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Free Radical Biology & Medicine Nov 2023Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the fourth leading cause of cancer-related death worldwide. Advanced or metastatic HCC is currently...
Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the fourth leading cause of cancer-related death worldwide. Advanced or metastatic HCC is currently managed using systemic drug therapy with unsatisfactory patient survival. Cold atmospheric plasma has emerged as a promising, physicochemical, and broad-spectrum oncotherapy. In this preclinical study, we investigated the anti-neoplastic functions and mechanism of piezoelectric direct discharge technology-based CAP, Piezo-CAP, on HCC in vitro and in vivo. Various HCC cells lines, such as SMMC7721, HepG2 and LM3, were used as in vitro cancer model for the phenotypic and mechanistic studies. Specifically, the cell counting Kit-8 and colony formation assay, flow cytometry, Transwell assay, Western blot, reactive oxygen species (ROS) assay, and glutathione to oxidized glutathione ratio (GSH/GSSG) assay were used to demonstrate plasma-induced changes in HCC cell proliferation, cell cycle progression, migration and invasion, epithelial-to-mesenchymal transition, intracellular ROS, and antioxidant capacity, respectively. In addition, the Acridine orange and ethidium bromide (AO/EB) staining and transmission electron microscopy were performed for cellular and subcellular assessment of HCC cell apoptosis. The Ad-mCherry-RFP-LC3B fluorescent double-labeled lentiviral system was used to detect autophagic flux. On the other hand, RNA-sequencing, quantitative real-time PCR, and Western blot were used to demonstrate plasma-induced metabolic and molecular disruption of tumor glycolysis and oncogenic proliferation, respectively. In vivo experiments using a human cell-line-derived xenograft model and immunohistochemistry (IHC) were utilized to investigate the mechanism. Piezo-CAP exerted anti-neoplastic functions through inhibiting cell proliferation, migration and invasion, and promote cell apoptosis and autophagy. Treatment of Piezo-CAP could suppress proliferation and induce autophagy of HCC cells through simultaneously disrupts cancer survival pathways of redox deregulation, glycolytic pathway, and PI3K/AKT/mTOR/HIF1α pathway signaling. Moreover, upon translation of these in vitro results into the tissue level, Piezo-CAP significantly suppressed in situ tumor growth. These findings collectively suggest that Piezo-CAP-induced apoptosis and autophagy of HCC cells though a multitargeted blockade of major cancer survival pathways of deregulated redox balance, glycolysis, and PI3K/AKT/mTOR/HIF-1α signaling.
Topics: Humans; Carcinoma, Hepatocellular; Proto-Oncogene Proteins c-akt; Liver Neoplasms; Phosphatidylinositol 3-Kinases; Reactive Oxygen Species; Cell Line, Tumor; TOR Serine-Threonine Kinases; Apoptosis; Cell Proliferation; Autophagy; Glycolysis
PubMed: 37543168
DOI: 10.1016/j.freeradbiomed.2023.07.036 -
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 and Protocols Aug 2023Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of...
Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.
PubMed: 37623923
DOI: 10.3390/mps6040072 -
Nigerian Journal of Clinical Practice Sep 2023Neutrophils continuously migrate into the oral cavity from various sources like gingival crevicular fluid and saliva both in health and in inflammation. The migration of...
BACKGROUND
Neutrophils continuously migrate into the oral cavity from various sources like gingival crevicular fluid and saliva both in health and in inflammation. The migration of the neutrophils into the various tissues and into the oral cavity occurs when the host microbial interplay tips the balance favoring the initiation of the inflammatory and immune reactions which depending on the amount of the microbial load results in the development of acute and chronic infections in the susceptible host.
AIM
The present study was designed to quantify and compare the oral salivary neutrophil levels in patients with gingivitis and chronic and aggressive periodontitis as well as in healthy controls, before and after scaling and root planing (SRP) and to compare the difference within the selected study groups.
MATERIALS AND METHODS
Forty subjects were classified into four groups, that is, healthy controls, gingivitis, and chronic and aggressive periodontitis. Oral rinse samples were collected using Hank's balanced salt solution from each patient before and after phase I periodontal therapy. Cells in the rinse samples were stained with Acridine orange, and neutrophil counts were carried out using a fluorescence microscope and a hemocytometer.
RESULTS
Baseline oral salivary neutrophil levels were maximum in the chronic periodontitis group followed by the aggressive group and then the gingivitis group. Oral salivary neutrophil levels also positively correlated to probing pocket depth, plaque index, calculus index, and gingival index in all four study groups. Maximum reduction in the oral salivary neutrophil levels after phase I periodontal therapy was seen in the gingivitis group.
