Late Apoptosis Research Articles

An arresten-derived anti-angiogenic peptide triggers apoptotic cell death in endothelial cells.

In recent years, anti-angiogenic peptides have received considerable attention as candidates for cancer treatment. Arresten is an angiogenesis inhibitor that cleaves from the α1 chain of type IV collagen and stimulates apoptosis in endothelial cells. We have recently indicated that a peptide corresponding to the amino acid 78 to 86 of arresten, so-called Ars, prevented the migration and tube formation of HUVECs and the colon carcinoma growth in mice significantly. The current study aimed to determine whether induction of apoptotic cell death in endothelial cells is one of the biochemical mechanisms of this anti-angiogenic peptide. This hypothesis was assessed using the MTT assay, cell cycle analysis, Annexin V-FITC/PI staining, BCL2, CASP8, CASP9, p53, and CDKN2A gene expression studies as well as evaluating apoptosis in tumor tissues by TUNEL assay. Results demonstrated that 40µM of Ars significantly stimulated 46.2% of early and late apoptosis in HUVECs compared to 13.6% in the untreated cells and did not significantly alter the cell cycle distribution. Moreover, BCL2 and CASP8 were down-regulated, while CASP9 and p53 were up-regulated in endothelial cells. CDKN2A gene expression, the regulator of G1 cell cycle arrest, was not significantly altered. It might be suggested that Ars induced apoptosis in endothelial cells through the mitochondrial pathway and had no effect on the cell cycle. Besides, Ars induced apoptosis significantly in vivo. However, further studies are required to confirm the detailed molecular mechanism of Ars, this peptide has the potential to be optimized for clinical translations.

In-Vitro Effects of Perfluorooctanoic Acid on Human Sperm Function: What Are the Clinical Consequences?

Background: Lifestyle and environmental pollution harm male fertility. Perfluoroalkyl substances (PFAS) are bio-accumulates in the environment as well as in several human tissues, and one of the most common PFAS is perfluorooctanoic acid (PFOA). Therefore, this study aimed to evaluate the in vitro effects of PFOA with hydrophobic and waterproofing properties on motility and bio-functional sperm parameters. Methods: To accomplish this, 50 healthy men with normozoospermia and not exposed to high doses of PFAS were enrolled. Their spermatozoa were incubated for 3 h with increasing concentrations of PFOA (0, 0.01, 0.1, and 1 mM) to evaluate its effects. In particular, we evaluated the effects of PFOA on total and progressive sperm motility and, by flow cytometry, on the following bio-functional sperm parameters: degree of chromatin compactness, viability, early and late apoptosis, mitochondrial membrane potential, the degree of lipoperoxidation, and concentrations of mitochondrial superoxide anion. Results: The results showed that PFOA decreased both total and progressive sperm motility, impaired chromatin compactness, and increased sperm lipid peroxidation and mitochondrial superoxide anion levels. Conclusions: This study showed that PFOA alters several sperm parameters and thus it may play a negative role in male fertility.

Promoting the Anticancer Activity with Multidentate Furan-2-Carboxamide Functionalized Aroyl Thiourea Chelation in Binuclear Half-Sandwich Ruthenium(II) Complexes.

