Acridine Orange/ethidium Bromide Research Articles

Copper (II) increases anti-Proliferative activity of thymoquinone in colon cancer cells by increasing genotoxic, apoptotic, and reactive oxygen species generating effects

Thymoquinone is the main active compound derived from the essential oil of the Nigella sativa plant seed. While thymoquinone is an antioxidant, it has been reported in several studies that thymoquinone has dose-dependent pro-oxidant activity with the Fenton reaction in the presence of transition elements such as iron and copper. This study aimed to investigate cytotoxic, apoptotic, genotoxic, and reactive oxygen species (ROS) generating effects of thymoquinone treated with copper in colon cancer cells. HT-29 cells were treated with pro-oxidant-acting doses of thymoquinone alone and together with the non-toxic dose of Copper (II) Sulfate for 24 h. Cytotoxic, apoptotic, genotoxic, and ROS production activities were analyzed by MTT viability test, Acridine Orange/Ethidium Bromide (AO/EB) staining, alkaline single cell gel electrophoresis and H2DCF-DA assay, respectively. Viability results showed that thymoquinone and copper synergistically affect cancer cells, and DNA damage was increased with the synergic effect. The intracellular ROS was increased when thymoquinone and copper were applied together. Applying redox-active copper (II) with thymoquinone increases DNA damage, apoptosis, and cell death by increasing the amount of intracellular ROS through pro-oxidant activity. Treatments targeting copper-related pathways may open new therapeutic avenues for cancer treatment.

Exploring the therapeutic applications of nano-therapy of encapsulated cisplatin and anthocyanin-loaded multiwalled carbon nanotubes coated with chitosan-conjugated folic acid in targeting breast and liver cancers

This study aimed to assess the targeted nano-therapy of encapsulated cisplatin (Cis) and anthocyanin (Ant)-loaded multiwalled carbon nanotubes (CNT) coated with chitosan conjugated folic acid on breast MCF7 and liver HepG2 cancer cells. Zeta potential, UV-spectroscopy, FTIR, TEM, and SEM were used to evaluate CNT, its modified form (CNT Mod), CNT-loaded Cis NPs, CNT-loaded Ant NPs, and CNT- Cis + Ant NPs. All treatments induced apoptosis-dependent cytotoxicity in both cell lines as revealed functionally by the MTT assay, morphologically (DNA degradation) by acridine orange/ethidium bromide (AO/EB) double staining, and molecularly (Bax upregulation and Bcl2 downregulation) by real-time PCR, with best effect for the combined treatment (CNT- Cis + Ant NPs). This combined treatment also significantly reduced inflammation (low TNFα), migration (low MMP9 and high TIMP1), and angiogenesis (low VEGF), while significantly increasing antioxidant status (high Nrf2 and OH-1) in MCF7 and HepG2 cells compared to other treatments. Interestingly, cells treated with CNT Mod exhibited higher cytotoxic, apoptotic, anti-migratory, and anti-angiogenic potentials relative to CNT-treated cells. In conclusion, targeted nano-therapy of encapsulated cisplatin and anthocyanin-loaded carbon nanotubes coated with chitosan conjugated folic acid can efficiently combat breast and liver cancers by sustained release, in addition to its apoptotic, antioxidant, anti-inflammatory, anti-metastatic, and anti-angiogenic effects.

Inhibitory Effects of Propolis Flavonoids on Migration and Invasion of Laryngeal Cancer Cell and Analysis of Related Signal Pathways

