Cell Viability Assay Research Articles

Inhibition of NLRP3 inflammasome by MCC950 under hypoxia alleviates photoreceptor apoptosis via inducing autophagy in Müller glia.

NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.

Bioinspired poly-dopamine/nano-hydroxyapatite: an upgrading biocompatible coat for 3D-printed polylactic acid scaffold for bone regeneration.

Poly-lactic acid (PLA) has been proposed in dentistry for several regenerative procedures owing to its biocompatibility and biodegradability. However, the presence of methyl groups renders PLA hydrophobic, making the surface less ideal for cell attachment, and it does not promote tissue regeneration. Upgrading PLA with inductive biomaterial is a crucial step to increase the bioactivity of the PLA and allow cellular adhesion. Our purpose is to evaluate biocompatibility, bioactivity, cellular adhesion, and mechanical properties of 3D-printed PLA scaffold coated with poly-dopamine (PDA) and nano-hydroxyapatite (n-HA) versus PLA and PLA/n-HA scaffolds. The fused deposition modelling technique was used to print PLA, PLA with embedded n-HA particles, and PLA scaffold coated with PDA/n-HA by immersion. After matrices characterization for their chemical composition and surface properties, testing the compressive strength was pursued using a universal testing machine. The bioactivity of scaffolds was evaluated by monitoring the formation of calcium phosphate compounds after simulated body fluid immersion. The PLA/PDA/n-HA scaffold showed the highest compressive strength which was 29.11 ± 7.58 MPa with enhancing calcium phosphate crystals deposition with a specific calcium polyphosphate phase formed exclusively on PLA/PDA/n-HA. With cell viability assay, the PDA/n-HA-coated matrix was biocompatible with increase in the IC50, reaching ⁓176.8 at 72 without cytotoxic effect on the mesenchymal stem cells, promoting their adhesion and proliferation evaluated by confocal microscopy. The study explored the biocompatibility, bioactivity, and the cell adhesion ability of PDA/n-HA coat on a 3D-printed PLA scaffold that qualifies its use as a promising regenerative material.

MiR-6839-5p inhibits cell proliferation, migration and invasion; a possible correlation with the suppressing VEGFA expression in human chondrosarcoma cells

MicroRNAs play an important role in the proliferation, invasion, and metastasis of malignancy. In previous studies (detailed in our previous paper), the expression of miR-6839-5p was significantly increased in SW1353 cells after 125I seed 6 Gy irradiation, which indicated miR-6839-5p may play a tumor suppression function in chondrosarcoma cells. This study aimed to identify the effects of miR-6839-5p on the human chondrosarcoma cells, and investigate the potential target genes of miR-6839-5p. Firstly, chondrosarcoma cells (SW1353 and CAL78) were transfected with hsa-miR-6839-5p specific mimic. Secondly, Cell viability assay (MTT assay), Colony formation assay, Wound healing assay, Transwell assay, TUNEL staining and Western blotting experiments were performed, and the results proved miR-6839-5p can inhibit chondrosarcoma cells proliferation, migration and invasion. Meanwhile, miR-6839-5p significantly down-regulated apoptosis facilitator Bcl-2 expression, and promoted apoptosis of chondrosarcoma cells. It is reasonable to speculate miR-6839-5p might downregulate Bcl-2 expression to induce apoptosis in SW1353 human chondrosarcoma cells. Lastly, RNA extraction and bioinformatic analysis was performed on SW1353 cells transfected with hsa-miR-6839-5p specific mimic to investigate the potential target genes of miR-6839-5p. A total of 253 differentially expressed mRNA genes (105 up-regulated genes and 148 down-regulated genes) were found, and 23 differentially expressed downregulated genes were identified. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to validate the results, which demonstrated the expression of BST2, VEGFA, FPR3 and PPARA was significantly downregulated by miR-6839-5p mimic. Furthermore, miR-6839-5p inhibitor can restore or partially restore the expression value of the above four genes. The analysis results of miRNA target gene prediction database indicated VEGFA was the most likely direct target gene of miR-6839-5p.

Extracellular matrix markerLAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway.