CONCLUSION
From our study, we conclude that the oral salivary neutrophil levels decreased significantly after SRP. Estimation of changes in the oral salivary neutrophil levels has the potential to aid in monitoring treatment outcomes. Thus, it suggests that it could be used as a simple, noninvasive laboratory technique to monitor the periodontal status and disease progression.
Topics: Humans; Neutrophils; Periodontal Pocket; Aggressive Periodontitis; Chronic Periodontitis; Gingivitis
PubMed: 37794540
DOI: 10.4103/njcp.njcp_3_23 -
International Journal of Molecular... Dec 2023Piperlongumine (PL) is an amide alkaloid with diverse pharmacological effects against cancer, bronchitis and asthma; however, research on its efficacy against melanoma...
Piperlongumine (PL) is an amide alkaloid with diverse pharmacological effects against cancer, bronchitis and asthma; however, research on its efficacy against melanoma is lacking. The present study investigated the anticancer effects of PL on A375SM and A375P human melanoma cells. PL decreased the survival rate of A375SM and A375P cells, as shown by MTT assay, increase of apoptotic cells by DAPI staining. And PL induced apoptosis by decreasing the expression of the anti‑apoptotic protein Bcl‑2 and increasing that of the pro‑apoptotic proteins cleaved‑PARP and Bax. PL also induced apoptosis in A375SM and A375P cells via the MAPK pathway, increasing expression of the MAPK pathway proteins, phosphorylated‑(p‑ERK), p‑JNK p‑p38. These proteins were confirmed by western blot. In addition, A375SM and A375P cells treated with PL showed an increased number of acidic vesicular organelles by acridine orange staining. Also, autophagy induced by the expression of 1A/1B‑light chain 3, Beclin 1and mTOR was investigated through western blot. When PL was applied following treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine, autophagy exhibited a cytoprotective effect against apoptosis in MTT assay. Pretreatment of A375P cells with the ERK inhibitor PD98059 and the JNK inhibitor SP600125 followed by treatment with PL confirmed that apoptosis and autophagy were mediated via the MAPK/ERK pathway by western blot. In summary, the present study provided empirical evidence supporting the anticancer effects of PL on human melanoma cells and indicated the potential of PL as a treatment for melanoma.
Topics: Humans; Cell Line, Tumor; Apoptosis; Melanoma; Apoptosis Regulatory Proteins; Autophagy
PubMed: 37830157
DOI: 10.3892/ijmm.2023.5318 -
Autophagy Feb 2024AO: acridine orange; ATM: ATM serine/threonine kinase; CHEK1: checkpoint kinase 1; CHEK2: checkpoint kinase 2; CI: combination index; DMSO: dimethyl sulfoxide; DSBs:...
AO: acridine orange; ATM: ATM serine/threonine kinase; CHEK1: checkpoint kinase 1; CHEK2: checkpoint kinase 2; CI: combination index; DMSO: dimethyl sulfoxide; DSBs: double-strand breaks; GBM: glioblastoma; HR: homologous recombination; H2AX: H2A.X variant histone; IHC: immunohistochemistry; LAPTM4B: lysosomal protein transmembrane 4 beta; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PARP: poly(ADP-ribose) polymerase; RAD51: RAD51 recombinase; SQSTM1: sequestosome 1; SSBs: single-strand breaks; RNF168: ring finger protein 168; XPO1: exportin 1.
Topics: Humans; Sequestosome-1 Protein; Glioblastoma; Autophagy; Phthalazines; Proteins; Poly(ADP-ribose) Polymerases; Membrane Proteins; Oncogene Proteins; Ubiquitin-Protein Ligases; Piperazines
PubMed: 37712615
DOI: 10.1080/15548627.2023.2252301 -
Journal of Oral and Maxillofacial... 2023Sex determination in forensic medicine is considered one of the first and foremost steps in personal identification. The need for identifying the exact sex of the...
UNLABELLED
Sex determination in forensic medicine is considered one of the first and foremost steps in personal identification. The need for identifying the exact sex of the individual arises when deciding whether a person can exercise certain civil rights reserved for one particular sex, for competing in sex-specific athletic and sports events, legitimacy, divorce, paternity disputes and also to some criminal offenses. Nuclear sexing by Barr body examination can be done using buccal smears to establish the sex of the individual when routine methods fail to disclose the exact gender of the individual.
AIM
To determine and compare the Barr bodies present in exfoliated buccal epithelial cells in males, females and transgender populations using light and fluorescence microscopy.