A new set of binuclear arene ruthenium complexes [Ru2(p-cymene)2(k4-N2OS)(L1-L3)Cl2] (Ru2L1-Ru2L3) encompassing furan-2-carboxamide-based aroylthiourea derivatives (H2L1-H2L3) was synthesized and characterized by various spectral and analytical techniques. Single-crystal XRD analysis unveils the N^O and N^S mixed monobasic bidentate coordination of the ligands constructing N, S, Cl/N, O, and Cl legged piano stool octahedral geometry. DFT analysis demonstrates the predilection for the formation of stable arene ruthenium complexes. In vitro antiproliferative activity of the complexes was examined against human cervical (HeLa), breast (MCF-7), and lung (A549) cancerous and noncancerous monkey kidney epithelial (Vero) cells. All the complexes are more efficacious against HeLa and MCF-7 cells with low inhibitory doses (3.86-11.02 μM). Specifically, Ru2L3 incorporating p-cymene and -OCH3 fragments exhibits high lipophilicity, significant cytotoxicity against cancer cells, and lower toxicity on noncancerous cells. Staining analysis indicates the apoptosis-associated cell morphological changes expressively in MCF-7 cells. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) analyses reveal that Ru2L3 can raise ROS levels, reduce MMP, and trigger mitochondrial dysfunction-mediated apoptosis. The catalytic oxidation of glutathione (GSH) to its disulfide form (GSSG) by the complexes may simultaneously increase the ROS levels, alluding to their observed cytotoxicity and apoptosis induction. Flow cytometry determined the quantitative classification of late apoptosis and S-phase arrest in MCF-7 and HeLa cells. Western blotting analysis confirmed that the complexes promote apoptosis by upregulating Caspase-3 and Caspase-9 and downregulating BCL-2. Molecular docking studies unfolded the strong binding affinities of the complexes with VEGFR2, an angiogenic signaling receptor, and BCL2, Cyclin D1, and HER2 proteins typically overexpressed on tumor cells.

Novel Tetracyclic Azaphenothiazines with the Quinoline Ring as New Anticancer and Antibacterial Derivatives of Chlorpromazine.

Phenothiazine derivatives are widely studied in various fields such as biology, chemistry, and medicine research because of their pharmaceutical effects. The first compound used successfully in the treatment of psychosis was a phenthiazine derivative, chlorpromazine. Apart from its activity in neurons, chlorpromazine has also been reported to display anticancer and antibacterial properties. In this study, we present the synthesis and research on the activity of A549, MDA, MiaPaCa, PC3, and HCT116 cancer cell lines and of S. aureus, S. epidermidis, E. coli, and P. aeruginosa bacterial strains against a series of new tetracyclic chlorpromazine analogues containing a quinoline scaffold in their structure instead of the benzene ring and various substituents at the thiazine nitrogen. The structure of these novel molecules has been determined by 1H NMR, 13C NMR, and HRMS spectral techniques. The seven most active of the twenty-four new chlorpromazine analogues tested were selected to study the mechanism of cytotoxic action. Their ability to induce apoptosis or necrosis in cancer cells was assessed by flow cytometry analysis. The results obtained confirmed the proapoptotic activity of selected compounds, especially in terms of inducing late apoptosis or necrosis in cancer cell lines A549, MiaPaCa-2, and HCT-116. Furthermore, studies on the induction of cell cycle arrest suggest that the new chlorpromazine analogues exert antiproliferative effects by inducing cell cycle arrest in the S phase and, consequently, apoptosis.

Transcriptome sequencing reveals the promotion of apoptosis and M1 polarization of microglia under simulated microgravity

The exponential progress in the field of space science and technology has facilitated the prolonged habitation of astronauts in the space environment. However, the neurological and cognitive impairments of astronauts have become major factor that directly impede the successful completion of space missions. Studies have demonstrated that microgravity can diminish neuronal functions and cognitive abilities by altering the microenvironment of the brain (BME). Microglia, vital immune cells responsible for regulating the integrity of the blood-brain barrier, have been identified as the principal contributing elements to the neurological deterioration observed in microgravity conditions. The primary objective of this study was to provide an understanding of the molecular processes underlying the behavior of microglia under conditions of microgravity. Using a Random Positioning Machine (RPM) to simulate microgravity, our study found that microgravity reduces BV2 microglial cell viability, induces M1 polarization, and significantly increases late apoptosis. Transcriptome analysis revealed 142 differentially expressed genes related to microglial phenotype and inflammation, with qPCR validating selected DEGs. These results elucidate the molecular mechanisms of microglial behavior under microgravity, highlighting gene expression changes and cellular damage. This study provides a conceptual framework for comprehending the influence of microgravity on the neurological system, establishing countermeasures against functional damage to the nervous system caused by space flight, and serving as a reference for future deep space exploration and long-term manned space medical support.

