ABSTRACT
BACKGROUND: Necroptosis has emerged as a novel molecular pathway that can be targeted by chemotherapy agents in the treatment of cancer. OSW-1, which is derived from the bulbs of Ornithogalum saundersiae Baker, exerts a wide range of pharmacological effects. AIM: To explore whether OSW-1 can induce necroptosis in colorectal cancer (CRC) cells, thereby expanding its range of clinical applications. METHODS: We performed a sequence of functional experiments, including Cell Counting Kit-8 assays and flow cytometry analysis, to assess the inhibitory effect of OSW-1 on CRC cells. We utilized quantitative proteomics, employing tandem mass tag labeling combined with liquid chromatography-tandem mass spectrometry, to analyze changes in protein expression. Subsequent bioinformatic analysis was conducted to elucidate the biological processes associated with the identified proteins. Transmission electron microscopy (TEM) and immunofluorescence studies were also performed to examine the effects of OSW-1 on necroptosis. Finally, western blotting, siRNA experiments, and immunoprecipitation were employed to evaluate protein interactions within CRC cells. RESULTS: The results revealed that OSW-1 exerted a strong inhibitory effect on CRC cells, and this effect was accompanied by a necroptosis-like morphology that was observable via TEM. OSW-1 was shown to trigger necroptosis via activation of the RIPK1/RIPK3/MLKL pathway. Furthermore, the accumulation of p62/SQSTM1 was shown to mediate OSW-1-induced necroptosis through its interaction with RIPK1. CONCLUSION: We propose that OSW-1 can induce necroptosis through the RIPK1/RIPK3/MLKL signaling pathway, and that this effect is mediated by the RIPK1-p62/SQSTM1 complex, in CRC cells. These results provide a theoretical foundation for the use of OSW-1 in the clinical treatment of CRC.
Subject(s)
Colorectal Neoplasms , Necroptosis , Plant Extracts , Receptor-Interacting Protein Serine-Threonine Kinases , Sequestosome-1 Protein , Signal Transduction , Humans , Cell Line, Tumor , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/genetics , HCT116 Cells , Necroptosis/drug effects , Plant Extracts/pharmacology , Protein Kinases/metabolism , Proteomics/methods , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Sequestosome-1 Protein/metabolism , Sequestosome-1 Protein/geneticsABSTRACT
Picornavirus hepatitis A virus (HAV) is a common cause of hepatitis worldwide. It is spread primarily through contaminated food and water or person-to-person contact. HAV I has been identified as the most common type of human HAV infection. Here, we have developed a cell-free toehold switch sensor for HAV I detection. We screened 10 suitable toehold switch sequences using NUPACK software, and the VP1 gene was used as the target gene. The optimal toehold switch sequence was selected by in vivo expression. The best toehold switch concentration was further found to be 20 nM in a cell-free system. 5 nM trigger RNA activated the toehold switch to generate visible green fluorescence. The minimum detection concentration decreased to 1 pM once combined with NASBA. HAV I trigger RNA could be detected accurately with excellent specificity. In addition, the cell-free toehold switch sensor was verified in HAV I entities. The successful construction of the cell-free toehold switch sensor provided a convenient, rapid, and accurate method for HAV I on-site detection, especially in developing countries, without the involvement of expensive facilities and additional professional operators.
