OBJECTIVE: To explore the effect and mechanism of action of bufalin in triple-negative breast cancer (TNBC) drug-resistant cell lines. METHODS: The normal human mammary epithelial cell line, TNBC cell line, TNBC adriamycin-resistant cell line, and TNBC docetaxel-resistant cell line were treated with different doses of bufalin (0-1,000 nmol/L) at different time points (0-72 h). Propidium iodide staining, AV-FITC/PI double staining, Hoechst 33342/PI double staining and transmission electron microscopy (TEM) were used to evaluate the death patterns of the cell lines. RESULTS: Bufalin killed the TNBC cell line and its drug-resistant cell lines in a dose/time-dependent manner (all P<0.01). After treatment with bufalin for 24 h, the adriamycin-resistant cell line showed a co-existing pattern of necroptosis and apoptosis. However, at 48 h, necroptosis was the main manifestation. After treatment with bufalin, the expressions of tumor necrosis factor α, phospho-tumor necrosis factor receptor 1, phospho-receptor interacting protein 1 and c-caspase 3 increased (all P<0.01), the killing effect of bufalin could be mostly inhibited by NEC-1, and by z-VAD-fmk (both P<0.01). Besides, the intracellular reactive oxygen species (ROS) levels increased considerably (P<0.01), the antioxidant N-acetyl cysteine or Nec-1 could inhibit the increase of ROS level and the killing effect of bufalin (all P<0.01). The adriamycin-resistant cell line exhibited necroptosis characteristic after 48 h of bufalin treatment under TEM. CONCLUSIONS Bufalin could induce necroptosis through RIP1/ROS-mediated pathway to kill the drug-resistant TNBC cell lines. This finding provides critical experimental data and theoretical basis for the clinical application of bufalin to overcome the difficulties in the treatment of TNBC.
Antioxidants/pharmacology* ; Apoptosis ; Bufanolides ; Caspase 3/metabolism* ; Cell Line ; Cell Line, Tumor ; Cysteine/pharmacology* ; Docetaxel/pharmacology* ; Doxorubicin/pharmacology* ; Fluorescein-5-isothiocyanate/pharmacology* ; Humans ; Necroptosis ; Propidium/pharmacology* ; Reactive Oxygen Species/metabolism* ; Receptors, Tumor Necrosis Factor ; Triple Negative Breast Neoplasms/drug therapy* ; Tumor Necrosis Factor-alpha/pharmacology*

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
A549 Cells ; Adenosine Diphosphate Ribose/pharmacology* ; Apoptosis ; Apoptosis Regulatory Proteins ; Arsenic ; Arsenites ; Cacodylic Acid/pharmacology* ; Caspase 3 ; Caspases/pharmacology* ; Cytochromes c/pharmacology* ; Humans ; Mitochondrial Proteins/pharmacology* ; Poisons ; Propidium/pharmacology* ; RNA, Messenger ; Sincalide/pharmacology* ; Sodium Compounds ; bcl-Associated Death Protein/metabolism*