Klebsiella pneumoniae-OMVs activate death-signaling pathways in Human Bronchial Epithelial Host Cells (BEAS-2B)

The programmed cell death pathways of apoptosis are important in mammalian cellular protection from infections. The activation of these pathways depends on the presence of membrane receptors that bind bacterial components to activate the transduction mechanism. In addition to bacteria, these mechanisms can be activated by outer membrane vesicles (OMVs). OMVs are spherical vesicles of 20–250 nm diameter, constitutively released by Gram-negative bacteria. They contain several bacterial determinants including proteins, DNA/RNA and proteins, that activate different cellular processes in host cells. This study focused on Klebsiella pneumoniae-OMVs in activating death mechanisms in human bronchial epithelial cells (BEAS-2B). Characterization of purified OMVs was achieved by scanning electron microscopy, nanoparticle tracking analysis and protein profiling. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while apoptotic induction was measured by flow cytometry and confirmed by western blotting. The OMVs produced showed a spherical morphology, with a diameter of 137.2 ± 41 nm and a vesicular density of 7.8 × 109 particles/mL Exposure of cell monolayers to 50 μg of K. pneumoniae-OMV for 14 h resulted in approximately 25 % cytotoxicity and 41.15–41.14 % of cells undergoing early and late apoptosis. Fluorescence microscopy revealed reduced cellular density, the presence of apoptotic bodies, chromatin condensation, and nuclear membrane blebbing in residual cells. Activation of caspases −3 and −9 and dysregulation of BAX, BIM and Bcl-xL indicated the activation of mitochondria-dependent apoptosis. Furthermore, a decrease in the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase involved endoplasmic reticulum stress with the potential formation of reactive oxygen species. These findings provide evidence for the role of OMVs in apoptosis and involvement in the pathogenesis of K. pneumoniae infections.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Effects of hypoxia on the growth of gastric cancer and the chemotherapeutic efficacy of 5-fluorouracil.

Hypoxia is an important cause of chemotherapy resistance in gastric cancer. However, little is known about the growth of gastric cancer under purely hypoxia conditions. This study aims to study the effect of hypoxia on the growth patterns of gastric cancer cells and explore the response of gastric cancer cells to the chemotherapeutic drug 5-fluorouracil (5-FU) in a hypoxic environment. Gastric cancer cells MKN45 were cultured under 1% oxygen hypoxia and conventional air conditions. An intervention group with the addition of the chemotherapeutic drug 5-FU was also established. The proliferation and apoptosis of gastric cancer cells under different oxygen conditions and intervention groups were detected using the cell counting kit-8 (CCK-8) method, JC-1 mitochondrial membrane potential assay, and Annexin-V/PI double staining method. Cell cycle changes were detected by flow cytometry, and mitochondrial changes were detected using electron microscopy. In the absence of 5-FU intervention, compared with the normoxia group, the hypoxia group showed higher rates of early and late apoptosis and higher cell death rates as indicated by the JC-1 mitochondrial membrane potential assay, Annexin-V/PI double staining, and CCK-8 results. Flow cytometry results showed that the cell cycle was arrested in the G0/G1 phase without progression. Electron microscopy revealed more severe mitochondrial destruction. However, with 5-FU intervention, the hypoxia group showed lower apoptosis rates, more cell cycle progression, and less mitochondrial destruction compared with the normoxia group. Hypoxic environments promote apoptosis and even death in gastric cancer cells, but hypoxia counteracts the efficacy of the chemotherapeutic drug 5-FU, which may contribute to 5-FU chemotherapy resistance.

