Serum-deprived Conditions Research Articles

Abstract 893: GD2 is a potential biomarker for glioblastoma cancer stem cells

Abstract Introduction: Treatment resistance and tumor recurrence of Glioblastoma (GBM) have been associated with cancer stem cells (CSCs), a subpopulation of cancer cells defined by capabilities of sphere-formation in 3D culture, self-renewal, migration, and survival in serum-deprived conditions. Therefore, therapies specifically targeting CSCs are urgently needed. Previous works have identified Ganglioside GD2 as a biomarker of triple negative breast cancer CSCs and suggested that GD2 is highly expressed in GBM cells. Here, we hypothesize that GD2 is also a potential biomarker of GBM CSCs and is involved with cancer stemness. Methods: GBM cells U-87 MG and U-118 MG were sorted as GD2- and GD2+ cells via FACS. Cells were cultured in 2D and 3D. 3D neurosphere size was measured and GD2 in 2D and 3D cultures was quantified via flow cytometry. Transwell migration assay was used to examine the migration capability of GD2- and GD2+ cells. Unsorted cells were cultured in either complete (10% serum) or deprived (1% serum) media and their GD2 was quantified via flow cytometry. Results: To test our hypothesis, we determined whether GD2+ cells express CSCs capabilities of sphere-formation in 3D culture, migration, and survival in serum-deprived conditions. First, we examined the sphere-forming capability by culturing GD2-, GD2+, and unsorted cells in 3D and found that they all formed neurospheres. However, GD2+ neurosphere size was significantly larger compared with unsorted and GD2- neurospheres for day 1 and 2. Interestingly, the significant difference was no longer seen on day 6. Analysis for GD2 expression in the spheres revealed that GD2- spheres contained GD2+ cells, suggesting that GD2- cells could spontaneously dedifferentiate to generate CSCs. To test this, we sorted GD2- and GD2+ cells into 2D conditions and quantified daily GD2 expression via flow cytometry. We found that GD2- seeded cells spontaneously expressed GD2 over six days, explaining the presence of GD2 in GD2- neurospheres. GD2+ cells also demonstrated the enhanced capability to migrate of CSCs with a significantly higher number of migrated cells compared with GD2- cells in the transwell migration assay. To examine the ability to survive in serum-deprived conditions, we cultured unsorted cells in either complete or deprived media. We found that these cells proliferated in the serum-deprived condition at approximately the same level as complete media. GD2 analysis by flow cytometry also showed a remarkably higher percentage of GD2+ cells in deprived media, suggesting that GD2 is involved in driving cell survival and maintaining cancer stemness in serum-deprived conditions. Conclusion: Our data suggests that GD2+ cells demonstrate characteristics of CSCs and GD2 is a potential biomarker of CSCs in GBM. Therefore, GD2 could be utilized as a differential therapeutic target for GBM. Further mechanistic studies are necessary to elucidate how GD2 is synthesized and maintains the stemness of GBM CSCs. Citation Format: Khoa Nguyen, Minh Tran, Thomas Cheng, Kurtis Willingham, Reza Izadpanah, Matthew Burow. GD2 is a potential biomarker for glioblastoma cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 893.

Influence of Umbelliferone on the Anticonvulsant and Neuroprotective Activity of Selected Antiepileptic Drugs: An In Vivo and In Vitro Study.