Laryngeal cancer (LGC) is a malignant tumor that occurs in the larynx, and it is mainly treated through chemotherapy, radiotherapy, and surgery. Nevertheless, the five-year survival rate for patients is poor. Bee propolis contains various bioactive compounds and abundant anti-tumor active ingredients. Nevertheless, research on the use of propolis extracts for the treatment of LGC is relatively limited. This research aimed to demonstrate the inhibitory effects of ethanol extracts of propolis on migration (Mig) and invasion (Inv ) of LGC cells, as well as the related signaling pathways. The effects of graded ethanol extraction of propolis on the proliferation (Pro), Inv, Mig, apoptosis (Apo), and related signaling pathways of Hep-2 cells were analyzed. Propolis was extracted using ethanol (0%, 25%, 50%, 75%, and 100%) for the graded extraction of crude propolis. The flavonoid content and yield of the extracts were determined. The effects of various concentrations of propolis flavonoids on the clearance of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, O2- radicals, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals were evaluated, as well as their effects on the Pro inhibition of normal human pancreatic ductal epithelial (hTERT-HPNE) cells. Hep-2 cells of LGC were cultured using media containing 0, 25, 50, and 100 μmol/L propolis flavonoids. The cell Pro activity, Inv, Mig, Apo, and expression of PI3K/Akt pathway-related proteins were evaluated using CCK-8 assay, Transwell chamber assay, acridine orange/ethidium bromide (AO/EB) double staining method, and Western blotting, respectively. It was revealed that extraction with 50% ethanol solution yielded a higher content and yield of flavonoids, which were 51.20% and 7.42%, respectively. As the concentration of propolis flavonoids increased, the clearance rates of DPPH, O2-, and ABTS radicals, as well as the inhibition of hTERT-HPNE Pro, gradually increased. The maximum clearance rates were 84.1%, 26.6%, and 92.3%, respectively, while the maximum cell Pro inhibition rate was only 8.6%. Relative to the 0 μmol/L propolis flavonoid treatment group, the Hep-2 cells treated with 25, 50, and 100 μmol/L propolis flavonoids exhibited decreased cell Pro activity, reduced number of invasive and migratory cells, increased Apo rate, decreased PI3K and p-Akt proteins, and demonstrated a concentration-dependent effect (P < 0.05). In summary, the extraction with 50% ethanol solution resulted in a higher yield of flavonoids. Propolis flavonoids demonstrated marked antioxidant activity and did not cause damage to normal hTERT-HPNE cells. They exhibited inhibitory effects on the Pro, Inv, and Mig of Hep-2 cells in LGC, and promoted cell Apo. These effects may be associated with PI3K/Akt signaling inhibition.

Chrysin-functionalized gold nanoparticles and paclitaxel exhibit synergistic impact on lung cancer cell lines via regulating the AKT/PPAR-ϒ/β-catenin pathway

Lung cancer has become progressively widespread, posing a challenge to traditional chemotherapeutic drugs such as platinum compounds and paclitaxel (PTX) owing to growing resistance. Along with that, the chemotherapeutic drugs infer major side effects. The usage of natural compounds as chemosensitizers to boost the efficacy of these chemotherapeutic drugs and minimizing their toxicity is a plausible approach. In our investigation, we employed PTX as the standard chemotherapeutic agent and utilized chrysin-functionalized gold nanoparticles (CHR-AuNPs) to augment its cytotoxicity. Gold nanoparticles were chosen for their inherent cytotoxic properties and ability to enhance chrysin’s bioavailability and solubility. Characterization of CHR-AuNP revealed spherical nanoparticles within the nano-size range (35–70 nm) with a stable negative zeta potential of −22 mV, confirmed by physicochemical analyses including UV–visible spectroscopy, Fourier transform infrared (FTIR) spectral analysis, and visual observation of the wine-red coloration. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay cytotoxicity studies demonstrated CHR-AuNP’s superior efficacy compared to CHR alone, with synergistic effects observed in combination with PTX, validated by Compusyn software. Morphological changes indicative of apoptosis were more pronounced with combined treatment, corroborated by acridine orange/ethidium bromide (AO/EtBr) staining and Annexin V assays. Furthermore, the combination treatment amplified reactive oxygen species (ROS) production and destabilized mitochondrial membrane potential, while altering the expression of pro-apoptotic and anti-apoptotic proteins. Exploring the mechanistic pathways, we found that the drugs upregulated PPAR-γ expression while suppressing Akt and overexpressing PTEN, thereby impeding the Wnt/β-catenin pathway commonly dysregulated in lung cancer. This highlights the potential of low-dose combination therapy with PTX and CHR-AuNP as a promising strategy for addressing lung cancer’s challenges.

