Changes In Nuclear Morphology Research Articles

Proteasome inhibition induces microtubule-dependent changes in nuclear morphology

Proteasome inhibition induces microtubule-dependent changes in nuclear morphology

Doxophylline, a Non-Selective Phosphodiesterase Inhibitor, Protects Against Chronic Fatigue-Induced Neurobehavioral, Biochemical, and Mitochondrial Alterations.

Chronic fatigue stress (CFS) is a multisystem disorder which exhibits multiple signs of neurological complications like brain fog, cognitive deficits and oxidative stress with no specific treatment. Doxophylline, a non-selective phosphodiesterase inhibitor (PDEI), has anti-inflammatory properties with enhanced blood-brain barrier penetration and tissue specificity. We have evaluated the neuroprotective potential of doxophylline in a murine model of forced swim test (FST) induced CFS and in H2O2 (hydrogen peroxide) induced oxidative stress in PC12 cells. An FST model to induce a state of CFS in mice was induced by forcing them to swim daily for 6min for 15 days. The drug was administered daily 30min prior to FST. The immobility period was compared for day 1 and day 15. Animals were sacrificed on day 16 for biochemical, mitochondrial, and histopathological estimations in the brain. Cytotoxicity assay, reactive oxygen species (ROS) and nuclear morphology determination were carried out in PC12 cells. A significant increase in immobility has been observed on the 15th day in CFS-induced mice compared to doxophylline treated group. Neurobehavioral studies revealed hypo locomotion, anxiety, motor incoordination, and memory deficit. Biochemical analysis showed a significant change in oxidative stress markers (superoxide dismutase (SOD), reduced glutathione (GSH), catalase, lipid peroxidation (LPO) and nitrite levels) and acetylcholinesterase enzyme activity (AChE) in brain homogenates. Doxophylline pre-treatment protects against these impairments. In PC12 cell lines, doxophylline exhibits alleviation against H2O2-induced oxidative stress, intracellular ROS generation, and changes in nuclear morphology. Doxophylline could be promising and possess therapeutic potential in CFS treatment. Further research is needed to test if doxophylline can be repurposed for neurological disorders.

Effect of Acacia concinna Extract on Apoptosis Induction Associated with Endoplasmic Reticulum Stress and Modulated Intracellular Signaling Pathway in Human Colon HCT116 Cancer Cells.

Colorectal cancer (CRC) stands as one of the most prevalent cancer types and among the most frequent causes of cancer-related death globally. Acacia concinna (AC) is a medicinal and edible plant that exhibits a multitude of biological properties, including anticancer properties. This study aimed to investigate the impact of the AC extract on apoptosis induction and the underlying mechanisms associated with this effect in KRAS-mutated human colon HCT116 cells. The effect of AC extract on cell cytotoxicity was evaluated using MTT assay. Nuclear morphological changes were visualized with Hoechst 33342 staining, while mitochondrial membrane potential (MMP) was assessed via JC-1 staining. Flow cytometry was employed for cell cycle analysis, and intracellular ROS levels were determined using DCFH-DA staining. The results showed that HCT116 cells exposed to AC extract showed reduced cell growth and prompted apoptosis, as indicated by an increase in chromatin condensation, apoptotic bodies, the sub-G1 apoptotic cell population, and disrupted MMP. Expression levels of apoptosis mediator proteins determined by Western blot analysis showed an increase in pro-apoptotic proteins (Bak and Bax) while decreasing anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1), leading to caspase-7 activation and PARP inactivation. AC extract was also found to enhance intracellular reactive oxygen species (ROS) levels and stimulate endoplasmic reticulum (ER) stress. Furthermore, AC extract increases the phosphorylation of ERK1/2, p38, and c-Jun while downregulating PI3K, Akt, β-catenin, and their downstream target proteins. These results demonstrate that AC extract could inhibit cancer cell growth via ROS-induced ER stress associated with apoptosis and regulate the MAPK, PI3K/Akt, and Wnt/β-catenin signaling pathways in HCT116 cells. Therefore, AC extract may be a novel candidate for natural anticancer resources for colon cancer treatment.

Heterogeneous Nuclear Ribonucleoprotein A1 Knockdown Alters Constituents of Nucleocytoplasmic Transport.

