AM Assay Research Articles

ATF5-Mediated Mitochondrial Unfolded Protein Response (UPRmt) Protects Neurons Against Oxygen-Glucose Deprivation and Cerebral Ischemia.

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved mitochondrial response that is critical for maintaining mitochondrial and energetic homeostasis under cellular stress after tissue injury and disease. Here, we ask whether UPRmt may be a potential therapeutic target for ischemic stroke. We performed the middle cerebral artery occlusion and oxygen-glucose deprivation models to mimic ischemic stroke in vivo and in vitro, respectively. Oligomycin and meclizine were used to trigger the UPRmt. We used 2,3,5-triphenyltetrazolium chloride staining, behavioral tests, and Nissl staining to evaluate cerebral injury in vivo. The Cell Counting Kit-8 assay and the Calcein AM Assay Kit were conducted to test cerebral injury in vitro. Inducing UPRmt with oligomycin protected neuronal cultures against oxygen-glucose deprivation. UPRmt could also be triggered with meclizine, and this Food and Drug Administration-approved drug also protected neurons against oxygen-glucose deprivation. Blocking UPRmt with siRNA against activating transcription factor 5 eliminated the neuroprotective effects of meclizine. In a mouse model of focal cerebral ischemia, pretreatment with meclizine was able to induce UPRmt in vivo, which reduced infarction and improved neurological outcomes. These findings suggest that the UPRmt is important in maintaining the survival of neurons facing ischemic/hypoxic stress. The UPRmt mechanism may provide a new therapeutic avenue for ischemic stroke.

Cysteine and glycine-rich protein 2 retards platelet-derived growth factor-BB-evoked phenotypic transition of airway smooth muscle cells by decreasing YAP/TAZ activity.

Cysteine and glycine-rich protein 2 (Csrp2) has emerged as a key factor in controlling the phenotypic modulation of smooth muscle cells. The phenotypic transition of airway smooth muscle cells (ASMCs) is a pivotal step in developing airway remodeling during the onset of asthma. However, whether Csrp2 mediates the phenotypic transition of ASMCs in airway remodeling during asthma onset is undetermined. This work aimed to address the link between Csrp2 and the phenotypic transition of ASMCs evoked by platelet-derived growth factor (PDGF)-BB in vitro. The overexpression or silencing of Csrp2 in ASMCs was achieved through adenovirus-mediated gene transfer. The expression of mRNA was measured by quantitative real-time-PCR. Protein levels were determined through Western blot analysis. Cell proliferation was detected by EdU assay and Calcein AM assays. Cell cycle distribution was assessed via fluorescence-activated cell sorting assay. Cell migration was evaluated using the scratch-wound assay. The transcriptional activity of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) was measured using the luciferase reporter assay. A decline in Csrp2 level occurred in PDGF-BB-stimulated ASMCs. Increasing Csrp2 expression repressed the PDGF-BB-evoked proliferation and migration of ASMCs. Moreover, increasing Csrp2 expression impeded the phenotypic change of PDGF-BB-stimulated ASMCs from a contractile phenotype into a synthetic/proliferative phenotype. On the contrary, the opposite effects were observed in Csrp2-silenced ASMCs. The activity of YAP/TAZ was elevated in PDGF-BB-stimulated ASMCs, which was weakened by Csrp2 overexpression or enhanced by Csrp2 silencing. The YAP/TAZ activator could reverse Csrp2-overexpression-mediated suppression of the PDGF-BB-evoked phenotypic switching of ASMCs, while the YAP/TAZ suppressor could dimmish Csrp2-silencing-mediated enhancement on PDGF-BB-evoked phenotypic switching of ASMCs. In summary, Csrp2 serves as a determinant for the phenotypic switching of ASMCs. Increasing Csrp2 is able to impede PDGF-BB-evoked phenotypic change of ASMCs from a synthetic phenotype into a synthetic/proliferative phenotype through the effects on YAP/TAZ. This work implies that Csrp2 may be a key player in airway remodeling during the onset of asthma.

Lectin-anticancer peptide fusion demonstrates a significant cancer-cell-selective cytotoxic effect and inspires the production of "clickable" anticancer peptide in Escherichia coli.

Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant in cancer cells can be valuable new tools for selective cancer cell killing. The lectin Shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in Escherichia coli, isolated them by Gb3-affinity chromatography, and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labeled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid Nε -((2-azidoethoxy)carbonyl)-L-lysine (AzK) at a selected position by amber stop codon suppression. The R-DIM-P-LF11-215 20AzK and the unlabeled R-DIM-P-LF11-215 parent peptide were produced as GST-fusion proteins for soluble expression in E. coli for the first time. We purified both variants by size-exclusion chromatography and analyzed their peptide masses. Finally, a cyanin 3 fluorophore was covalently conjugated to R-DIM-P-LF11-215 20AzK by strain-promoted alkyne-azide cycloaddition. Our results showed that the recombinant lectin-peptide fusion R-DIM-P-LF11-215-Stx1B killed >99% Gb3+ HeLa cells while Gb3-negative cells were unaffected. The peptides R-DIM-P-LF11-215 and R-DIM-P-LF11-215 20AzK were produced recombinantly in E. coli in satisfactory amounts and were tested functional by cytotoxicity and cell-binding assays, respectively.