BACKGROUND: Fluoranthene (FR) is a common environmental pollutant that exists in a complex mixture with other polycyclic aromatic hydrocarbons (PAHs). We identified biomarkers for monitoring FR exposure and investigated the rescue effect of FR-induced cellular toxicity via aryl hydrocarbon receptor (AHR) antagonist activity in bone marrow derived mesenchymal stem cells (BM-MSCs).METHODS: Morphological changes, viability, and rescue effects of an AHR antagonist (CH223191) were examined in BM-MSCs after exposure to FR. Cytotoxic effects were assayed using the tetrazolium-based colorimetric assay. Apoptosis was measured by annexin V and propidium iodide dye-based flowcytometry assay, mitochondrial membrane potential assay, and nuclear DNA fragmentation assay. Molecular signaling pathways of apoptosis and autophagy were investigated using immunoblotting. Proteomics were performed in order to reveal the spectra of cellular damage and identify biomarkers for FR exposure.RESULTS: Exposing BM-MSCs to FR (IC₅₀=50 µM) induced cell death and morphological changes, while the AHR antagonist showed rescue effects. Autophagy was activated and mitochondrial membrane potential was decreased. Proteomic analysis identified 48 deregulated proteins (26 upregulated and 22 downregulated). Among them, annexin A6, pyruvate kinase, UDP-glucose dehydrogenase, and phospholipase A2 could be potential biomarkers for FR exposure.CONCLUSION: The exposure of BM-MSCs to FR induced remarkable alterations in cellular biology and the proteome, allowing for identification of novel biomarkers for FR exposure. Furthermore, AHR antagonists might be able to prevent cellular damage due to FR exposure.
Annexin A5 ; Annexin A6 ; Apoptosis ; Autophagy ; Biomarkers ; Bone Marrow ; Cell Death ; DNA Fragmentation ; Immunoblotting ; Membrane Potential, Mitochondrial ; Mesenchymal Stromal Cells ; Oxidoreductases ; Phospholipases A2 ; Polycyclic Hydrocarbons, Aromatic ; Propidium ; Proteome ; Proteomics ; Pyruvate Kinase ; Receptors, Aryl Hydrocarbon

Contaminated liver fluke egg in the environment has led to the high prevalence of human opisthorchiasis associated with cholangiocarcinoma in Southeast Asia. To find the effective lessening methods of Opisthorchis viverrini eggs in the contaminated environment, we investigated the temperature conditions for killing of these trematode eggs in vitro. Numerous O. viverrini eggs were obtained in the proximal part of uteri of adult worms from experimental hamsters. Mature eggs with miracidium were allocated by experimental groups (2 control: positive and negative and 4 treatment: 50, 60, 70, and 80°C) with 0.85% saline, and treated by the experimental plan. Eggs in each experimental groups were observed under the confocal microscope after stain with Propidium Iodide (PI) to evaluate the effect of temperatures. Eggs in 70 and 80°C groups were all killed after over 10 min heated. Majority of eggs in 60°C (10, 15, and 30 min heated), 70 and 80°C (5 min heated) groups were inactivated. However in 50°C group, below half of eggs were to be killed in all time lapse (10, 15 and 30 min). In order to prevent O. viverrini infection and cholangiocarcinoma, direct treatment of sewage by heating at 70 or 80°C at least 10 min is essential. Therefore, treatment of O. viverrini eggs at a high temperature is a potential method for controlling egg contamination in sewage.
Adult ; Animals ; Asia, Southeastern ; Cholangiocarcinoma ; Cricetinae ; Eggs ; Fasciola hepatica ; Heating ; Homicide ; Hot Temperature ; Humans ; In Vitro Techniques ; Methods ; Opisthorchiasis ; Opisthorchis ; Ovum ; Prevalence ; Propidium ; Sewage ; Uterus

BACKGROUND: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. METHODS: Mouse AE II cell line MLE-12 were exposed to 1–10 µg/mL BLM and 0.01–100 µM baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. RESULTS: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor α, and transforming growth factor β1. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. CONCLUSION: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.
Animals ; Apoptosis ; Bisbenzimidazole ; Bleomycin ; Cell Cycle Checkpoints ; Cell Cycle ; Cell Line ; Cell Survival ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Gene Expression ; Genes, vif ; Humans ; Idiopathic Pulmonary Fibrosis ; Interleukin-6 ; Lung ; Mice ; Propidium ; RNA, Messenger ; Transforming Growth Factors ; Tumor Necrosis Factor-alpha