Changes in nuclear morphology, alterations to the nuclear pore complex (NPC), including loss, aggregation, and dysfunction of nucleoporins (Nups), and nucleocytoplasmic transport (NCT) abnormalities have become hallmarks of neurodegenerative diseases. Previous RNA sequencing data utilizing knockdown of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) identified enrichment for pathways and changes in RNAs related to nuclear morphology and showed differential expression of key nuclear targets. This suggests that dysfunction of hnRNP A1, which is observed in neurodegenerative diseases, may contribute to abnormalities in nuclear morphology, NPC, and NCT. We performed knockdown of hnRNP A1 in Neuro-2A cells, a neuronal cell line, to examine nuclear morphology, NPC, and NCT. First, we examined nuclear morphology using Lamin B, wherein we observed increased nuclear envelope abnormalities in cells with hnRNP A1 knockdown as compared to control. To quantify changes in Lamin B, we designed and validated an automated computer-based model, which quantitatively confirmed our observations. Next, we investigated the impact of hnRNP A1 knockdown on components of the NPC and NCT. In line with the previous literature, we found changes in Nups, including altered distribution and reduced protein expression, as well as disrupted NCT. Finally, we validated our findings in multiple sclerosis (MS) brains, a disease with a significant neurodegenerative component caused by hnRNP A1 dysfunction, where neuronal nuclear envelope alterations were significantly increased as compared to controls. Together, these data implicate hnRNP A1 as an important contributor to nuclear morphology, Nup expression and distribution, and NCT and suggest that hnRNP A1 dysfunction may lead to defects in these processes in neurodegenerative diseases.

Effect of crude ethanolic seed extract from Mucuna pruriens on proliferation, apoptosis, and cell cycle arrest in gastric adenocarcinoma (AGS) cells

Abstract Background Gastric cancer is a prevalent form of malignancy among many common carcinoma cases globally. This study was designed to assess the anticancer potential of the crude ethanolic seed extract from Mucuna pruriens against the gastric cancer cell line (AGS). Various assays were employed to assess the anticancer properties, including examinations of cell viability, nuclear morphology, apoptosis using AO/EB staining, changes in mitochondrial membrane potential, lactate dehydrogenase activity, DNA fragmentation, and cell cycle arrest. Results The crude extract exhibited significant anticancer activity against the human gastric cancer cell line (AGS), as determined by the MTT assay, with an inhibition concentration (IC50) of 600 µg/mL at 24 h. Distinct cellular and nuclear morphological changes were observed with different concentrations of crude ethanolic seed extract. The LDH release assay reveals cell death in AGS cells, as evidenced by a significant increase in the release of LDH enzyme. DNA fragmentation analysis and flow cytometry results indicate that the extract induces chromatin condensation, apoptotic cell death, and cell cycle arrest at the G0/G1 phase in the AGS cancer cell line. These results highlight the potential therapeutic advantages of Mucuna pruriens seed extract against gastric cancer cells. Conclusion This study could pave the way for identifying diverse natural bioactive compounds sourced from Mucuna pruriens seed, leading to the development of novel drug with potential anticancer properties.

Capsaicin Promotes Apoptosis and Inhibits Cell Migration via the Tumor Necrosis Factor-Alpha (TNFα) and Nuclear Factor Kappa B (NFκB) Signaling Pathway in Oral Cancer Cells.

