Real-time Cell Analysis Research Articles

Absence of Functional Autoantibodies Targeting Angiotensin II Receptor Type 1 and Endothelin-1 Type A Receptor in Circulation and Purified IgG From Patients With Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare but severe autoimmune disease characterized by immune dysregulation, fibrosis, and vasculopathy. Although previous studies have highlighted the presence of functional autoantibodies targeting the angiotensin II receptor type 1 (AT1) and endothelin-1 type A receptor (ETAR), leading to autoantibody-mediated receptor stimulation and subsequent activation of endothelial cells (ECs), a comprehensive understanding of the direct interaction between these autoantibodies and their receptors is currently lacking. Moreover, existing data confirming the presence of these autoantibodies in SSc often rely on similar methodologies and assays. Our aim was to replicate previous findings and to investigate the functional effects of IgG derived from patients with SSc (SSc IgG) on AT1 and ETAR signaling, the downstream EC response, and the presence of AT1-binding autoantibodies in circulation. Quantitative polymerase chain reaction and cytokine enzyme-linked immunosorbent assay, alongside a real-time cell analyzer, were used to assess receptor-specific functional characteristics of purified SSc IgG (n = 18). Additionally, a novel protein capture assay using solubilized epitope-tagged AT1 was developed to detect AT1-binding autoantibodies in plasma samples from patients with SSc (n = 28) and healthy donors (n = 14). No evidence for EC activation in an AT1- or ETAR-dependent manner was revealed. Furthermore, stimulation with SSc IgG did not induce receptor activation or alter G protein-coupled receptor signaling on agonist stimulation in a model with receptor overexpression. Lastly, no AT1-binding autoantibodies were detected in plasma samples from patients with SSc when using epitope-tagged solubilized AT1. Overall, our study did not provide evidence to support the presence of AT1- or ETAR-activating autoantibodies in purified SSc IgG or AT1-binding autoantibodies in the circulation of patients with SSc.

Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

Background: Chimeric antigen receptor (CAR) T cell therapy has shown significant promise in treating hematological malignancies, yet its application in solid tumors, particularly those expressing the epidermal growth factor receptor (EGFR), remains limited. This study investigates the potential of CAR-engineered peripheral blood mononuclear cells (PBMCs) as a novel adoptive cell therapy against EGFR-positive cancers. Methods: Lentiviral transduction at an MOI of 50 was performed to generate specific anti-EGFR second generation CAR-effector cells. The transduced PBMCs were stimulated with cytokines and CD3/CD28 beads to enhance their proliferation and activation. Flow cytometric and real-time cell analysis were performed at various effector-to-target ratios to explore the cytotoxic potential of CAR-PBMCs. Results: CAR-PBMCs exhibited improved targeting and cytotoxicity against EGFR-positive cancer cell lines MDA-MB-468 and SK-BR-3, compared to untransduced controls, with unsignificant effects on allogeneic PBMCs. Conclusion: CAR-PBMCs hold considerable potential as a therapeutic strategy for EGFR-positive solid tumors, warranting further clinical investigation.

Impedance-based method for the quantification of infectious SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent involved in the coronavirus disease 2019 (COVID-19) pandemic. The development of infectious titration methods is crucial to provide data for a better understanding of transmission routes, as well as to validate the efficacy of inactivation treatments. Nevertheless, the low-throughput analytical capacity of traditional methods may be a limiting factor for a large screening of samples. The aim of the study was to develop a Real-Time Cell Analysis (RTCA) assay based on the measurement of cell impedance to quantify infectious SARS-CoV-2. The kinetics of cell impedance showed a virus-specific Cell Index (CI) drop. This enabled the correlation between viral concentrations and time at which a 50% drop in CI values was observed (tCI50), with establishment of a standard curve. In parallel, the improved Spearman and Kärber method was used to quantify infectious titer since the virus-induced CI drop is correlated to the Cytopathic Effect. The estimated uncertainty was respectively 0.57, 0.36, and 0.26 log10 with 4, 8, and 16 wells per dilution. Thus, the RTCA assay is a powerful tool with a greatly simplified workflow for effective risk assessment in the field of food and environmental virology.

Cell-Interface-Deciphering Lipid Nanotablet for Nanoparticle Logic Gate-Based Real-Time Single-Cell Analysis.