Triple negative breast cancer (TNBC) is a heterogeneous and aggressive disease characterized by a high risk of mortality and poor prognosis. It has been reported that Laminin γ2 (LAMC2) is highly expressed in a variety of tumors, and its high expression is correlated with cancer development and progression. However, the function and mechanism by which LAMC2 influences TNBC remain unclear. Kaplan-Meier survival analysis and Immunohistochemical (IHC) staining were used to examine the expression level of LAMC2 in TNBC. Subsequently, cell viability assay, wound healing and transwell assay were performed to detect the function of LAMC2 in cell proliferation and migration. A xenograft mouse model was used to assess tumorigenic function of LAMC2 in vivo. Luciferase reporter assay and western blot were performed to unravel the underlying mechanism. In this study, we found that higher expression of LAMC2 significantly correlated with poor survival in the TNBC cohort. Functional characterization showed that LAMC2 promoted cell proliferation and migration capacity of TNBC cell lines via up-regulating CD44. Moreover, LAMC2 exerted oncogenic roles in TNBC through modulating the expression of epithelial-mesenchymal transition (EMT) markers. Luciferasereporterassay verified that LAMC2 targeted ZEB1to promote its transcription.Interestingly, LAMC2 regulated cell migration in TNBC via STAT3 signaling pathway. LAMC2 targeted ZEB1 via activating CD44/STAT3 signaling pathway to promote TNBC proliferation and migration, suggesting that LAMC2 could be a potential therapeutic target in TNBC patients.

P04 How fibroblasts and azathioprine prepare the soil for cutaneous squamous cell carcinoma

Abstract Introduction and aims DNA damage-inducing immunosuppressive drugs such as azathioprine (AZA) act synergistically with ultraviolet (UV)A radiation and photosensitize the skin of immunosuppressed individuals, such as organ transplant recipients. These individuals have an increased risk of developing cutaneous squamous cell carcinoma (cSCC). The specific combination of AZA and UVA causes oxidative DNA damage that leaves behind a characteristic mutational signature in tumour keratinocytes of patients treated with AZA. However, as UVA radiation can penetrate the dermis, the impact of AZA/UVA on cells of the tumour microenvironment, such as fibroblasts, also need to be addressed. Fibroblasts are crucial for cSCC development and progression as they can switch to a protumourigenic phenotype. Methods Cell viability and senescence assays were performed to investigate the impact of AZA in combination with or without UVA on neonatal (nonsun-exposed) and adult (sun-exposed) human dermal fibroblasts. In addition, the impact of AZA/UVA-treated fibroblasts on the clonal growth properties of different cSCC cell models was investigated using three-dimensional (co)culture models. Results We found that AZA and UVA synergistically reduce the viability of both UV-naïve neonatal and sun-exposed adult dermal fibroblasts. In addition, combined AZA and UVA treatments lead to an increased percentage of senescent fibroblasts. We hypothesize that the aberrant secretome of these senescent fibroblasts can impact the growth behaviour of tumour cells. In a soft agar assay, the more malignant cSCC cells (MET-1) formed more and larger clonal spheroids and were less dependent on growth factors compared with spheroids formed by premalignant (PM-1) keratinocytes. Conclusions AZA and UVA act synergistically and potentiate a UVA-induced senescent fibroblast phenotype. The changed characteristics of these fibroblasts can impact the stem cell-like properties from premalignant and malignant keratinocytes. Combining keratinocyte and fibroblast culture models enables us to study how AZA/UVA promote the initiation and progression of cSCC.

Bcl-2 dependent modulation of Hippo pathway in cancer cells

IntroductionBcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2.MethodsWe performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.ResultsWe demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.ConclusionsWe discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells

Ethnopharmacological relevanceAdinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. Aim of the studyTo explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. Materials and methodsHuman NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0–160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0–40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0–1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0–10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. ResultsTFAN significantly inhibited A549 cells (wild-type p53) but not NCI–H1299 cells (p53-deficient), NCI–H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. ConclusionThe disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

Quantitative systems pharmacology modeling of tumor heterogeneity in response to BH3-mimetics using virtual tumors calibrated with cell viability assays.

Both primary and acquired resistance mechanisms that involve intra-tumoral cell heterogeneity limit the use of BH3-mimetics to trigger tumor cell apoptosis. This article proposes a new quantitative systems pharmacology (QSP)-based methodology in which cell viability assays are used to calibrate virtual tumors (VTs) made of virtual cells whose fate is determined by simulations from an apoptosis QSP model. VTs representing SU-DHL-4 and KARPAS-422 cell lines were calibrated using invitro data involving venetoclax (anti-BCL2), A-1155463 (anti-BCLXL), and/or A-1210477 (anti-MCL1). The calibrated VTs provide insights into the combination of several BH3-mimetics, such as the distinction between cells eliminated by at least one of the drugs (monotherapies) from the cells eliminated by a pharmacological combination only. Calibrated VTs can also be used as initial conditions in an agent-based model (ABM) framework, and a minimal ABM was developed to bridge invitro SU-DHL-4 cell viability results to tumor growth inhibition experiments in mice.