Background Oral squamous cell carcinoma (OSCC) is a highly prevalent cancer worldwide. Microbial infections, poor oral hygiene, and chronic viral infections such as human papillomavirus (HPV) contribute to its incidence. Capsaicin, known for its presence in chili peppers, has demonstrated potential antiproliferative effects in cancer cells. It operates by inducing programmed cell death, regulating the expression of transcription factors, halting cell cycle progression, and influencing growth signal transduction pathways. These findings suggest capsaicin's promising role as a candidate for further exploration in combating oral cancer. Aim This study intends to identify and evaluate the anticancer properties of capsaicin on oral cancer cells through in vitro investigations. Methodology Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique, the cell viability of oral cancer cells treated with capsaicin was evaluated. Capsaicin was applied to the KB1 cells in a range of concentrations (25-150 µg/mL) over 24 hours. The morphological alterations of the cells were assessed using a phase contrast microscope. Nuclear factor kappa B (NFκB) and tumor necrosis factor-alpha (TNFα) were subjected to quantitative real-time polymerase chain reaction (PCR) gene expression analysis. To investigate nuclear morphological changes, oral cancer cells were stained with acridine orange/ethidium bromide (AO/EtBr). The apoptotic nuclei were visualized using a fluorescent microscope. A scratch wound healing test was performed to check for capsaicin's anti-migratory potential. Result In our investigation of oral cancer cells treated with capsaicin, there was a significant drop in cell viability between the control and treatment groups (p < 0.05). The inhibitory concentration (IC50) was found to be 74.4 μM/mL in oral cancer cells. Following treatment, fewer cells were present, and those that were present shriveled and exhibited cytoplasmic membrane blebbing. Under AO/EtBr staining, treated cells exhibited chromatin condensation and nuclear disintegration. Furthermore, the migration of capsaicin-treated cells was significantly lower than that of control cells. The results of gene expression analysis demonstrated a considerable downregulation of TNFα and NFκB following capsaicin administration. Conclusion The study's findings suggest that capsaicin may have anti-tumor properties in oral cancer cells. More research is desperately needed to fully understand the mechanism underlying capsaicin's anticancer potential and therapeutic applicability.

Resveratrol Protects Müller Cells Against Ferroptosis in the Early Stage of Diabetic Retinopathy by Regulating the Nrf2/GPx4/PTGS2 Pathway.

The aim of this study was to investigate the anti-ferroptotic effect of resveratrol (RSV) on retinal Müller cells (RMCs) in the early stages of diabetic retinopathy (DR) via the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPx4)/prostaglandin-endoperoxide synthase 2 (PTGS2). The retina was obtained from normal and diabetic Sprague-Dawley rats or wild-type and Nrf2 knockout (KO) diabetic mice, with or without RSV (10mg/kg/d) treatment for 12weeks. RMCs transfected with or without SiNrf2 were cultured with high glucose and RSV (20mM). The retinal neurofunctional changes were measured by electroretinogram (ERG). The retinal inner nuclear layer cell mitochondrial morphological changes were detected by transmission electron microscopy. The cell viabilities were measured by cell counting kit-8 (CCK-8) assay. The levels of Fe2+, malonic dialdehyde (MDA), and glutathione (GSH) were measured by colorimetric method. The expression of Nrf2, GPx4, and PTGS2 was detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunocytochemistry. In vivo, RSV inhibited retinal neurofunctional changes and mitochondrial morphological changes; decreased Fe2+, MDA, and PTGS2; and increased GSH, Nrf2, and GPx4 in retina of DM rats. In vitro, RSV decreased MDA and PTGS2 and increased cell viability, GSH, Nrf2, and GPx4. In vivo and vitro, the role of Nrf2-regulated signaling pathway in anti-ferroptosis by RSV was further confirmed using Nrf2 KO mice and pre-transfected SiNrf2 in RMCs. These findings indicated that RSV is a potential therapeutic option for DR and that Nrf2/GPx4/PTGS2 plays a role in the anti-ferroptosis mechanism of RSV on RMCs.

CYP1B1 promotes PARPi-resistance via histone H1.4 interaction and increased chromatin accessibility in ovarian cancer

IntroductionOvarian cancer is the most lethal gynecological cancer and presents significant therapeutic challenges. The discovery of synthetic lethality between PARP inhibitors (PARPi) and homologous recombination deficiency marked a new era in treating BRCA1/2-mutated tumors. However, PARPi resistance remains a major clinical challenge. MethodsRNA sequencing was used to identify genes altered by PARPi treatment and LC-MS was used to detect proteins interacting with CYP1B1. Resistance mechanisms were explored through ATAC-seq and gene expression manipulation. Additional techniques, including micrococcal nuclease digestion assays, DAPI staining, and fluorescence microscopy, were used to assess changes in nuclear morphology and chromatin accessibility. ResultsThe gradual exposure of Olaparib has developed a PARPi-resistant cell line, A2780-OlaR, which exhibits significant upregulation of CYP1B1 at both RNA and protein levels. Down-regulating CYP1B1 expression or using specific inhibitors decreased the cellular response to Olaparib. Linker histone H1.4 was identified as associated with CYP1B1. ATAC-seq showed differential chromatin accessibility between A2780-OlaR and parental cells, indicating that the downregulation of H1.4 was associated with increased chromatin accessibility and higher cell viability after Olaparib treatment. ConclusionOur findings reveal a novel role for CYP1B1 in driving PARPi resistance through distinct molecular mechanisms in A2780-OlaR. This study highlights the importance of chromatin accessibility in PARPi efficacy and suggests the CYP1B1/H1.4 axis as a promising therapeutic target for overcoming drug resistance in ovarian cancer, offering potentially therapeutic benefits.