Unravelling the tissue regenerative nature of marine polysaccharide chitosan embedded halloysite reinforced poly (vinyl alcohol) nanocomposite films

The study focuses on developing the poly (vinyl alcohol) (PVA) nanocomposite films reinforced with Halloysite Nano-Tubes (HNT) which were surface functionalised with chitosan forming a Schiff base structure. The work was aimed to traverse its way in tissue engineering. Modification of halloysite with sea polysaccharide chitosan enhanced its ability to bind to the PVA matrix. The modification was assisted by FTIR, XRD, FESEM, TEM, AFM and DSC-TGA techniques. The films could withstand a higher temperature and exhibited high ranges of tensile strength and Young’s modulus. In addition, biocompatible studies such as in-vitro swelling, enzymatic degradation, water contact angle and hemolysis presented extremely well compatibilities proving it to be viable in physiological pH (in phosphate buffered saline). The cell adhesion and proliferation studies conducted on NIH3T3 mouse fibroblasts revealed the cell proliferation and tissue regeneration properties of the films. Both the tests performed for cell growth- Trypan blue dye exclusion and Acridine Orange Ethidium bromide assays showed a doubled rate of cell growth on the films which proves its biomedical nature.Graphical

Organotin(IV) complexes derived from 2,6-diacetylpyridine bis(2-hydroxybenzoylhydrazone) as prospective anti-proliferative agents: Synthesis, characterization, structures and in vitro anticancer activity

Six organotin(IV) complexes, viz., [Me2Sn(L)] (1), [n-Bu2Sn(L)] (2), [n-Oct2Sn(L)] (3), [Bz2Sn(L)]·0.5C7H8 (4), [n-BuSn(L)Cl] (5), and [PhSn(L)Cl] (6), were synthesized using a 2,6-diacetylpyridine bis(2-hydroxybenzoylhydrazone), H2L. Compounds were characterized by Fourier transform infrared (FT-IR), High-resolution mass spectrometry (HRMS), and solutions Fourier transform nuclear magnetic resonance (FT-NMR) spectroscopies. The structures 1–6 were established by single-crystal X-ray diffraction (SC-XRD) analysis. Diffraction results evidenced that complexes 1–6 were seven-coordinated mononuclear species with the equatorial plane comprising the pentagonal N3O2 chelate ring of the doubly deprotonated L and two axial ligands, either R (R = Me, n-Bu, n-Oct, Bz) or R (n-Bu or Ph) and Cl ligands. Additionally, the photophysical properties were examined due to the enhanced conjugation and rigidity of the molecules while thermogravimetric analysis was carried out to evaluate the thermal stabilities of compounds. The anti-proliferative activity of the complexes 1–6 was tested against prostate cancer cells (DU-145) and normal human embryonic kidney cells (HEK-293). Among the compounds, dibutyltin compound 2 exhibited increased anti-proliferative activity, with an IC50 value of 6.16 ± 1.56 μM. The investigation of its mechanism of action involves using AO/EB (acridine orange/ethidium bromide) and ROS (reactive oxygen species) generation assays. This likely detects apoptotic morphological alterations in the nucleus of the cells, with ROS generation ultimately leading to apoptosis and cell death. The superior activity of 2 may be attributed to the C···H contacts and respective higher de outside and di inside distances from the Hirshfeld surface. Thus, these compounds could be a promising alternative to classical chemotherapy agents.

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.

Investigation of cytotoxic effect and action mechanism of a synthetic peptide derivative of rabbit cathelicidin against MDA-MB-231 breast cancer cell line

Antimicrobial peptides (AMPs) have sparked significant interest as potential anti-cancer agents, thereby becoming a focal point in pursuing novel cancer-fighting strategies. These peptides possess distinctive properties, underscoring the importance of developing more potent and selectively targeted versions with diverse mechanisms of action against human cancer cells. Such advancements would offer notable advantages compared to existing cancer therapies. This research aimed to examine the toxicity and selectivity of the nrCap18 peptide in both cancer and normal cell lines. Furthermore, the rate of cellular death was assessed using apoptosis and acridine orange/ethidium bromide (AO/EB) double staining at three distinct incubation times. Additionally, the impact of this peptide on the cancer cell cycle and migration was evaluated, and ultimately, the expression of cyclin-dependent kinase 4/6 (CDK4/6) genes was investigated. The results obtained from the study demonstrated significant toxicity and selectivity in cancer cells compared to normal cells. Moreover, a strong progressive increase in cell death was observed over time. Furthermore, the peptide exhibited the ability to halt the progression of cancer cells in the G1 phase of the cell cycle and impede their migration by suppressing the expression of CDK4/6 genes.