BACKGROUND: Respiratory viral infections are the leading cause of asthma exacerbations. Eosinophil activation results in the formation of eosinophil extracellular traps (EETs), which release web-like structures of DNA and proteins that bind, disarm and extracellularly kill pathogens. OBJECTIVE: We investigated whether the respiratory syncytial virus (RSV) in vitro could induce EETs in bronchoalveolar lavage fluid eosinophils in a murine model of asthma. METHODS: BALB/cJ mice (6–8 weeks old) were sensitized with 2 subcutaneous injections of ovalbumin (20 μg) on days 0 and 7, followed by three intranasal challenges with ovalbumin (100 μg) on days 14, 15, and 16 of the protocol. The control group received Dulbecco's phosphate-buffered saline. Bronchoalveolar lavage fluid eosinophils of ovalbumin group or control group were stimulated with RSV (103 PFU/mL) in vitro for 3 hours. After that, culture supernatant was collected to perform the analyses proposed in this study. RESULTS: We verified an increase in extracellular DNA concentration in bronchoalveolar lavage fluid eosinophils from ovalbumin group stimulated with RSV (10³ PFU/mL) in vitro, which was confirmed by confocal microscopy. We demonstrated that most cells are negative for annexin V and propidium iodide in all groups evaluated. Also, RSV in vitro decreased interferon-ɣ in culture supernatant when compared to the ovalbumin group. CONCLUSION: In this study, we demonstrated for the first time that RSV in vitro induces EETs formation in eosinophils from asthmatic mice.
Animals ; Annexin A5 ; Asthma ; Bronchoalveolar Lavage Fluid ; DNA ; Eosinophil Peroxidase ; Eosinophils ; Extracellular Traps ; In Vitro Techniques ; Inflammation ; Injections, Subcutaneous ; Mice ; Microscopy, Confocal ; Ovalbumin ; Propidium ; Respiratory Syncytial Viruses

Wet wipes are being increasingly used because of their convenience. Particularly, oral wet wipes are useful for regular cleaning of a baby's mouth after birth. Therefore, the consumption of oral wet wipes has increased over the past few years and a variety of products are commercially available. However, product information on safety is not sufficiently provided and still raises doubts regarding adverse effects. To confirm the safety of wet wipes as an oral hygiene item and provide information for their use, we investigated the cytotoxicity of oral wet wipes and verified the underlying mechanism. The anti-bacterial effect of oral wet wipes was analyzed using the disk diffusion method. The cytotoxic effects of oral wet wipes were observed based on morphological changes using microscopy and determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in gingival epithelial cells and gingival fibroblasts. Evaluation of apoptosis by oral wet wipes was explored using propidium iodide flow cytometric analysis and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. Apoptosis-related molecules were also analyzed using western blotting. Five types of oral wet wipes were tested, and two products from Fisher-Price and Dr. Kennedy revealed strong cytotoxic effects on gingiva epithelial cells and gingiva fibroblasts, although they also showed intense anti-bacterial effects on oral bacteria. Cell cycle arrest in the G2/M phase and apoptosis were observed based on treatment of extracts from Fisher-Price and Dr. KENNEDY. Relatively high TUNEL levels, reduction of proliferating cell nuclear antigen and cyclin-dependent kinase 4 expression, and fragmentation of poly (ADP-ribose) polymerase were also elucidated. These results suggest that commercial oral wet wipes could exert cytotoxic influences on oral tissue, although there are anti-bacterial effects, and careful attention is required, especially for infants and toddlers.
Apoptosis ; Bacteria ; Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Survival ; Cyclin-Dependent Kinase 4 ; Deoxyuridine ; Diffusion ; DNA Nucleotidylexotransferase ; Epithelial Cells ; Fibroblasts ; Gingiva ; Humans ; In Situ Nick-End Labeling ; Infant ; Methods ; Microscopy ; Mouth ; Oral Hygiene ; Parturition ; Proliferating Cell Nuclear Antigen ; Propidium

