Propofol enhances stem-like properties of glioma via GABAAR-dependent Src modulation of ZDHHC5-EZH2 palmitoylation mechanism

Abstract

Background: Propofol is a commonly used anesthetic. However, its effects on glioma growth and recurrence remain largely unknown.

Methods: The effect of propofol on glioma growth was demonstrated by a series of in vitro and in vivo experiments (spheroidal formation assay, western blotting, and xenograft model). The acyl-biotin exchange method and liquid chromatography-mass spectrometry assays identified palmitoylation proteins mediated by the domain containing the Asp-His-His-Cys family. Western blotting, co-immunoprecipitation, quantitative real-time polymerase chain reaction, co-immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays were used to explore the mechanisms of the γ-aminobutyric acid receptor (GABA_AR)/Src/ZDHHC5/EZH2 signaling axis in the effects of propofol on glioma stem cells (GSCs).

Results: We found that treatment with a standard dose of propofol promoted glioma growth in nude mice compared with control or low-dose propofol. Propofol-treated GSCs also led to larger tumor growth in nude mice than did vector-treated tumors. Mechanistically, propofol enhances the stem-like properties of gliomas through GABA_AR to increase Src expression, thereby enhancing the palmitoylation of ZDHHC5-mediated EZH2 and Oct4 expression.

Conclusion: These results demonstrate that propofol may promote glioma growth through the GABA_AR-Src-ZDHHC5-EZH2 mechanism and are helpful in guiding the clinical use of propofol to obtain a better patient prognosis after the surgical resection of tumors.

Keywords: EZH2; GABAAR; Glioma stem cells; Propofol; Src; ZDHHC5.

Fig. 1

Propofol increases glioma growth by enhancing the stem-like properties of tumors. A GBM patient-derived xenografts (PDX; GBM0378) mouse model injected with low and standard doses of propofol by ex vivo bioluminescent assay. Quantification of bioluminescent photon intensity (n = 5 in each group). B Detection of GFAP, Sox2, and Oct4 in the tumor of a PDX mouse model injected with or without low or standard doses of propofol by western blot. C Detection of CD44 and CD133 in the tumor of a PDX mouse model injected with or without low or standard doses of propofol by flow cytometry. D Ex vivo bioluminescent assay comparing glioma growth in mice injected with propofol-treated or DMSO-treated glioma stem cells (GSCs). Quantification of bioluminescent photon intensity (n = 5 in each group). E Detection of glioma spheres formed for U118-dervied and LN18-dervied GSCs treated with or without propofol and cultured for a second passage. Scale bars, 100 μm. Quantification of glioma spheres formation capacity (n = 5 in each group). Ctrl, control; LD, low dose; and SD, standard dose

Fig. 2

Propofol increases EZH2 palmitoylation mediated by ZDHHC5. A RT-PCR analysis of the mRNA levels of 24 known DHHCs in U118-derived GSCs treated with or without propofol (n = 5 in each group). B Lysates of propofol-treated U118-derived GSCs were subjected to the acyl-biotinyl exchange method (ABE) with a streptavidin biotinylated antibody. Palmitoylated proteins were subjected to LC–MS/MS analysis. The identified peptide sequences of EZH2 are shown. C Acyl-biotinyl exchange method and western blotting for palmitoylated EZH2 and western blotting for ZDHHC5, p-EZH2 S21, and H3K27me3 in U118-derived GSCs treated with or without propofol (n = 5 in each group). GAPDH was used as a loading control. D The mRNA levels of CDKN1B, RUNX3, and HOXA5 in U118-derived GSCs treated with or without propofol were analyzed by RT-PCR (n = 5 in each group). β-actin was used as the loading control. E Western blotting for CDKN1B, RUNX3, and HOXA5in U118-derived GSCs treated with or without propofol (n = 5 in each group). GAPDH was used as a loading control. F Detection of glioma spheres formed for U118-derived GSCs treated with or without propofol, and EZH2 inhibitor, tazemetostat, and cultured for a second passage. Scale bars, 100 μm. Quantification of glioma spheres formation capacity (n = 5 in each group). Ctrl, control; Taz, tazemetostat