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.

Synergistic effects of quercetin-loaded CoFe2O4@Liposomes regulate DNA damage and apoptosis in MCF-7 cancer cells: based on biophysical magnetic hyperthermia

Introduction Breast cancer (BC) is the most common malignancy in women globally. Significant progress has been made in developing structural nanoparticles (NPs) and formulations for targeted smart drug delivery (SDD) of pharmaceuticals, improving the precision of tumor cell targeting in therapy. Significance Magnetic hyperthermia (MHT) treatment using magneto-liposomes (MLs) has emerged as a promising adjuvant cancer therapy. Methods CoFe2O4 magnetic NPs (MNPs) were conjugated with nanoliposomes to form MLs, and the anticancer drug quercetin (Que) was loaded into MLs, forming Que-MLs composites for antitumor approach. The aim was to prepare Que-MLs for DD systems (DDS) under an alternating magnetic field (AMF), termed chemotherapy/hyperthermia (chemo-HT) techniques. The encapsulation efficiency (EE), drug loading capacity (DL), and drug release (DR) of Que and Que-MLs were evaluated. Results The results confirmed successful Que-loading on the surface of MLs, with an average diameter of 38 nm and efficient encapsulation into MLs (69%). In vitro, experimental results on MCF-7 breast cells using MHT showed high cytotoxic effects of novel Que-MLs on MCF-7 cells. Various analyses, including cytotoxicity, apoptosis, cell migration, western blotting, fluorescence imaging, and cell membrane internalization, were conducted. The Acridine Orange-ethidium bromide double fluorescence test identified 35% early and 55% late apoptosis resulting from Que-MLs under the chemo-HT group. TEM results indicated MCF-7 cell membrane internalization and digestion of Que-MLs, suggesting the presence of early endosome-like vesicles on the cytoplasmic periphery. Conclusions Que-MLs exhibited multi-modal chemo-HT effects, displaying high toxicity against MCF-7 BC cells and showing promise as a potent cytotoxic agent for BC chemotherapy.

Arrhythmogenic cardiomyopathy related phenotypes are detected and pharmacologically modulated in Galectin-3 zebrafish models

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of University and Research Introduction Arrhythmogenic Cardiomyopathy (AC) is a rare inherited cardiac disorder characterized by fibro-fatty replacement and progressive loss of the ventricular myocardium, causing life-threatening arrhythmias, syncope and sudden cardiac death in young and athletes. About 50% of AC cases carry one or more mutations in genes encoding for desmosomal proteins. However, mutations in non-desmosomal genes have been identified in 1-3% of AC patients, such as Galectin-3 (Gal-3). Purpose For a better understanding of Gal-3 role in AC pathogenesis, we have generated a stable Gal-3 knock-out (KO) zebrafish line, able to reproduce cardiac abnormalities and cell signaling dysregulation characteristic of AC, on which environmental factors and candidate drugs can be tested. Methods The zebrafish Gal-3 mutant line was generated by CRISPR/Cas9 strategy and then phenotypically characterized. Phenotyping included heart activity characterization, by pyHeart4Fish software, and gene expression analysis using Real Time PCR and signaling pathway reporters. Immunofluorescence analysis was performed using an antibody against L-plastin, a leukocyte marker for the detection of inflammatory cells, and Acridine Orange/Ethidium Bromide (AO/EB) staining to check for apoptotic and necrotic events. Transmission Electron Microscopy (TEM) analysis was carried out to detect possible ultrastructural alterations. A pathway-directed pharmacological treatment was applied to try to rescue pathological phenotypes. Results The Gal-3 KO zebrafish line, analysed at larval stage, presented structural abnormalities, pericardial effusion and/or hemopericardium associated with reduced contractility, bradycardia and presence of arrhythmia events. In addition to Wnt/β-catenin signaling dysregulation, as seen in other AC forms, we also observed cell death and inflammation. Specifically, the analysis of the L-plastin inflammatory marker showed remarkable infiltration of inflammatory cells in the cardiac region of Gal-3 mutants, whereas AO/EB staining demonstrated elevated number of apoptotic and necrotic cells. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes in mutant adult hearts. Of note, pharmacological treatment at larval stage, using a Wnt/β-catenin signaling agonist, partially rescued a set of AC cardiac phenotypes (contractility, bradycardia, arrhythmia). Conclusions Overall, the Gal-3 KO zebrafish line confirms in zebrafish the strict connection between Gal-3 mutation and AC-like cardiac alterations. Moreover, cell death and inflammation, known to be involved in the remodelling of the myocardium, were observed. The pharmacological rescue of the Wnt/β-catenin signaling partially recovered a set of cardiac phenotypes, confirming this pathway as a key modulator of AC progression. In conclusion, our results strongly support the usefulness of the Gal-3 zebrafish mutant to clarify Gal-3 involvement in AC, offering future perspectives for AC treatment.