Analyzing the cell interface is of paramount importance in understanding how cells interact and communicate with other cells, but an advanced analytical platform that can process complex and networked interactions between cell surface ligands and receptors is lacking. Herein, we developed the cell-interface-deciphering lipid nanotablet (CID-LNT) for multiplexed real-time cell analysis. LNT is a nanoparticle-tethered lipid bilayer chip where freely diffusing plasmonic nanoparticles induce scattering signal changes. The CID-LNT transduces cell surface protein information into DNA data, which operate as nanoparticle logic gates. As a proof of concept, we detected and analyzed programmed death-ligand 1 (PD-L1) and associated immune signals (TNF-α, EGF, and IFN-γ). PD-L1 is an immune checkpoint that suppresses T cell activity with inflammatory biomolecules facilitating its expression. The CID-LNT can serve as a dynamic nanoparticle logic board, enabling the logic gate-based analysis of membrane proteins, and can be expanded to immunological synapse analysis, cell interface engineering, and molecular diagnostics.

AZD1390, an Ataxia telangiectasia mutated inhibitor, enhances cisplatin mediated apoptosis in breast cancer cells.

Genomic instability is often caused by deficiencies in DNA damage repair pathways, making therapeutic targeting of DDR beneficial for cancer patients with specific DDR functions. ATM kinase plays a critical role in various cellular processes and its deficiency increases sensitivity to DDR-targeted agents in different cancers. Recent studies highlight ATM inhibition as a potential clinical target, with AZD1390 being a notable ATM inhibitor due to its potent and selective inhibition, ability to accumulate at DNA breaks. The study aimed to evaluate the potential anti-cancer effects of AZD1390, a key component of the DNA damage response, in breast cancer cells. The impact of the combination of AZD1390 and cisplatin on various parameters such as cell viability, proliferation, colony formation capacity, DNA damage, reactive oxygen species (ROS) levels, mitochondrial membrane potential, cell cycle progression, and cell death in breast cancer cells was evaluated using several methodologies, including WST-1 assays, real-time cell analysis, colony formation assays, comet assays, DCF-DA, MMP/JC-1 staining assays, flow cytometry along with Western blot analysis. We found that AZD1390 and cisplatin displayed synergistic antitumor effects in breast cancer cells at low doses. Addinationaly exhibited significant anti-proliferative effects in colony formation and real-time cell proliferation experiments, increasing intracellular ROS levels and mitochondrial membrane potential.The combined treatment also arrested the cell cycle at the G2-M point. Furthermore, combination of AZD1390 with cisplatin enhances its apoptotic effects in MCF-7 and MDA-MB-231cells. These findings could aid in developing new treatments for breast cancer that exploit the genomic instability of cancer cells.

Effect of Propolis on PPP2R1A and Apoptosis in Cancer Cells.

Recently, it has been shown that protein phosphatase 2A (PP2A) dysfunction was common in many cancer types and was mediated by various inactivation mechanisms. Although many research studies observed antitumor effect of propolis extracts in various types of cancer, the mechanism of effect are still obscure. In this study, we investigated the effect of propolis on PPP2R1A expression and its relationship with apoptosis in the SW-620 (colorectal cancer), DU-145 and PC-3 (prostate cancer), and MCF-7 (breast cancer) cell lines, with WI-38 (healthy fibroblast) cells serving as the control. Moreover, we aimed to investigate the impact of propolis on apoptosis by analyzing apoptosis markers such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), APAF-1, and caspases-3, -8, and -9. Propolis samples were extracted, and their phenolic compounds were quantified using LC-MS/MS. The RealTime Cell Analysis System-xCELLigence (RTCA-SP) device and software were employed to assess cell viability and cytotoxicity of the propolis samples. The IC50 values for propolis were determined (298 μg/mL for SW-620, 185.6 μg/mL for DU-145, 250.7 μg/mL for PC - 3, 292.9 μg/mL for MCF-7, and 311.2 μg/mL for WI-38). Subsequently, the effects of propolis on PPP2R1A expression and apoptosis markers (TRAIL, Apaf-1, and caspases-3, -8, and -9) were analyzed. When we compared the healthy cell lines to cancer cell lines, a statistically significant increase in caspase-3 (3.62-fold) and in TRAIL (4.38-fold) was observed in the SW-620 cell line after the application of propolis. In addition, in the PC-3 cell line, a 1.4-fold increase in caspase-8 was observed compared with the healthy cell line, which is also statistically significant. Our findings indicated that propolis increased the PPP2R1A levels and apoptosis markers in cancer cell lines. It has been suggested that high PPP2R1A levels induced by propolis treatment might activate the apoptosis pathway. In this study, the inducible effect of propolis on PPP2R1A levels, identified as a new target for cancer treatment, was demonstrated for the first time. The findings suggest that propolis holds promise as a potential cancer therapy by increasing PPP2R1A levels, a key molecule in cancer treatment.