Umbelliferone (7-hydroxycoumarin; UMB) is a coumarin with many biological properties, including antiepileptic activity. This study evaluated the effect of UMB on the ability of classical and novel antiepileptic drugs (e.g., lacosamide (LCM), levetiracetam (LEV), phenobarbital (PB) and valproate (VPA)) to prevent seizures evoked by the 6-Hz corneal-stimulation-induced seizure model. The study also evaluated the influence of this coumarin on the neuroprotective properties of these drugs in two in vitro models of neurodegeneration, including trophic stress and excitotoxicity. The results indicate that UMB (100 mg/kg, i.p.) significantly enhanced the anticonvulsant action of PB (p < 0.01) and VPA (p < 0.05), but not that of LCM orLEV, in the 6-Hz test. Whether alone or in combination with other anticonvulsant drugs (at their ED50 values from the 6-Hz test), UMB (100 mg/kg) did not affect motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; grip strength; or passive avoidance tests, respectively. Pharmacokinetic characterization revealed that UMB had no impact on total brain concentrations of PB or VPA in mice. The in vitro study indicated that UMB has neuroprotective properties. Administration of UMB (1 µg/mL), together with antiepileptic drugs, mitigated their negative impact on neuronal viability. Under trophic stress (serum deprivation) conditions, UMB enhanced the neurotrophic abilities of all the drugs used. Moreover, this coumarin statistically enhanced the neuroprotective effects of PB (p < 0.05) and VPA (p < 0.001) in the excitotoxicity model of neurodegeneration. The obtained results clearly indicate a positive effect of UMB on the anticonvulsant and neuroprotective properties of the selected drugs.

Tofacitinib Decreases Autophagy of Fibroblast-Like Synoviocytes From Rheumatoid Arthritis Patients.

The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.

Serum deprivation enhanced monocrotophos mediated cellular damages in human lung carcinoma and skin keratinocyte

IntroductionPesticides are used extensively to ensure food security for the ever-growing population around the globe. Monocrotophos (MCP) is a low-cost organophosphate pesticide extensively used in Indian agriculture. People received exposure to pesticides occupationally or by other means. Human lungs and skin are the highest exposure locations for pesticides in agricultural activities. AimThe present study aims to decipher the cytotoxicity of monocrotophos on the lung carcinoma and skin keratinocytes cell lines. MethodsThe representative cancer cell lines A549, human lung adenocarcinoma and HaCaT, human skin keratinocytes cultured under complete media were exposed to different doses of monocrotophos (MCP- 50 μM and MCP-100 μM). Both cells were also cultured under serum-deprived conditions (0% FBS) and provided similar treatment to analyze the toxic responses. In addition, the endpoints associated with cell viability, oxidative stress, cell cycle analysis and micronuclei formations were investigated. ResultsOur present study exhibited significant cytotoxic effects of monocrotophos on A549 and HaCaT cells via ROS generation. In addition, the cell cycle arrest in the G0 phase and micronuclei formation finding highlights the upraised effect of monocrotophos (MCP) in serum-deprived samples compared to experimental controls cultured under serum-containing media. ConclusionMonocrotophos enhanced cytotoxicity in serum-deprived A549 and HaCaT cells through cell viability depletion, ROS generation, cell cycle arrest, and micronuclei formation compared to complete medium (10% FBS).

Serum deprivation enhanced ethanol-induced toxic responses in A549, lung carcinoma cells

Aim: The current study explores the toxic consequences of ethanol on human lung carcinoma cell, A549 in serum-deprived condition. Methodology: Human lung carcinoma cells, A549, were cultured in complete and serum-deprived medium for 6 hr. Subsequently, they were exposed to 50 mM and 100 mM concentrations of ethanol. Cytotoxicity studies linked with cell viability, oxidative stress, cell cycle arrest and micronuclei formation were performed using various toxicological parameters, namely MTT assay, DCFDA based ROS generation, cell cycle analysis and micronuclei formation assay. The cytotoxicity of ethanol in complete and serum deprived medium were compared at similar doses and time duration. Results: The metabolic viability assay demonstrated that 50 mM and 100 mM concentration of ethanol did not induce significant levels of cytotoxic alteration in A549 lung carcinoma cells in complete medium. However, in serum-deprived conditions, 50 mM and 100 mM ethanol concentration significantly altered cell viability. Further, exposure of 50 mM and 100 mM concentration of ethanol enhanced reactive oxygen species levels in A549 cells more significantly in serum-deprived conditions than in complete medium. In addition to cytotoxicity and oxidative stress, 50 mM and 100 mM ethanol also arrested the cells at G0 phase more significantly in serum deprived conditions compared to complete medium. Interpretation: Both 50 mM and 100 mM ethanol concentration enhanced the cell cytotoxicity and reactive oxygen species, cell cycle arrest and micronuclei formation more severely in serum-deprived medium than in complete medium (containing 10% FBS) under similar treatment conditions.