Multifunctional FFP2 Face Mask for White Light Disinfection and Pathogens Detection using Hybrid Nanostructures and Optical Metasurfaces.

A new generation of an FFP2 (Filtering Face Piece of type 2) smart face mask is achieved by integrating broadband hybrid nanomaterials and a self-assembled optical metasurface. The multifunctional FFP2 face mask shows simultaneously white light-assisted on-demand disinfection properties and versatile biosensing capabilities. These properties are achieved by a powerful combination of white light thermoplasmonic responsive hybrid nanomaterials, which provide excellent photo-thermal disinfection properties, and optical metasurface-based colorimetric biosensors, with a very low limit of pathogens detection. The realized system is studied in optical, morphological, spectroscopic, and cell viability assay experiments and environmental monitoring of harmful pathogens, thus highlighting the extraordinary properties in reusability and pathogens detection of the innovative face mask.

Somatic structural variants drive distinct modes of oncogenesis in melanoma.

The diversity of structural variants (SVs) in melanoma and how they impact oncogenesis are incompletely known. We performed harmonized analysis of SVs across melanoma histological and genomic subtypes, and we identified distinct global properties between subtypes. These included the frequency and size of SVs and SV classes, their relation to chromothripsis events, and the role of topologically associated domain (TAD) boundary altering SVs on cancer-related genes. Following our prior identification of double-stranded break repair deficiency in a subset of triple wild-type cutaneous melanoma, we identified MRE11 and NBN loss-of-function SVs in melanomas with this mutational signature. Experimental knockouts of MRE11 and NBN, followed by olaparib cell viability assays in melanoma cells, indicated that dysregulation of each of these genes may cause sensitivity to PARPi in cutaneous melanomas. Broadly, harmonized analysis of melanoma SVs revealed distinct global genomic properties and molecular drivers, which may have biological and therapeutic impact.

Development of a label‐free, impedance‐based biosensor to identify harmful effects of pesticides on insect cells

AbstractInsects are a major part of the planet's ecosystem and their vital role as pollinators for agriculture is undisputed. Alongside factors as climate change or loss of habitats, rising use of pesticides emerges as a key threat to insect populations. For fighting this man‐made problem, development of an easy, fast, sensitive, and non‐invasive biosensor for determining pesticide toxicity may help to ban harmful substances and formulations. Here, a biosensor based on Sf21 (Spodoptera frugiperda) insect cells as sensors and electric cell‐substrate impedance sensing (ECIS) as physical transducer is described. Sf21 cell suspensions and well‐defined pesticide solutions were mixed immediately before seeding on planar gold‐film electrodes. The capacitance at 20 kHz was recorded as a function of time as a measurand for cell adhesion providing dose–response profiles of pesticide impact. For future in‐field applications, decoupling of the cell culture routines from the actual cytotoxicity assay is mandatory. Thus, suspensions of Sf21 cells were cryopreserved at –80°C in the wells of multielectrode arrays and thawed anytime for conducting the assays. Five pesticides were tested for their concentration‐dependent cytotoxicity expressed as EC50 values by ECIS and validated using the well‐established WST‐1 cell viability assay. Results were found to be in good agreement. Our studies revealed cytotoxic effects of some pesticides sold for home usage far below the recommended concentration and were found to be more toxic than formulations sold for agricultural industry only.

