Dichlorofluorescein Diacetate Research Articles

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.

Inhibitory effects of progesterone on the human acute lymphoblastic leukemia cell line

IntroductionAcute lymphoblastic leukemia (ALL) is a rare form of blood cancer that can quickly advance if left untreated. Research has suggested that progesterone (P4) may be effective in treating certain types of tumors. Specifically, the membrane progesterone receptors may play a role in either enhancing or inhibiting cell growth in various tumors. This study aimed to investigate the impact of P4 on inhibition of NALM6 cells. MethodsNALM6 cells were exposed to different concentrations of P4 (ranging from 10 to 50 μM) at 24,48 and 72 h intervals. The cell survival rate was then evaluated using an MTT assay. Additionally, the study assessed the rate of mPR expression in the cells using flow cytometry at 48 and 72 h after P4 administration (at concentrations of 20 and 10 μM, respectively). Furthermore, the level of ROS was evaluated using the dichlorofluorescein diacetate (DCFDA) flow cytometry technique. ResultsThe study found that mPR-β was expressed in NALM6 cells and that P4 had a significant inhibitory effect on the growth of tumor cells in a time and concentration-dependent manner. Furthermore, P4 was found to reduce mPR-β expression at 48 and 72 h. The treatment also resulted in a decrease in ROS levels compared to untreated cells (P ≤ 0.05). ConclusionThe study suggests that p4 may be effective in growth-inhibiting NALM6 cells by decreasing cell viability and reducing ROS levels. However, further research is needed to understand the mechanism of action and interactions with various receptors and to confirm its effectiveness in treating NALM6 leukemia.

Yttrium oxide nanoparticles ameliorates calcium hydroxide and calcium titanate nanoparticles induced genomic DNA and mitochondrial damage, ROS generation and inflammation

Calcium hydroxide (Ca(OH)2NPs), calcium titanate (CaTiO3NPs) and yttrium oxide (Y2O3NPs) nanoparticles are prevalent in many industries, including food and medicine, but their small size raises concerns about potential cellular damage and genotoxic effects. However, there are very limited studies available on their genotoxic effects. Hence, this was done to investigate the effects of multiple administration of Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs on genomic DNA stability, mitochondrial membrane potential integrity and inflammation induction in mouse brain tissues. Mice were orally administered Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs at a dose level of 50 mg/kg b.w three times a week for 2 weeks. Genomic DNA integrity was studied using Comet assay and the level of reactive oxygen species (ROS) within brain cells was analyzed using 2,7 dichlorofluorescein diacetate dye. The expression level of Presenilin-1, tumor necrosis factor-alpha (TNF-α) and Interleukin-6 (IL-6) genes and the integrity of the mitochondrial membrane potential were also detected. Oral administration of Ca(OH)2NPs caused the highest damage to genomic DNA and mitochondrial membrane potential, less genomic DNA and mitochondrial damage was induced by CaTiO3NPs administration while administration of Y2O3NPs did not cause any remarkable change in the integrity of genomic DNA and mitochondrial membrane potential. Highest ROS generation and upregulation of presenilin-1, TNF-α and IL-6 genes were also observed within the brain cells of mice administrated Ca(OH)2NPs but Y2O3NPs administration almost caused no changes in ROS generation and genes expression compared to the negative control. Administration of CaTiO3NPs alone slightly increased ROS generation and the expression level of TNF-α and IL-6 genes. Moreover, no remarkable changes in the integrity of genomic DNA and mitochondrial DNA potential, ROS level and the expression level of presenilin-1, TNF-α and IL-6 genes were noticed after simultaneous coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs. Coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs mitigated Ca(OH)2NPs and CaTiO3NPs induced ROS generation, genomic DNA damage and inflammation along with restoring the integrity of mitochondrial membrane potential through Y2O3NPs scavenging free radicals ability. Therefore, further studies are recommended to study the possibility of using Y2O3NPs to alleviate Ca(OH)2NPs and CaTiO3NPs induced genotoxic effects.

