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Nanomedicine (London, England) Dec 2021To develop a tumor-targeted chemo-photothermal nanomedicine through the functionalization of acridine orange (AO)-loaded gold-core mesoporous silica shell...
To develop a tumor-targeted chemo-photothermal nanomedicine through the functionalization of acridine orange (AO)-loaded gold-core mesoporous silica shell (AuMSS) nanorods with polyethylenimine (PEI) and hyaluronic acid (HA). Functionalization of the AuMSS nanorods was achieved through the chemical linkage of PEI followed by electrostatic adsorption of HA. HA functionalization improved AuMSS' cytocompatibility by decreasing blood hemolysis, and PEI-HA inclusion promoted a controlled and sustained AO release. assays revealed that HA functionalization increased the internalization of nanoparticles by human negroid cervix epithelioid carcinoma cancer (HeLa) cells, and the combinatorial treatment mediated by AuMSS/PEI/HA_AO nanorods presented an enhanced effect, with >95% of cellular death. AuMSS/PEI/HA_AO formulations can act as tumor-targeted chemo-photothermal nanomedicines for the combinatorial therapy of cervical cancer.
Topics: Acridine Orange; Doxorubicin; Gold; Humans; Hyaluronic Acid; Nanoparticles; Nanotubes; Neoplasms; Polyethyleneimine; Silicon Dioxide
PubMed: 34854343
DOI: 10.2217/nnm-2021-0270 -
Journal of Cell Science Dec 2016Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red...
Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.
Topics: Acids; Acridine Orange; Animals; Autophagy; Cell Size; Cytological Techniques; Flow Cytometry; Fluorescence; HEK293 Cells; Humans; Microscopy, Confocal; Organelles; Rats, Wistar; Spectrometry, Fluorescence
PubMed: 27875278
DOI: 10.1242/jcs.195057 -
Journal of Cellular Biochemistry Sep 2022Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal...
Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy.
Topics: Acridine Orange; Anthraquinones; Apoptosis; Autophagy; Caspase 3; Cathepsin D; Chloroquine; HeLa Cells; Humans; Lysosomes; Neutral Red; Oxides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species
PubMed: 35901236
DOI: 10.1002/jcb.30311 -
Anticancer Research Jun 2024The increasing incidence of renal cell carcinoma (RCC) and its associated bone metastasis pose challenges in surgical interventions, warranting the exploration of novel...
BACKGROUND/AIM
The increasing incidence of renal cell carcinoma (RCC) and its associated bone metastasis pose challenges in surgical interventions, warranting the exploration of novel therapeutic approaches. Therefore, this study aimed to assess the impact of hematogenously administering acridine orange (AO) alone and in combination with zoledronic acid (ZA) on bone metastasis in RCC.
MATERIALS AND METHODS
RENCA cells (1.0×10 cells/10 μl) were directly injected into the right femur of male BALB/c mice. The mice were categorized into four groups based on the applied therapeutic intervention and were euthanized after five weeks. Micro-computed tomography was performed to quantify the extent of periosteal reaction, indicative of bone metastasis, along the entire length of the femur. Tumor weight and volume were measured at euthanization. Hematoxylin and eosin staining was used to examine the extent of tumor development in the bone. Apoptotic cell, osteoclast, and vascular endothelial growth factor (VEGF)-positive cell counts were assessed using TdT-mediated dUTP-biotin nick end labeling, tartrate-resistant acid phosphatase staining, and VEGF staining, respectively.
RESULTS
The periosteal reaction was significantly reduced in the intervention groups compared to the control group (p<0.05). The apoptotic cell numbers in the intervention groups surpassed that in the control group (p<0.05), whereas those of osteoclasts and VEGF-positive cells in the intervention groups were lower than those in the control group (p<0.05).
CONCLUSION
AO hinders bone metastasis progression in RCC, and combination therapy with ZA may be more effective than AO administration alone.