Arctiin attenuates iron overload‑induced osteoporosis by regulating the PI3K/Akt pathway.

Iron overload is a prevalent pathological factor observed among elderly individuals and those with specific hematological disorders, and is frequently associated with an elevated incidence of osteoporosis. Although arctiin (ARC) has been shown to possess antioxidant properties and the ability to mitigate bone degeneration, its mechanism of action in the treatment of iron overload‑induced osteoporosis (IOOP) remains incompletely understood. To explore the potential molecular mechanisms underlying the effects of ARC, the MC3T3‑E1 cell osteoblast cell line was used. Cell Counting Kit was used to assess MC3T3‑E1 cell viability. Alkaline phosphatase staining and alizarin red staining were assessed for osteogenic differentiation. Calcein AM assay was used to assess intracellular iron concentration. In addition, intracellular levels of reactive oxygen species (ROS), lipid peroxides, mitochondrial ROS, apoptosis rate and mitochondrial membrane potential changes in MC3T3‑E1 cells were examined using flow cytometry and corresponding fluorescent dyes. The relationship between ARC and the PI3K/Akt pathway was then explored by western blotting and immunofluorescence. In addition, the effects of ARC on IOOP was verified using an iron overload mouse model. Immunohistochemistry was performed to evaluate expression of osteogenesis‑related proteins. Micro-CT and H&E were used to analyze bone microstructural parameters and histomorphometric indices in the bone tissue. Notably, ARC treatment reversed the decreased viability and increased apoptosis in MC3T3‑E1 cells originally induced by ferric ammonium citrate, whilst promoting the formation of mineralized bone nodules in MC3T3‑E1 cells. Furthermore, iron overload induced a decrease in the mitochondrial membrane potential, augmented lipid peroxidation and increased the accumulation of ROS in MC3T3‑E1 cells. ARC not only positively regulated the anti‑apoptotic and osteogenic capabilities of these cells via modulation of the PI3K/Akt pathway, but also exhibited antioxidant properties by reducing oxidative stress. Invivo experiments confirmed that ARC improved bone microarchitecture and biochemical parameters in a mouse model of iron overload. In conclusion, ARC exhibits potential as a therapeutic agent for IOOP by modulating the PI3K/Akt pathway, and via its anti‑apoptotic, antioxidant and osteogenic properties.

Mera: A scalable high throughput automated micro-physiological system.

There is an urgent need for scalable Microphysiological Systems (MPS's)1 that can better predict drug efficacy and toxicity at the preclinical screening stage. Here we present Mera, an automated, modular and scalable system for culturing and assaying microtissues with interconnected fluidics, inbuilt environmental control and automated image capture. The system presented has multiple possible fluidics modes. Of these the primary mode is designed so that cells may be matured into a desired microtissue type and in the secondary mode the fluid flow can be re-orientated to create a recirculating circuit composed of inter-connected channels to allow drugging or staining. We present data demonstrating the prototype system Mera using an Acetaminophen/HepG2 liver microtissue toxicity assay with Calcein AM and Ethidium Homodimer (EtHD1) viability assays. We demonstrate the functionality of the automated image capture system. The prototype microtissue culture plate wells are laid out in a 3×3 or 4×10 grid format with viability and toxicity assays demonstrated in both formats. In this paper we set the groundwork for the Mera system as a viable option for scalable microtissue culture and assay development.

Cellular responses to 8-methyl nonanoic acid, a degradation by-product of dihydrocapsaicin, in 3T3-L1 adipocytes

BackgroundCapsaicinoids, such as dihydrocapsaicin (DHC), exert the health-promoting effects of chili peppers on energy metabolism. The metabolic responses to capsaicinoids are primarily mediated through transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the varying contributions of their metabolites to beneficial health outcomes remain unclear. 8-methyl nonanoic acid (8-MNA), a methyl-branched medium chain fatty acid (MCFA), is an in vivo degradation by-product of DHC. Since MCFAs have emerged as metabolic modulators in adipocytes, here we examined various cellular responses to 8-MNA in 3T3-L1 adipocytes.MethodsThe viability of 3T3-L1 adipocytes exposed to various concentrations of 8-MNA was assessed by the Calcein AM assay. Biochemical assays for lipid accumulation, AMP-activated protein kinase (AMPK) activity, lipolysis and glucose uptake were performed in 3T3-L1 adipocytes treated with 8-MNA during 48-h nutrient starvation or 5-day maturation.Results8-MNA caused no impact on cell viability. During nutrient starvation, 8-MNA decreased lipid amounts in association with AMPK activation, a molecular event that suppresses lipogenic processes. Moreover, 3T3-L1 adipocytes that were treated with 8-MNA during 5-day maturation exhibited a reduced lipolytic response to isoproterenol and an increased glucose uptake when stimulated with insulin.ConclusionsThese results suggest that 8-MNA derived from DHC modulates energy metabolism in adipocytes and also support the idea that the metabolic benefits of chili consumption are partly attributable to 8-MNA.

