ABSTRACT
BACKGROUND: Emerging evidence suggests that pyroptosis, a form of programmed cell death, has been implicated in cancer progression. The involvement of specific proteins in pyroptosis is an area of growing interest. TOM20, an outer mitochondrial membrane protein, has recently garnered attention for its potential role in pyroptosis. Our previous study found that NBT could induce pyroptosis by ROS/JNK pathway in esophageal cancer cells. PURPOSE: This study aims to investigate whether NBT induces pyroptosis and verify whether such effects are involved in up-regulation of TOM20 in esophageal cancer cells. METHODS: The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) was used to analyze the clinical significance of GSDME in esophageal cancer. MTT assay, morphological observation and Western blot were performed to verify the roles of TOM20 and BAX in NBT-induced pyroptosis after CRISPR-Cas9-mediated knockout. Immunofluorescence was used to determine the subcellular locations of BAX and cytochrome c. MitoSOX Red was employed to assess the mitochondrial reactive oxygen species (ROS) level. KYSE450 and TOM20 knockout KYSE450-/- xenograft models were established to elucidate the mechanisms involved in NBT-induced cell death. RESULTS: In this study, NBT effectively upregulated the expression of TOM20 and facilitated the translocation of BAX to mitochondria, which promoted the release of cytochrome c from mitochondria to the cytoplasm, leading to the activation of caspase-9 and caspase-3, and finally induced pyroptosis. Knocking out TOM20 by CRISPR-Cas9 significantly inhibited the expression of BAX and the downstream BAX/caspase-3/GSDME pathway, which attenuated NBT-induced pyroptosis. The elevated mitochondrial ROS level was observed after NBT treatment. Remarkably, the inhibition of ROS by N-acetylcysteine (NAC) effectively suppressed the activation of TOM20/BAX pathway. Moreover, in vivo experiments demonstrated that NBT exhibited potent antitumor effects in both KYSE450 and TOM20 knockout KYSE450-/- xenograft models. Notably, the attenuated antitumor effects and reduced cleavage of GSDME were observed in the TOM20 knockout model. CONCLUSION: These findings reveal that NBT induces pyroptosis through ROS/TOM20/BAX/GSDME pathway, which highlight the therapeutic potential of targeting TOM20 and GSDME, providing promising prospects for the development of innovative and effective treatment approaches for esophageal cancer.
Subject(s)
Esophageal Neoplasms , Gasdermins , Mitochondrial Precursor Protein Import Complex Proteins , Pyroptosis , Reactive Oxygen Species , Signal Transduction , bcl-2-Associated X Protein , Animals , Humans , Male , Mice , bcl-2-Associated X Protein/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/metabolism , Mice, Nude , Mitochondria/drug effects , Mitochondria/metabolism , Phosphate-Binding Proteins/metabolism , Pyroptosis/drug effects , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
The vigorous growth of the new shoots of the peach tree was not beneficial to high quality and efficient cultivation. High concentration of amino acids can inhibit plant growth, but the mechanism is not clear. In this study, we explored the regulatory effects of seven amino acids (phenylalanine, valine, leucine, isoleucine, serine, D-alanine, and proline) (10 gâ L-1) on the growth of peach trees. The results showed that phenylalanine, valine, and proline inhibited peach seedling growth and valine has the most significant effect and it can promote the root growth of peach seedlings. Compared with paclobutrazol, valine treatment improves net photosynthetic rate and fruit quality without reducing shoot diameter or puncture strength, and it does not affect leaf morphology. Valine enhanced the expression of PpSnRK1 (sucrose non-fermenting-1-related protein kinase) and inhibited the expression of PpTOR (Target of Rapamycin) and PpS6K (Ribosomal S6 kinase). The gibberellin content was significantly reduced in the valine treatment group. The endogenous valine content of peach seedlings was increased, acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) activity was inhibited by feedback, isoleucine synthesis was decreased, the relative amounts of branched chain amino acids were unbalanced, and growth was inhibited. However, isoleucine spraying after valine treatment could increase the content of isoleucine and alleviate the inhibition of valine on the shoot growth. In conclusion, valine is environmentally friendly to inhibit the growth of new shoots of peach trees by regulating the balance of PpSnRK1 and PpTOR and the synthesis of isoleucine.