Synthesis of triazole AD-1 derivatives and its mechanism of mediating DNA damage of ROS in lung cancer cells

Based on the significant biological activities and the remarkable physical and chemical properties of 1H-1,2,3-triazole pharmacophore, we herein adopted the strategy of click chemistry to combine the triazole fragment and the unique scaffold of 25-OCH3-PPD (AD-1) to design a series of potent compounds inducing apoptosis and DNA damage. The anti-proliferative effect was verified by MTT assay and colony formation assay. DNA double-stand breaks (DSBs) were obtained by observing the nuclear focus formation and the protein expression of γ-H2AX. Cell cycle arrest was evaluated by the cycle-related proteins such as CDK2, CDK4, CDK6, Cyclin D1 and P21. Apoptosis was assessed by flow cytometry, mitochondrial membrane potential (MMP) detection and the expression of apoptosis-related proteins. Reactive oxygen species (ROS) generation was measured with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) staining. According to SAR analysis, the most potent compound 6a exhibited great inhibitory effect against A549 cells, which IC50 value of 2.84 ± 0.68 μM. Furthermore, 6a remarkably induced DNA damage, cell cycle arrest and apoptosis in A549 cells. 6a treatment increased the levels of ROS. Network pharmacology and molecular docking predicted the potential signaling pathways and ligand-receptor interactions, and the results of western blotting showed that 6a inhibited the PI3K/Akt/Bcl-2 signaling pathway by decreasing PI3K and Bcl-2 and total level of Akt expression, while Bax and Cyt c were increasing in 6a-treated A549 cells. As mentioned above, 6a has a potent inhibitory effect in A549 cells through induction of DNA damage, apoptosis via ROS generation and modulation of PI3K/Akt/Bcl-2 signaling pathway.

Roles of AhR/CYP1s signaling pathway mediated ROS production in uremic cardiomyopathy

PurposeUremic cardiomyopathy (UCM) is the leading cause of chronic kidney disease (CKD) related mortality. Uremic toxins including indoxyl sulfate (IS) play important role during the progression of UCM. This study was to explore the underlying mechanism of IS related myocardial injury. MethodsUCM rat model was established through five-sixths nephrectomy to evaluate its effects on blood pressure, cardiac impairment, and histological changes using echocardiography and histological analysis. Additionally, IS was administered to neonatal rat cardiomyocytes (NRCMs) and the human cardiomyocyte cell line AC16. DHE staining and peroxide-sensitive dye 2′,7′-dichlorofluorescein diacetate (H2DCFDA) was conducted to assess the reactive oxygen species (ROS) production. Cardiomyocyte hypertrophy was estimated using wheat germ agglutinin (WGA) staining and immunofluorescence. Aryl hydrocarbon receptor (AhR) translocation was observed by immunofluorescence. The activation of AhR was evaluated by immunoblotting of cytochrome P450 1 s (CYP1s) and quantitative real-time PCR (RT-PCR) analysis of AHRR and PTGS2. Additionally, the pro-oxidative and pro-hypertrophic effects were evaluated using the AhR inhibitor CH-223191, the CYP1s inhibitor Alizarin and the ROS scavenger N-Acetylcysteine (NAC). ResultsUCM rat model was successfully established, and cardiac hypertrophy, accompanied by increased blood pressure, and myocardial fibrosis. Further research confirmed the activation of the AhR pathway in UCM rats including AhR translocation and downstream protein CYP1s expression, accompanied with increasing ROS production detected by DHE staining. In vitro experiment demonstrated a translocation of AhR triggered by IS, leading to significant increase of downstream gene expression. Subsequently study indicated a close relationship between the production of ROS and the activation of AhR/CYP1s, which was effectively blocked by applying AhR inhibitor, CYP1s inhibitor and siRNA against AhR. Moreover, the inhibition of AhR/CYP1s/ROS pathway collectively blocked the pro-hypertrophic effect of IS-mediated cardiomyopathy. ConclusionThis study provides evidence that the AhR/CYP1s pathway is activated in UCM rats, and this activation is correlated with the uremic toxin IS. In vitro studies indicate that IS can stimulate the AhR translocation in cardiomyocyte, triggering to the production of intracellular ROS via CYP1s. This process leads to prolonged oxidative stress stimulation and thus contributes to the progression of uremic toxin-mediated cardiomyopathy.

Assessment of the toxicity of different antiretroviral drugs and their combinations on Sertoli and Leydig cells

The human immunodeficiency virus continues to pose a significant global public health challenge, affecting millions of individuals. The current treatment strategy has incorporated the utilization of combinations of antiretroviral drugs. The administration of these drugs is associated with many deleterious consequences on several physiological systems, notably the reproductive system. This study aimed to assess the toxic effects of abacavir sulfate, ritonavir, nevirapine, and zidovudine, as well as their combinations, on TM3 Leydig and TM4 Sertoli cells. The cell viability was gauged using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays. Reactive oxygen species (ROS) production was assessed via the 2’,7’-dichlorofluorescein diacetate (DCFDA) test, and DNA damage was determined using the comet assay. Results indicated cytotoxic effects at low drug concentrations, both individually and combined. The administration of drugs, individually and in combination, resulted in the production of ROS and caused damage to the DNA at the tested concentrations. In conclusion, the results of this study suggest that the administration of antiretroviral drugs can lead to testicular toxicity by promoting the generation of ROS and DNA damage. Furthermore, it should be noted that the toxicity of antiretroviral drug combinations was shown to be higher compared to that of individual drugs.