S-27-4: OXIDIZED LDL POTENTIATES ANGIOTENSIN II-INDUCED GALPHAQ ACTIVATION THROUGH THE AT1-LOX1 RECEPTOR COMPLEX IN KIDNEY CELLS

Objective: We have reported that the binding of oxidized LDL (oxLDL) to its receptor, LOX-1 activates the G protein-coupled angiotensin II(AII) type 1 receptor (AT1) through membrane complex of the receptors. oxLDL-induced activation of AT1 is characterized by the beta-arrestin-biased signaling with no activation of Galphaq, the main effector of AII-induced cellular response (2021 iScience). In this study, we performed cell experiments to investigate how the co-treatment of oxLDL and AII activates AT1 particularly by focusing on Galphaq-dependent pathway. Design and Methods: We used CHO-cells stably transfected with AT1 (CHO-AT1), LOX-1 and AT1 (CHO-LOX-1-AT1) and two rat kidney cells (NRK49F and NRK52E). Activation of Galphaq and the subsequent induction of Ca influx were quantified by accumulation of IP1 and Fura-2 AM assay, respectively. We also utilized Galphaq-BRET biosensor vector to detect the activity of Galphaq. We assessed mRNA expression of genes associated with oxidative stress, inflammation, and fibrosis in NRK49F and NRK52E. We also assessed protein expression of alphaSMA as a molecular marker of epithelial mesanchymal transition (EMT) after 3 days of the indicated stimulation in NRK49F and NRK52E.The proliferative activity was measured using BrdU assay in NRK49F. Results: The activation of Galphaq was not observed by oxLDL treatment alone but was enhanced by the co-treatment of oxLDL and AII compared to AII alone in CHO-LOX-1-AT1 but not in CHO-AT1. Ca influx was induced not by oxLDL alone but by the co-treatment of oxLDL and a low concentration of AII that was insufficient to induce Ca influx when treated alone in CHO-LOX-1-AT1. We also found that the activation of Galphaq and the induction of Ca influx were detected by the co-treatment of oxLDL and AII but not by single treatment in rat kidney cells. These phenomenons were inhibited by ARB, a Gq inhibitor or the siRNA knockdown of AT1 or LOX-1. Furthermore, combination treatment enhanced the expression of the majority of genes associated with renal injury compared to the single treatment, and EMT was induced only by the combination treatment. These phenomenons were inhibited either by ARB or the Gq inhibitor. The combination treatment enhanced the proliferative activity of kidney fibroblasts compared with single treatment, that was inhibited by ARB, the Gq inhibitor or the siRNA knockdown of AT1 or LOX-1. Conclusions: These findings suggest the synergistic role of the AT1-LOX-1 interaction whereby oxLDL potentiates Ang II-induced Galphaq signaling, leading to the enhancement of renal injury-associated cellular response.

Abstract P115: Oxidized Ldl Potentiates Angiotensin Ii-induced gαQ Activation Through The At1-lox1 Receptor Complex In Kidney Cells

Introduction: In this study, we performed cell experiments to investigate how the co-treatment of oxLDL and AII activates AT1 particularly by focusing on Gαq-dependent pathway. Methods: We used CHO-cells stably transfected with AT1 (CHO-AT1), LOX-1 and AT1 (CHO-LOX-1-AT1) and two rat kidney cells (NRK49F and NRK52E). Activation of Gαq and the subsequent induction of Ca influx were quantified by accumulation of IP1 and Fura-2 AM assay, respectively. We also assessed protein expression of αSMA as a molecular marker of epithelial mesanchymal transition (EMT) after 3 days of the indicated stimulation in NRK49F and NRK52E.The proliferative activity was measured using BrdU assay in NRK49F. Results: The activation of Gαq was not observed by oxLDL treatment alone but was enhanced by the co-treatment of oxLDL and AII compared to AII alone in CHO-LOX-1-AT1 but not in CHO-AT1. Ca influx was induced not by oxLDL alone but by the co-treatment of oxLDL and a low concentration of AII that was insufficient to induce Ca influx when treated alone in CHO-LOX-1-AT1. We also found that the activation of Gαq and the induction of Ca influx were detected by the co-treatment of oxLDL and AII but not by single treatment in rat kidney cells. These phenomenons were inhibited by ARB, a Gq inhibitor or the siRNA knockdown of AT1 or LOX-1. Furthermore, the EMT was induced only by the combination treatment compared to the single treatment. These phenomenons were inhibited either by ARB or the Gq inhibitor. The combination treatment enhanced the proliferative activity of kidney fibroblasts compared with single treatment, that was inhibited by ARB, the Gq inhibitor or the siRNA knockdown of AT1 or LOX-1.. Conclusions: These findings suggest the synergistic role of the AT1-LOX-1 interaction whereby oxLDL potentiates Ang II-induced Gαq signaling, leading to the enhancement of renal injury-associated cellular response.