Elesclomol-copper synergizes with imidazole ketone erastin by promoting cuproptosis and ferroptosis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) encompass a collection of clonal hematopoietic malignancies distinguished by the depletion of peripheral blood cells. The treatment of MDS is hindered by the advanced age of patients, with a restricted repertoire of drugs currently accessible for therapeutic intervention. In this study, we found that ES-Cu strongly inhibited the viability of MDS cell lines and activated cuproptosis in a copper-dependent manner. Importantly, ferroptosis inducer IKE synergistically enhanced ES-Cu-mediated cytotoxicity both in vitro and in vivo. Of note, the combination of IKE and ES-Cu intensively impaired mitochondrial homeostasis with increased mitochondrial ROS, MMP hyperpolarized, down-regulated iron-sulfur proteins and declined oxygen consumption rate. Additionally, ES-Cu/IKE treatment could enhance the lipoylation-dependent oligomerization of the DLAT. To elucidate the specific order of events in the synergistic cell death, inhibitors of ferroptosis and cuproptosis were utilized to further characterize the basis of cell death. Cell viability assays showed that the glutathione and its precursor N-acetylcysteine could significantly rescue the cell death under either mono or combination treatment, demonstrating that GSH acts at the crossing point in the regulation network of cuproptosis and ferroptosis. Significantly, the reconstitution of xCT expression and knockdown of FDX1 cells have been found to contribute to the tolerance of mono treatment but have little recovery impact on the combined treatment. Collectively, these findings suggest that a synergistic interaction leading to the induction of multiple programmed cell death pathways could be a promising approach to enhance the effectiveness of therapy for MDS.

Blueberry extract and its bioactive compounds mitigate oxidative stress and suppress human lung cancer cell (A549) growth by modulating the expression of p53/EGFR/STAT3/IL6-mediated signaling molecules.

Bioactive phytocompounds are crucial components in all plants. Since the time of traditional medicine, the utilization of plants has been grounded in the potential of these bioactive compounds to treat or manage specific illnesses. These natural bioactive compounds have sparked growing interest in employing medicinal plants for addressing various conditions, such as inflammatory diseases, diabetes, and cancer. This study focuses on assessing the qualitative phytochemical composition, antioxidant potential, and cytotoxic effects of blueberry (Vaccinium sect. Cyanococcus) extract using three different solvents, namely water, ethanol, and methanol. The extract exhibited notable antioxidant activities, as evidenced by DPPH and H2O2 free radical scavenging assays. The cell viability assay also demonstrated cell growth inhibition in A549 cells. Furthermore, nine specific phytocompounds sourced from existing literature were selected for molecular docking studies against CDK6 and, AMPK key protein kinases which enhance the cancer progression. The molecular docking results also revealed favorable binding scores, with a high score of -9.5 kcal/mol in CDK6 protein and a maximum score of AMPK with targets of -8.8 kcal/mol. The selected phytocompounds' pharmacodynamic properties such as ADMET also supported the study. Furthermore, rutin stated that pre-dominantly present in blueberry plants shows a potent cytotoxicity effect in A549 cells. Functional annotations by bioinformatic analysis for rutin also revealed the strong enrichment in the involvement of PI3K/AKT1/STAT, and p53 signaling pathways. Based on this analysis, the identified rutin and other compounds hold a promising anticancer activity. Overall, the comprehensive evaluation of both in vitro and in silico data suggests that the Vaccinium sect. Cyanococcus extract could serve as a valuable source of pharmaceutical agents and may prove effective in future therapeutic applications.

Phthalazine sulfonamide derivatives as carbonic anhydrase inhibitors. Synthesis, biological and in silico evaluation.

Carbonic Anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide involved in several biological processes. They show a wide diversity in tissue distribution and their subcellular localization. Twenty-two novel phthalazine derivatives were designed, synthesized, and evaluated against four human isoforms: hCA I, hCA II, hCA IX, and hCA XII. Compounds appeared to be very active mostly against hCA IX (7) and hCA I (6) isoforms being more potent than reference drug acetazolamide (AAZ). Some compounds appeared to be very selective with a selectivity index up to 13.8. Furthermore, docking was performed for some of these compounds on all isoforms to understand the possible interactions with the active site. Additionally, the most active compounds against hCA IX were subjected to cell viability assay. The anticancer activity of the compounds (3a-d, 5d, 5i, and 5m) was investigated using two human breast cancer cell lines, i.e. MCF-7 and MDA-MB-231 cells, and the normal counterpart, namely MCF10-A cells.