Heat-Killed Lactilactobacillus sakei WB2305 and Lactiplantibacillus plantarum WB2324 Inhibited LPS-Induced Inflammation in Human Airway Epithelial Cells.

Asthma is characterized by inflammation of the airways, including the inflammatory and airway structural cells. Probiotics, which have diverse effects, even within the same species, are being studied to prevent and mitigate the severity of asthma. Lactilactobacillus sakei WB2305 and Lactiplantibacillus plantarum WB2324 were isolated from kimchi. These strains have acceptable probiotic properties and are safe. In addition, the anti-inflammatory potential of the heat-killed isolates against lipopolysaccharide (LPS)-induced inflammation in the human pulmonary epithelial cell line (A549) was investigated. The heat-killed Lact. sakei WB2305 and Lact. plantarum WB2324 reduced the chemokine and cytokines mRNA expression levels, as shown by the results of using real-time polymerase chain reaction. Western blotting results showed that the nuclear factor-kappa B (NF-κB) activation and mitogen-activated protein kinases (MAPK) signaling pathways were suppressed by treatment with the heat-killed strains. The production amounts of eotaxin, tumor necrosis factor-ɑ (TNF-α), and interleukin-6 (IL-6) were lower than those in LPS-only treated cells. Additionally, 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining confirmed decreased reactive oxygen species (ROS) production in A549 cells. Therefore, the results of present study demonstrate the anti-inflammatory and anti-asthmatic activities of heat-killed Lact. sakei WB2305 and Lact. plantarum WB2324 in human airway epithelial cells.

Bifenox induces hepatotoxicity and vascular toxicity in zebrafish embryos via ROS production and alterations in signaling pathways

Existing evidence shows that currently used pesticides pose toxicological risks to exposed wildlife. Chemically, bifenox belongs to diphenyl ethers, a well-known group of herbicides. Its mechanism of action primarily involves inducing lipid peroxidation and blocking protoporphyrinogen oxidases. Toxicity of diphenyl ether herbicides has been elucidated in animal cells; however, in vivo toxicological evaluations of bifenox are required to determine its unexpected effects. This study aimed to determine the negative effects of bifenox, and its effects on higher eukaryotes. We found that early stages of zebrafish embryo exposed to bifenox demonstrated increased mortality and physiological defects, based on the LC50 value. Bifenox severely inhibited blood vessel growth by reducing key elements of complex connectivity; fluorescently tagged transgenic lines (fli1a:EGFP) showed morphological changes. Additionally, transgenic lines that selectively identified hepatocytes (fabp10a:DsRed) showed reduced fluorescence, indicating that bifenox may inhibit liver development. To evaluate the level of oxidative stress, we used 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probes in zebrafish embryos to identify the underlying mechanisms causing developmental damage. Our findings demonstrate that exposure to bifenox causes abnormalities in the hepatic and cardiovascular systems during zebrafish embryogenesis. Therefore, this study provides new information for the evaluation of toxicological risks of bifenox in vertebrates.

Dihydroquercetin improves experimental acute liver failure by targeting ferroptosis and mitochondria-mediated apoptosis through the SIRT1/p53 axis