Development and Bioavailability Assessment of an Estriol-Containing Vaginal Hydrogel.

Genitourinary syndrome of menopause (GSM) affects a significant percentage of postmenopausal women and manifests as vaginal dryness, irritation, and urinary discomfort, typically treated with vaginal estrogens. Hydrogels are preferred over creams due to their superior comfort and mucoadhesive properties. This study introduces a novel vaginal gel formulation containing hydroxyethyl cellulose (HEC) and estriol-hydroxypropyl-β-cyclodextrin complex (E3-HPBCD) for the treatment of GSM. The estriol (E3) release profile of the gel was evaluated using a Franz diffusion cell system, and its permeability was tested on reconstructed human vaginal epithelium. Biocompatibility was assessed using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT), lactate dehydrogenase (LDH) assays, and real-time cell analysis (RTCA) on human skin keratinocyte (HaCaT) cells, which showed increased cell viability and no obvious cytotoxicity. The results indicated that efficient E3 release and satisfactory epithelial permeability with HPBCD provide the bioavailability of E3. These results suggest the potential of the gel as a biocompatible and effective alternative for the treatment of GSM. Further studies are required to assess the long-term safety and clinical efficacy.

Advances in diagnostic assays for Clostridioides difficile infection in adults

Clostridioides difficile (C. difficile) was a gram-positive anaerobic bacterium in the gut, exhibiting clinical manifestations ranging from mild diarrhoea to fatal pseudomembranous colitis. C. difficile infection (CDI) remains a serious public health problem and accounted for an estimated 360,075 cases in the United States in 2021. It has attracted the utmost attention of the world health organization (WHO). Since publication of a review of the diagnosis of CDI in adults, new clinical diagnostic assays have become available and clinical practice guidelines were updated. This paper presents a comprehensive review of contemporary laboratory diagnostic approaches for CDI in adult patients, with a focus on the utilisation and potential advancements of five sophisticated methodologies, CRISPR in conjunction with nucleic acid amplification tests (NAATs), gene sequencing technology, ultra-high performance liquid chromatography-mass spectrometry, Raman spectroscopy, and real-time cell analysis (RTCA). It can provide new perspectives and ideas for the early diagnosis of CDI in clinical settings.

Antiproliferative effects of hazelnut cell culture extract on the different cancer cell lines.

The increasing incidence of cancer has necessitated the discovery of novel anticancer compound sources. The presence of taxanes in hazelnut cell cultures has promoted new promising pharmacotherapeutic applications. The antiproliferative properties of hazelnut (Corylus avellana cv. 'Kalınkara') cell culture extracts against different human cancer cell lines (HeLa, MCF-7, MDA-MB-231, A549) with Beas-2B as control were evaluated. The cytotoxicity of C. avellana culture extract (5 µM, 10 µM, and 20 µM) on all cell lines was evaluated with xCELLigence Real Time Cell Analysis System. Mitotic activity (450-655 nm), BrdU activity (450-550 nm) and caspase 3,7 activity (490-520 nm) were analyzed with a spectrophotometer through 24, 48, and 72 hours. Based on the values obtained from the xCELLigence Cell Analysis System, a 10 µM concentration of the culture extract was assigned as the IC50 dose. Culture extracts at 10 µM enhanced the reduction in the proliferation of all cancer cells assayed. The highest decrease in mitotic (59.32%) and BrdU (53.77%) activity was observed in A549 lung cancer cells. However, caspase 3,7 activity (35.08%) was the highest in aggressive MDA-MB-231 breast cancer cells. The culture extracts decreased the viability of A549 cells to a greater extent than that of MCF-7 and MDA-MB-231 breast, and HeLa cervical cancer cells. C. avellana cv. 'Kalınkara' cell culture extracts have potential use in the treatment of lung and, to a lesser extent, breast and cervical cancers.