Ecofriendly and high-performance flexible pressure sensor derived from natural plant materials for intelligent audible and silent speech recognition

Flexible pressure sensors (FPSs) are widely used in human–computer interaction (HCI) and medical diagnoses, but their further development faces several challenges. Integrating artificial intelligence (AI) with FPSs is indispensable for advancing the cognitive capabilities of HCI systems, but the FPS must also be ecofriendly, cost-effective, and high-performance to meet the demands of environmental protection and sustainable development. Here, a biodegradable capacitive FPS is presented that is fabricated from natural plant materials (i.e., cornstarch and lignocellulose) while exhibiting remarkable attributes such as high sensitivity (2.427 kPa−1 at 0–2 kPa), rapid response/recovery time (20/24 ms), extremely low detection limit (∼10 Pa), outstanding stability (> 6000 fatigue tests), excellent biocompatibility (survival rate exceeding 99 % in cell viability assays), and prominent biodegradability (complete degradation within 24 h). Prepared sensors were integrated with optimized AI algorithms, embedded systems, and wireless transmission technologies to develop a dual-functional (i.e., audible and silent) speech recognition system that achieved recognition rates exceeding 95 %. The developed sensor is expected to play a crucial role in future HCI and medical assistance applications.

Green synthesis of copper oxide nanoparticles using Simarouba glauca leaf extract, characterization and screening for their biological applications

The biological synthesis of metallic nanoparticles using plants has received a lot of attention because of their ability to decrease metallic ions by using the phytochemicals in them. The current study emphasizes the green synthesis of copper oxide nanoparticles (CuO NPs) with Simarouba glauca leaf extract. The synthesis of CuO NPs was characterized using UV-vis spectrophotometry, FTIR indicated the presence of different metabolites involved in the formation of nanoparticles, XRD showed the crystalline nature, SEM images indicated the rod shaped CuO NPs with the average size from 190 to 265 nm, EDS and mapping analysis showed the formation of CuO NPs with purity, and TEM analysis indicated the agglomerated and rod shaped CuO NPs. The average size of the nanoparticle was found to be 85 nm with zeta potential of −5.2 mV. TGA and DSC were performed to know the stability of the nanoparticle. Further, CuO NPs were assessed for their applications like antioxidant, antimicrobial properties. The anti-angiogenic, in vitro cell viability assay was performed using NPs against HCT-116, IC50 value to be 17.48 ± 0.6 µg/mL and HeLa cell lines, IC50 values to be 20.21 ± 0.33 µg/mL. The anti-ROS properties for both cell lines were assessed using histogram.

NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

BackgroundGenetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models.ResultsWe treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells.ConclusionsNB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.

Chemokine‑ and chemokine receptor-based subtypes predict prognosis, immunotherapy and chemotherapy response in colorectal cancer patients

BackgroundThe clinical significance and comprehensive characteristics of chemokines and chemokine receptors in colorectal cancer (CRC) have not been previously reported. Our study aims to investigate the expression profiles of chemokines and chemokine receptors, as well as establish subtypes in CRC. Methods1009 CRC samples were enrolled in our study. Consensus unsupervised clustering analysis was conducted to establish subtypes, and a risk score model was developed using univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses. 36 pairs of tissue specimens of CRC patients and two CRC cell lines were used to validate the subtypes and risk score in vitro. Quantitative real-time PCR and western blotting were employed to validate mRNA and protein expression levels, respectively. Flow cytometry was utilized for analyzing cell apoptosis, while cell viability assay and EdU assay were conducted to assess cell proliferation ability. ResultsThe Cluster B group shares similarities with the low-risk group in terms of exhibiting a higher level of immune cell infiltration and belonging to hot tumor. Patients CRC in the Cluster B group demonstrate a more favorable prognosis and exhibit better response to immunotherapy and chemotherapy. On the other hand, the Cluster A group resembles the high-risk group as it displays lower levels of immune cell infiltration, indicating a cold tumor phenotype. CRC patients in the Cluster A group have poorer prognoses and show less therapeutic efficacy towards immunotherapy and chemotherapy. Furthermore, we utilized a total of 36 pairs of tissue samples obtained from patients with CRC, along with two CRC cell lines for validation in vitro. This comprehensive approach further enhances the scientific validity and reliability of the identified subtypes and risk score in their ability to predict prognosis, response to immunotherapy, and response to chemotherapy among CRC patients. ConclusionWe first established robust prognostic subtypes based on chemokines and chemokine receptors, which could potentially serve as a novel biomarker for guiding individualized treatment in patients with CRC undergoing immunotherapy and chemotherapy.