Novel indole Schiff base β-diiminato compound as an anti-cancer agent against triple-negative breast cancer: In vitro anticancer activity evaluation and in vivo acute toxicity study

Breast cancer is the most prevalent cancer among women globally, with triple-negative breast cancer (TNBC) associated with poor prognosis and low five-year survival rates. Schiff base compounds, known for their extensive pharmacological activities, have garnered significant attention in cancer drug research. This study aimed to evaluate the anticancer potential of a novel β-diiminato compound and elucidate its mechanism of action. The compound’s effect on cell viability was assessed using MTT assays in breast cancer cell lines including MCF-7 and MDA-MB-231. Cytotoxic effects were further analyzed using trypan blue exclusion and lactate dehydrogenase (LDH) release assays. In order to assess the mechanism of inhibitory activity and mode of cell death induced by this compound, flow cytometry of cell cycle distribution and apoptosis analysis were carried out. Apoptosis incidence was initially assessed through cell and nuclear morphological changes (Hoechst 33342/Propidium iodide (PI) staining) and further confirmed by Annexin V/PI staining and flow cytometry analysis. In addition, the effect of this compound on the disruption of mitochondrial membrane potential (MMP) and generation of the reactive oxygen species (ROS) was determined using the JC-1 indicator and DCFDA dye, respectively. The results demonstrated that the 24 h treatment with β-diiminato compound significantly suppressed the viability of MDA-MB-231 and MCF-7 cancer cells in a dose-dependent manner with the IC50 value of 2.41 ± 0.29 and 3.51 ± 0.14, respectively. The cytotoxic effect of the compound was further confirmed with a dose-dependent increase in the number of dead cells and enhanced LDH level in the culture medium. This compound exerted its anti-proliferative effect by G2/M phase cell growth arrest in MDA-MB-231 breast cancer cells and induced apoptosis-mediated cell death, which involved characteristic changes in cell and nuclear morphology, phosphatidylserine externalization, mitochondrial membrane depolarization, and increased ROS level. Neither hepatotoxicity nor nephrotoxicity was detected in the biochemical and histopathological analysis confirming the safety characterization of this compound usage. Therefore, the results significantly confirmed the potential anticancer activity of a novel β-diiminato compound, as evidenced by the induction of cell cycle arrest and apoptosis, which might be driven by the ROS‑mediated mitochondrial death pathway. This compound can be a promising candidate for future anticancer drug design and TNBC treatment, and further preclinical and clinical studies are warranted.

Induction of Apoptosis and ROS Production in Liver Cancer Cells by Saponin Fraction from Alcea rosea L. Seeds

Background: Natural compounds are essential targets in anticancer research due to the toxic effects of current chemotherapy and drug resistance. Methods: Saponin extraction from Alcea rosea L. was carried out using 20% methanol and liquid-liquid extraction coupled with the Solid phase extraction (SPE) approach. The saponin fraction was explored for its cytotoxic activity against Huh-7 and MDA-MB-231 cancer cell lines and a non–cancer cell line (HUVEC). The cytotoxic activity and oxidative stress were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2,7 dichlorofluorescein diacetate dyes respectively. The apoptotic potential was investigated using DAPI and acridine orange-ethidium bromide dual staining. Result: The saponin fraction exhibited cytotoxic potential against the cell lines investigated. The IC50 values of the saponin fraction were 49.02, 300 and 325 µg\mL against Huh-7, MDA-MB-231 and HUVEC cell lines, respectively. The cytotoxicity for the extract was dose-dependent. The saponin fraction calculated selectivity index (SI) value indicated high selectivity towards Huh-7 cells (SI = 6.62) compared with normal HUVEC cells. Huh-7 cells treated with saponin fraction showed cellular and nuclear morphological changes, such as apoptotic body formation and chromatin condensation, as observed using light and fluorescent microscopy. However, further investigation is required to assess the the saponin fraction as a potential therapeutic agent.