Exploring the molecular mechanism of Epimedium brevicornu Maxim. in treating breast cancer via network pharmacology and in vitro experiments

ObjectiveTo evaluate the therapeutic effects of Epimedium brevicornu Maxim. (EBM, Yin Yang Huo) on breast cancer using network pharmacology and in vitro validation. It also aimed to explore the novel targets and mechanisms of EBM in the treatment of breast cancer to facilitate the discovery of new drugs and their clinical application. MethodsNetwork pharmacology was used to identify and screen the components and targets of EBM for breast cancer treatment. Molecular docking was further screened the effective components and targets of EBM. Wound-healing assays and flow cytometry analysis were used to detect the ability of two compounds to intervene in the migration and apoptosis of MDA-MB-231 cells, and their mechanism of action was further explored using western blotting experiments. ResultsEBM contained 19 active components. Among them were β-anhydroicaritin (Anhy) and isoliquiritigenin (Iso), which were selected for in vitro experiments. Treatment resulted in a dose-dependent suppression of MDA-MB-231 cell viability, with an IC50 of 23.73 μmol/L for Iso and 21.28 μmol/L for Anhy. In the wound healing assay, cells in Anhy and Iso groups exhibited considerable inhibition of migration at 48 h. In flow cytometry analysis, treatment with Iso (20 μmol/L) for 96 h resulted in significantly higher levels of both early and late apoptosis in the Iso group than that in the control group (P = .004 and P = .014, respectively). Additionally, both Iso (20 μmol/L) and Anhy (10 and 20 μmol/L) induced cell necrosis at 96 h. Western blotting revealed that Anhy and Iso increased the expression of Bax and TBK1/NAK. ConclusionThese findings suggested that Anhy and Iso, the two components of EBM, inhibit MDA-MB-231 cell proliferation and migration of and induce their apoptosis, providing substantial support for future studies on breast cancer.

Co-delivery of curcumin and resveratrol by folic acid-conjugated poly(glycerol adipate) nanoparticles for enhanced synergistic anticancer effect against osteosarcoma

This study explored the co-delivery of curcumin (CUR) and resveratrol (RV) using folic acid-conjugated poly(glycerol adipate)-based nanoparticles (FPPC NPs) to enhance their synergistic anticancer effects against osteosarcoma. Based on synergistic toxicity experiments against Saos-2 cells, the optimal synergistic CUR:RV ratios were 1:2 and 1:3, which were used for co-encapsulation. Increasing the amount of RV in the co-loaded NPs did not affect the properties of the nanocarriers, but predominantly increased the loading capacity of RV, especially at the 1:3 ratio, by 1.8–2.0 times, mediated by their interaction. All co-loaded NPs demonstrated sustained release of CUR with a burst release of RV, and the presence of RV accelerated the initial release of CUR from the carriers. Furthermore, the co-encapsulated NPs maintained CUR and RV synergism and greatly enhanced their toxicity against osteosarcoma by at least 1.8 times compared to their corresponding solutions through profound accumulation of Saos-2 cells in the sub G1 phase and late apoptosis. The internalization of FPPC NPs into cells via endocytosis was dose- and time-dependent. This study offers a proof-of-concept for a potential co-delivery system using tumor-targeted poly(glycerol adipate)-based NPs to enhance the anticancer activity of CUR and RV against osteosarcoma.

Abstract 4590: Antitumor effects of histone deacetylase inhibitors on breast cancer cell viability and mammosphere formation