Topics: Zoledronic Acid; Animals; Carcinoma, Renal Cell; Bone Neoplasms; Kidney Neoplasms; Male; Acridine Orange; Mice; Mice, Inbred BALB C; Apoptosis; Cell Line, Tumor; Humans; Vascular Endothelial Growth Factor A; Imidazoles; X-Ray Microtomography; Xenograft Model Antitumor Assays
PubMed: 38821618
DOI: 10.21873/anticanres.17055 -
Heliyon Jul 2023Chemotherapy can often cause a variety of side effects including bone marrow (BM) suppression, termed as myelosuppression. Accordingly, facile and effective management...
Chemotherapy can often cause a variety of side effects including bone marrow (BM) suppression, termed as myelosuppression. Accordingly, facile and effective management of chemotherapy-induced myelosuppression is currently a pivotal task for experimental pathologists and oncologists. Here, we chose to use activated carbon (AC) with an extensive surface area for studying its possible protective effectiveness with respect to BM in doxorubicin (DOX)-treated rats. Spherical AC with an extended surface area up to 4490 m/g was prepared for (p/o) delivery, whereas for intraperitoneal (i/p) delivery we used the powdered form of AC that was derived from the aforementioned spherical AC. During the monthly treatment of animals with AC and DOX these two components were delivered alternately (not in the same day). After treatment, BM cells were isolated from femurs of sacrificed animals, stained with acridine orange (AO) and analyzed by flow cytometry. Regardless of the route of AC delivery (p/o or i/p), apparent myeloprotection with a possible regenerative effect was observed in animals that received DOX, as evidenced by recovery of the populations of total nucleated cells (TNC) and polychromatic (immature) erythrocytes accompanied by a considerable reduction of the number of apoptotic/dead cells among TNC (≤2.0%). Moreover, as a result of AC administrations, there was a significant increase of AO green and far-red fluorescence intensities in the population of TNC, which is suggestive of the ongoing quantitative and conformational changes in DNA and RNA associated with cell recovery and proliferation. Thus, AC preparations under the present experimental conditions can effectively tackle DOX-induced myelosuppression via mechanisms not necessarily associated with adsorptive detoxification.
PubMed: 37539240
DOI: 10.1016/j.heliyon.2023.e18414 -
Methods in Molecular Biology (Clifton,... Mar 2024Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several...
Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown that Leishmania modulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy in Leishmania is inconclusive because of the limited tools available to study the Leishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy in Leishmania major. Transmission electron microscopy (TEM) allowed us to visualize Leishmania autophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments in Leishmania major by detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation in Leishmania parasites that could provide insights into developing improved therapeutic targets against leishmaniasis.
PubMed: 38441724
DOI: 10.1007/7651_2024_517 -
Pakistan Journal of Pharmaceutical... Mar 2023Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations...
Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations and transmitted horizontally to other species of bacteria. The elimination of such resistance plasmid is of great importance to contain dispersal of antibiotics resistance. E. coli strains were identified, screened for the presence of antibiotics resistance by disc diffusion method, and cured by sub-lethal concentrations of Ethidium bromide and Acridine orange. After curing, again antibiotic resistance was determined. Before and after curing, plasmids were extracted by column spin Kit and subjected to 1% agarose gel electrophoresis and antibiotic resistance genes were identified by PCR. The Ethidium bromide was more effective than Acridine orange in eliminating antibiotics resistance and resistance genes bearing plasmids (4, 5, 6, 8, 9, 10 and <10kb). The most frequently eliminated antibiotic resistance was against Imipenem and Meropenem followed by Cefoperazone-sulbactam, Amikacin and cephalosporins in sequence. The loss of antibiotic resistance was associated with the elimination of plasmid-borne antibiotic resistance genes; bla-TEM, bla-SHV, bla-CTX-M, qnrA, qnrB, qnrC and qnrD. Some E. coli strains did not show the removal of antibiotics resistance and plasmids, suggesting the presence of resistance genes on main chromosome and or non-curable plasmids.
Topics: Anti-Bacterial Agents; Escherichia coli; Ethidium; Acridine Orange; Microbial Sensitivity Tests; Plasmids; Drug Resistance, Microbial; beta-Lactamases
PubMed: 37548194
DOI: No ID Found -
Chemical Biology & Drug Design Mar 2020Monoamine oxidase (MAO) is an important drug target as the MAO isoforms play key roles in neurodegenerative disorders such as Alzheimer's disease and Parkinson's...