414.6: Porcine ULBP1 Disruption Does Not Significantly Reduce Human NK Cell Activation and Cytotoxicity in an in Vitro Model

Background: Pig-to-human xenotransplantation has the potential to address the critical organ shortage. Genetically-engineered pig organs have significantly prolonged survival in human and nonhuman primates by overcoming hyperacute rejection. Human natural killer (NK) cell-mediated acute xenograft rejection of porcine endothelial cells has been established. Porcine UL-Binding Protein (ULBP1) has been identified as a porcine ligand of human NK cell activating receptor NKG2D; interactions presumably lead to NK cell activation and ultimately porcine cell cytotoxicity. We sought to improve pig-to-human compatibility by eliminating ULBP1 ligand in an immortalized porcine liver-derived endothelial cell line (ipLDEC) with five-gene knockout (5GKO). The 5GKO cell line has mutations in GGTA1/CMAH/β4galNT2/SLA-I α-chain/β2M: three genes encoding enzymes responsible for xenoantigen production and two genes encoding swine leukocyte antigen class I. Methods: CRISPR/Cas9 technology was used to knockout the porcine ULBP1 gene in the 5GKO cell line to create a ULBP1-KO/5GKO cell line. Human peripheral blood mononuclear cells (PBMCs) from four donors were activated with human IL-2 for five days. Human PBMCs were co-cultured with 5GKO, ULBP1-KO/5GKO, and Wild-Type (WT) ipLDECs. Cells were stained with antibodies to CD45, CD3, CD56, and CD107a. Flow cytometry analysis was gated on the CD3-CD56+ NK cell population and monitored for expression of CD107a, a marker of NK cell degranulation. Calcein AM cytotoxicity assay was performed to determine human NK cell-mediated ipLDECs cell death. WT, 5GKO, ULBP1-KO/5GKO, HLA-G+/5GKO (positive control) were incubated in a calcein solution for 30 minutes at a concentration of 106 cells/mL. Stained porcine cells were co-cultured with activated human PBMCs at a 10:1 E:T ratio for 4 hours. Co-culture supernatant was harvested and calcein release was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm to calculate cytotoxicity percentages. Results: Porcine ULBP1 gene mutation was confirmed by Sanger sequencing (Figure 1a), establishing a porcine ULBP1-KO/5GKO cell line. There is no statistically significant difference in NK cell activation between co-cultures of human PBMCs (n=4) with pULBP1-KO/5GKO and 5GKO ipLDEC using CD107a. (Figure 1b). There is no statistically significant difference in NK-induced cytotoxicity between co-cultures of human PBMCs (n=6) with porcine ULBP1-KO/5GKO and 5GKO cell lines using Calcein AM assays (Figure 1c). As expected, HLA-G+/5GKO cytotoxicity was decreased (p = 0.0168) compared to WT (Figure 1c). Conclusion: We established a porcine ULBP1-KO/5GKO cell line. Our studies demonstrate no statistically significant difference in NK cell activation and NK cell-mediated cytotoxicity between 5GKO and ULBP1-KO/5GKO cell lines. Our studies show that ULBP1 is not a crucial ligand for human NKG2D. Future studies will target different human NK cell activating porcine ligands.

Decellularized Human Umbilical Tissue-Derived Hydrogels Promote Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells

Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissue. Appropriate three-dimensional scaffolding materials that mimic cartilage are ideal for the repair of chondral defects. The emerging decellularized tissue-based scaffolds have the potential to provide essential biochemical signals and structural integrity, which mimics the natural tissue environment and directs cellular fate. Umbilical cord-derived hydrogels function as 3D scaffolding material, which support adherence, proliferation, migration, and differentiation of cells due to their similar biochemical composition to cartilage. Therefore, the present study aimed to establish a protocol for the formulation of a hydrogel from decellularized human umbilical cord (DUC) tissue, and assess its application in the proliferation and differentiation of UC-MSCs along chondrogenic lineage. The results showed that the umbilical cord was efficiently decellularized. Subsequently, DUC hydrogel was prepared, and in vitro chondral differentiation of MSCs seeded on the scaffold was determined. The developed protocol efficiently removed the cellular and nuclear content while retaining the extracellular matrix (ECM). DUC tissue, pre-gel, and hydrogels were evaluated by FTIR spectroscopy, which confirmed the gelation from pre-gel to hydrogel. SEM analysis revealed the fibril morphology and porosity of the DUC hydrogel. Calcein AM and Alamar blue assays confirmed the MSC survival, attachment, and proliferation in the DUC hydrogels. Following seeding of UC-MSCs in the hydrogels, they were cultured in stromal or chondrogenic media for 28 days, and the expression of chondrogenic marker genes including TGF-β1, BMP2, SOX-9, SIX-1, GDF-5, and AGGRECAN was significantly increased (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Moreover, the hydrogel concentration was found to significantly affect the expression of chondrogenic marker genes. The overall results indicate that the DUC-hydrogel is compatible with MSCs and supports their chondrogenic differentiation in vitro.

Immunomodulatory Effects by Photodynamic Treatment of Glioblastoma Cells In Vitro.