BackgroundFerroptosis and mitochondria-mediated apoptosis are both involved in the pathogenesis of acute liver failure (ALF). Ferroptosis-produced reactive oxygen species (ROS) trigger the chain oxidation of polyunsaturated phospholipids and promote mitochondrial apoptosis. Dihydroquercetin (DHQ) also plays an important protective role against liver injury. PurposeHere, we aimed to investigate the protective effects of DHQ on ALF. We also explored the underlying mechanism. MethodsWe established a Lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced ALF mouse model and tumor necrosis factor-α (TNF-α)/D-Gal-induced ALF LO2 cell model. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and Dihydroethidium (DHE) were used to detect total ROS levels. Lipid ROS was assessed using C11-BODIPY flow cytometry. Lipid peroxidative products levels were detected using MDA ELISA assay and 4-hydroxynonenal (4-HNE) immunohistochemistry. QRT-PCR and western blots were used to test mRNA and protein expression levels, respectively. Cell viability was evaluated with CCK8 assay, and apoptosis was analyzed using flow cytometry. ResultsDHQ treatment improved LPS/D-Gal-induced ALF, as well as TNF-α/D-Gal-induced reductions in LO2 viability and increased sirtuin 1 (SIRT1) expression. DHQ pretreatment also reduced the accumulation of ROS, reduced lipid peroxidation, elevated mitochondrial membrane potentials (ΔΨm), and decreased liver cell apoptosis both in vivo and in vitro. Additionally, the knockdown of SIRT1 and p53 activator (Tenovin-6) treatment reversed DHQ's inhibitory effects on ferroptosis and mitochondria-mediated apoptosis in vitro. DHQ enhanced p53 deacetylation by both up-regulating SIRT1 expression and directly bonding to SIRT1. We also found that Tenovin-6′s stimulatory effects on ferroptosis and mitochondria-mediated apoptosis in the DHQ-treated LO2 ALF cell model were partially attenuated by overexpression of solute carrier family 7member 11 (SLC7A11), as well as by apoptotic protease activating factor 1 (Apaf-1) knockdown. ConclusionOur results suggest that DHQ alleviated ALF by inhibiting both ferroptosis and mitochondria-mediated apoptosis by regulating the SIRT1/p53 axis. Thus, DHQ may serve as a novel therapy for ALF.

Pemetrexed loaded gold nanoparticles as cytotoxic and apoptosis inducers in lung cancer cells through ROS generation and mitochondrial dysfunction pathway.

Supramolecular nanoparticles containing peptides and drugs have recently gained recognition as an effective tumor treatment drug delivery system. A multitarget drug termed pemetrexed is effective against various cancers, including nonsmall cell lung cancer. The work aims to establish the capability of pemetrexed gold nanoparticles (PEM-AuNPs) to induce apoptosis and explore molecular changes. X-ray diffraction, Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, scanning electron microscope, and transmission electron microscope were used to investigate the synthesized nanoparticles. The MTT assay was utilized to investigate the anticancer properties of PEM-AuNPs at varying concentrations (50, 100, and 200µM). PEM-AuNPs demonstrated a decrease in cell viability with 55.87%, 43.04%, and 25.59% for A549 cells and 54.31%, 37.40%, and 25.84% for H1299 cells at the respective concentrations. To assess apoptosis and perform morphological analysis, diverse biochemical staining techniques, including acridine orange-ethidium bromide and 4',6-diamidino-2-phenylindole nuclear staining assays, were employed. Additionally, 2',7'-dichlorofluorescein diacetate staining confirmed the induction of reactive oxygen species generation, while JC-1 staining validated the impact on the mitochondrial membrane at the IC50 concentration of PEM-AuNPs. Thus, the study demonstrated that the synthesized PEM-AuNPs exhibited enhanced anticancer activity against both A549 and H1299 cells.

Water extract of moschus alleviates erastin-induced ferroptosis by regulating the Keap1/Nrf2 pathway in HT22 cells

Ethnopharmacological relevanceMoschus, first described in the Shennong's Classic of the Materia medicine, is a scarce and precious animal medicine. Modern pharmacological researches have suggested that Moschus has neuroprotective actions, and its mechanism is related to anti-inflammatory, antioxidant, and anti-apoptosis effects. Ferroptosis is one of the major pathologies of Alzheimer's disease (AD) and is widely implicated in the pathogenesis and progression of AD. Although previous studies have suggested that Moschus possesses neuroprotective effect, whether Moschus could mitigate neuronal damages by inhibiting the onset of ferroptosis is unknown in model cells of AD. Aim of the studyThe aim of study was to explore the water extract of Moschus (WEM) on ferroptosis caused by erastin and the potential mechanism. Materials and methodsErastin was used to stimulate HT22 cells to form ferroptosis model to evaluate the anti-ferroptosis effect of WEM by cell counting kit-8 and lactic dehydrogenase (LDH) tests. The malondialdehyde (MDA) and glutathione (GSH) kits are used for detection of MDA and GSH levels, and 2′,7′-dichlorofluorescein diacetate and C11 BODIPY 581/591 fluorescence probe are used for evaluation of reactive oxygen species (ROS) and lipid peroxide (LOOH) levels. And Western blot was used to test nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), and ferroptosis associated proteins including glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter subunit (SLC7A11), ferritin heavy chain 1 (FTH1), ferroportin1 (FPN1), transferrin receptor (TFRC). In addition, the Nrf2 inhibitor ML385 was applied to verify whether WEM prevents erastin-induced ferroptosis by activating the Keap1/Nrf2 pathway. ResultsAfter WEM treatment, erastin-induced HT22 cell survival was significantly elevated, the accumulation of intracellular MDA, ROS, and LOOH were significantly reduced, the level of GSH and expressions of ferroptosis inhibitors GPX4 and SLC7A11 were significantly increased, and iron metabolism-related proteins TFRC, FPN1, and FTH1 were regulated. These effects of WEM are implemented by activating the Keap1/Nrf2 pathway. ConclusionsThis study demonstrated that WEM could perform neuroprotective effects by alleviating ferroptosis, verified that WEM treatment of AD can be mediated by the Keap1/Nrf2 pathway, and provided theoretical support for the application of WEM in the treatment of AD.