Abstract Histone deacetylases (HDACs) are overexpressed in various types of primary human cancer and have become attractive targets for cancer therapy. This study aimed to evaluate the cytotoxic properties of three HDAC inhibitors (suberoylanilide hydroxamic acid (SAHA, or Vorinostat), Valproic Acid (VPA) and CAY10603) and compare them to Paclitaxel (PTX), a chemotherapy agent used to treat Triple Negative Breast Cancers (TNBCs). In the present study, drug response in the MDA-MB-231, MDA-MB-468, and MCF-10A cell lines was evaluated by cell viability assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Mammospheres generated from MDA-MB-231 and MDA-MB-468 breast cancer cells were also subjected to drug treatment; their response was assessed under both normoxic and hypoxic conditions. We found that SAHA and CAY10603 inhibited breast cancer cell viability without significantly affecting MCF-10A cells. SAHA, CAY10603, and VPA all arrested a significant percentage of MDA-MB-231 cells at G2/M phase after 24h compared to controls. TNBC cells experienced an increase in both early and late apoptosis following treatment with the most effective concentrations of SAHA, CAY10603, and VPA (1.8µM, 2µM, and 0.4mM, respectively). SAHA proved to be the most effective drug for induction of apoptosis compared to the control, followed by CAY10603 (8.26% and 6%, 3.67%). In both TNBCs and mammospheres under hypoxic conditions, SAHA and CAY10603 down regulated HIF-1α expression significantly, but PTX did not. In contrast, VPA and PTX down regulated CTNNB1 gene expression. This study indicates that epi-drugs exert an anti-tumor effect on TNBCs, suggesting a potential role for HDAC inhibitors as novel therapeutics for breast cancer patients. Citation Format: Golnaz Asaadi Tehrani, Becca Kubick, Meenal Datta. Antitumor effects of histone deacetylase inhibitors on breast cancer cell viability and mammosphere formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4590.

Abstract 3162: Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin

Abstract Introduction: Epidemiological studies showed that beta-adrenergic antagonists caused a low incidence of several cancer types. Glioblastoma highly expresses beta-2-adrenergic receptors. GBM accounts for 50% of central nervous system cancers. After standard-of-care therapeutic intervention, there is a survival time of 15 months. Our goal is to develop a translational therapy that will trigger the death of GBM cells while preserving healthy cells Methodology: Salmeterol xinafoate is a biased agonist that activates the G-protein pathway. Biased agonists activate signaling through the G-protein or the beta-arrestin pathway of G-protein coupled receptors. Dose-response experiments were performed using beta1 and beta2-adrenergic receptor antagonists and biased beta2-adrenergic receptor agonists to determine loss of glioma cell viability in GBM5, GBM12, and LN229 glioma cell lines. Western immunoblotting gauged survivin expression levels in all three lines. Epoxomicin was used to inhibit the proteasome complex. Annexin V and propidium iodide were employed to measure apoptotic or necrotic cell death. Results: Disrupting beta2-adrenergic receptor signaling by salmeterol xinafoate led to cell death of glioma cells but spared healthy neuron-glia cells using similar doses. Salmeterol xinafoate revealed greater efficacy for killing glioma cells than beta1 and beta2-adrenergic receptor antagonists. To determine the mechanism of glioma cell death, LN229 cells were treated with escalating doses of Salmeterol xinafoate, and at 24 hours, annexin V staining showed early apoptosis, and Annexin V/propidium iodide co-staining also showed late apoptosis. Propidium iodide revealed minor necrosis in LN229 glioma cells. Western immunoblotting showed salmeterol xinafoate downregulated survivin in a dose-dependent manner. However, the epoxomicin's inhibition of the proteasome complex prevented dose-escalated downregulation of survivin for all three glioma cell lines. Conclusion: The bitopic structure of salmeterol enables this drug to bind to the beta2-adrenergic receptors ligand binding site and an exosite site by its aryloxyalkyl tail that may contribute to the increased efficacy. Salmeterol xinafoate triggers glioma cell death by apoptosis at least in one glioma cell line. Salmeterol Xinafoate down-regulates survivin through protease degradation. Promising results show that the FDA-approved drug salmeterol xinafoate enables a favorable transition to the clinic. Repurposing this drug for GBMs may be combined with the current standard-of-care chemotherapies to enhance more significant loss of tumor volume and increase patient survival. Citation Format: Orli Algranatti, Ji Yae Lee, James M. Angelastro. Antagonizing beta-2 adrenergic receptor’s function triggers death in glioma cells and down-regulates survivin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3162.