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

P88. FAT GRAFT PROCESSING USING THEREVOLVE ENVI™ SYSTEM VERSUS DECANTATION: IN VITRO PROPERTIES AND TISSUE QUALITY

PURPOSE: Evaluate the in vitro properties and tissue quality of fat grafts using the REVOLVE ENVI™ 600 system (Allergan Aesthetics, an AbbVie Company) compared with decantation and evaluate the properties of ENVI waste. METHODS: Lipoaspirate (N=6 patients) was aliquoted and processed using ENVI or decantation. The volume composition of each layer (fat content, free oil, aqueous, cellular debris), hematocrit content, particle size, fat cell viability (calcein AM assay), and activity (lipolysis assay) were analyzed. Stromal vascular fraction (SVF) was analyzed for CD45−/CD31−/CD34+ cells and colony-forming units (CFU). RESULTS: ENVI grafts had a higher mean (±SD) fat content than decanted grafts (85.6%±6.1% vs 72.1%±4.0%, respectively; p<0.001); ENVI waste contained primarily aqueous fluid with negligible viable fat. ENVI grafts had significantly lower contaminants (free oil and cellular debris; p<0.001), as well as lower hematocrit content than decanted grafts (0.22±0.41 vs 5.18±6.88 optical density [OD]) or ENVI waste (2.81±2.74 OD). ENVI grafts contained a significantly greater population of large-particle fat globules than decanted grafts or ENVI waste (p<0.001), with the percentage of tissue particles >1000 µm being highest for ENVI (ENVI: 61.6%±9.2%; decantation: 52.5%±13.4%; ENVI waste: 0.49%±1.50%), and the percentage of particles <200 µm being lowest for ENVI (ENVI: 15.7%±2.6%; decantation: 32.2%±8.9%; ENVI waste: 97.9%±4.5%). Relative to decantation, ENVI produced 145.2%±36.0% (p=0.01) more viable adipocytes with 145.7%±46.2% (p=0.04) greater activity, 195.5%±104.2% (p=0.24) more CD45−/CD31−/CD34+ cells, and 363.5%±161.2% (p=0.04) more SVF CFU/cc of graft. CONCLUSION: Fat grafts processed using the ENVI system demonstrated greater viability and better fat quality than those processed by decantation.

BODIPY-Tagged Platinum(II) Curcumin Complexes for Endoplasmic Reticulum-Targeted Red Light PDT.

[Pt(RB)(Cur)]NO3 (RBC), [Pt(IRB)(Cur)]NO3 (IRBC), and [Pt(L)(Cur)]NO3 (PBC), where HCur is curcumin, L is 1-benzyl-2-(2-pyridyl)benzimidazole, and RB and IRB are red-light-active non-iodo and diiodo-BODIPY tagged to L, respectively, were synthesized and characterized, and their anticancer activities were studied (BODIPY, boron-dipyrromethene). RBC and IRBC displayed BODIPY-centered absorption bands within 615-635 nm along with the respective curcumin bands at 445 and 492 nm in 10% dimethyl sulfoxide (DMSO)-Dulbecco's phosphate-buffered saline (DPBS). Emission bands were observed at 723 and 845 nm for RBC and IRBC, respectively, in 10% DMSO-DPBS. RBC (ΦΔ, 0.27) and IRBC (ΦΔ, 0.40) generated singlet oxygen in red light (λ = 642 nm) as evidenced from 1,3-diphenylisobenzofuran (DPBF) titrations. The formation of 1O2 from BODIPY and HO• from the curcumin was evidenced from the mechanistic pUC19 DNA photocleavage studies. The BODIPY complexes showed photocytotoxicity in A549, HeLa, and MDA-MB-231 cells while being less toxic in the dark [IC50: 1.3-6.9 μM, red light; 7.2-12.8 μM, 400-700 nm visible light]. The emissive RBC displayed localization in the endoplasmic reticulum (ER). Apoptotic cell death was evidenced from the Annexin-V/fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay and green fluorescence in red light in the Fluo-4 AM assay due to ER stress, and mitochondrial dysfunction was evidenced from the 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay in A549 cells.

Cetylpyridinium chloride inhibits human breast tumor cells growth in a no-selective way.

Analyze the antitumor capacity of cetylpyridinium chloride (CPC) on human breast tumor cells, and the possible action mechanism. The human breast tumor cells MCF-7 and no-tumor breast cells MCF-10A were exposed to CPC under various condition (concentration and duration). Cell viability was measured with MTT assay, the LIVE/DEAD assay, and fluorescence microscopy. Membrane permeability after CPC exposure was evaluated by Calcein AM assay, mitochondrial morphology with a MitoView staining, and genotoxicity with the comet assay and fluorescence microscopy. CPC was cytotoxic to both MCF-7 and MCF-10A as of a 24-h exposure to 0.1 µM. Cytotoxicity was dose-dependent and reached 91% for MCF-7 and 78% for MCF-10A after a 24-h exposure to 100 µM CPC, which outperformed the positive control doxorubicin in effectiveness and selectivity. The LD50 of CPC on was 6 µM for MCF-7 and 8 µM for MCF-10A, yielding a selectivity index of 1.41. A time response analysis revealed 64% dead cells after only 5 min of exposure to 100 µM CPC. With respect to the action mechanisms, the comet assay did not reveal genome fragmentation. On the other hand, membrane damage was dose-dependent and may also affect mitochondrial morphology. Cetylpyridinium chloride inhibits MCF-7 cell growing in a non-selective way as of 5 min of exposure. The action mechanism of CPC on tumor cells involves cell membrane damage without change neither mitochondrial morphology nor genotoxicity.