Anti-bacterial and anti-biofilm activities of arachidonic acid against the cariogenic bacterium Streptococcus mutans.

Streptococcus mutans is a Gram-positive, facultative anaerobic bacterium, which causes dental caries after forming biofilms on the tooth surface while producing organic acids that demineralize enamel and dentin. We observed that the polyunsaturated arachidonic acid (AA) (ω-6; 20:4) had an anti-bacterial activity against S. mutans, which prompted us to investigate its mechanism of action. The minimum inhibitory concentration (MIC) of AA on S. mutans was 25 μg/ml in the presence of 5% CO2, while it was reduced to 6.25-12.5 μg/ml in the absence of CO2 supplementation. The anti-bacterial action was due to a combination of bactericidal and bacteriostatic effects. The minimum biofilm inhibitory concentration (MBIC) was the same as the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies showed decreased expression of biofilm-related genes, suggesting that AA also has a specific anti-biofilm effect. Flow cytometric analyses using potentiometric DiOC2(3) dye, fluorescent efflux pump substrates, and live/dead SYTO 9/propidium iodide staining showed that AA leads to immediate membrane hyperpolarization, altered membrane transport and efflux pump activities, and increased membrane permeability with subsequent membrane perforation. High-resolution scanning electron microscopy (HR-SEM) showed remnants of burst bacteria. Furthermore, flow cytometric analysis using the redox probe 2',7'-dichlorofluorescein diacetate (DCFHDA) showed that AA acts as an antioxidant in a dose-dependent manner. α-Tocopherol, an antioxidant that terminates the radical chain, counteracted the anti-bacterial activity of AA, suggesting that oxidation of AA in bacteria leads to the production of cytotoxic radicals that contribute to bacterial growth arrest and death. Importantly, AA was not toxic to normal Vero epithelial cells even at 100 μg/ml, and it did not cause hemolysis of erythrocytes. In conclusion, our study shows that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans.

Evaluation of Saudi Juniperus procera Extracts Cytotoxicity and Regulatory Mechanisms of Tumorigenesis Against Two Breast Cancer Cell Lines

Background Breast cancer poses a major threat to female health worldwide. One of the medicinal plants used in traditional Saudi medicine is the Juniperus procera (Arar) plant, which is used to cure various illnesses, including cancer. Objectives The cytotoxic properties of Saudi J. procera extracts and their regulatory mechanisms against tumorigenesis in breast cancer were investigated. Methods The antioxidant capacities of the fruit and leaf extracts were detected by the 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay and 2,2-diphenyl-1-1picrylhydrazyl (DPPH) assay. The cell viability of the extracts was determined by 3-(4,5-dimethylthiazoyl)-2,5-diphenyl-tetrazolium bromide assay. The cell migration was detected by a wound healing assay. The cell cycle and cell apoptosis were analyzed by flow cytometry. The reactive oxygen species (ROS) was evaluated using a DCFDA-cellular (the cell permeant reagent 2',7' –dichlorofluorescein diacetate) reactive oxygen species detection assay. The protein and gene expression levels were estimated by western blotting and Reverse transcription polymerase chain reaction (RT-PCR), respectively. Results Phytochemical analysis revealed a high content of phenolic and flavonoids in fruit and leaf extracts. Relative to leaf extract, the fruit extract showed higher antioxidant activities and exhibited more potent cytotoxic effects against two breast cancer cell lines: MCF-7 and MDA-MB-231. The fruit extract arrested the cell cycle of the tested cancer cell lines in S phase and G1 phase, and downregulated PI3K (phosphoinositide 3-kinase-protein kinase)/Akt (protein kinase B)/mTOR (Mammalian target of rapamycin) pathway. It also suppressed aromatase expression in MCF-7 but upregulated it in MDA-MB-231. Conclusion J procera fruit and leaf extracts have significant antitumor activities against MCF-7 and MDA-MB-231 breast cancer cell lines.

