WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line

Abstract

Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1-baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line.

Keywords: WNK2; autophagic flux; autophagy; glioblastoma cell line; inhibition.

Figure 1

Evaluation of LC3B and p62 proteins by western blot. A172 WT, A172 EV, and A172 WNK2 cell lines were treated with bafilomycin A1 (BAF, 20 nM), starvation (EBSS medium), everolimus (EVERO, 10 nM), or the combination BAF+EVERO for 4 (A) or 6 h (B). The graphs are representative of three independent biological experiments. β-actin protein was used as an endogenous loading control. Symbols mean (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Figure 2

Evaluation of proteins involved in the mammalian target of rapamycin (mTOR) pathway by western blot. A172 WT, A172 EV, and A172 WNK2 cell lines were treated with bafilomycin A1 (BAF, 20 nM), starvation (EBSS medium), or everolimus (EVERO, 10 nM) for 4 (A) and 6 h (B). The protein extract was evaluated for phosphorylation of mTOR and its substrates p-p70S6K and p-4EBP1 by western blot. Normalized densitometric band intensities of mTOR activity used α-tubulin as an endogenous loading control. For the substrates p-p70S6K and p-4EBP1, β-actin was used as an endogenous control. The graphs are representative of two independent biological experiments. n.s.: Not significant.

Figure 3

Evaluation of LC3B and p62 proteins by using transient transfection with pDest.mCherry-GFP-LC3B and pDest.mCherry-GFP-p62 plasmids. A172 WT, A172 EV, and A172 WNK2 cell lines were transfected with plasmid pDest.mCherry-GFP-LC3B (A) or pDest.mCherry-GFP-p62 (B) and then treated for 24 h with bafilomycin A1 (baf A1). Hoechst (DAPI) treatment indicates nuclear staining by blue fluorescence. FITC indicates green fluorescence and Texas Red indicates red fluorescence wavelengths by the In Cell Analyzer platform. In the figures, yellow dots indicate the presence of LC3B or p62 in autophagosomes, whereas red dots indicate autophagolysosomes due to loss of green fluorescence in an acidic environment. In the graphics, the yellow and red bars indicate the quantification of autophagossomos and autophagolysosomes, respectively, observed in the merged. The graphs are representative of two independent biological experiments. Images were quantified using Image-Pro software. Symbols mean (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. (+) means presence of baf A1; (-) means absence of baf A1.

Figure 3

Evaluation of LC3B and p62 proteins by using transient transfection with pDest.mCherry-GFP-LC3B and pDest.mCherry-GFP-p62 plasmids. A172 WT, A172 EV, and A172 WNK2 cell lines were transfected with plasmid pDest.mCherry-GFP-LC3B (A) or pDest.mCherry-GFP-p62 (B) and then treated for 24 h with bafilomycin A1 (baf A1). Hoechst (DAPI) treatment indicates nuclear staining by blue fluorescence. FITC indicates green fluorescence and Texas Red indicates red fluorescence wavelengths by the In Cell Analyzer platform. In the figures, yellow dots indicate the presence of LC3B or p62 in autophagosomes, whereas red dots indicate autophagolysosomes due to loss of green fluorescence in an acidic environment. In the graphics, the yellow and red bars indicate the quantification of autophagossomos and autophagolysosomes, respectively, observed in the merged. The graphs are representative of two independent biological experiments. Images were quantified using Image-Pro software. Symbols mean (*) p < 0.05; (**) p < 0.01; (***) p < 0.001. (+) means presence of baf A1; (-) means absence of baf A1.

Figure 4

Evaluation of the effect of WNK2 overexpression on the autophagic process after chloroquine (CQ) treatment. (A) A172 WT, A172 EV, and A172 WNK2 cell lines were treated with DMEM 0.5% FBS for control conditions and to induce autophagy with the autophagy flux inhibitor chloroquine (CQ, 30 μM) for 16 h after a 3-h starvation period. Images were acquired at DAPI (blue, nuclei), FITC (green, LC3 dots), and CY3 (red, cytoplasm) wavelengths by the In Cell Analyzer platform. (B) Graph of the quantification of LC3B vesicles in merged after control condition and treatment with CQ for 16 h. The graphs are representative of three independent biological experiments. The quantification of images was realized in Image-Pro software. Symbols mean (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.