Evodiamine induces ROS-Dependent cytotoxicity in human gastric cancer cells via TRPV1/Ca2+ pathway

Evodiamine (EVO), a key active ingredient of the fruit of Evodiae fructus, is provided with antitumor effects (mainly cytotoxic effect) including proliferation inhibition, cell cycle arrest, apoptosis, and metastasis inhibition. Our study aims to explain the underlying role of TRPV1/Ca2+ in EVO-induced cytotoxicity in human gastric cancer cells. Human gastric cancer line BGC-823 was used to study EVO-induced cytotoxicity. Cell viability was examined using CCK-8 assay. Apoptosis was examined using Annexin V-FITC/PI staining assay. Intracellular ROS ([ROS]i) levels were examined using DCFH-DA assay. Mitochondrial morphology was examined using Mitotracker Green staining. Mitochondrial membrane potential (Δψm) were examined using JC-1 assay. Intracellular Ca2+ levels ([Ca2+]i) were examined using Fluo-4 AM assay. Mitochondrial ROS ([ROS]m)levels were examined using Mitotracker Green/MitoSOX Red staining. Mitochondrial Ca2+ ([Ca2+]m)levels were examined using Mitotracker Green/Rhod-2 Red staining. The protein levels was detected by Western blot. EVO exposure causes significant ROS generation and apoptotic cell death. Pretreatment of EUK134 significantly ameliorated EVO-induced apoptotic cell death. Furthermore, EVO exposure induced [ROS]i generation and mitochondrial dysfunction, including [ROS]m generation and Δψm dissipation, which can be significantly attenuated by pre-incubation of rotenone indicating that [ROS]m is the main source of EVO-induced intracellular ROS generation. Importantly, EVO-induced cytotoxicity was significantly ameliorated by intracellular Ca2+ chelation, confirming that EVO induces cell death through Ca2+ overload. Pharmacological and genetic inhibition of TRPV1 could significantly attenuate Ca2+ influx, ROS generation and apoptotic cell death induced by EVO exposure, while exogenous TRPV1 overexpression could augment the EVO-induced cytotoxicity. Moreover, genetic inhibition of mitochondrial calcium uniporter (MCU) attenuated EVO-induced cell death and mitochondrial dysfunction. EVO exposure induced endoplasmic reticulum (ER) stress demonstrated by the activation of PERK/CHOP in cells exposed to EVO, and PERK/CHOP activation was depleted by EUK134 pre-treatment. Our results support the concept that EVO induces ROS-dependent cytotoxicity via TRPV1/Ca2+ Pathway.

Abstract 1404: Loss of adenomatous polyposis coli induces DOX resistance through upregulation of ABC transporters

Abstract Chemoresistance is a leading cause of breast cancer related deaths. Therefore, understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement improving patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is lost in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we were the first to show that APC loss decreased doxorubicin (DOX) induced apoptosis. Therefore, we examined the mechanisms contributing to DOX resistance with APC loss to identify combination therapy options. We previously made the novel observation that APC loss in MMTV-PyMT;ApcMin/+ cells activated signal transducer and activator of transcription 3 (STAT3) thereby increasing the expression of the drug efflux pump, multidrug resistance protein 1 (MDR1). ATP-binding cassette (ABC) transporters, such as MDR1, are one of the most well-known contributors to drug resistance. These ABC transporters are capable of exporting drugs from the cell preventing apoptosis. Decreased intracellular DOX accumulation was observed in APC-deficient cells suggesting enhanced drug efflux. To investigate this decreased intracellular DOX accumulation, we measured the expression and activity of the drug export pumps, MDR1 and multidrug resistance protein 1 (MRP1). APC-deficient cells also have increased expression of MRP1. We used a calcein incorporation assay and demonstrated that APC loss increased MDR1 activity, which was restored by an MDR1 inhibitor. In addition, MDR1 inhibition sensitized the MMTV-PyMT;ApcMin/+ cells to DOX-mediated apoptosis. MRP1 inhibition through genetic manipulation will be used to assess MRP1 activity using the calcein AM assay and its contribution to DOX resistance. MDR1 and MRP1 inhibition will also be used to restore intracellular DOX accumulation. Future studies including evaluating human tumor protein lysates will for APC, MDR1, and MRP1 expression. Correlation studies will be used to determine whether loss of APC correlates with either high expression of MDR1 or MRP1. Taken together, APC loss mediates DOX resistance via exporting DOX demonstrating the potential use of combination therapy to overcome chemoresistance. Citation Format: Casey D. Stefanski, Jenifer R. Prosperi, Lauren Milac. Loss of adenomatous polyposis coli induces DOX resistance through upregulation of ABC transporters [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 1404.