Effect of M2 Macrophage-Derived Soluble Factors on Behavioral Patterns and Cytokine Production in Various Brain Structures in Depression-Like Mice.

We studied the effect of soluble factors derived from human macrophages polarized to M2 phenotype under conditions of serum deprivation (M2-SF) on behavioral pattern and cytokine production in various brain structures in mice with modeled stress-induced depression. Intranasal administration of M2-SF for 7 days led to stimulation of locomotor and exploratory activities and a decrease in emotional reactivity in the open-field test as well as reduction in depression-like behavior in Porsolt forced swimming test and a decrease in anxiety and anhedonia. Correction of depression-like behavior was accompanied by down-regulation of proinflammatory cytokines (IL-1β, IL-6, TNFα, and IFNγ) in pathogenetically important brain structures (striatum, hippocampus, and frontal cortex). These data indicate that the antidepressant potential of M2 type macrophages can be mediated by the anti-inflammatory effects of M2-SF.

Establishment of Doxorubicin Resistant Stem-like Cell line from MCF-7 Cells

Background Conventional therapy of Breast cancer (BCa) is associated with numerous challenges, relapse being the topmost. It has been shown that disease relapse occurs due to the existence of cancer stem cells (CSCs), residing within the tumor. Objectives To develop a cancer stem cell line model, exploiting the property of stem cells to resist treatment with anti-cancer therapeutic agents and to develop novel therapeutic interventions targeting the elimination of CSCs. Methods Single-cell clones (SCC) from MCF-7 cells were established in 96-well plates using a culture medium containing 5% FBS. Enrichment of cancer stem cells was carried out from established single cell clones which were initially cultured in a serum-deprived nutrient medium for six weeks followed by Doxorubicin treatment. Doxorubicin-resistant clones were established and evaluated for their growth and sphere-forming abilities. Further, these clones were characterized based on the presence of stem-cell markers using a semi-quantitative reverse transcription-polymerase chain reaction and results were compared with the parental MCF-7 cell line. Results In a complete medium, these spheres differentiated and started growing as a monolayer with differential expression levels of genes involved in stemness and cellular differentiation. When these spheres were sub-cultured again in stem-cell medium and detached to give rise to single cells, these clones retained sphere-forming ability. Doxorubicin-resistant clones showed a tendency to grow in spheres in a stem-cell medium with serum-deprived culture conditions. Conclusion Our results provide undeviating evidence of the successful establishment of clones of MCF-7 cell line exhibiting stem cell-like properties. These cell lines have been reprogrammed as stem cell-like models and can be used to delineate recurrent breast cancer attributed to CSCs.

Hemin enhances the cardioprotective effects of mesenchymal stem cell-derived exosomes against infarction via amelioration of cardiomyocyte senescence