Abstract 956: NCX 4040 and napabucasin synergistically inhibits stemness in human prostate cancer (PCa) cellular models and potently targets transgenic murine PCa cells via inducing oxidative stress

Abstract Prostate cancer (PCa) continues to have a substantial impact on men’s wellbeing and quality of life regardless of recent progress in diagnosis and treatments and it continues to have the second highest occurrence rate following lung cancer. The strategy of combination drug therapy has gradually become a valuable choice for treating various cancer types, addressing the heterogeneity within tumors, and surpassing the constraints of existing treatment methods. In our previous work, when we combined two drugs NCX 4040 (an oxidative stress inducer) and napabucasin (a STAT3 signaling inhibitor), we discovered that the combination treatment synergistically works to inhibit the malignant characteristics of human PCa cells. Also, the combination upregulated the cGAS-STING immunogenic pathway in PCa cells which could potentially activate the cancer-targeting cytotoxic T-cells. In the current study, we aimed at investigating the anti-stemness properties of the drug duo which can not only inhibit cancer cell differentiation but also can be effective in preventing cancer relapse. We used two cell lines i.e., BPH-Cd which served as a model of cadmium induced PCa and DU145 as a model of advanced PCa to examine the anti-stemness properties of the drug combination. Based on western-blot analysis, we found the downregulation of transcription factors such as Oct4, Sox2, Nanog, β-catenin and cell surface markers like CD44, CD133 and ALDHA1, which are responsible for stem-cell like properties. In addition, we have tested the combinatory anticancer effects in transgenic mouse derived PCa cell line (Myc-Cap) as a prelude to conduct future studies in FVB mouse syngeneic model to evaluate the tumor suppressive and the antitumor immunogenic responses of combination treatment as it activated cGAS-STING pathway. By performing the cell viability assay, we found that the IC50 value of NCX 4040 is ~10µM and that of napabucasin is ~1μM, however when used in combination, ~90% inhibition is seen at 10µM of NCX and 250nM of napabucasin dual treatment. The drug combination also showed synergy in inhibiting tumorigenicity in clonogenic assay and migratory potential in wound-healing assay. By investigating the ROS production, cell death and cell cycle mechanism using flow cytometric analysis, it was observed that the combination therapy synergistically induces cellular oxidative stress which causes cancer cells to undergo late apoptosis and halts the growth of Myc-Cap cells in G2M phase of cell cycle. In conclusion, our data suggests that combination therapy of NCX 4040 and napabucasin can be potential treatment strategy for PCa warranting further evaluation in in vivo models. Citation Format: Shrushti Shah, Gnanasekar Munirathinam. NCX 4040 and napabucasin synergistically inhibits stemness in human prostate cancer (PCa) cellular models and potently targets transgenic murine PCa cells via inducing oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 956.

Antiproliferative and Apoptosis-Inducing Effects of Ethanolic Extract of Morus Alba Leaves on Human Chronic Myeloid Leukaemia K-562 Cell Lines

The rate of occurrence and prevalence of chronic myeloid leukaemia (CML) are increasing globally, despite it being a rare type of cancer. Morus alba also known as mulberry, has been used in Chinese traditional medicine for a long time to treat various conditions and diseases like hypertension and arthritis. Modern scientific studies have also recognized its medicinal properties, including antioxidant, antidiuretic, antidiabetic, antimicrobial and anticancer. However, its antiproliferative effects on specifically human CML K-562 cell lines remain poorly elucidated. This study aims to determine the antiproliferative effects of M. alba on K-562 cells. In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Acridine orange and propidium iodide (AO/PI) dual staining were then employed to assess the method of cell death. The ethanolic extract of M. alba leaves exhibited significant antiproliferative activity, with the lowest maximal half inhibitory concentration (IC50) value observed at 72 hours being 23 µg/mL. Notably, AO/PI staining revealed significant morphological changes, such as membrane blebbing, nuclear fragmentation, and indicator of early apoptosis, and late apoptosis. These results demonstrated that ethanolic extract of M. alba leaves induces apoptotic cell death in K-562 cells. Overall, these findings suggested that ethanolic extract of M. alba leaves exerts potent antiproliferative effects on human CML K-562 cells a in time- and dose-dependent manner.