Targeting CD38 with Daratumumab Suppresses B-Cell Receptor Signaling and Enhances the Activity of Ibrutinib in Waldenstrom Macroglobulinemia Cells

Introduction: Waldenstrom macroglobulinemia (WM) is characterized by malignant B-lymphoplasmacytic cells that rely on B-cell receptor (BCR) signaling, which is clinically exploited with ibrutinib (Ibr). Although effective, Ibr tx. mostly results in partial remission of disease. This signifies the need to examine combination tx. strategies that can lead to superior disease eradication and counteract onset of Ibr resistance. WM cells express several surface antigens, including CD38 and Daratumumab (Dara) is a first-in-class monoclonal antibody that binds CD38; inducing an immune effector-mediated cytolytic reaction. CD38 also functions as a molecular hub for integration of signals transmitted through the B-cell receptor complex (BCR) to promote cell proliferation. We hypothesized that cotargeting BTK (with Ibr) and CD38 (with Dara) would induce both an immune-mediated and cell intrinsic mode of death to result in enhanced WM cell kill.Methods: Wildtype WM cell lines (BCWM.1, RPCI.WM1) and ibrutinib-resistant (IR) subclones were used in experiments. CD38 MFI and surface antibody binding (sAbc) was determined by flow cytometry. Ibr (1uM), Dara (0.1ug/mL) or Ibr + Dara-mediated death through antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) was measured using the Calcein AM assay at an effector to target (E:T) ratio of 50:1. Antibody-dependent cellular phagocytosis (ADCP) was assessed using human macrophages at an E:T ratio of 2:1. An IgG1 isotype Ab was used as a control in experiments.Results: CD38 sAbc in BCWM.1 and RPCI-WM1 cells was 4864 and 35892, respectively; consistent with previously noted MFI's (Paulus et al PLOS One, 2016). Specific lysis induced by single agent Dara in wildtype WM cells, showed a mean ADCC of 24.46%, CDC: 20.29% and ADCP: 20.12%. Dara can also induce apoptosis and we noted an average of 33.35% annexin-V/PI+ WM cells after 24h Dara tx. We surmised the anti-WM activity of Dara could be enhanced by Ibr as 1.) the BTK inhibitor has been shown to improve anti-tumor T-cell responses and 2.) the combined effects of each drug could synergistically attenuate BCR-associated signaling. As anticipated, mean ADCC, CDC, ADCP and apoptosis increased to 52.7%, 38.41%, 30.19% and 57.53%, respectively in Ibr + Dara-treated wildtype WM cells. While potential mechanisms of the enhanced immune-mediated specific lysis from the combination tx. are being investigated, we observed cell signaling changes that may account for the increased cell death. As expected, Dara decreased pLyn, pSyk, pBTK, pPLCG2 and pERK as well as pAKT, pmTOR and pS6K levels in WM cells. And these proteins decreased further in Ibr + Dara treated cells. Next, we asked the question whether Dara +/- Ibr would be active in IR WM cells, which had lower CD38 sAbc (mean 8400, range 1024 - 15776). Mean ADCC by Dara in IR WM cells was 19.52%, CDC: 31.85%, ADCP: 27.5% and apoptosis at 31.3%. While ADCC was expectedly lower in IR vs WT cells, CDC and ADCP were significantly higher. We then tested the combination of Ibr + Dara in IR WM cells and noted greater ADCC (63.97%), CDC (60.34%) and apoptosis (60.23%), whereas ADCP did not increase (29.45%). We also analyzed BCR-associated protein expression and noted single agent Dara decreased pBTK, pPLCG2, pERK and pS6K, whereas the effects of Ibr + Dara were more cell line specific. As IR WM subclones are less reliant on BTK/BCR-mediated signaling, cell death from Dara (+/- Ibr) may be due to other mechanisms possibly related to alterations in the mitochondria (intrinsic to IR cells, Paulus et al BCJ 2017).Conclusion: We demonstrate in vitro activity of Dara and Ibr in WM cells, showing that 1.) Dara induces cell death through various mechanisms (ADCC, CDC, ADCP and apoptosis); 2.) while higher CD38 sAbc appears to correlate with greater ADCC, this was not the case for CDC, ADCP or apoptosis; 3.) Dara significantly modulates BCR-associated signaling; an effect preserved in IR WM cells and documented for the first time and 4.) the combination tx. exerts significantly more WM cell death in nearly all the aforementioned assays than either single agent alone and in some cases more so in IR WM cells. With further preclinical results supporting these data, to be shared at the conference, our investigations provide rationale for clinical testing of Ibr + Dara in WM. DisclosuresSher:LAM Therapeutics, Inc: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria.

Cypermethrin induces Sertoli cell apoptosis through mitochondrial pathway associated with calcium.