Metabolomic insights and bioactive efficacies of Tragopogon dubius root fractions: Antioxidant and antiproliferative assessments

Tragopogon dubius is commonly consumed as a vegetable and used in traditional medicine for treating inflammatory skin conditions and cutaneous swelling. Despite known pharmacological properties of its leaves and roots, many of its biological characteristics and active phytochemicals remain unexplored. The present study investigates the phytochemical composition, antioxidant, and anticancer properties of methanolic root extracts and isolated fractions (TdRM-1 and TdRM-2) of T. dubius. Utilizing preparative thin-layer chromatography, the crude extract was successfully separated into TdRM-1 and TdRM-2, characterized by GC-MS and FTIR analysis, revealing a diverse range of bioactive compounds including terpenes, flavonoids, and phenolic acids. Qualitative phytochemical screening indicated the presence of carbohydrates, tannins, alkaloids, and other phytoconstituents. Advanced UPLC-ESI-QTOF-MS analysis identified 54 metabolites, significantly contributing to the chemical profiling of the extract. The antioxidant activities of the fractions were quantitatively assessed using ABTS, DPPH, and superoxide radical scavenging assays, where TdRM-2 exhibited superior activity with IC50 values ranging from 51.29 to 60.03 μg/mL. Anticancer potential was evaluated against A549, LN-18, and MCF-7 cancer cell lines, demonstrating that TdRM-2 significantly inhibited cell proliferation with GI50 values as low as 31.62 μg/mL for A549 cells. Additionally, fluorescence microscopy revealed that TdRM-2 induces apoptosis, indicated by changes in nuclear morphology and loss of mitochondrial membrane potential. Annexin V-FITC/PI double staining indicate that the TdRM-2 fractions from T. dubius can significantly inhibit the growth of A-549, LN-18, and MCF-7 cancer cell lines by inducing apoptosis These findings suggest that T. dubius root extracts, particularly the TdRM-2 fraction, hold promising therapeutic potential due to their significant antioxidant and anticancer activities, underpinned by their rich phytochemical composition. This study underscores the importance of T. dubius as a source of natural bioactive compounds with potential health benefits.

Cytotoxic Potential of Betulinic Acid Fatty Esters and Their Liposomal Formulations: Targeting Breast, Colon, and Lung Cancer Cell Lines.

Betulinic acid is a lupane-type pentacyclic triterpene mostly found in birch bark and thoroughly explored for its wide range of pharmacological activities. Despite its impressive biological potential, its low bioavailability has challenged many researchers to develop different formulations for achieving better in vitro and in vivo effects. We previously reported the synthesis of fatty acid esters of betulinic acid using butyric, stearic, and palmitic acids (But-BA, St-BA, and Pal-BA) and included them in surfaced-modified liposomes (But-BA-Lip, St-BA-Lip, Pal-BA-Lip). In the current study, we evaluated the cytotoxic effects of both fatty acid esters and their respective liposomal formulations against MCF-7, HT-29, and NCI-H460 cell line. The cytotoxic assessment of BA derivatives revealed that both the fatty esters and their liposomal formulations acted as cytotoxic agents in a dose- and time-dependent manner. But-BA-Lip exerted stronger cytotoxic effects than the parent compound, BA and its liposomal formulation, and even stronger effects than 5-FU against HT-29 cells (IC50 of 30.57 μM) and NCI-H460 cells (IC50 of 30.74 μM). BA's fatty esters and their respective liposomal formulations facilitated apoptosis in cancer cells by inducing nuclear morphological changes and increasing caspase-3/-7 activity. The HET-CAM assay proved that none of the tested compounds induced any irritative effect, suggesting that they can be used safely for local applications.

Tyrosine Kinase Inhibitor Lenvatinib Causes Cardiotoxicity by Inducing Endoplasmic Reticulum Stress and Apoptosis through Activating ATF6, IRE1α and PERK Signaling Pathways.