Anti-Leishmania major activity of Calotropis procera extract by increasing ROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions

BackgroundLeishmaniasis, caused by protozoan parasites of the genus Leishmania, is a neglected tropical disease with 700,000 to 1,000,000 global new cases annually. Adverse effects associated with expense, long-term treatment and drug resistance have made conventional therapies unfavorable, encouraging the search for alternative drugs based on plant products. In this study, the effect of Calotropis procera (Asclepiadaceae) extract against viability of promastigotes and amastigotes of Leishmania major was evaluated in vitro.MethodsThe extract from the leaves of C. procera seedlings was prepared using a methanol maceration method. The colorimetric cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth-inhibitory effect of the extract on promastigotes. The level of reactive oxygen species (ROS) in promastigote cultures was determined after treatment with the extract using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method and compared with untreated cultures (control). After exposure to the extract the expression levels of tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) genes were determined and compared to control in peripheral blood mononuclear cells (PBMCs) infected with L. major.ResultsBased on the MTT assay, the C. procera extract significantly reduced the proliferation of L. major promastigotes with IC50 values of 377.28 and 222.44 μg/mL for 24 and 72 h, respectively (p < 0.01). After treatment with 222.44 and 377.28 μg/mL of C. procera extract, ROS production in L. major promastigote cultures increased 1.2- to 1.65-fold and 2- to 4-fold compared to the control, respectively (p < 0.05). C. procera extract induced significant increases in gene expression of TNF-α (2.76–14.83 fold), IFN-γ (25.63–threefold) and iNOS (16.32–3.97 fold) in infected PBMCs compared to control (p < 0.01).ConclusionsOn the basis of its anti-leishmanial activity, C. procera can be considered as a promising new plant source for the potential treatment of leishmaniasis.

Role of heat shock protein 90 in hypoxia-induced angiogenesis and epithelial-mesenchymal transition for esophageal squamous cancer cells.

395 Background: Hypoxia is known as an important trigger for the development and progression of human cancers. Heat shock proteins (Hsps) may be up-regulated in response to cellular stressors including hypoxia. To date, the functional role of Hsps within hypoxic tumor microenvironment for esophageal squamous cell cancer (ESCC) remains poorly defined. Methods: CoCl2 was used to induce hypoxia that mimics a hypoxic tumor microenvironment in cultured ESCC cells. Successful induction of hypoxia was confirmed by 2’,7’ –dichlorofluorescein diacetate (DCFDA) assay by detection of cellular reactive oxygen species (ROS). 7-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a selective Hsp90 inhibitor, was used to treat 2 ESCC cell lines, KYSE-170 and -510 cells pretreated with or without CoCl2, an agent to induce a hypoxic tumor microenvironment. Cytotoxicity (MTT) and migration assays were conducted in KYSE-17 and -510 ESCC cells in response to different concentrations of CoCl2, followed by protein expression of Hsp 90, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1a (HIF-1a), mTOR as well as markers related epithelial-mesenchymal transition (EMT) such as snail or E-cadherin, by immunoblot or ELISA. In parallel, cell proliferation and migration assays of ESCC cells were analyzed. Results: CoCl2 induced hypoxia was supported by an induction of reactive oxygen species (ROS). CoCl2 (200 mM) significantly suppressed cell viability and proliferation with a concomitant up-regulation of VEGF and HIF-1a in a dose-dependent fashion. In contrast, cell migration was significantly increased in response to CoCl2, while down-regulating E-cadherin with concomitant increase in Snail expression. 17-DMAG decreased expression of VEGF and HIF-1a. In addition, it inhibited cell migration and invasion of ESCC cells were analyzed. Conclusions: Our data have shown that CoCl2 induced hypoxia promotes EMT and angiogenesis, which are inhibited by 17-DMAG, suggesting that hypoxia induced EMT and angiogenesis is Hsp 90 dependent in ESCC.