BackgroundApplication of mesenchymal stem cell-derived exosomes (MSC-EXO) has emerged as a novel therapeutic strategy for myocardial infarction (MI). Our previous study showed that pretreatment with hemin, a potent heme oxygenase-1 (HO-1) inducer, enhanced the cardioprotective effects of MSCs in a mouse model of MI. This study aimed to investigate the therapeutic effects of EXO derived from hemin-pretreated MSCs (Hemin-MSC-EXO) in MI and explore the potential mechanisms.MethodsMSC-EXO and Hemin-MSC-EXO were collected and characterized. MSC-EXO and Hemin-MSC-EXO were intramuscularly injected into the peri-infarct region in a mouse model of MI. Heart function of mice was assessed by echocardiography. The mitochondrial morphology of neonatal mice cardiomyocytes (NMCMs) under serum deprivation and hypoxic (SD/H) conditions was examined by Mitotracker staining. The cellular senescence of NMCMs was determined by senescence-associated-β-galactosidase assay. A loss-of-function approach was adopted to determine the role of Hemin-MSC-exosomal-miR-183-5p in the regulation of cardiomyocyte senescenceResultsEXO were successfully isolated from the supernatant of MSCs and Hemin-pretreated MSCs. Compared with MSC-EXO, injection of Hemin-MSC-EXO significantly improved cardiac function and reduced fibrosis. Both MSC-EXO and Hemin-MSC-EXO ameliorated cardiomyocyte senescence and mitochondrial fission in vitro and in vivo, and the latter exhibited better protective effects. MicroRNA sequencing revealed a higher level of miR-183-5p in Hemin-MSC-EXO than in MSC-EXO. MiR-183-5p knockdown partially abrogated the protective effects of Hemin-MSC-EXO in attenuating mitochondrial fission and cellular senescence of cardiomyocytes induced by SD/H. High mobility group box-1 (HMGB1) abundance was lower in Hemin-MSC-EXO-treated than MSC-EXO-treated mouse hearts, and HMGB1 was identified as one of the potential target genes of miR-183-5p. Mechanistically, Hemin-MSC-EXO inhibited SD/H-induced cardiomyocyte senescence partially by delivering miR-183-5p into recipient cardiomyocytes via regulation of the HMGB1/ERK pathway. Furthermore, knockdown of miR-183-5p reduced the Hemin-MSC-EXO-mediated cardioprotective effects in a mouse model of MI.ConclusionOur results reveal that Hemin-MSC-EXO are superior to MSC-EXO in treating MI. Exosomal miR-183-5p mediates, at least partially, the cardioprotective effects of Hemin-MSC-EXO by inhibiting cardiomyocyte senescence via regulation of the HMGB1/ERK pathway. This study highlights that MSC-EXO have high translational value in repairing cardiac dysfunction following infarction.Graphic abstract

EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p &lt; 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation.

M1 macrophage-derived exosomes transfer miR-222 to induce bone marrow mesenchymal stem cell apoptosis

In the myocardial infarction microenvironment, the effect of macrophages on the function of bone marrow mesenchymal stem cells (BMSCs) is unclear. In this study, we investigated the role of hypoxia/serum deprivation (H/SD)-induced M1-type macrophage-derived exosomes on BMSC viability, migration, and apoptosis. We found that H/SD reduced BMSC viability and migration, increased BMSC apoptosis, and induced macrophage polarization toward the M1 phenotype. BMSCs were cultured by the supernatant of H/SD-induced THP-1 cells (M1-type macrophages) with or without exosome inhibitor treatment. The results show that BMSC apoptosis is increased in the H/SD-induced THP-1 cell supernatant group and is decreased by GM4869 treatment, indicating that M1-type macrophages induce BMSC apoptosis through exosomes. In addition, we confirm that miR-222 plays an important role in promoting BMSC apoptosis by targeting B-cell lymphoma (Bcl)-2. M1-type macrophage-derived exosomes significantly decrease BMSC viability and migration and increase BMSC apoptosis, and these effects are partly abolished by a miR-222 inhibitor. Our findings suggest that under H/SD conditions, exosomes derived from M1-type macrophages can induce BMSC apoptosis by delivering miR-222 to BMSCs.Under conditions of hypoxia and serum deprivation, M1 macrophages secrete exosomes and transfer miR-222 to bone marrow mesenchymal stem cells (BMSC), which inhibits the expression of the antiapoptotic gene Bcl-2. This results in BMSC apoptosis and inhibition of mesenchymal stem cell proliferation and migration, which may affect the efficacy of BMSC in the treatment of acute myocardial infarction.