Lenvatinib is a tyrosine kinase inhibitor that can improve progression-free survival in patients with thyroid cancer and hepatocellular carcinoma. However, it is limited by adverse cardiovascular events, including hypertension and cardiac dysfunction. Activation of endoplasmic reticulum stress is involved in cardiomyocyte apoptosis. This study aimed to confirm whether the cardiotoxicity of lenvatinib is associated with endoplasmic reticulum stress by targeting the activating transcription factor 6 (ATF6), inositol- requiring enzyme 1α (IRE1α) and protein kinase RNA-like ER kinase (PERK) signaling pathways. Male C57/BL6 mice were intragastric administration with 30 mg/kg/day lenvatinib. Electrocardiography (ECG) and echocardiography were used to detect arrhythmias and cardiac function. Neonatal rat cardiomyocytes were treated with lenvatinib for 48h. Cell counting kit (CCK8), 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFHDA), Hoechst 33258 and dihydrorhodamine 123 were respectively used for evaluating cell viability, the level of reactive oxygen species (ROS), nuclear morphological changes and mitochondrial membrane potential (MMP) level. Lenvatinib remarkably decreased the posterior wall thickness of left ventricle during diastole and systole but caused little decrease to the left ventricular ejection fraction (LVEF, %). Furthermore, lenvatinib greatly prolonged the corrected QT interval (QTc) and altered the morphology of cardiomyocytes. No dramatic difference in fibrosis was found in mouse cardiac slices. Lenvatinib upregulates apoptosis-related protein expression. In addition, lenvatinib increased ERS-related protein expression (GRP78, CHOP, and ATF6) and enhanced PERK phosphorylation. In neonatal rat cardiac myocytes, lenvatinib markedly decreased the viability of cardiomyocytes and induced apoptosis. Furthermore, ROS production increased and MMP decreased. Similar to the mice experiment, lenvatinib caused upregulation of apoptosis-related and ERS-related proteins and increased the phosphorylation levels of PERK and IRE1α. Lenvatinib-induced cardiotoxicity is associated with ERS-induced apoptosis by targeting the ATF6, IRE1α, and PERK signaling pathways.

O-172 Live imaging reveals key events in preimplantation embryo development

Abstract Preimplantation development encompasses the zygote to blastocyst stages, occurring in vivo within the Fallopian tube and in vitro within embryology labs worldwide. During these first five days of life, several key morphologic changes take place facilitating cell fate specification and resulting in blastocyst assembly containing ICM and trophectoderm lineages. These include cell polarization, embryo compaction and inner-outer segregation, which transform the collection of blastomeres into a morula. Subsequently, the blastocoel cavity forms and expands followed by hatching from the zona pellucida prior to implantation. This ability to self-organize has enabled live imaging of these events within intact embryos. Moreover, this has revealed the dynamics of early mitoses, nuclear morphology and cytoskeletal changes driving early development. Although a great deal has been learned in the mouse, events in the human embryo remain relatively unknown. Recent achievements in stem cell-based embryoid models and non-invasive live imaging now poise researchers to make significant advancements unveiling human embryo development. Furthermore, a comprehensive understanding of human embryogenesis is essential to improving IVF efficiency and outcomes. Preimplantation development in mouse and human will be reviewed with a focus on recent discoveries made possible by live imaging.

Expression of ITPR2 regulated by lncRNA-NONMMUT020270.2 in LPS-stimulated HT22 cells