Abstract PO1-23-09: A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC)

Abstract Background: CDK4/6i are part of the standard management of HR+ MBC. The MAPK pathway has been implicated in mediating resistance to CDK4/6 blockade (Wander 2020). A variety of molecular alterations dysregulate MAPK signaling, including NF1 mutations (NF1-MUT) (Wallace 2012), though NF1 has not been implicated in CDK4/6i resistance. To assess this, we analyzed retrospective clinical data from multiple institutions, real-world data (RWD) from the GuardantINFORM database, and preclinical models of HR+ breast tumors. The primary objective of this study was to characterize clinical outcomes of CDK4/6i in patients (pts) with NF1-MUT MBC, and to validate those findings in laboratory models. Methods: Pts with pathogenic (p)NF1-MUT were identified via sequencing of circulating tumor DNA (ctDNA) or tumor tissue. Pathogenic was defined as nonsense, insertion/deletion, frameshift, or oncogenic missense mutations. Progression free survival (PFS) on CDK4/6i was analyzed, and intrinsic resistance was defined as progression < 6 months on 1st line (1L) CDK4/6i or < 3 mo on 2nd line and beyond (2L+). Acquired resistance was defined as PFS >6 mo (1L) or >3 mo (2L+) with a NF1 alteration detected post-progression. RWD from the GuardantINFORM dataset had Guardant360 (G360) ctDNA testing linked with claims data on >37,000 pts with advanced or MBC tested between June 2014 and March 2023. Pts on CDK4/6i within 90 days pre-G360 were stratified as NF1-MUT (n = 28) vs. NF1-non-mutant (NF1-nonMUT) (n = 1133) and analyzed for real-world time-to-next-treatment (rwTTNT) and real-world overall survival (rwOS). Propensity score weighting for age, sex, line of therapy, and year of G360 were used to create a balanced control group. To determine whether NF1 loss-of-function alterations were causal of CDK4/6i resistance, we used CRISPR/Cas9 to delete NF1 in MCF7 breast cancer cells. MCF7 wild-type (WT) and two NF1-knockout (KO) clones were treated with palbociclib ± estrogen deprivation, followed by cell viability assays. MAPK and PI3K/AKT pathway activation were measured by immunoblot analysis. Results: Across 4 institutions, 40 pts with HR+, HER2- MBC expressing pNF1-MUT and prior CDK4/6i were included. The mean age at MBC diagnosis was 56 years, and the majority of pts received no prior treatment in the metastatic setting before CDK4/6i (range 0 - 5 prior lines). Intrinsic or acquired resistance to CDK4/6i was seen in 29/40 (73%) tumors harboring pNF1-MUT. We analyzed pts with baseline pNF1-MUT who received 1L CDK4/6i plus endocrine therapy (n = 13) and found that the median PFS was 7.7 months (range 2 - 18 mo). We next examined GuardantINFORM RWD and discovered a significant difference in rwTTNT and rwOS on CDK4/6i for NF1-MUT (n = 28) vs. weighted NF1-nonMUT (n = 28) tumors. Mean age (62 years) and level of pretreatment (majority 2L+) were similar between the groups. Compared to NF1-nonMUT, NF1-MUT pts had a shorter median rwTTNT on CDK4/6i regimens (4.2 vs. 12.4 mo, weighted log-rank p< 0.0001) and worse rwOS (15.8 vs 45.2 mo, weighted log-rank p=0.016). In MCF7 cells, NF1-KO exhibited a palbociclib IC50 11- to 13-fold higher than NF1-WT controls. In estrogen-deprived media, treatment with 250 nM palbociclib for 6 days reduced MCF7 NF1-WT cell viability 75% but only 5-20% in NF1-KO cells. As expected, phosphorylation of ERK and AKT were strongly induced in NF1-KO cells relative to NF1-WT. Conclusions: Using multicenter clinical and real-world evidence, we demonstrate that NF1-MUT is associated with inferior duration and worse outcomes on CDK4/6i in MBC. Pts with baseline pNF1-MUT on 1L CDK4/6i and endocrine therapy had a median PFS below typical responses expected in clinical practice. A link between NF1 loss and CDK4/6i resistance was supported by in vitro experiments in HR+ breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, therefore, future directions include investigating alternative or combination treatment strategies targeting the MAPK and/or PI3K/AKT pathways in these tumors. *MRL and RCP contributed equally. ABH and SAW are co-senior authors. Citation Format: Maxwell Lloyd, M Rosario Chica-Parrado, Todd Knepper, Chang-Ching Lin, Emily Podany, Christine Walko, Fabiana Napolitano, Caroline Weipert, Jiemin Liao, Arielle Medford, Lianne Ryan, Jennifer Keenan, Nicole Zhang, Shiyuan Liu, Gerburg Wulf, Katherine Clifton, Cynthia Ma, Hyo Han, Leif Ellisen, Aditya Bardia, Carlos Arteaga, Ariella Hanker, Seth Wander. A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-09.