Fig. 3

Propofol increases Oct4 expression via the downregulation of the methylation levels of its promoter. A The mRNA levels of Oct4 in U118-derived GSCs treated with or without propofol and EZH2 inhibitor, tazemetostat were analyzed by RT-PCR (n = 5 in each group). β-actin was used as the loading control. B The DNA methylation levels of the Oct4 promoter in U118-derived GSCs treated with or without propofol were determined by bisulfite genomic sequencing (n = 7). Black and white circles represent methylated and unmethylated sites, respectively. C The mRNA levels of Oct4 in U118-derived GSCs transfected with control, Dnmt1, Dnmt2, Dnmt3A, or Dnmt3B siRNA (n = 5 in each group). β-actin was used as the loading control. D Luciferase reporter assays with the wild-type Oct4 reporter vector and DNMT3A, EZH2 (or EZH2 palmitoylation mutants), and ZDHHC5, alone or in combination, in U118-derived GSCs. The luciferase values were relative to Renilla luciferase activity (n = 7 in each group). E Lysates from 293 T cells expressing Flag-EZH2 palmitoylation mutants (EZH2 C571, 576A), HA-ZDHCH5, and Myc-DNMT3A were subjected to immunoprecipitation, followed by immunoblotting with anti-Flag, anti-Myc, and anti-HA antibodies. Ctrl, control; Taz, tazemetostat

Fig. 4

Propofol upregulates ZDHHC5 transcripts by increasing the Src and p53 interaction. A The mRNA levels of ZDHHC5 in U118-derived GSCs treated with propofol in different concentrations were analyzed by RT-PCR (n = 5 in each group). β-actin was used as the loading control. B ChIP was performed on U118-derived GSCs with the indicated p53 and Src antibodies. PCR analysis was performed on the endogenous promoters of ZDHHC5 and cdk1 genes; cdk1 was used as a positive control; and the primers for unrelated target sites of ZDHHC5 (ZDHHC5 NT) were used as a negative control. C An immunoprecipitation assay was performed on U118-derived GSCs with the indicated p53 and Src antibodies. D Luciferase reporter assays with the wild-type ZDHHC5 reporter vector, p53 and Src, alone or in combination, in U118-derived GSCs. The luciferase values were relative to the Renilla luciferase activity (n = 7 in each group). E Detection of glioma spheres formed for U118-derived GSCs treated with or without propofol, Src siRNA or Src inhibitor, PP2, and cultured for a second passage. Quantification of the glioma sphere-formation capacity (n = 5 in each group). Western blotting for p-Src and Src in GSCs treated with or without propofol, Src siRNA or Src inhibitor, PP2 (n = 5 in each group). GAPDH was used as a loading control

Fig. 5

Propofol increases Src expression via GABA_AR. A Western blotting for p-Src and Src in wild-type or GABA_AR β3/δ knockout (KO) U118-derived GSCs treated with propofol (n = 5 in each group). GAPDH was used as a loading control. B The mRNA levels of Src in U118-derived GSCs treated with or without propofol were analyzed by RT-PCR (n = 5 in each group). β-actin was used as the loading control. C CHX chase assay indicates that the half-life of Src protein was markedly prolonged, by western blot analysis, with the treatment with muscimol or propofol compared to control conditions. GAPDH was used as the loading control. D Western blot analysis of Src ubiquitylation in U118-derived GSCs following the overexpression of Src-Flag or control vector and treatment with propofol or control condition. Src was immunoprecipitated with anti-Flag and immunoblotted with an anti-HA antibody. E Schematic representation demonstrating that propofol can enhance the stem-like properties of gliomas through the interaction of the GABA_AR-Src-ZDHCH5-EZH2 signaling axis. Ctrl, control; p-Src, phosphorylated Src; and CHX, cycloheximide