P–801 Effects of Lonomia obliqua venom components on human endometrial stromal cells: A potential source for new cytoprotective biomolecules against recurrent pregnancy loss

Abstract Study question Could new molecules like Lonomia obliqua lipocalins and hemolins have cytoprotective effects on endometrial stem cells (hESC)? Summary answer Lonomia obliqua venom-induced hESC viability, proliferation and migration occurred mainly by protection against oxidative damage and ERK-dependent pathway activation What is known already Recurrent pregnancy loss (RPL) is associated with severe physical and psychological morbidity, for which there is no treatment options. The pathophysiology involves deficiency in proliferation and migration capacities of endometrial stromal cells (hESCs) impairing embryo implantation and development. Animal venoms are rich sources of bioactive molecules and despite its known toxic effects, they also have protective components such as pro-proliferative molecules, growth factor-like, anti-apoptotic and anti-oxidant. Study design, size, duration This study was an experimental in vitro with endometrial stem cells. Treatment duration was 8–72h. Every assay had control cells exposed to phosphate buffered saline (PBS). Participants/materials, setting, methods hESCs were isolated from fresh human endometrial biopsies and characterized according standard protocols. Then the effects of L. obliqua venomous secretions on cell viability, proliferation and migration were determined using MTT, wound-healing assay, sulforhodamine B (SRB) assay and measuring the immunocontent of Ki67. Venom components involved in cell enhancing effects were also identified by classical chromatographic methods and proteomic analysis. Assays were conducted in triplicate. Main results and the role of chance The hESCs in culture showed adhesiveness properties, presented a fusiform fibroblastoid morphology and ability to in vitro differentiate into adipocytes, osteocytes and chondrocytes. The expression of cell surface markers was also characterized by flow cytometry. hESCs were positive for mesenchymal markers (CD105, CD90 and CD73) and negative for hematopoietic markers (CD45 and CD11), indicating that isolated cells have potential for multilineage differentiation. L. obliqua bristle extract (LOBE) increased dose-dependently hESCs viability in a concentration range varying from 0.001 to 0.1 µg/mL, independently of the cell isolation bath. For some cell isolates (patient ID 1, 3, 4, 6 and 7) it was observed a slightly reduction in hESC viability at highest LOBE concentrations (10 µg/mL). Treatment increased hESC viability in the presence of low concentrations of fetal bovine serum (1% FBS) and even in its complete absence. This effect was long lasting, being significant up to 72 h of incubation with LOBE in serum deprivation conditions. r to identify the potential molecules involved in the cytoprotective action, a mass spectrometry-based proteomic analysis was performed. It was identified a total number of 430 proteins in LOBE and 312 proteins in L. obliqua hemolymph. Limitations, reasons for caution This study was only conducted in vitro. Wider implications of the findings: In this work we reported the identification of at least six protein classes with cytoprotective properties through proteomic analysis and isolated one fraction enriched in this cytoprotective factors. L. obliqua secretions induced increase in hESCs viability, proliferation and migration mainly by the protection against oxidative damage and ERK-dependent pathway activation. Trial registration number Not applicable