BackgroundLong non-coding RNA (lncRNA)-NONMMUT020270.2 is downregulated and co-expressed with inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) in the hippocampus of Alzheimer's disease (AD) mice. However, whether the expression of ITPR2 was regulated by lncRNA-NONMMUT020270.2 remains unclear. we aimed to investigate regulating relationship of lncRNA-NONMMUT020270.2 and ITPR2. MethodsHT22 cells were firstly transfected with the pcDNA3.1-lncRNA-NONMMUT020270.2 overexpression plasmid or with the lncRNA-NONMMUT020270.2 smart silencer, and then were stimulated with lipopolysaccharide (LPS) for 24h. The mRNA expression levels of lncRNA-NONMMUT020270.2 and ITPR2 were measured by reverse transcription-quantitative PCR. Cell viability was assessed using a Cell Counting Kit 8 assay. The expression of Aβ1-42 was detected by ELISA. The expression levels of p-tau, caspase-1, and inositol trisphosphate receptor (IP3R) proteins were detected by western-blotting. Nuclear morphological changes were detected by Hoechst staining. Flow cytometry and Fluo-3/AM were carried out to determine cell apoptosis and the intracellular Ca2+. ResultsLPS significantly decreased cell viability, and ITPR2 mRNA and IP3R protein expression levels. While it markedly enhanced the expression levels of p-tau and Aβ1-42, cell apoptosis rate, as well as intracellular Ca2+ concentration (P < 0.05). In addition, lncRNA-NONMMUT020270.2 overexpression significantly increased the expressions levels of ITPR2 mRNA and IP3R protein (P < 0.05), and inhibited expression of p-tau and Aβ1-42, cell apoptosis rate, and reduced intracellular Ca2+ concentration (P < 0.05). By contrast, lncRNA-NONMMUT020270.2 silencing notably downregulated expressions levels of ITPR2 mRNA and IP3R protein (P < 0.05), and elevated expression levels of p-tau and Aβ1-42, cell apoptosis rate, and intracellular Ca2+ concentration (P < 0.05). ConclusionlncRNA-NONMMUT020270.2 was positively correlated with ITPR2 expression in LPS-induced cell. Downregulating the lncRNA-NONMMUT020270.2 and ITPR2 may promote cell apoptosis and increase intracellular Ca2+ concentration.

Green and chemical synthesized zinc oxide nanoparticles: Evaluation of their anti-proliferative activity against breast cancer cell line – An in vitro and in silico approach

Zinc Oxide nanoparticles (ZnO NPs) were synthesized through both chemical and green approaches using wheat grass (WG) extract. Characterization techniques included Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM) with Energy-Dispersive X-ray Spectroscopy (EDX), Fourier-transform infrared Spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and Ultraviolet-visible (UV-Vis) spectroscopy. CS-ZnO NPs and WG-ZnO NPs exhibited characteristic surface plasmon resonance (SPR) peaks at 360 nm and 320 nm, respectively, with low polydispersity index (PDI) values indicating uniform nanoparticle dispersion. FTIR analysis revealed distinct functional groups on the ZnO NPs’ surface for stabilization. XPS confirmed atomic composition and oxidation state, while XRD confirmed their hexagonal wurtzite structure. SEM images revealed rod-shaped structures, supported by EDX analysis confirming ZnO NP formation through elemental content. The synthesized ZnO NPs demonstrated significant cytotoxicity against MDA-MB 231 breast cancer cells, causing changes in nuclear morphology, increased reactive oxygen species levels, and membrane damage. Wound healing assay highlighted their potential in inhibiting cancer metastasis. Molecular docking between WG compounds and breast cancer targets revealed that the enhanced efficacy of green-synthesized ZnO NPs could be attributed to covalent linkage of active molecules of WG onto the nanoparticle surface. Overall, ZnO NPs synthesized using the green method outperformed their chemically synthesized counterparts.

Fumaria vaillantii extract protects PC12 cells against neurotoxicity induced by 6-OHDA.

Parkinson's disease is a neurological disorder caused by the loss of dopaminergic neurons in the midbrain. Various mechanisms are involved in the incidence of the disease including oxidative stress. Several herbs and natural products may interfere with the oxidative-stress pathway due to their antioxidant effects. Herein, we aimed to investigate the neuroprotective role of F. vaillantii extract on Parkinson's in vitro and in vivo model owing to the presence of the bioactive agents with antioxidant properties. In vitro experments showed that 6-hydroxydopamine could induce toxicity in PC12 cells. The impact of F. vaillantii extract on cell viability was measured by using MTT assay. Nuclear morphological changes were qualitatively evaluated employing Hoechst staining. The antioxidant activity of the extract was determined by ROS and lipid peroxidation assays. Tyrosine hydroxylase protein expression was measured by western blotting in PC12 cells. For in vivo study, movement parameters were evaluated. The results indicated that 75 µΜ of 6-OHDA induced 50% toxicity in PC12 cells for 24h. Following post-treatment with F. vaillantii extract (0.1mg/ml) for 72h, we observed that the extract effectively prevented cell toxicity induced by 6-OHDA and reduced the apoptotic cell population. Furthermore, the extract attenuated the ROS level, lipid peroxidation and increased protein expression of TH after 72h of treatment. In addition, oral administration of 300mg/kg of F. vaillantii extract for 14 days improved locomotor activity, catalepsy, bradykinesia, motor coordination and reduced the apomorphine-caused rotation in 6-OHDA- induced Parkinson's disease-like symptoms in male rats. The present study suggests a protective role for the extract of F. vaillantii against oxidative stress-induced cell damage in the PC12 cells exposed to neurotoxin 6-OHDA which was verified in in vivo model by reducing the motor defects induced by 6-OHDA. This extract could be a promising therapeutic agent for the prevention of PD progression.