The protective mechanism of Tao Hong Si Wu decoction against breast cancer through regulation of EGFR/ERK1/2 signaling

Ethnopharmacological relevanceTao Hong Si Wu Decoction (THSWD), a traditional Chinese herbal medicine, is widely utilized in clinical settings, either alone or in combination with other medications, for the treatment of breast cancer.Aim of the study: The specific targeting molecule(s) of THSWD and its associated molecular mechanisms remain unclear. This research aims to elucidate the underlying molecular mechanisms of THSWD in the treatment of breast cancer. Materials and methodsThe pharmacological properties of THSWD were investigated in breast cancer cells and tumor tissues using a range of methods including Acridine Orange/Ethidium Bromide (AO/EB) staining, Transwell assay, flow cytometry, immunofluorescence assay, and breast cancer mice models. ResultsOur findings demonstrate that THSWD induces necrosis and/or apoptosis in breast cancer cells, while significantly inhibiting cell migration. Target proteins of THSWD in anticancer activity include EGFR, RAS, and others. THSWD treatment for breast cancer is associated with the EGFR/ERK1/2 signaling pathway. ConclusionOur findings offer initial insights into the primary mechanism of action of THSWD in breast cancer treatment, indicating its potential as a complementary therapy deserving further investigation.

Biogenic silver nanoparticle synthesis using orange peel extract and its multifaceted biomedical application.

The aim of this study was to employ an agro-industrial byproduct, specifically Citrus sinensis peels, as a reservoir of polyphenols. The natural chemicals present in C. sinensis peels serve as reducing agents in an environmentally benign method for synthesizing silver nanoparticles (AgNPs). This methodology not only provides a more environmentally friendly method for synthesizing nanoparticles but also enhances the value of agricultural waste, emphasizing the sustainable utilization of resources. In our study, AgNPs were successfully synthesized using peel aqueous exact of C. sinensis and then their various biological activity has been investigated. The synthesized AgNPs were characterized by UV-vis spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and transmission electron microscopy (TEM) analysis. Furthermore, their effectiveness in inhibiting growth and biofilm formation of Escherichia coli, Staphylococcus aureus, and Candida albicans has been investigated. The minimum inhibitory concentrations (MIC) for E. coli and S. aureus were both 32μg/mL, and for C. albicans, it was 128µg/mL. At 250µg/mL of AgNPs, 94% and 92% biofilm inhibition were observed against E. coli and S. aureus, respectively. Furthermore, AgNPs demonstrated significant toxic effects against human prostate cancer cell line DU145 as investigated by anti-apoptotic, 4',6-diamidino-2-phenylindole (DAPI), reactive oxygen species (ROS), and acridine orange/ethidium bromide (AO/EtBr) assays. We also conducted uptake analysis on these pathogens and cancer cell lines to preliminarily investigate the mechanisms underlying their toxic effects. These findings confirm that AgNPs can serve as a cost-effective, non-toxic, and environmentally friendly resource for green synthesis of medicinal AgNPs. Moreover, this approach offers an alternative recycling strategy that contributes to the sustainable use of biological by-products.