Construction of a stable 4T1 cell line expressing UL19 by the PiggyBac transposon system

To investigate the mechanism of the major capsid protein VP5 (encoded by the UL19 gene) of oncolytic herpes simplex virus type Ⅱ (oHSV2) in regulating the antitumor function of immune cells, we constructed a mouse breast cancer cell line 4T1-iRFP-VP5-GFP stably expressing VP5 protein, near-infrared fluorescent protein (iRFP), and green fluorescent protein (GFP) by using the PiggyBac transposon system. Flow cytometry and Western blotting were employed to screen the monoclonal cell lines expressing both GFP and VP5 and examine the expression stability of UL19 in the constructed cell line. The results of SYBR Green I real-time PCR and Western blotting showed that the copies of UL19 and the expression level of VP5 protein in the 15th passage of 4T1-iRFP-VP5-GFP cells were significantly higher than those in the 4T1 cells transiently transfected with UL19, demonstrating the stable insertion of UL19 into the 4T1 cell genome. The real-time cell analysis (RTCA) was employed to monitor the proliferation of 4T1-iRFP-VP5-GFP cells, which showed similar proliferation activity to their parental 4T1 cells. Further studies confirmed that NK92 cells exhibited stronger cytotoxicity against 4T1-iRFP-VP5-GFP cells than against 4T1 cells. This study layed a foundation for elucidating the role of VP5 protein in regulating immune cells, including T cells and NK cells, via HLA-E in 4T1 cells to exert the anti-tumor function.

Differential Biological Effects of Trifolium pratense Extracts-In Vitro Studies on Breast Cancer Models.

The increasing popularity of herbal supplements emphasizes the need of scientific data regarding their health benefits and possible toxicological concerns. The complexity of botanical extracts, which include thousands of distinct compounds, contributes to the challenging nature of this endeavor. In this study, we explored the hormetic effects of two Trifolium pratense extracts on breast cell lines. Using a wide range of concentrations (0.1 to 3.33 mg/mL), we analyzed how extracts modulate cellular processes such as viability, proliferation, and oxidative stress on breast adenocarcinoma highly invasive estrogen receptor negative (ER-) and noninvasive ER+ cells, as well as on non-tumorigenic ER- normal cells. The cytotoxicity and real-time cell analysis (RTCA) assays showed that both extracts exercised a biphasic dose effect on adenocarcinoma ER+ and normal ER- cell proliferation and oxidative stress. We report a monotonic dose-dependent cytotoxicity on highly invasive adenocarcinoma ER- cells; the induced apoptosis was based on the pro-oxidant activity of extracts. The reactive oxygen species (ROS) generation by high-dose ethanolic extract was observed in all cells, followed by mitochondria dysfunction. Oxidative stress parameters, such as malondialdehyde (MDA) and reduced glutathione (GSH) levels, and superoxide dismutase (SOD) activity were affected. Our study demonstrates that T. pratense extracts have chemoprevention potential in normal and tumorigenic breast cells by modulating cellular proliferation and oxidative stress.

Investigation of the efficacy of siRNA-mediated KRAS gene silencing in pancreatic cancer therapy.

Pancreatic carcinoma is an aggressive cancer that progresses without many symptoms. The difficulty of early diagnosis and an inadequate response to traditional treatments also cause the survival rate of pancreatic cancer to be low. Current research is focusing on methods of diagnosis and treatment, such as gene therapy, to increase survival rates. Small interfering ribonucleic acid (siRNA) has emerged as a promising advanced therapeutic strategy for cancer treatment. This study sought to silence the KRAS gene in the human pancreatic carcinoma cell line using a complex of small interfering ribonucleic acid (siRNA) and gold nanoparticles (AuNP). In this study, 25 nM siRNA and gold nanoparticles at 0.5 mg/ml, 0.25 mg/ml, and 0.125 mg/ml concentrations were used to silence the KRAS gene in the CAPAN-1 cell line. Real-time PCR analysis, agarose gel electrophoresis, and double staining were carried out, and xCelligence real-time cell analysis (RTCA) was used to measure proliferation. The PCR analysis revealed crossing point (CP) values of actin beta (ACTB) ranging from 33.04 to 35.98, which was in the expected range for all samples. The interaction between the gold nanoparticle/siRNA complex in the double staining analysis revealed that the most effective concentration of gold nanoparticle was 0.125 mg/ml. The WST-1 technique showed that siRNA/AuPEI cells in application groups had a viability rate of over 90%, indicating no toxicity or side effects. The xCELLigence RTCA®showed that at hour 72, there was a significant difference in proliferation in the 0.5 mg/mL PEI/AuNP-siRNA, 0.25 mg/mL PEI/AuNP-siRNA, and 0.125 mg/mL PEI/AuNP-siRNA application groups compared to the control and siRNA groups (p<0.05). By hour 96, all three groups were statistically different from the control and siRNA groups in terms of proliferation (p<0.05). The results of this analysis suggest that the AuPEI/siRNA complex can be effectively used to silence the target gene, but more studies are needed to verify these results.