Insight into the inhibitory activity and mechanism of bovine cathelicidin BMAP 27 against Salmonella Typhimurium

This study synthesized an antimicrobial peptide based on the bovine cathelicidin BMAP 27 sequence. It was found to have a broad spectrum of antibacterial activity, with exceptionally high activity against Salmonella. However, the antibacterial mechanism of BMAP 27 against Salmonella remains unclear. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of BMAP 27 against Salmonella enterica serovar Typhimurium were determined to be 2 μM and 4 μM, respectively. After treatment with 2 MIC of BMAP 27, the absorbance of DNA in centrifugal supernatant increased from 0.244 to 1.464, and that of protein rose from 0.174 to 0.774, respectively. BMAP 27 has compromised the cell membrane as observed through field emission scanning electron microscope (FESEM) and transmission electron microscopy (TEM), and confirmed by the propidium iodide (PI) test. The alkaline phosphatase (AKP) enzyme activity in the supernatant of the 2 MIC treatment group was 2.15 times higher than the control group, indicating extracellular membrane damage. BMAP 27 treatment increased intracellular ROS levels as tested by dichlorofluorescein diacetate (DCFH) staining. DNA interaction analysis revealed that BMAP 27 has a binding affinity towards DNA, causing its characteristic bands to disappear and peak intensity at 260 nm to reduce. Molecular docking identified its potential binding mode with DNA. The crystal violet biofilm staining results demonstrated that BMAP 27 inhibited S. Typhimurium biofilm formation by 43.1 % and cleared mature biofilms by 53.62 %. Confocal Laser scanning electron microscopy (CLSM) observed that BMAP 27 could kill bacteria within the biofilm and dislodge bacteria from the surface of glasses. Swimming tests identified that the motor capacity of S. Typhimurium was diminished by BMAP 27. By counting the total bacteria, BMAP 27 was revealed to exert bacteriostatic effects in chilled pork and orange juice, which might provide a basis for its application in the inhibition of Salmonella.

Cola rostrata K. Schum. constituents induce cytotoxicity through reactive oxygen species generation and mitochondrial membrane depolarisation

Aim: While the traditional use of Cola rostrata in treating illnesses and diseases has not been reported, the presence of cytotoxic principles has been reported in phylogenetically and biogeographically related species within the Cola genus. This study, therefore, evaluated the cytotoxic potential of extracts of the plant, and the associated cellular and molecular mechanisms. Methods: Activity-based fractionation of the extracts was carried out and cytotoxicity was assessed in the human cervical cancer cell line, HeLa, and the transformed human lung cell line, MRC5-SV2, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay complemented with brightfield imaging. The 2ʼ,7ʼ-dichlorofluorescein diacetate (DCFDA) assay was used to assess induction of cellular reactive oxygen species (ROS), while flow cytometry of 5,5ʼ,6,6ʼ-tetrachloro-1,1ʼ,3,3ʼ-tetraethyl-imidacarbocyanine iodide (JC-1)-stained cells assessed the loss of mitochondrial membrane potential (∆ΨM). Gas chromatography-mass spectrometry (GC-MS) analysis was carried out on an active fraction. Results: Extracts of the fruit epicarp and leaf were cytotoxic against the cell lines. Half-maximal inhibitory concentration (IC50) values for the 48 h cytotoxicity of the ethanol extract of the epicarp against HeLa and MRC5-SV2 cells were 48.0 μg/mL ± 12.1 μg/mL and 40.4 μg/mL ± 7.2 μg/mL, respectively, while fractions from second-level partitioning of the hexane fraction of the leaf extract elicited cytotoxicity with IC50 values ranging from 12.8 μg/mL ± 1.0 μg/mL to 39.6 μg/mL ± 7.2 μg/mL in both cell lines, following 48 h treatment. GC-MS revealed the presence of seventeen compounds in a hexane fraction of the leaf extract, including even- and odd-chain fatty acids, the most abundant of which were n-hexadecanoic acid, decanoic acid 10-(2-hexylcyclopropyl); and octadecanoic acid. The mechanisms of cytotoxicity of most active fractions involved generation of ROS and mitochondrial membrane depolarisation. Conclusions: The findings show that C. rostrata is rich in cytotoxic phytochemicals which could be isolated for developing new anti-cancer agents.

Histidine-Based Supramolecular Nanoassembly Exhibiting Dual Enzyme-Mimetic Functions: Altering the Tautomeric Preference of Histidine to Tailor Oxidative/Hydrolytic Catalysis.