PURPOSE: Dieckol, a phlorotannin compound isolated from Ecklonia cava, has been reported to have antioxidant, antiviral, anti-inflammatory, and anticancer properties. The purpose of this study was to investigate its anticancer effects on human breast cancer cell lines. METHODS: In this study, the viability of two human breast cancer cell lines SK-BR-3 and MCF-7 was investigated after dieckol treatment using a WST-1 assay. Apoptosis and cell cycle distribution were assayed via Annexin V-fluorescein isothiocyanate and propidium iodide staining followed by flow cytometric analysis. Immunoblotting analysis was also performed using Bax/Bcl-2 to determine whether the dieckol-induced apoptosis was mediated by the intrinsic apoptotic pathway. RESULTS: In a dose dependent manner, dieckol reduced the number of viable cells and increased the number of apoptotic cells. The effect of dieckol on the cell cycle distribution was analyzed using flow cytometry. Dieckol treatment significantly increased the percentage of MCF-7 and SK-BR-3 in the G2/M phase. Immunoblot analysis revealed that 24 hours of dieckol exposure increased the Bax/Bcl-2 ratio. CONCLUSION: Dieckol induced cytotoxicity in MCF-7 and SK-BR-3 human breast cancer cells inducing apoptosis and cell cycle arrest. Therefore, it is suggested that dieckol may be a potential therapeutic agent for breast cancer.
Apoptosis* ; Breast Neoplasms* ; Breast* ; Cell Cycle Checkpoints* ; Cell Cycle* ; Cell Line* ; Flow Cytometry ; Humans* ; Immunoblotting ; Propidium

OBJECTIVE: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells. METHODS: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair. RESULTS: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. CONCLUSIONS The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
Apoptosis ; Caspases/metabolism* ; Cell Proliferation ; Cell Survival/drug effects* ; Doxorubicin/pharmacology* ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Membrane Potential, Mitochondrial ; Oligonucleotide Array Sequence Analysis ; Oxidative Stress/drug effects* ; Phosphorylation ; Propidium/chemistry* ; RNA, Small Interfering/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Rhodamines/chemistry* ; Signal Transduction/drug effects* ; Transcriptome ; Tumor Suppressor Protein p53/metabolism* ; Vitreoretinopathy, Proliferative/drug therapy*

PURPOSE: We previously found that the histone methyltransferase suppressor of variegation, enhancer of zeste, trithorax and myeloid-nervy-deformed epidermal autoregulatory factor-1 domain-containing protein 3 (SMYD3) is a potential independent predictive factor or prognostic factor for overall survival in gastric cancer patients, but its roles seem to differ from those in other cancers. Therefore, in this study, the detailed functions of SMYD3 in cell proliferation and migration in gastric cancer were examined. MATERIALS AND METHODS: SMYD3 was overexpressed or suppressed by transfection with an expression plasmid or siRNA, and a wound healing migration assay and Transwell assay were performed to detect the migration and invasion ability of gastric cancer cells. Additionally, an MTT assay and clonogenic assay were performed to evaluate cell proliferation, and a cell cycle analysis was performed by propidium iodide staining. Furthermore, the expression of genes implicated in the ataxia telangiectasia mutated (ATM) pathway and proteins involved in cell cycle regulation were detected by polymerase chain reaction and western blot analyses. RESULTS: Compared with control cells, gastric cancer cells transfected with si-SMYD3 showed lower migration and invasion abilities (P<0.05), and the absence of SMYD3 halted cells in G2/M phase and activated the ATM pathway. Furthermore, the opposite patterns were observed when SMYD3 was elevated in normal gastric cells. CONCLUSIONS: To the best of our knowledge, this study provides the first evidence that the absence of SMYD3 could inhibit the migration, invasion, and proliferation of gastric cancer cells and halt cells in G2/M phase via the ATM-CHK2/p53-Cdc25C pathway. These findings indicated that SMYD3 plays crucial roles in the proliferation, migration, and invasion of gastric cancer cells and may be a useful therapeutic target in human gastric carcinomas.
Ataxia Telangiectasia ; Blotting, Western ; Cell Cycle ; Cell Proliferation ; G2 Phase Cell Cycle Checkpoints ; Histones ; Humans ; Plasmids ; Polymerase Chain Reaction ; Propidium ; RNA, Small Interfering ; Stomach Neoplasms* ; Transfection ; Wound Healing