Cypermethrin, one kind of pyrethroid pesticides, has been shown to act as endocrine-disrupting chemicals (EDCs). The purpose of this study was to explore the roles of Sertoli cell apoptosis through mitochondrial pathway associated with calcium (Ca2+) in cypermethrin-induced male reproductive toxicology. The mouse Sertoli cells TM4 were cultured with 0μM, 10μM, 20μM, 40μM and 80μM of cypermethrin. We used flow cytometry, Fluo-4 AM, western blot and JC-1 Assay Kit to examine apoptosis, intracellular Ca2+, expressions of mitochondrial apoptotic pathway-related proteins and mitochondrial membrane potential. We found cypermethrin increased apoptosis rate of TM4 cells significantly and with a significant increase in intracellular Ca2+ concentration. Cypermethrin significantly decreased the protein expressions of cytosolic B-cell lymphoma-2 (Bcl-2) and mitochondrial cytochrome c (Cyt-c). The protein expressions of cytosolic Bcl-2-associated x (Bax), Cyt-c, cleaved caspase-3, calmodulin (CaM), Ca2+/CaM-dependent protein kinases II (CaMKII) and phosphorylated CaMKII were increased significantly in cypermethrin-exposed TM4 cells. Cypermethrin decreased mitochondrial membrane potential significantly. Then, Bcl-2 family and Ca2+/CaM/CaMKII pathway participate in cypermethrin-induced homeostasis. Ca2+ overload activates mitochondrial pathway by increasing permeability of mitochondrial membrane and decreasing mitochondrial membrane potential. We suggest cypermethrin induces Sertoli cell apoptosis involving mitochondrial pathway associated with Ca2+ regulated by Bcl-2 family and Ca2+/CaM/CaMKII pathway. The study provides a new insight into mechanisms involved in cypermethrin-induced male reproductive toxicology.

BOUNDING MIDDLE PERIOD CEMETERY USE IN SAN PEDRO DE ATACAMA, CHILE

ABSTRACTThe San Pedro de Atacama oases, located in northern Chile’s hyperarid Atacama Desert, have been occupied for at least 3000 years. Here, we examine cemetery use in the oases, with emphasis on the Middle Period (ca. AD 400–1000). By modeling of a large corpus (n=243) of radiocarbon dates, over 90% of which are direct AMS assays of human bone collagen, we attempt to establish a temporal framework by which to explore the establishment of formalized social inequality in this period. Modeling of these dates at three locally defined scales (allayllus, inter-ayllu, and intra-ayllu) permit heretofore unavailable insights into the chronological and spatial dimensions of life and mortuary activity in the oases and allow us to better contextualize patterns of social inequality during the dynamic Middle Period. The results of this modeling indicate two distinct peaks of occupation during the Middle Period in San Pedro and document significant temporal variability in cemetery use patterns on both inter- and intra-aylluscales. These results stress the importance of local social and environmental factors to the occupation of the oases and provide crucial chronological structure for future archaeological and bioarchaeological research in the region.

Chronic GPER1 Activation Protects Against Oxidative Stress-Induced Cardiomyoblast Death via Preservation of Mitochondrial Integrity and Deactivation of Mammalian Sterile-20-Like Kinase/Yes-Associated Protein Pathway.

Introduction: Estrogen (17β-estradiol, E2) is well-known to induce cardioprotective effects against ischemia/reperfusion (I/R) injury. We recently reported that acute application of E2 at the onset of reperfusion in vivo induces cardioprotective effects against I/R injury via activation of its non-steroidal receptor, G protein-coupled estrogen receptor 1 (GPER1). Here, we investigated the impact and mechanism underlying chronic GPER1 activation in cultured H9c2 rat cardiomyoblasts.Methods: H9c2 rat cardiomyoblasts were cultured and pretreated with the cytotoxic agent H2O2 for 24 h and incubated in the presence of vehicle (control), GPER1 agonists E2 and G1, or GPER1 agonists supplemented with G15 (GPER1 antagonist) for 48 or 96 h. After treatment, cells were collected to measure the rate of cell death and viability using flow cytometry and Calcein AM assay or MTT assay, respectively. The resistance to opening of the mitochondrial permeability transition pore (mPTP), the mitochondrial membrane potential, and ATP production was assessed using fluorescence microscopy, and the mitochondrial structural integrity was observed with electron microscopy. The levels of the phosphorylation of mammalian sterile-20-like kinase (MST1) and yes-associated protein (YAP) were assessed by Western blot analysis in whole-cell lysate, while the expression levels of mitochondrial biogenesis genes, YAP target genes, and proapoptotic genes were measured by qRT-PCR.Results: We found that after H2O2 treatment, chronic E2/G1 treatment decreased cell death effect was associated with the prevention of the S phase of the cell cycle arrest compared to control. In the mitochondria, chronic E2/G1 activation treatment preserved the cristae morphology, and increased resistance to opening of mPTP, but with little change to mitochondrial fusion/fission. Additionally, chronic E2/G1 treatment predominantly reduced phosphorylation of MST1 and YAP, as well as increased MST1 and YAP protein levels. E2 treatment also upregulated the expression levels of TGF-β and PGC-1α mRNAs and downregulated PUMA and Bim mRNAs. Except for ATP production, all the E2 or G1 effects were prevented by the cotreatment with the GPER1 antagonist, G15.Conclusion: Together, these results indicate that chronic GPER1 activation with its agonists E2 or G1 treatment protects H9c2 cardiomyoblasts against oxidative stress-induced cell death and increases cell viability by preserving mitochondrial structure and function as well as delaying the opening of mPTP. These chronic GPER1 effects are associated with the deactivation of the non-canonical MST1/YAP mechanism that leads to genetic upregulation of cell growth genes (CTGF, CYR61, PGC-1α, and ANKRD1), and downregulation of proapoptotic genes (PUMA and Bim).