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Dcf1 induces glioblastoma cells apoptosis by blocking autophagy
Guanghong Luo1,2,3, Ruili Feng1, Wengang Li4
1Laboratory of Molecular Neural Biology, School of Life Sciences, Shanghai University, Shanghai, China.
Background:
Dcf1 has been demonstrated to play vital roles in many CNS diseases, it also has a destructive role on cell mitochondria in glioma cells and promotes the autophagy. Hitherto, it is unclear whether the viability of glioblastoma cells is affected by Dcf1, in particular Dcf1 possesses broad localization on different organelles, and the organelles interaction frequently implicated in cancer cells survival.
Methods:
Surgically excised WHO grade IV human glioblastoma tissues were collected and cells isolated for culturing. RT-PCR and DNA sequencing assay to estimate the abundance and mutation of Dcf1. iTRAQ sequencing and bioinformatic analysis were performed. Subsequently, immunoprecipitation assay to evaluate the degradation of HistoneH2A isomers by UBA52 ubiquitylation. Transmission electron microscopy (TEM) was applied to observe the structure change of mitochondria and autophagosome. Organelle isolated assay to determine the distribution of protein. Cell cycle and apoptosis were evaluated by flow cytometric assays.
Results:
Dcf1 was downregulated in WHO grade IV tumor without mutation, and overexpression of Dcf1 was found to significantly regulate glioblastoma cells. One hundred and seventy-six differentially expressed proteins were identified by iTRAQ sequencing. Furthermore, we confirmed that overexpression of Dcf1 destabilized the structure of the nucleosome via UBA52 ubiquitination to downregulate HistoneH2A.X but not macroH2A or HistoneH2A.Z, decreased the mitochondrial DNA copy number and inhibited the mitochondrial biogenesis, thus causing mitochondrial destruction and dysfunction in order to supply cellular energy and induce mitophagy preferentially but not apoptosis. Dcf1 also has disrupted the integrity of lysosomes to block autolysosome degradation and autophagy and to increase the release of Cathepsin B and D from lysosomes into cytosol. These proteins cleaved and activated BID to induce glioblastoma cells apoptosis.
Conclusions:
In this study, we demonstrated that unmutated Dcf1 expression is negatively related to the malignancy of glioblastoma, Dcf1 overexpression causes nucleosomes destabilization, mitochondria destruction and dysfunction to induce mitophagy preferentially, and block autophagy by impairing lysosomes to induce apoptosis in glioblastoma.
Insights
Dcf1 (Doublecortin-like kinase 1) downregulation correlates with glioblastoma malignancy. Its overexpression destabilizes nucleosomes, destroys mitochondria, and impairs lysosomes, ultimately inducing mitophagy and apoptosis in glioblastoma cells.
Area of Science:
- Neuro-oncology
- Cancer Cell Biology
- Mitochondrial Biology
Background:
- Doublecortin-like kinase 1 (Dcf1) plays roles in CNS diseases and affects glioma cell mitochondria and autophagy.
- The precise impact of Dcf1 on glioblastoma cell viability and its organelle interactions remain unclear.
Purpose of the Study:
- To investigate the role of Dcf1 in glioblastoma viability.
- To elucidate the molecular mechanisms by which Dcf1 influences glioblastoma cell fate.
Main Methods:
- Analysis of Dcf1 expression and mutation in glioblastoma tissues.
- Proteomic analysis (iTRAQ) to identify differentially expressed proteins.
- Assays for nucleosome stability, mitochondrial function, and lysosomal integrity.
- Flow cytometry for cell cycle and apoptosis analysis.
Main Results:
- Dcf1 was downregulated in WHO grade IV glioblastoma without mutation.
- Dcf1 overexpression destabilized nucleosomes via UBA52 ubiquitination, decreasing HistoneH2A.X levels.
- Overexpression led to mitochondrial dysfunction, reduced mitochondrial DNA copy number, and induced mitophagy.
- Lysosomal integrity was disrupted, blocking autophagy and releasing cathepsins, which promoted apoptosis.
Conclusions:
- Unmutated Dcf1 expression is inversely related to glioblastoma malignancy.
- Dcf1 overexpression induces nucleosome destabilization, mitochondrial dysfunction, and mitophagy.
- Dcf1 impairs lysosomes, blocking autophagy and promoting apoptosis in glioblastoma.
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