Challenges persist in replicating enzyme-like active sites with functional group arrangements in supramolecular catalysis. In this study, we present a supramolecular material comprising Fmoc-modified histidine and copper. We also investigated the impact of noncanonical amino acids (δmH and εmH), isomers of histidine, on the catalytic process. The Fmoc-δmH-based nanoassembly exhibits an approximately 15-fold increase in oxidative activity and an ∼50-fold increase in hydrolytic activity compared to Fmoc-εmH (kcat/Km). This distinction arises from differences in basicity and ligation properties between the ε- and δ-nitrogen of histidine. The addition of guanosine monophosphate further enhances the oxidative activity of the histidine- and methylated histidine-based catalysts. The Fmoc-δmH/Cu2+-based nanoassembly catalyzes the oxidation/hydrolysis cascade of 2',7'-dichlorofluorescein diacetate, benefiting from the synergistic effect between the copper center and the nonligating ε-nitrogen of histidine. These findings advance the biomimetic catalyst design and provide insights into the mechanistic role of essential residues in natural systems.

Phytochemical analysis of carotenoid profile in Mentha piperita and Artemisia vulgaris: cytotoxicity in tumoral cells and evaluation of plasmid DNA cleavage

ABSTRACT Several medicinal plants have been administered to cancer patients attributed to their anticarcinogenic and chemoprotective properties, in addition to lower toxicity compared to traditional therapies. The aim was to investigate the antioxidant properties and carotenoid composition of aqueous extracts of Mentha piperita or Artemisia vulgaris which were previously found to exert beneficial effects on human health through diet. aqueous extracts exhibited potent antioxidant activity. A diversity of carotenoids was identified in these extracts using HPLC-PDA-MS/MS. Both extracts contained predominantly all-trans-lutein as the main component within this class. In order to investigate antioxidant properties, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) techniques were used. The (3–4,5 dimethylthiazol-2, 5 diphenyl tetrazolium bromide) (MTT) and Crystal Violet assays assessed cellular cytotoxicity. Assessments of presence of reactive species were carried out following exposure of oral squamous cell carcinoma cell line (SCC-4) to various aqueous extracts of M piperita or A vulgaris utilizing dichlorofluorescein diacetate (DCFH-DA) and nitric oxide (NO) assays. Exposure to these extracts induced severe cytotoxic effects, which led to investigation of the biochemical and molecular mechanisms underlying this observed effect. Data demonstrated that both solutions induced oxidative stress and DNA damage, especially at higher concentrations using agarose gel subjected to electrophoresis. It is known that exposure to excess amounts of antioxidants results in a prooxidant effect which is beneficial in cancer therapy. Further, the extracts were found to reduce viability of SCC-4 in culture, indicating that this antitumoral activity may be of therapeutic importance and requires further study.

Evaluation of the effects of simulated hypoxia by CoCl2 on radioresistance and change of hypoxia-inducible factors in human glioblastoma U87 tumor cell line

PurposeGlioblastoma (GBM) is considered the most common and lethal type of brain tumor with a poor prognosis. GBM treatment has challenges due to its aggressive nature, which often causes treatment failure and recurrence. Hypoxia is one of the characteristics of glioblastoma tumors that contribute to radioresistance and malignant phenotypes of GBM. In this study, we aimed to determine the effects of hypoxia on the radiosensitivity of U87 GBM cells by the hypoxia-mimicking model. MethodsFollowing the treatment of cells with different concentrations of CoCl2, an MTT assay was used to evaluate the cytotoxicity of CoCl2. To understand the effects of Ionizing radiation on CoCl2-treated groups, cells were exposed to irradiation after pretreating with 100 μM CoCl2, and a clonogenic survival assay was performed to determine the radiosensitivity of U87 cells. Also, the intracellular Reactive oxygen level was measured by 2′,7′–dichlorofluorescein diacetate (DCFDA) probe staining. Additionally, the expression of hypoxia-associated genes, including HIF-1α, HIF-2α, and their target genes (GLUT-1), was monitored by reverse transcription polymerase chain reaction (RT-PCR). ResultsOur study revealed that the cell viability of CoCl2-treated cells was decreased in a concentration-dependent manner. Also, CoCl2 did not cause any cytotoxicity on U87 cells at a concentration of 100 μM after treatment for 24 h. Colony formation assay showed that CoCl2 pretreatment induced radioresistance of tumor cells compared to non-treated cells. Also, CoCl2 can protect cells against irradiation by the clearance of ROS. Moreover, Real-time results showed that the mRNA expression of HIF-1α and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2α mRNA did not change significantly in hypoxia or irradiation alone conditions, but it increased significantly only in hypoxia + irradiation conditions. ConclusionTaken together, our results indicated that simulating hypoxia by CoCl2 can effectively increase hypoxia-associated genes, specially HIF-1α and GLUT-1, but did not affect HIF-2α gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.