Dcf1 induces glioblastoma cells apoptosis by blocking autophagy

Abstract

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.

Keywords: Dcf1; apoptosis; glioblastoma; lysosome; mitochondria; mitophagy.

FIGURE 1

Dcf1 destabilized the structure of nucleosomes and damaged DNA. (A) Scatter plot of the protein expression (n = 3). Red: upregulated proteins; green: downregulated proteins; gray: unchanged proteins. (B) Detection of HistoneH2A isomer expression using Western blotting (n = 3). (C) Immunoprecipitation of UBA52 and HistoneH2A isomers. (D) Summary of nucleosome‐related protein changes determined by iTRAQ (n = 3). (E) Evaluation of DNA damage with γ‐H2A.X (n = 3). (F) Immunofluorescence image of γ‐H2A.X. Scale bar: 50 μm. Data were presented as mean ± SEM. Significance between every two groups was calculated by the Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 2

Dcf1 destroyed mitochondria. (A) The mtDNA/nDNA ratio detected with RT‐PCR (n = 6). (B) Mitochondrial staining with MitoTracker Green. (C) Detection of mitochondrial membrane potential with a JC‐1 kit. (D) Western blotting results for the mitochondrial biogenesis pathway (n = 3). (E) Detection of isolated mitochondrial membrane permeability transition pores (MPTPs) with Ca²⁺ absorbance examination. (F) Immunofluorescence images of mitochondrial structure. (G) ATP concentrations in glioblastoma cells determined using an ATP Assay Kit (n = 4). Scale bars: 50 μm. Data were presented as mean ± SEM. Significance between every two groups was calculated by the Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 3

Dcf1 activated mitophagy in glioblastoma cellss. (A) PARL expression determined by Western blotting (n = 4). (B) Western blotting results for mitophagy receptors (n = 4). (C) Immunofluorescence image of mitophagy. (D) Western blotting of LC3‐II/LC3‐I (n = 4). (E) Representative images of GFP‐LC3 puncta in glioblastoma cells. (F) Ultrastructural evidence showing elevated autophagy levels in glioblastoma cells. (G) Dcf1 promoted the fusion of APs and lysosomes. Scale bars: 50 μm. Data were presented as mean ± SEM. Significance between every two groups was calculated by the Student's t‐test. *p < 0.05, **p < 0.01

FIGURE 4

Dcf1 disrupted the integrity of lysosomes and blocked the process of autophagy. (A) Representative images of LysoTracker Red staining. (B) Representative images and summary of the results of lysosome staining with acridine orange. (C) Western blotting detection of lysosomal proteins (n = 4). (D) Image of pH determination via flow cytometry. (E) Detection of acid phosphatase activity with an acid phosphatase kit (n = 4). (F) Western blotting detection of PS1 (n = 4). (G) Western blotting detection of Cathepsin B and Cathepsin D release from lysosomes into the cytosol (n = 3). (H) Western blotting detection of cleaved BID and Bcl‐2 (n = 4). Scale bars: 50 μm. Data were presented as mean ± SEM. Significance between every two groups was calculated by the Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 5

Dcf1 regulated apoptosis of glioblastoma cells via the extrinsic death receptor apoptotic pathway. (A) Western blotting detection of apoptosis‐related proteins (n = 4). (B) Western blotting detection of the extrinsic death receptor apoptosis pathway (n = 4). Data were presented as mean ± SEM. Significance between every two groups was calculated by the Student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001