Abstract 2322: Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer

Abstract Nutrient deprivation is one of the hallmark conditions of a rapidly growing tumor. Rapid proliferation of tumor cells leads to increased consumption of metabolites such as glucose and glutamine resulting in metabolic stress. However, phenotypic and functional status of tumor cells under these conditions is not clear. We hypothesize that nutrient deprivation-induced metabolic stress results in a breast cancer stem cell-like (BCSC) phenotype in triple negative breast cancer (TNBC). We found that serum-deprived conditions result in significant upregulation of GD2+ cells compared to serum rich conditions: from 9%±2% to 26%±5% in MDA-MB-231 and 7%±2% to 29%±5% in SUM159 cells. Interestingly, this affect was compensated when the cells were supplemented with serum-rich medium. In addition, a positive correlation was observed between GD2+ cell number and tumor volume in vivo, suggesting metabolic stress in the growing tumors results in GD2+ phenotype in TNBC cells. Next, TNBC cells cultured under glucose deprivation or in the presence of glycolysis inhibitor, 2-deoxy glucose (2DG) showed a 2-fold increase in GD2+ cells compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 expression. Nutrient deprived conditions also showed a significant increase in the expression of stress markers ATF4 and SESN2 demonstrating TNBC cells under metabolic stress. In addition, the above conditions also showed a 2.5-fold upregulation of the mammosphere formation in TNBC cells compared to nutrient rich conditions. Global metabolomics profiling identified glutathione biosynthesis to be most highly upregulated in GD2+ cells. Furthermore, we found that supplementation of glutamine (precursor for glutathione biosynthesis) increases the percentage of GD2+ cells. Carbon and nitrogen tracing using 13C- and 15N-labeled glutamine by mass spectrometry showed its direct contribution to GD2 biosynthesis. Treatment with glutamine transporter (ASCT2) inhibitor V9302 reduced GD2+ cell number by 70%-80% in TNBC cell lines and dramatically inhibited mammosphere and soft agar colony formation, and migration of TNBC cell lines. Mechanistic studies revealed that V9302 inhibits glutathione pathway and induces ROS levels in TNBC cells. Additionally, V9302 treatment inhibited AKT-ERK-mTOR-4E-BP1 pathway and combination of V9302 with paclitaxel synergizes TNBC cell killing. We also identified that, V9302 treatment induced ferroptosis (iron-dependent cell death) in TNBC cells. Finally, V9302 treatment significantly inhibited tumor growth (p&amp;lt;0.0001) in TNBC-PDX model. In conclusion, metabolic stress results in GD2+ BCSC phenotype in TNBC cells and glutamine directly contributes to GD2 biosynthesis. Targeting ASCT2 could complement conventional chemotherapy in TNBC. Citation Format: Appalaraju Jaggupilli, Stanley Ly, Khoa Nguyen, Roshan Borkar, Bin Yuan, Danthasinghe Waduge Piyarathna, Yuanqing Yan, Helen Piwnica-Worms, Henry Charles Manning, Michael Andreeff, Nagireddy Putluri, Lokesh Battula. Targeting glutamine transporter (ASCT2) inhibits metabolic stress-induced GD2+ cancer stem cell-like phenotype in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2322.

Ischemia Injury induces mPTP opening by reducing Sirt3

Mitochondrial permeability transition pore (mPTP) opening is critical to mitochondrial apoptosis during ischemic injury. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and cell death. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species. However, it is unclear the interaction between Sirt3 and mPTP and the roles they play in ischemic stroke. We used the middle cerebral artery occlusion (MCAO) model, a mouse model of stroke, to examine Sirt3 and mPTP-related protein levels. We then applied lentivirus packaged Sirt3 overexpression in HT22 cells, a mouse hippocampal neuronal cell line, to investigate the underlying mechanism. We found Sirt3 protein level was decreased in the penumbra area in MCAO mice, along with an increase in mPTP related proteins, namely voltage-dependent anion channel 1 (VDAC1) and adenine nucleotide translocator 1 (ANT1). Sirt3 overexpression suppressed the increase in VDAC1, ANT1 and cleaved caspase 3 that were induced by the serum and glucose deprivation (SGD) condition. Our studies suggest that ischemic injury induced mPTP opening and apoptosis by reducing Sirt3. It helps to identify new therapeutic targets for ischemic stroke.

Adhesion-growth factor crosstalk regulates AURKB activation and ERK signalling in re-adherent fibroblasts

Aurora kinases despite their similarity have distinct roles in the cell cycle, which is regulated by cell-matrix adhesion and growth factors. This study reveals loss of adhesion and re-adhesion to differentially regulate Aurora kinases. AURKB activation that drops on the loss of adhesion recovers on re-adhesion in serumdeprived conditions but not in the presence of serum growth factors. A rapid 30 min serum treatment of serumdeprived cells blocks the adhesion-dependent recovery of AURKB, which negatively correlates with Erk activation. AZD mediated inhibition of AURKB in serum-deprived re-adherent cells promotes Erk activation and membrane ruffling, comparable to presence of serum. These studies thus define a novel adhesion-growth factor-dependent regulation of AURKB that controls adhesion-dependent Erk activation in re-adherent fibroblasts.

Effect of M2 Macrophage-Derived Soluble Factors on Proliferation and Apoptosis of SH-SY5Y Cells.