Cytotoxic and apoptotic effects of Prunus spinosa fruit extract on HT-29 colon cancer line

Colon cancer holds the position of the third most common type of cancer and stands as the third leading cause of cancer-related deaths for both men and women. Modern strategies in cancer prevention center around the use of natural compounds, which demonstrate a range of effects, including preventive, inhibitory, and latency-inducing impacts on the progression of cancer. In the present study, aqueous extracts derived from the fruits of Prunus spinosa L. (blackthorn, Rosaceae) are employed to assess their cytotoxic potential against the HT-29 colon cancer cell line. The fruit extract is administered to the HT29 cell line in different concentrations over 24 and 48-hours to evaluate the induction of apoptosis. The MTT cell viability test is employed to quantify the cytotoxic effect, indicating the extent of the impact. Additionally, the EB/AO (ethidium bromide/acridine orange) dual staining method is utilized to gather supplementary information regarding the cytotoxic effects. Observations after 24 hours of exposure showed no significant cytotoxic effect; however, 48-hour exposure revealed IC20, IC50, and IC80 values of 1.27, 173.7, and > 1000 µg/ml, respectively, as determined by MTT analysis. Correspondingly, values of 5.06, 123.8, and > 1000 µg/ml were recorded by the EB/AO dual staining method. Our results show that P. spinosa fruit water extract has an inhibitory effect on the HT-29 cell viability by exerting cytotoxic and apoptotic effects in a concentration-dependent and time-dependent manner. Toxicity studies have shown that MTT and EB/AO support each other and achieve similar results. Further extensive research into the metabolic and functional effects of P. spinosa could illuminate its potential and increase its economic importance in the field of anticancer treatments as a natural drug.

Preparation of Ag@3D-TiO2 Scaffolds and Determination of its Antimicrobial Properties and Osteogenesis-promoting Ability.

The micro-nano structure of 3D-printed porous titanium (Ti) alloy with excellent performance in avoiding stress shielding and promoting bone tissue differentiation provides a new opportunity for the development of bone implants, but it necessitates higher requirements for bone tissue differentiation and the antibacterial properties of bone implants in clinical practice. This study investigated the preparation, antimicrobial properties, and osteogenesis-promoting ability of the 3D printed porous Ti alloy anodic oxidized Ag-carrying (Ag@3D-TiO2) scaffolds. The 3D printed porous Ti alloy (3D-Ti), anodized 3D printed porous Ti alloy (3D-TiO2), and Ag@3D-TiO2 scaffolds were synthesized using electron beam melting. The antimicrobial properties of the scaffolds were examined using antibacterial tests and their cytocompatibility was assessed using a cell proliferation assay and acridine orange/ethidium bromide (AO/EB) staining. In vitro cellular assays were used to investigate the effects of the scaffold microstructural features on cell activity, proliferation, and osteogenesis-related genes and proteins. In vivo animal experiments were used to evaluate the anti-inflammatory and osteogenesis-promoting abilities of the scaffolds. The Ag@3D-TiO2 scaffolds exhibited sustained anti-microbial activity over time, enhanced cell proliferation, facilitated osteogenic differentiation, and increased extracellular matrix mineralization. In addition, alkaline phosphatase (ALP), collagen type I (COL-I), and osteocalcin (OCN)-related genes and proteins were upregulated. In vivo animal implantation experiments, the anti-inflammatory effect of the Ag@3D-TiO2 scaffolds were observed using histology, and a large amount of fibrous connective tissue was present around it; the Ag@3D-TiO2 scaffolds were more bio-compatible with the surrounding tissues compared with 3D-Ti and 3D-TiO2; a large amount of uniformly distributed neoplastic bone tissue existed in their pores, and the chronic systemic toxicity test showed that the 3D-Ti, 3D-TiO2, and Ag@3D-TiO2 scaffolds are biologically safe. The goal of this study was to create a scaffold that exhibits antimicrobial properties and can aid bone growth, making it highly suitable for use in bone tissue engineering.

