Decitabine combined with cold atmospheric plasma induces pyroptosis via the ROS/Caspase-3/GSDME signaling pathway in Ovcar5 cells.

BACKGROUND: High methylation of the DFNA5 gene results in the absence of GSDME, a key protein that mediates pyroptosis, while decitabine demethylates the DFNA5 gene, resulting in high expression of the GSDME protein. Cold atmospheric plasma (CAP) is a novel anti-cancer method that induces tumor cell death. METHODS: The pyroptosis induced by decitabine in combination with CAP in Ovcar5 cells was evaluated. In particular, mitochondrial membrane potential was estimated by JC-1 staining, dehydrogenase (LDH) release was assessed by ELISA, Annexin V/PI staining was detected by flow cytometry, the cell cycle changes were evaluated using PI staining followed by detection by flow cytometry, and Caspase-9 cleavage, Caspase-3 cleavage and GSDME expression were evaluated by western blot. RESULTS: Decitabine resulted in high expression of the GSDME in Ovcar5 in a concentration-dependent manner and increased tumor cell sensitivity to CAP. CAP induced mitochondrial damage and activated the Caspase-9/Caspase-3 pathway. Therefore, decitabine combined with CAP induced Ovcar5 cell pyroptosis through Caspase-3 mediated GSDME cleavage. Reactive oxygen species (ROS) generated by CAP treatment played an important role in the CAP/decitabine combination-induced production of ROS, activation of Caspase-9/Caspase-3, GSDME cleavage and pyroptosis that ROS scavenger NAC inhibited all these processes. CONCLUSIONS: CAP combined with decitabine induced Caspase-3 activation, which cleaved decitabine-upregulated GSDME and ediated pyroptosis.

Proteomics analysis of resistance mechanism of Trichoderma harzianum under U(VI) stress.

Trichoderma harzianum has a certain resistance to Hexavalent Uranium (U(VI)), but its resistance mechanism is unknown. Based on proteomics sequencing using DIA mode, differentially expressed proteins (DEPs) of Trichoderma harzianum under U(VI) stress were identified. GO enrichment, KEGG annotation analysis and DEPs annotation were performed. The results showed that 8 DEPs, 8 DEPs and 15 DEPs were obtained in the low-dose, medium-dose and high-dose groups, respectively. The functional classification of GO demonstrated that DEPs were associated with 17 molecular functions, 5 biological processes, and 5 cellular components. Furthermore, DEPs were enriched in transport and catabolism, energy metabolism, translation, and signal transduction. These findings showed that Trichoderma harzianum was significantly changed in protein expression and signaling pathway after U(VI) exposure. Therefore, these results have provided Trichoderma harzianum with a theoretical background that can be applied to environmental cleanup.

Transcriptome analysis of damage mechanism of Candida utilis under U(VI) stress.

Marine radioactive pollution has a great impact on Marine microorganisms, but the damage mechanism by hexavalent uranium (U(VI)) exposure has been rarely known. In this study, Candida utilis (C. utilis) were exposed to U(VI) for 50, 100 and 150 mg/L, and then morphologic change and RNA-Seq in C. utilis were determined. U(VI) exposure significantly induced the changes of morphological characteristics of C. utilis. There were 39 DEGs in the 50 mg/L treated group, including 30 up-regulated genes and 9 down-regulated genes. There were 196 DEGs, 31 up-regulated and 165 down-regulated in the 100 mg/L treated group. The 150 mg/L treated group had 272 DEGs, 74 up-regulated and 198 down-regulated, compared with the control group. The results showed that the number of DEGs increased dose-dependently with U(VI) treatment. The results of this study provide a theoretical basis for the mechanism of radioactive wastewater damage to Marine microorganisms.

U(VI) exposure induces apoptosis and pyroptosis in RAW264.7 cells.

U(VI) pollution has already led to serious harm to the environment and human health with the increase of human activities. The viability of RAW264.7 cells was assessed under various U(VI) concentration stress for 24 and 48 h. The reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD) activities of RAW264.7 cells under U(VI) stress were measured. The results showed that U(VI) decreased cell activity, induced intracellular ROS production, abnormal MMP, and increased SOD activity. The flow cytometry with Annexin-V/PI double labeling demonstrated that the rate of late apoptosis increased with the increase of U(VI) concentration, resulting in decreased Bcl-2 expression and increased Bax expression. The morphology of RAW264.7 cells dramatically changed after 48 h U(VI) exposure, including the evident bubble phenomenon. Besides, U(VI) also increased the proportion of LDH releases and increased GSDMD, and Ras, p38, JNK, and ERK1/2 protein expression, which indicated that the MAPK pathway was also involved. Therefore, U(VI) ultimately led to apoptosis and pyroptosis in RAW264.7 cells. This study offered convincing proof of U(VI) immunotoxicity and established the theoretical framework for further fundamental studies on U(VI) toxicity.

Low-Temperature Plasma-Activated Medium Inhibits the Migration of Non-Small Cell Lung Cancer Cells via the Wnt/ß-Catenin Pathway.

This study explored the molecular mechanism of the plasma activation medium (PAM) inhibiting the migration ability of NSCLC (non-small cell lung cancer) cells. The effect of PAM incubation on the cell viability of NSCLC was detected through a cell viability experiment. Transwell cells and microfluidic chips were used to investigate the effects of PAM on the migration capacity of NSCLC cells, and the latter was used for the first time to observe the changes in the migration capacity of cancer cells treated with PAM. Moreover, the molecular mechanisms of PAM affecting the migration ability of NSCLC cells were investigated through intracellular and extracellular ROS detection, mitochondrial membrane potential, and Western blot experiments. The results showed that after long-term treatment with PAM, the high level of ROS produced by PAM reduced the level of the mitochondrial membrane potential of cells and blocked the cell division cycle in the G2/M phase. At the same time, the EMT process was reversed by inhibiting the Wnt/ß-catenin signaling pathway. These results suggested that the high ROS levels generated by the PAM treatment reversed the EMT process by inhibiting the WNT/ß-catenin pathway in NSCLC cells and thus inhibited the migration of NSCLC cells. Therefore, these results provide good theoretical support for the clinical treatment of NSCLC with PAM.

Transcriptome Analysis of the Immortal Human Keratinocyte HaCaT Cell Line Damaged by Tritiated Water.

Radioactive elements, such as tritium, have been released into the ocean in large quantities as a result of the reactor leakage accident. In this study, an MTT assay demonstrated that the viability of HacaT cells decreased after tritiated water treatment. Bioinformatics analysis was used to analyze gene changes in the HacaT cells. The sequencing results showed 267 significantly differentially expressed genes (DEGs), and GO enrichment analysis showed that the DEGs were mainly divided into three parts. The KEGG pathway analysis showed that the up-regulated DEGs were involved in Wnt and other pathways, while the down-regulated DEGs were involved in Jak-STAT and others. A Western blot assay was used to verify the parts of the sequencing results. This study was the first to explore the mechanism of tritiated water on HacaT cells using Transcriptome analysis. The results will provide a theoretical basis for the study of tritiated water hazard mechanisms.