Abstract PO4-23-09: Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells

Abstract In HER2-enriched (HER2E) breast cancer (BC), the dual HER2/EGFR inhibitor Lapatinib is a standard of care for advanced staged disease. Anti-HER2 therapies disrupt tumor progression through inhibition of the PI3K/AKT pathway. Some treated tumors will override inhibition by shifting to MAPK/ERK signaling to fuel cell growth, thus fostering resistance to anti-HER2 therapy. The tyrosine receptor kinase FGFR4 is included in the profile for a HER2E subtype. FGFR4 is overexpressed in ~30% of BC and can signal through PI3K/AKT and MAPK/ERK. Inhibitors for FGFR4 such as BLU554 are being investigated in phase I/II clinical trials for hepatocellular carcinoma, showing success in tumors that are positive for FGFR4’s preferred ligand FGF19. According to cBioPortal patient data, FGF19 is amplified in ~20% of BC, and has been shown to trigger an autocrine loop in triple negative BC through FGFR4. We examined BLU554 as a dual therapeutic with Lapatinib and dissected intracellular signaling stemming from the combination in HER2E BC. MDA-MB-453 BC cells (HER2+FGFR4+) were used for a dual strategy with BLU554 and Lapatinib. Cell viability assays were performed at matched doses, then as a dose response matrix, ranging from 0.1-20 µM. Findings were used for synergy analysis using synergyfinder.fimm.fi. Western blots determined protein activity in response to treatments. Ridaforolimus (mTOR inhibitor) was used in combination with either BLU554 or Lapatinib to assess MAPK/ERK and PI3K/AKT pathway crosstalk in MB-453 cells. Additionally, FGF19 was added to cells after a 2-hour pre-treatment to challenge BLU554 + Lapatinib and compare changes in signaling with FGF19 present. MB-453 cells treated with BLU554 decreased cell viability similarly to Lapatinib at 10 µM (65% [SD ± 3.3] and 63% [SD ± 2.1], respectively). Dual treatment decreased viability compared to single agents at all concentrations but the greatest difference was observed at 20 µM (38.5% in dual compared to Lapatinib [p-value = 0.00006]; 45.1% in dual compared to BLU554 [p-value = 0.00016]). Dose-response matrix of dual treatment yielded a ZIP synergy score of 16.852 ± 2.97 (>10 is synergistic), with the greatest synergy and inhibition at 10 µM and 20 µM of both drugs. BLU554 caused a therapeutic potency shift of Lapatinib, such that lower doses of Lapatinib (0.1 µM, 1 µM) are effective at higher doses of BLU554 (10 µM, 20 µM). Westerns of MB-453 with one dose of BLU554 increased phospho-ERK1/2 (pERK1/2) at 16- or 48-hr time points versus control, but when BLU554 was re-dosed then pERK1/2 was successfully inhibited. Both pAKT and pS6RP were increased by re-dosing BLU554. Cells treated for 2 hrs with Lapatinib decreased pAKT and pS6RP and did not change pERK, whereas a 2-hr treatment of BLU554 increased pAKT and decreased pERK. Dual treatment exhibited inhibition of ERK, AKT, 4EBP1, and S6RP phosphorylation all to a greater extent than single agents in combination with Ridaforolimus. Challenge by ectopic FGF19 of BLU554 + Lapatinib was unable to restore downstream signaling of AKT and ERK. BLU554 synergistically interacts with Lapatinib to decrease cell viability while increasing its therapeutic potency. Lapatinib primarily inhibits AKT signaling, whereas BLU554 targets ERK. Dual treatment simultaneously blocks MAPK/ERK and PI3K/AKT signaling and yields decreases of mTOR downstream effectors pS6RP and p4EBP1. These effects are maintained even when challenged with FGF19, suggesting that dual inhibition of FGFR4 and HER2 could prevent pathway activation from external stimuli. Citation Format: Jordan Beardsley, Joseph Goode, Deborah Altomare. Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-09.