Macrophages play the key role in the regulation of neuroregeneration. For evaluation of the neuroregenerative potential of M2 macrophages, we studied the effect of macrophages polarized with IL-4 (M2a (IL-4)) and by efferocytosis under conditions of serum deprivation (LS, Low Serum; M2(LS)) on proliferative activity and apoptosis of SH-SY5Y cells under conditions of deficiency of growth/serum factors. Conditioned media of both M2(LS) and M2a(IL-4) stimulated proliferation of SH-SY5Y cells. Moreover, soluble factors of M2(LS) and M2a(IL-4) reduced the degree of early apoptosis of SH-SY5Y cells and the protective effect of M2(LS) was observed at earlier terms of culturing. Our findings suggest that M2 macrophages have high neuroregenerative potential that is mediated through soluble factors and manifests itself both in stimulation of proliferation and inhibition of apoptosis of SH-SY5Y cells.

Human type 2 macrophages biologically active soluble products in the editing of stress-induced depressive-like behavior

IntroductionIn the scientific world widely discussed phenomenon of “cytokine-induced depression”. Macrophages have high plasticity and are able to control the inflammatory response; in particular, anti-inflammatory type-2 macrophages have a pronounced potential due to complex soluble factors production.ObjectivesWe have developed an original method for the type-2 macrophages generation; the resulting macrophages are characterized by the high level of a whole range of neurotrophic, neuroprotective, proangiogenic and anti-inflammatory factors production. The aim of the study was to investigate effects of human type-2 macrophages soluble products on behavioral phenotype and brain cytokines synthesis in depressive-like animals.MethodsType-2 macrophages were generated by culturing an adherent fraction of mononuclear cells with 50 ng/ml recombinant human GM-CSF in serum deprivation conditions for 7 days. (CBA x C57Bl/6)F1 depressive-like male mice, developed under the long-term social stress, were undergoing the human type-2 macrophages conditioned medium intranasal administration (60 ml twice daily for one animal) for 6 days. Mice behavioral phenotyping was carried out using an automatic registration system (Noldus Information Technology). Cytokines were determined by ELISA.ResultsDepressive-like mice behavioral phenotyping after type-2 macrophages conditioned medium administration revealed anhedonia decrease, motor activity stimulation in the open field and forced swimming tests, anxiety reduction in elevated plus maze. Behavioral changes were recorded against the pro-inflammatory cytokines (TNF-α, IL-1β, IL- 6, INFγ) decrease in striatum and hippocampus, as well as anti-inflammatory IL- 10 increase in hippocampus and hypothalamus.ConclusionsResults demonstrated the effectiveness of human type-2 macrophages biologically active soluble products in relation to the stress-induced depressive-like behavior editingDisclosureNo significant relationships.

Correlation between the expression of cancer stem cell marker BMI1 and glioma prognosis

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) appears to be essential for promoting certain types of cancer, and its inhibition effectively reduced the stemness of cancer cells. Therefore, this study aimed to investigate the potential role of BMI1 in glioma. To this end, we first investigated BMI1 expression in brain tumors using microarray datasets in ONCOMINE, which indicated that BMI1 levels were not commonly increased in clinical brain tumors. Moreover, survival plots in PROGgeneV2 also showed that BMI1 expression was not significantly associated with reduced survival in glioma patients. Interestingly, stressful serum deprivation and anchorage independence growth conditions led to an increased BMI1 expression in glioma cells. A stress-responsive pathway, HDAC/Sp1, was further identified to regulate BMI1 expression. The HDAC inhibitor vorinostat (SAHA) prevented Sp1 binding to the BMI1 promoter, leading to a decreased expression of BMI1 and attenuating tumor growth of TMZ-resistant glioma xenografts. Importantly, we further performed survival analysis using PROGgeneV2 and found that an elevated expression of HDAC1,3/Sp1/BMI1 but not BMI1 alone showed an increased risk of death in both high- and low-grade glioma patients. Thus, HDAC-mediated Sp1 deacetylation is critical for BMI1 regulation to attenuate stress- and therapy-induced death in glioma cells, and the HDAC/Sp1 axis is more important than BMI1 and appears as a therapeutic target to prevent recurrence of malignant glioma cells persisting after primary therapy.