Monoamine oxidase (MAO) is an important drug target as the MAO isoforms play key roles in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, as well as in neuropsychiatric diseases such as depression. Methylene blue is an inhibitor of MAO-A, while azure B, the major metabolite of methylene blue, and various other structural analogues retain the ability to inhibit MAO-A. Based on this, the present study evaluated 22 dyes, many of which are structurally related to methylene blue, as potential inhibitors of human MAO-A and MAO-B. The results highlighted three dye compounds as good potency competitive and reversible MAO inhibitors, and which exhibit higher MAO inhibition than methylene blue: acridine orange, oxazine 170 and Darrow red. Acridine orange was found to be a MAO-A specific inhibitor (IC = 0.017 μM), whereas oxazine 170 is a MAO-B specific inhibitor (IC = 0.0065 μM). Darrow red was found to be a non-specific MAO inhibitor (MAO-A, IC = 0.059 μM; MAO-B, IC = 0.065 μM). These compounds may be advanced for further testing and preclinical development, or be used as possible lead compounds for the future design of MAO inhibitors.
Topics: Acridine Orange; Anthraquinones; Azure Stains; Coloring Agents; Drug Design; Humans; Methylene Blue; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Neurodegenerative Diseases; Neuroprotective Agents; Oxazines; Structure-Activity Relationship
PubMed: 31834986
DOI: 10.1111/cbdd.13654 -
Animal Biotechnology Dec 2023Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We...
Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man probe for detection and quantification of and simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically -suspected cows of three -endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed and in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of and simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of spp.
Topics: Humans; Female; Cattle; Animals; Theileriasis; Theileria annulata; Cattle Diseases; RNA, Ribosomal; Membrane Proteins; Acridines
PubMed: 36695009
DOI: 10.1080/10495398.2023.2168197 -
European Journal of Obstetrics,... Dec 2021Cryopreservation refers to the cooling of cells and tissues to sub-zero temperatures in order to stop all biologic activity and preserve them for future use. Human sperm...
OBJECTIVE
Cryopreservation refers to the cooling of cells and tissues to sub-zero temperatures in order to stop all biologic activity and preserve them for future use. Human sperm cryopreservation is an important tool for assisted reproductive technology and male fertility preservation. However, cryopreservation significantly reduces the quality of spermatozoa. The antioxidant effects of curcumin on different cells have been widely reported. This study was aimed to evaluate changes in post-thaw viability, morphology, motility, chromatin condensation and DNA integrity in response to the addition of curcumin to human sperm freezing extender.
MATERIALS AND METHODS
Semen of 23 normozoospermic men was collected and each sample was divided into three equal aliquots: Control, DMSO, Curcumin. The samples were analyzed freshly for viability (Eosin Y), morphology (Diff-Quick), motility (following WHO standarts), sperm chromatin packaging (aniline blue) and DNA integrity (acridine orange). The control group remained untreated and was mixed with cryopreservation medium (in-house 1:1). The DMSO group was mixed with cryopreservation medium containing 0.1% DMSO. The curcumin group was mixed with cryopreservation medium containing 10 µM curcumin. Samples stained with Diff-Quick and aniline blue were examined under light microscope, samples stained with Eosin Y were examined under phase-contrast microscope and samples stained with acridine orange were examined under fluorescence microscope. Ten days after cryopreservation, samples were thawed and pre-freeze analyses repeated.
RESULTS
Obtained results showed that cryopreservation significantly (P < 0.001) reduces sperm parameters. In Curcumin group, progressive motility, sperm chromatin condensation and DNA integrity significantly (P < 0.001) increased after the thawing process, as compared with the control and the DMSO group.
CONCLUSION
These results suggest that the addition of curcumin to cryopreservation medium improves post-thaw progressive motility, sperm chromatin condensation and DNA integrity. It seems that curcumin ameliorates detrimental effects of cryopreservation on human spermatozoa. Further research is needed on the use of curcumin and other antioxidant substances in sperm cryopreservation.
Topics: Cryopreservation; Curcumin; Humans; Male; Semen Preservation; Sperm Motility; Spermatozoa
PubMed: 34773879
DOI: 10.1016/j.ejogrb.2021.10.027