Lily Polysaccharide Nano‐Selenium Promotes A549 Cells Apoptosis through Cell Cycle Inhibition

AbstractThis study uses Lilium davidii var. unicolor (Lanzhou lily) polysaccharides extracted from bulbs to prepare nano‐selenium (nano‐Se) polysaccharide (LPS). Here, the study explores the molecular structure characterization, inhibition effects, and mechanisms of action of prepared LPS on A549 lung cancer cells. The LPS structure is characterized using Fourier‐transform infrared spectroscopy (FTIR), UV spectroscopy (UV–vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), X‐ray photoelectron spectroscopy (XPS), etc. Results show that selenized polysaccharides bonded through electrostatic adsorption, with an average LPS particle size of 243.75 nm and zeta potential of −38.3 mV, which can be stably preserved at 4 °C for more than 3 months. The in vitro antitumor activity experiment shows that the prepared LPS inhibited A549 cell proliferation while affecting cell migration. Cell cycle analysis shows that apoptosis is promoted by blocking the G1 phase, while during the late apoptosis stage cells accounted for 60%. RNA‐Seq data analysis shows that the main mechanisms of action on A549 cells are to inhibit DNA replication initiation genes, block the cell cycle, and regulate the expression of apoptosis‐related genes and proteins in promoting cell apoptosis.

Unique fluorescent probe for the recognition of late apoptosis via translocation from plasma membrane to nucleus

Cell apoptosis is a crucial physiological process playing central roles in key biological and pathological activities. However, the current fluorescent probes for the detection of late apoptosis were “off–on” probes, which were facilely interfered by false positive signals caused by inhomogeneous staining and other factors. Herein, a unique fluorescent probe (NPn) discriminating late apoptosis from early apoptosis and heathy status with two different sets of fluorescent signals have been prepared, to overcome the possible false positive signals. NPn was designed impermeable to biomembranes and simultaneously with high affinity to DNA/RNA, which localized on the plasma membranes of living and early apoptotic cells, while relocated to the nucleus in late apoptotic cells. The hydrophilic amine unit and small ion radius were responsive for its membrane impermeability, which was confirmed with two control molecules without amine group. Using the probe, we have successfully evaluated the cell apoptosis induced by ultraviolet irradiation, rotenone, colchicine, and paclitaxel, demonstrating its potential application in biological researches.

The effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on human cervical, colorectal, and mammary carcinoma cell lines

Targeting the inflammation-related molecules with nonsteroidal anti-inflammatory drugs (NSAIDs) represents a promising approach for cancer prevention/therapy. We evaluated the in vitro anticancer effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on the proliferation, migration, and apoptosis of human cervical, colorectal, and mammary carcinoma cells. The antiproliferative activity and cytotoxicity of tested NSAIDs on HeLa, HT-29, and MCF-7 cell lines were assessed by the MTT test. The apoptosis-inducing potential was analyzed by fluorescent staining with acridine orange/ethidium bromide and DAPI. Migration activity was assessed by a wound-healing scratch assay. The tested NSAIDs reduced the viability of the used tumor cell lines. The cytomorphological analysis revealed reduced cell density and mitotic activity and the presence of cells with morphological features of early and late apoptosis. Significant inhibition of the migration capacity was established as well. In conclusion, NSAIDs could be candidates for the development of new pharmacological strategies for the treatment and prevention of cancer.

Exploring the therapeutic potential of SGLT2 inhibitors in cancer treatment: integrating in silico and in vitro investigations.

