A Ploidy Increase Promotes Sensitivity of Glioma Stem Cells to Aurora Kinases Inhibition
Multiple rounds of exposure to Danusertib can sensitize the resistant cell line, inducing senescence/autophagy. (a) Cell lines were subjected to multiple rounds of 48h treatment with Danusertib 500 nM. The sensitization of the resistant cell line (G166) to Aurora inhibition was reached already after two rounds of treatment with Danusertib 500 nM. This was confirmed by MTT assay (b) and the clonogenic assay (c). There was a significant decrease of the cell viability and the clonogenic potential in G166, which was similar to the one observed in GBM2, already after 48 h. Results are the means of three independent experiments. Error bars represent SEM. t- test was performed on raw data: p<0,001. (d) GBM2 (sensitive) and G166 (resistant) cell lines were treated with 500 nM Danusertib for two rounds of 48 h each. After the incubation, they were fixed and stained for β-galactosidase, to evaluate induction of senescence. Representative images showing β-galactosidase staining at baseline and after Danusertib treatment are reported. The graph shows the percentages of senescent cells in the two cell lines. An average of 100 cells/condition/experiment were randomly imaged and scored. After two rounds of exposure to Danusertib, there was a significant increase in the percentages of β–gal positive cells also in the resistant cell line (G166). Results are representative of three independent experiments. Error bars represent SEM. t- test was performed on raw data: p<0,01; p<0,001. (e) Beclin level and LC3-I and LC3-II ratio were determined in untreated and two rounds treated GBM2 and G166 cell lines in order to evaluate the activation of an autophagic process. Representative images of Western blot analysis and quantitative data are reported. Beclin, LCR-I, and II levels were determined and normalized on GAPDH. All the values are expressed in Arbitrary Unit (AU). After two rounds of Danusertib exposure, an increase of Beclin level and LC3-I and LC3-II ratio was detected also in G166 cell line. Results are representative of three independent experiments. t- test was performed on raw data: p<0,05. (f) Representative images of FISH analysis performed on GBM2 (sensitive) and G166 (resistant) cell lines after one and two rounds of 48 h treatment with Danusertib 500 nM are reported. Red signals correspond to chromosome 21, while green signals indicate chromosome 13. Scale bar = 100 μm. Quantitative analysis shows that Danusertib exposure induced a steady increase of the number of signals detected in both cell lines. The chromosome content detected in G166 after two rounds of Danusertib exposure is comparable to the one observed in GBM2 cell line already after one round of treatment. Results are expressed as mean of two independent experiments. At least 50 cells were analyzed in each experiment. t- test was performed on raw data: p<0,001.
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