Dual blockage of PI3K-mTOR and FGFR induced autophagic cell death in cholangiocarcinoma cells

PurposeTo assess the impact of concurrent inhibition of the FGFR and PI3K/mTOR signaling pathways on oncogenic characteristics in cholangiocarcinoma (CCA) cells, including proliferation, autophagy, and cell death. Materials and methodsKKU-213A, KKU-100, and KKU-213C cells were treated with either infigratinib or PKI-402 alone or in combination. Cell viability and cell death were evaluated using the sulforhodamine B (SRB) assay and acridine orange/ethidium bromide (AO/EB) staining. Cell cycle progression and apoptotic cell death were analyzed by flow cytometry. Western blotting was performed to assess the expression of proteins involved in cell cycle regulation and autophagy. Additionally, AO staining was employed to assess autophagic induction. ResultsThe combination of infigratinib and PKI-402 showed a remarked synergistic suppression in cell viability in both CCA cell lines compared to treatment with single inhibitors. This antiproliferative effect was associated with cell cycle arrest in the G2-M phase and a decrease in the expression of cyclin A and cyclin B1 in CCA cells. Furthermore, the combination treatment induced apoptotic cell death to a greater extent than treatment with a single inhibitor. Infigratinib enhanced the induction of autophagy by PKI-402, as evidenced by marked increases of autophagic vacuoles stained acridine orange, levels of LC3B-II and suppression of levels of p-mTOR and. Notably, inhibition of autophagic flux by chloroquine prevented cell death induced by the combination treatment. ConclusionsThis study demonstrated that concurrent inhibition of the key FGFR/PI3K/mTOR pathways in CCA carcinogenesis enhances the suppression of CCA cells. The present findings indicate potential clinical implications for using combination treatment modalities in CCA therapy.

In vitro cytotoxicity assessment of biosynthesized nanoceria against MCF-7 breast cancer cell lines

Breast cancer poses a significant health concern due to its increasing prevalence and mortality rates. Hence, prompt action is needed to develop innovative therapeutic interventions, as conventional treatments exhibit severe adverse effects and have limited efficacy. This study aims to develop an innovative therapeutic intervention for breast cancer using biosynthesized nanoceria. Nanoceria has gained significant importance in the medicinal field due to its biocompatibility, dual capabilities as both antioxidant and prooxidant, and specific cytotoxicity towards cancer cells. Herein, nanoceria was biosynthesized using Echinochloa frumentaceae grain extract (EFNC) due to its eco-friendly and cost-efficient nature, and it was systematically investigated using standard characterization techniques. Furthermore, their anticancer efficacy against MCF-7 breast cancer cell lines was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay, and the occurrence of apoptosis was determined using acridine orange/ethidium bromide (AO/EtBr) staining. Significantly, EFNC exhibited superior anticancer efficacy even at lower concentrations (10 µg/mL), and the IC50 value was found at 47.32 µg/mL. In addition, the observed bright green and orange-red fluorescence, along with fragmented/condensed chromatin features in AO/EtBr staining clearly indicated that the EFNC caused cell death through the apoptotic route. One potential factor contributing to the observed anticancer effectiveness of EFNC could be its prooxidant properties within cancer cells. These properties ultimately enhanced oxidative stress, induced the accumulation of reactive oxygen species (ROS), and led to apoptosis. Hence, the in vitro analysis substantiated the significant anticancer efficacy of EFNC, suggesting its potential utility as a promising therapeutic agent in anticancer treatment. These findings pave the way for further exploration and development of EFNC as a valuable candidate for addressing cancer-related challenges.

Ethanolic extract of Euphorbia Gypsicola induces differentiation and apoptosis in human myeloid leukemia K562 cells

IntroductionThe Euphorbiaceae family, widely distributed and long utilized in traditional medicine, has been recognized for its potential in developing anti-cancer drugs. The current study investigates the capacity of Euphorbia gypsicola extract to induce apoptosis and differentiation in the human myeloid leukemia K562 cell line. MethodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic activity was visually evaluated through acridine orange/ethidium bromide (AO/EB) dye staining and annexin V/PI double labeling procedures. Induction of differentiation was examined using nitro blue tetrazolium (NBT) reduction and Wright-Giemsa staining assays. The expression of apoptosis-related proteins including Bax, Bcl2 and Survivin was evaluated by western blotting method. ResultsThe E. gypsicola extract exhibited potent medication properties with IC50 values of 207.59, 48.58, and 7.08 ng/mL at 24, 48, and 72 h, respectively. Fluorescence microscopy, DNA fragmentation, annexin V/PI double staining, and sub-G1 cell cycle arrest confirmed apoptosis occurrence in K562 cells treated with the E. gypsicola extract. Our findings demonstrated that differentiated cells reduced NBT yellow solution after endocytosis, forming dark crystals. Wright Giemsa staining observations indicated a decrease in nuclear volume-to-cytoplasm ratio in K562 cells treated with the crude extract, signifying Induction of differentiation. Furthermore, western blot analysis revealed down-regulation of Survivin protein expression and up-regulation of Bax protein level following treatment with the E. gypsicola extract. However, no significant decrease in Bcl2 protein expression level was observed in K562 cells. ConclusionThese findings indicate the growth inhibitory effects of the E. gypsicola extract on leukemia K562 cells, which supports the further study of this plant to discover novel therapeutic compounds for the treatment of leukemia.