The present study aimed to investigate the anti-cancer mechanism of canagliflozin (CANA) and dapagliflozin (DAPA), sodium-glucose co-transporter-2 (SGLT2) inhibitors, using in silico and in vitro approaches. Network pharmacology was employed to predict the targets of the inhibitors and GO gene enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation conducted to explore the interacting pathways. Molecular docking and molecular dynamic (MD) simulation studies were performed to confirm the important targets and assess conformational stability. In vitro cytotoxicity assays, MIA-PaCa-2 and DU-145 cell lines CANA and DAPA was performed. Protein-protein interaction (PPI) network analysis indicated that CANA and DAPA exert anticancer effects through MAPK, mTOR, EGFR-KRAS-BRAF, FGFR, and PI3KA pathways. KEGG analysis revealed that these inhibitors could be used in the treatment of various cancers, including breast, prostate, pancreatic, chronic myeloid leukemia, thyroid, small cell lung, gastric, and bladder cancer. Docking results showed highest affinity for MAPK1 for CANA (- 9.60kcal/mol) and DAPA (- 9.58kcal/mol). MD simulation results showed that RMSD values for the MAPK1-compound exhibit remarkable stability over a timeframe of 100ns. In cytotoxicity assays using MIA-PaCa-2 and DU-145 cell lines, CANA demonstrated a potential antiproliferative effect on the pancreatic cell line MIA-PaCa-2 after 48h of treatment at a concentration of 100µg/ml. Furthermore, CANA arrested the cell cycle in the sub-G1 phase and induced late apoptosis and necrosis in MIA-PaCa-2 cell line. Based on these findings, CANA shows promise as a potential novel treatment strategy for pancreatic cancer.

Endotoxin Inflammatory Action on Cells by Dysregulated-Immunological-Barrier-Linked ROS-Apoptosis Mechanisms in Gut-Liver Axis.

Our study highlighted the immune changes by pro-inflammatory biomarkers in the gut-liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammations when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis. In duodenal tissue samples, gut immune barrier dysfunction was analyzed by pro-inflammatory biomarker expressions, oxidative stress, and cell death by flow cytometry methods. A significant innate and adaptative immune system reaction was observed as result of persistent endotoxin action in gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with the A-B child stage. Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression. Interesting mechanisms of ROS-cell death were observed in chronic and acute inflammation samples when gut cells were exposed to endotoxins and immune changes in the gut-liver axis. Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and liver-dysregulated barrier. Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation. Flow cytometric analysis of pro-inflammatory biomarkers linked to oxidative stress-cell death mechanisms shown in our study recommends laboratory techniques in diagnostic fields.

Design, synthesis, molecular modelling and biological evaluation of novel 6-amino-5-cyano-2-thiopyrimidine derivatives as potent anticancer agents against leukemia and apoptotic inducers

Herein, a novel series of 6-amino-5-cyano-2-thiopyrimidines and condensed pyrimidines analogues were prepared. All the synthesized compounds (1a-c, 2a-c, 3a-c, 4a-r and 5a-c) were evaluated for in vitro anticancer activity by the National Cancer Institute (NCI; MD, USA) against 60 cell lines. Compound 1c showed promising anticancer activity and was selected for the five-dose testing. Results demonstrated that compound 1c possessed broad spectrum anti-cancer activity against the nine cancerous subpanels tested with selectivity ratio ranging from 0.7 to 39 at the GI50 level with high selectivity towards leukaemia. Mechanistic studies showed that Compound 1c showed comparable activity to Duvelisib against PI3Kδ (IC50 = 0.0034 and 0.0025 μM, respectively) and arrested cell cycle at the S phase and displayed significant increase in the early and late apoptosis in HL60 and leukaemia SR cells. The necrosis percentage showed a significant increase from 1.13% to 3.41% in compound 1c treated HL60 cells as well as from 1.51% to 4.72% in compound 1c treated leukaemia SR cells. Also, compound 1c triggered apoptosis by activating caspase 3, Bax, P53 and suppressing Bcl2. Moreover, 1c revealed a good safety profile against human normal lung fibroblast cell line (WI-38 cells). Molecular analysis of Duvelisib and compound 1c in PI3K was performed. Finally, these results suggest that 2-thiopyrimidine derivative 1c might serve as a model for designing novel anticancer drugs in the future.