Evaluation of molecular effects associated with apoptosis, tumour progression, angiogenesis and metastasis by a novel combination of drugs with ormeloxifene in triple negative breast cancer cells.

To investigate the molecular effects of a novel combination [sertraline and plumbagin (comb) with ormeloxifene (Orm)] for anticancer activity in triple negative breast cancer cell line "MDA-MB-231". The cytotoxic effect of the drugs was analyzed by the MTT assay and nuclear morphological changes by acridine orange/ethidium bromide (AO/EB) staining. Induction of apoptosis by annexin V-FITC staining, active caspase-3 detection and cell cycle analysis were studied in vitro on "MDA-MB-231" cells. The qRT-PCR was done to explore the upregulation and down regulation of targeted genes for angiogenesis, metastasis, tumor suppression and protein folding on the triple negative breast cancer cells. The preliminary anti-angiogenic effect of the drugs was assessed by chorioallantoic membrane (CAM) assay. Orm showed inhibitory effects in "MDA-MB-231" cells in a dose and time dependent manner whereas; the drugs in combination gave better cytotoxic effects in the screening MTT assay. Orm + comb was more effective than Orm alone in eliciting apoptosis as well as inhibited the single cell to grow into a colony. CAM assay using Orm and Orm + comb suggested the anti-angiogenic potential which was further confirmed by the downregulation of VEGF in "MDA-MB-231" cells by qRT-PCR studies. The combination was found to effectively upregulate the expression of P53 and P21 and downregulate the gene expression of zinc finger E-box binding homeobox 1 (ZEB1) and heat shock protein 70 (HSP70) in "MDA-MB-231" cancer cells. Collectively this study reveals the efficacy of Orm + comb as more significant than the clinically used tamoxifen (Tam). The study elucidates the promising novelty of the combination as a potential chemotherapeutic intervention for mitigating the aggressiveness of triple negative breast cancer and it addresses the intrinsic resistance caused by single drug treatments.

Pickering Emulsion of Oleoresin from Dipterocarpus alatus Roxb. ex G. Don and Its Antiproliferation in Colon (HCT116) and Liver (HepG2) Cancer Cells.

Oleoresin of Dipterocarpus alatus Roxb. ex G. Don (DA) has been traditionally used for local medicinal applications. Several in vitro studies have indicated its pharmacological potential. However, the low water solubility hinders its use and development for pharmaceutical purposes. The study aimed to (1) formulate oil-in-water (o/w) Pickering emulsions of DA oleoresin and (2) demonstrate its activities in cancer cells. The Pickering emulsions were formulated using biocompatible carboxylated cellulose nanocrystal (cCNC) as an emulsifier. The optimized emulsion comprised 3% (F1) and 4% (v/v) (F2) of oleoresin in 1% cCNC and 0.1 M NaCl, which possessed homogeneity and physical stability compared with other formulations with uniform droplet size and low viscosity. The constituent analysis indicated the presence of the biomarker dipterocarpol in both F1 and F2. The pharmacological effects of the two emulsions were demonstrated in vitro against two cancer cell lines, HepG2 and HCT116. Both F1 and F2 suppressed cancer cell viability. The treated cells underwent apoptosis, as demonstrated by distinct nuclear morphological changes in DAPI-stained cells and Annexin V/PI-stained cells detected by flow cytometry. Our study highlights the prospect of Pickering emulsions for oleoresin, emphasizing enhanced stability and potential pharmacological advantages.

Macromolecules with predominant β-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 μg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.