Biomedicine and pharmacotherapeutic effectiveness of combinatorial atorvastatin and quercetin on diabetic nephropathy: An in vitro study

IntroductionDiabetic nephropathy is a type of kidney disorder that develops as a complication of multifactorial diabetes. Diabetic nephropathy is characterized by microangiopathy, resulting from glucose metabolism, oxidative stress, and changes in renal hemodynamics. This study strived to evaluate the in vitro cytoprotective activity of atorvastatin (ATR), and quercetin (QCT) alone and in combination against diabetic nephropathy. MethodsThe MTT assay was utilized to analyze the effects of the test compounds on NRK-52E rat kidney epithelial cells. The detection of apoptosis and ability to scavenge free radicals was assessed via acridine orange-ethidium bromide (AO-EB) dual fluorescence staining, and 2,2-diphenyl-1-picrylhydrazyfree assay (DPPH), respectively. The ability of anti-inflammatory effect of the test compounds and western blot analysis against TGF-β, TNF-α, and IL-6 further assessed to determine the combinatorial efficacy. ResultsAtorvastatin and quercetin treatment significantly lowered the expression of TGF-β, TNF-α, and IL-6 indicating the protective role in Streptozotocin-induced nephrotoxicity. The kidney cells treated with a combination of atorvastatin and quercetin showed green fluorescing nuclei in the AO-EB staining assay, indicating that the combination treatment restored cell viability. Quercetin, both alone and in combination with atorvastatin, demonstrated strong DPPH free radical scavenging activity and further encountered an anti-oxidant and anti-inflammatory effect on the combination of these drugs. ConclusionNevertheless, there is currently no existing literature that reports on the role of QCT as a combination renoprotective drug with statins in the context of diabetic nephropathy. Hence, these findings suggest that atorvastatin and quercetin may have clinical potential in treating diabetic nephropathy.

Anticancer activity of silver nanoparticles synthesized from aqueous leaf extract of Solanum trilobatum (Purple fruit pea eggplant) on human oral cancer cells

Introduction: This research explores the capability of silver nanoparticles (AgNPs) produced via friendly methods by using Solanum trilobatum leaf extract. The choice of S. trilobatum was supported by its diverse phytochemical makeup, which comprises recognized bioactive elements known for their anti-inflammatory and anti-cancer attributes. This research explores the effect of AgNPs derived from this plant as a promising eco-friendly strategy in oral cancer treatment. Methods: The synthesis of AgNPs involved employing S. trilobatum leaf extract, with observable color changes and spectral analyses confirming the unique characteristics of the nanoparticles. Fourier transform infrared spectroscopy detected distinct functional groups, whereas scanning electron microscopy (SEM) confirmed the presence of biocapped nanoparticles exhibiting various shapes and sizes spanning from 100 to 300 nm. Results: Cytotoxicity assessments via the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay revealed a half-minimal inhibitory concentration (IC50) value of 3.715 ± 0.242 μg/mL against the oral cancer (KB) cell line, indicating a significant inhibition. Acridine orange/ethidium bromide (AO/EtBr) and reactive oxygen species (ROS) assays further supported the AgNPs’ anti-cancer potential, affirming their promise for oral cancer therapy. Conclusion: AgNPs synthesized from S. trilobatum leaf extract showed substantial potential as a therapeutic agent for oral cancer. This research adds to the increasing evidence endorsing the use of environmentally friendly synthesized nanoparticles in medical treatments.