ◽

Fusion Protein ◽

Cell Line ◽

Receptor Agonist ◽

B Cell Lymphoma ◽

Synergistic Activity ◽

Trail Receptor ◽

Phase I Clinical Trials ◽

Single Chain ◽

Publicly Traded

Cell death can be initiated through activation of the extrinsic and intrinsic apoptotic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, preferentially triggers the extrinsic apoptotic pathway by binding as a trimer to two closely related cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Receptor trimerization leads to the formation of the death-inducing signaling complex (DISC) to recruit and activate downstream caspases that ultimately leads to apoptotic cell death. Because TRAIL signaling induces apoptosis, several TRAIL receptor agonists have been developed for the treatment of cancer. ABBV-621 is a novel, second generation TRAIL receptor agonist that is an engineered fusion protein consisting of an IgG1-Fc linked to a single chain trimer of TRAIL subunits resulting in a total of six death receptor binding sites per molecule to maximize receptor clustering that is currently being tested in Phase I clinical trials (NCT03082209). To expand upon the potential therapeutic utility of ABBV-621, we tested the combinatorial activity of ABBV-621 with numerous standard-of-care (SoC) therapeutics and targeted agents in diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Thein vitroresults led to selection of agents to combine with ABBV-621 forin vivostudies. In DLBCL cell line-derived xenograft (CDX) preclinical models, we observed combination activity of ABBV-621 with pevonedistat (PEV) a selective NEDD8 inhibitor. Additionally, synergistic activity was observed with ABBV-621 with either bendamustine (BED) or rituximab (RTX) alone, or BED/RTX together. In AML, we observed compelling combination activity of ABBV-621 with PEV in cell line-derived xenograft (CDX) models. In MM, combination of ABBV-621 plus bortezomib (BTZ) resulted in deeper anti-tumorigenic activity than either agent alone in several CDX models. The pre-clinical data presented here support expanding the indications and settings where ABBV-621 may have utility. A clinical trial assessing the activity of ABBV-621 in combination with bortezomib and dexamethasone in R/R MM patients is planned. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Smith: AbbVie:Current Employment, Current equity holder in publicly-traded company.Jin:AbbVie:Current Employment, Current equity holder in publicly-traded company.Chen:AbbVie:Current Employment, Current equity holder in publicly-traded company.Zhang:AbbVie:Current Employment, Current equity holder in publicly-traded company.Huska:AbbVie:Current Employment, Current equity holder in publicly-traded company.Widomski:AbbVie:Current Employment, Current equity holder in publicly-traded company.Bontcheva:AbbVie:Current Employment, Current equity holder in publicly-traded company.Buchanan:AbbVie:Current Employment, Current equity holder in publicly-traded company.Morgan-Lappe:AbbVie:Current Employment, Current equity holder in publicly-traded company.Phillips:AbbVie:Current Employment, Current equity holder in publicly-traded company.Tahir:AbbVie:Current Employment, Current equity holder in publicly-traded company.

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Effect of Hydroalcoholic Extract of Ephedramajor, Memordia charantia, and Resveratrol on Cytotoxicity and Caspase-3 Genes Expression Level in MCF-7 Breast Cancer Cell Line

Gene Cell and Tissue ◽

10.5812/gct.110658 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Seyed Kazem Sabbagh ◽

Ehsan Ghodrati ◽

Alireza Hajibeiki ◽

Mahta Mazaheri ◽

Mohammad Reza Sarafraz Ardakani ◽

...

Keyword(s):

Gene Expression ◽

Medicinal Plants ◽

Cell Viability ◽

Cell Line ◽

Plant Extracts ◽

Caspase 3 ◽

Cell Toxicity ◽

Hydroalcoholic Extract ◽

Expression Levels ◽

Mcf 7

Background: To increase the therapeutic effect of drugs to combat diseases, combination therapy with current chemical drugs and new medicines derived from medicinal plants is necessary. Objectives: The present work aimed to investigate the effect of hydroalcoholic extract of two medicinal plants, Ephedra major and Momordi cacharantia (Carla), and resveratrol drug on cell viability and expression levels of caspase-3 gene in MCF-7 cell line. Methods: In this experimental study, the hydroalcoholic extraction of tested plants was done with a Soxhlet extractor. The MTT assay and real-time PCR were used to determine cell toxicity and caspase-3 gene expression levels, respectively. Results: The highest and lowest cytotoxic effects of plant extracts and resveratrol were observed at concentrations of 500 and 150 µg/mL, respectively. The highest level of the caspase-3 gene expression was observed after 72 h of incubation by different concentrations of plant extracts and resveratrol. Conclusions: It can be concluded that both plant extracts could influence cell viability in MCF-7 cells via the increase of cell toxicity and expression of caspase3 gene. Thus, these species could be used in the pharmaceutical industry.

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