Potential PDE4A inhibition-mediated neuroprotective effects of psoralidin.

This study aimed to investigate the effect of psoralidin, a natural phenolic coumarin compound, on MK-801-induced neurotoxicity that may cause Alzheimer's disease and to determine the phosphodiesterase(PDE)-related molecular mechanism of action. In this study, neurotoxicity was performed using the MK-801 in the HT-22 cell line. The effects of compounds on the proliferation of HT-22 cells were determined by Real-Time Cell Analysis (RTCA). After measuring the total protein concentration, the PDE4A protein level was determined using the Western blot method. Psoralidin (100, 200, 400 µM) has been shown to have a neuroprotective effect against MK-801-induced neurotoxicity, as indicated by Real-Time Cell Analysis. In HT-22 cells, the half maximal effective concentration (EC50) value of psoralidin was calculated to be 230.4 µM, IC50 value of MK-801 was calculated to be 62.4 µM at 24 hours. It has been determined that psoralidin (200, 400 µM) inhibits PDE4A by using the Western blot method. This research uncovers that psoralidin has neuroprotective effects in MK801-associated accumulation of the excitatory amino acid glutamate neurodegeneration and Alzheimer's disease.

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p&amp;lt;0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Drug-Free Mesoporous Silica Nanoparticles Enable Suppression of Cancer Metastasis and Confer Survival Advantages to Mice with Tumor Xenografts.

Despite advancements in nanomedicine for drug delivery, many drug-loaded nanoparticles reduce tumor sizes but often fail to prevent metastasis. Mesoporous silica nanoparticles (MSNs) have attracted attention as promising nanocarriers. Here, we demonstrated that MSN-PEG/TA 25, with proper surface modifications, exhibited unique antimetastatic properties. In vivo studies showed that overall tumor metastasis decreased in 4T1 xenografts mice treated with MSN-PEG/TA 25 with a notable reduction in lung tumor metastasis. In vitro assays, including wound-healing, Boyden chamber, tube-formation, and real-time cell analyses, showed that MSN-PEG/TA 25 could modulate cell migration of 4T1 breast cancer cells and interrupt tube formation by human umbilical vein endothelial cells (HUVECs), key factors in suppressing cancer metastasis. The synergistic effect of MSN-PEG/TA 25 combined with liposomal-encapsulated doxorubicin (Lipo-Dox) significantly boosted mouse survival rates, outperforming Lipo-Dox monotherapy. We attributed the improved survival to the antimetastatic capabilities of MSN-PEG/TA 25. Moreover, Dox-loaded MSN-PEG/TA 25 suppressed primary tumors while retaining the antimetastatic effect, thereby enhancing therapeutic outcomes and overall survival. Western blot and qPCR analyses revealed that MSN-PEG/TA 25 interfered with the phosphorylation of ERK, FAK, and paxillin, thus impacting focal adhesion turnover and inhibiting cell motility. Our findings suggest that drug-free MSN-PEG/TA 25 is highly efficient for cancer treatment via suppressing metastatic activity and angiogenesis.

Hybrid Albumin-Decorated Lipid-Nanocarrier-Mediated Delivery of Polyphenol-Rich Sambucus nigra L. in a Potential Multiple Antitumoural Therapy.

The current research attempted to address the suitability of bioactive Sambucus nigra extract entrapped in albumin-decorated nanostructured lipid carriers (NLCs) as a promising "adjuvant" in improving tumour penetration for multiple antitumour therapy. The new hybrid albumin-decorated NLCs were characterised based on, e.g., the particle size, zeta electrokinetic potential, SambucusN entrapment efficiency, and fluorescence spectroscopy and tested for different formulation parameters. The antioxidant activity of NLC-SambucusN was significantly enhanced by a bovine serum albumin (BSA) polymer coating. According to the real-time cell analysis (RTCA) results, NLC-I-SambucusN-BSA behaved similarly to the chemotherapeutic drug, cisplatin, with cell viability for LoVo tumour cells of 21.81 ± 1.18%. The new albumin-NLC-SambucusN arrested cancer cells in G1 and G2 cycles and intensified the apoptosis process in both early and late phases. An advanced induction, over 50% apoptosis in LoVo colon cells, was registered for 50 μg/mL of NLC-II-SambucusN-BSA, a fourfold increase compared to that of untreated cells. RTCA and flow cytometry results showed that concentrations of the hybrid NLC-SambucusN up to 50 μg/mL do not affect the proliferation of normal HUVEC cells. This approach provides insightful information regarding the involvement of phytochemicals in future therapeutic strategies. Albumin-decorated NLCs can be considered a noteworthy strategy to be connected to antitumour therapeutic protocols.

KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic.MethodsSiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter.ResultsKLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation.ConclusionOur data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.

Ruthenium complexes with abiraterone acetate as antiproliferative agents

This study is dedicated to the development of multimodal anticancer agents. We have obtained ruthenium complexes conjugated with the steroid-type antitumor drug abiraterone acetate in order to take advantage of the dual antitumor properties of both ruthenium and abiraterone. The compounds exhibit good antiproliferative activity against cancer cells, with selectivity over primary fibroblasts. Real-time cell analysis revealed that compound dichlorido(η6-p-cymene)(abiraterone acetate)ruthenium(II) had pronounced antiproliferation activity compared to abiraterone acetate. Flow cytometric studies on the mechanism of cell death have revealed that the most active compound induces apoptosis more effectively than abiraterone acetate. Our findings demonstrate the potential of this novel dual-action compound as promising candidates for further development as anticancer agents.

The N-terminal fragment of histone deacetylase 4 (1-669aa) promotes chondrocyte apoptosis via the p53-dependent endoplasmic reticulum stress pathway.

Exogenous administration of the histone deacetylation 4 (HDAC4) protein can effectively delay osteoarthritis (OA) progression. However, HDAC4 is unstable and easily degrades into N-terminal (HDAC4-NT) and C-terminal fragments, and the HDAC4-NT can exert biological effects, but little is known about its role in chondrocytes and cartilage. Thus, the roles of HDAC4-NT fragments (1-289aa, 1-326aa and 1-669aa) in chondrocytes and cartilage were evaluated via real-time cell analysis (RTCA), safranin O staining, Sirius Red staining and nanoindentation. Molecular mechanisms were profiled via whole-transcriptome sequencing (RNA-seq) and verified invitro and invivo by a live cell real-time monitoring system, flow cytometry, western blotting and immunohistochemistry. The results showed that 1-669aa induced chondrocyte death and cartilage injury significantly, and the differentially expressed genes (DEGs) were enriched mainly in the apoptotic term and p53 signalling pathway. The validation experiments showed that 1-669aa induced chondrocyte apoptosis via the endoplasmic reticulum stress (ERS) pathway, and up-regulated p53 expression was essential for this process. Thus, we concluded that the HDAC4-NT fragment 1-669aa induces chondrocyte apoptosis via the p53-dependent ERS pathway, suggesting that in addition to overexpressing HDAC4, preventing it from degradation may be a new strategy for the treatment of OA.

Analysis of antioxidant nutrients, anti-HIV and anticancer metabolic fingerprints of Pelargonium quercifolium (L.f) L'Hér

Pelargonium quercifolium is an aromatic herb with a strong resinous scent. It is used traditionally to treat heart disease, hypertension, stress, rheumatism, combat hidden hunger, and inhibit HIV-1. The present study aimed to evaluate the toxicity, anti-HIV, antioxidant nutrients and anticancer potentials of the ethanol extract of P. quercifolium. In this study, high-resolution ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), high-performance liquid chromatography with diode-array detection (HPLC-D AD), xCELLigence real-time cell analyser single plate (RTCA-SP), MTT assays and luciferase-based assay were employed to elucidate the phytochemicals, cytotoxicity, anti-HIV, antioxidant nutrients and anticancer metabolites in P. quercifolium. Results show that the characterized metabolites in P. quercifolium most of which were flavonoids, phenolic derivatives, alkaloids and coumarins could arrest cancer cell proliferation and inhibit HIV infection given the high level of cytotoxicity of the tested plant extract. It was also observed and reported for the first time, the nutrient profile of P. quercifolium is higher than most vegetables consumed as a staple food. This study underpins the potential of using anticancer and antiviral metabolites from leaf extract of P. quercifolium as a pharmacological option.