MATERIALS AND METHODS
A total of 90 patients were recruited for the study. Group I consisted of 30 female patients. Group II consisted of 30 male patients and group III consisted of 30 transgender patients. The buccal mucosa was then scraped using a wooden spatula and the cells obtained were fixed in 95% ethanol. Two smears per individual were made and stained. One smear was stained with papanicolaou (PAP) stain and the other with Acridine orange and viewed under light microscopy and fluorescent microscopy, respectively.
RESULTS
When PAP stained slides were examined, the percentage of Barr-bodies in females ranged from 3% to 5% and in males it was 0% and in transgenders, it ranged from 0% to 5%. In Acridine orange stained smears, the percentage of Barr bodies in females ranged from 1% to 3% and in males it was 0% and in transgenders, it was 0%. Kruskal-Wallis test to study the relation of Barr body percentage in females, males and transgender subjects demonstrated significant differences between the groups ( < 0.001). Wilcoxon signed rank test was done for pairwise comparison, which showed that the distribution of percentage of positive cells in females are statistically significant from males and transgenders ( < 0. 001).
CONCLUSION
Nuclear sexing using Barr bodies offers a simple yet effective method for determining the sex of transgender patients which could help them in understanding their gender identity better and diagnose any underlying chromosomal aberration.
PubMed: 38304528
DOI: 10.4103/jomfp.jomfp_342_23 -
Antibiotics (Basel, Switzerland) Jul 2023The development and implementation of diagnostic methods that allow rapid assessment of antibiotic activity against pathogenic microorganisms is an important step...
The development and implementation of diagnostic methods that allow rapid assessment of antibiotic activity against pathogenic microorganisms is an important step towards antibiotic therapy optimization and increase in the likelihood of successful treatment outcome. To determine whether fluorescence microscopy with acridine orange can be used for rapid assessment (≤8 h) of the meropenem activity against , six isolates including three OXA-48-carbapenemase-producers were exposed to meropenem at different levels of its concentration (0.5 × MIC, 1 × MIC, 8 or 16 µg/mL) and the changes in the viable counts within 24 h were evaluated using fluorescence microscopy and a control culture method. The approach was to capture the regrowth of bacteria as early as possible. Within the first 8 h fluorescence microscopy allowed to categorize 5 out of 6 strains by their meropenem susceptibility (based on the MIC breakpoint of 8 mg/L), but meropenem activity against three isolates, two of which were OXA-48-producers, could not be accurately determined at 8 h. The method proposed in our study requires improvement in terms of accelerating the bacterial growth and regrowth for early meropenem MIC determination. Volume-dependent elevation in meropenem MICs against OXA-48-producers was found and this phenomenon should be studied further.
PubMed: 37508266
DOI: 10.3390/antibiotics12071170 -
Bioinorganic Chemistry and Applications 2023Multiple chemodrugs with nanotechnology have proven to be an effective cancer treatment technique. When taken combined, cabazitaxel (CTX) and cisplatin (PT) have more...
Multiple chemodrugs with nanotechnology have proven to be an effective cancer treatment technique. When taken combined, cabazitaxel (CTX) and cisplatin (PT) have more excellent cytotoxic effects than drugs used alone in the chemotherapy of several different cancers. However, several severe side effects are associated with using these chemotherapy drugs in cancer patients. Gold nanomaterials (AuNMs) are promising as drug carriers because of their small diameter, easy surface modifications, good biocompatibility, and strong cell penetration. This work aimed to determine the CTX and PT encapsulated with AuNMs against human glioma U87 cancer cells. The fabrication of the AuNMs achieved a negative surface charge, polydispersity index, and the mean sizes. The combined cytotoxic effect of CTX and PT bound to AuNMs was greater than that of either drug alone when tested on U87 cells. The half inhibitory concentration (IC) values for free PT were 54.7 g/mL (at 24 h) and 4.8 g g/mL (at 72 h). Results acquired from the MTT assay show cell growth decreases time- and concentration-dependent AuNMs, free CTX, free PT, and AuNMs@CTX/PT-induced cytotoxicity and, ultimately, the cell death of U87 cells via apoptosis. The biochemical apoptosis staining techniques investigated the cells' morphological changes of the cells (acridine orange and ethidium bromide (AO-EB) and nuclear staining (DAPI) techniques). The AO-EB and nuclear staining results reveal that the NPs effectively killed cancer cells. Furthermore, the flow cytometry analysis examined the mode of cell death. Therefore, AuNMs@CTX/PT has excellent potential in the cancer therapy of different cancer cells.
PubMed: 37920234
DOI: 10.1155/2023/8892099