Human Chorionic Gonadotropin-Mediated Induction of Breast Cancer Cell Proliferation and Differentiation.

Human chorionic gonadotropin (hCG) is a hormone that specifically binds to luteinizing hormone receptor (LHR) and exerts several roles, including the support of pregnancy and fetal gonadal steroidogenesis. Since hCG is also expressed by some tumor types, like breast cancer, many efforts have been made to study its role in neoplesia, with some studies showing a cancer-supportive role and others showing a cancer-protective role. A critical examination of the literature highlighted that the in vitro effect of hCG has been tested in the presence of fetal serum, which contains other gonadotropins, in the culture medium. Thus, we hypothesized that the use of serum in the cell culture medium might influence the cell response to the hCG treatment due to the presence of other hormones. Thus, we analyzed the in vitro effect of highly purified hCG on cell proliferation and the activation of the down-stream signal transduction pathway in three breast cancer cell lines, particularly focusing on MCF7, cultured in serum-deprived conditions. Our data show that hCG increases cell proliferation and activates the down-stream target Akt, together with a decrease of the LHR mRNA expression level. Finally, we also tested the differentiation capacity of hCG on MCF7 cancer stem cells (CSCs) and show that it favors the proliferation and differentiation of these cells, thus suggesting that hCG also renders cells more able to colonize and invade the organs.

Влияние растворимых факторов макрофагов М2-фенотипа на пролиферацию и апоптоз клеток линии SH-SY5Y

Macrophages play the key role in the regulation of neuroregeneration. For evaluation of the neuroregenerative potential of M2 macrophages, we studied the effect of macrophages polarized with IL-4 (M2a (IL-4)) and by efferocytosis under conditions of serum deprivation (LS, Low Serum; M2(LS)) on proliferative activity and apoptosis of SH-SY5Y cells under conditions of deficiency of growth/serum factors. Conditioned media of both M2(LS) and M2a(IL-4) stimulated proliferation of SH-SY5Y cells. Moreover, soluble factors of M2(LS) and M2a(IL-4) reduced the degree of early apoptosis of SH-SY5Y cells and the protective effect of M2(LS) was observed at earlier terms of culturing. Our findings suggest that M2 macrophages have high neuroregenerative potential that is mediated through soluble factors and manifests itself both in stimulation of proliferation and inhibition of apoptosis of SH-SY5Y cells.

Neuroprotective Potential of a Small Molecule RET Agonist in Cultured Dopamine Neurons and Hemiparkinsonian Rats.

Background:Parkinson’s disease (PD) is a progressive neurological disorder where loss of dopamine neurons in the substantia nigra and dopamine depletion in the striatum cause characteristic motor symptoms. Currently, no treatment is able to halt the progression of PD. Glial cell line-derived neurotrophic factor (GDNF) rescues degenerating dopamine neurons both in vitro and in animal models of PD. When tested in PD patients, however, the outcomes from intracranial GDNF infusion paradigms have been inconclusive, mainly due to poor pharmacokinetic properties.Objective:We have developed drug-like small molecules, named BT compounds that activate signaling through GDNF’s receptor, the transmembrane receptor tyrosine kinase RET, both in vitro and in vivo and are able to penetrate through the blood-brain barrier. Here we evaluated the properties of BT44, a second generation RET agonist, in immortalized cells, dopamine neurons and rat 6-hydroxydopamine model of PD.Methods:We used biochemical, immunohistochemical and behavioral methods to evaluate the effects of BT44 on dopamine system in vitro and in vivo.Results:BT44 selectively activated RET and intracellular pro-survival AKT and MAPK signaling pathways in immortalized cells. In primary midbrain dopamine neurons cultured in serum-deprived conditions, BT44 promoted the survival of the neurons derived from wild-type, but not from RET knockout mice. BT44 also protected cultured wild-type dopamine neurons from MPP+-induced toxicity. In a rat 6-hydroxydopamine model of PD, BT44 reduced motor imbalance and seemed to protect dopaminergic fibers in the striatum.Conclusion:BT44 holds potential for further development into a novel, possibly disease-modifying, therapy for PD.