Fig 1.
EdU incorporation and cell viability measurements on proliferating and quiescent TG1 and OB1 GSCs maintained in vitro.
(A) In the absence of serum (NS34 culture medium), proliferating TG1 GSCs (P TG1) grow as neurospheres (left panel). Non-proliferating quiescent TG1 GSCs are generated in vitro by leaving cells without medium change for 9–16 days (middle panel: Q9 quiescent TG1 cells; right panel: Q16 quiescent TG1 cells). Scale bars, 100 μm. (B) EdU incorporation into TG1 (black triangles) and OB1 GSCs’ DNA (blue triangles) grown without medium renewal for 1–16 days or into cells grown without medium renewal for 9 days and then subjected to freshly prepared culture medium at days 9 and 13 (TG1 cells: orange triangles; OB1 cells: green triangles). Results are from at least two independent experiments. (C) Survival of TG1 cells grown in culture for 1–16 days without medium renewal. Survival was assessed using 7-AAD. Mean ± SD (n = 3).
Fig 2.
Cell cycle analysis and Ki-67 expression in TG1 and OB1 GSCs under different in vitro culture conditions.
(A) TG1 (upper panel) and OB1 (lower panel) GSCs maintained in culture for 1, 9 and 14 days without medium renewal (d1, d9, d14, respectively) or cells obtained after 9 days without medium renewal and subjected to freshly prepared culture medium for 5 days (Q+5) were permeabilized and stained with anti-Ki-67 antibodies conjugated to FITC and propidium iodide (PI). (B-E) Histograms represent the percentage of TG1 (B, C) and OB1 (D, E) cells present in the G1, S, G2/M phases of the cell cycle as well as the percentage of quiescent cells in G0 and of cells showing DNA fragmentation (sub-G1). Cells in the G2/M phase were further distinguished as a function of the Ki-67 proliferation marker expression levels (G2/M high Ki-67 and G2/M low Ki-67). Analysis was performed on cells maintained for 1–14 days without medium renewal (B, D for TG1 and OB1 cells, respectively) or on cells left without medium renewal for 9 days (Q) and re-introduced into freshly prepared culture medium for 1 (Q+1), 2 (Q+2), 3 (Q+3), 4 (Q+4) or 5 (Q+5) days (C, E for TG1 and OB1 cells, respectively).
Fig 3.
Expression of apoptosis and cell cycle related genes and absence of senescence related activity in TG1 and OB1 cells under different in vitro culture conditions.
(A-B) Histograms representing mRNA expression levels of p53, Bax and p21 genes in proliferating (P) and quiescent (Q) TG1 (A) and OB1 (B) GSCs obtained after 9 days without medium renewal or quiescent cells after 9 days without medium renewal reintroduced in proliferating culture medium for 1–4 days (Q+1-Q+ 4). Results were expressed as fold change (2∧-ΔΔCt) taking proliferating TG1 (A) or OB1 (B) cells as calibrator. 18S rRNA was used as housekeeping gene. Data were from two independent experiments, each performed in duplicate. (C-E) Senescence-related β-galactosidase activity measurements in U-87 MG cells treated with TMZ (100 μM) for 5 days (C; positive control) and in quiescent TG1 and OB1 GSCs obtained after 9 days without medium renewal (D, E, respectively). Scale bars: 100 μm.
Fig 4.
Expression of stemness, pluripotency or differentiation related genes in TG1 and OB1 cells under different in vitro culture conditions.
(A-B) Histograms representing mRNA expression levels of stemness, pluripotency or differentiation associated genes included in the TaqMan Human Stem Cell Pluripotency Array from Life Technologies in proliferating (P) and quiescent (Q) (9 days without medium renewal) TG1 (A) and OB1 (B) GSCs. Results were normalized to the 18S rRNA levels and expressed as ΔΔCt taking proliferating TG1 (A) and OB1 (B) GSCs as calibrator samples. Data are from three independent experiments, each performed in duplicate. (C-D) Histograms representing mRNA expression levels of Nanog, GBX2 and IFITM1 genes in proliferating (P) and quiescent (Q) TG1 (C) and OB1 (D) GSCs obtained after 9 days without medium renewal or quiescent cells after 9 days without medium renewal reintroduced in proliferating culture medium for 1–4 days (Q+1-Q+4). Results are expressed as in A-B. Data are from two independent experiments, each performed in duplicate.
Fig 5.
Prestwick Library screen on GSCs using the ATP-Glo cell based assay.
(A) Schematic representation of the assay design and protocol. GBM: glioblastoma. (B) Results of the primary screen are represented as histograms of the relative ATP signal levels obtained for each compound screened against proliferating (black bars) and quiescent (open bars) TG1 cells. Molecules producing ATP levels that exceeded 200% of the control levels are not shown. Zoom in of results for compounds with an ATP level below 65% of the control levels is provided. A compound was considered as a hit either if it reduced relative ATP levels to less than 5% of control levels (compounds on the left of the orange dotted line) or if the corresponding luminescent signal was lower than the mean signal of negative control wells minus 5 times the standard deviation from this value (number of molecules indicated on the top of each bar). (C) 86 compounds reducing relative ATP levels and 16 molecules increasing relative ATP levels were tested in a secondary screen at concentrations of 5 and 50 μM. Results of the secondary screen performed at 50 μM are shown following the same representation as in (B).
Table 1.
EC50 values of Prestwick Library hit compounds on the different cell types tested.
Fig 6.
Dose-response curves for compounds exhibiting the strongest cytotoxic effect on GSCs.
(A) Chemical structures of selected compounds and dose-response curves of suloctidil (left), zuclopenthixol HCl (middle) and bisacodyl (right) with representative activity profiles on proliferating (─) and quiescent (▲) TG1 GSCs. The fitted sigmoidal logistic curve to ATP-Glo cell survival assay readouts is shown on each plot (n = 3). (B) Dose-response curves of suloctidil (left), zuclopenthixol HCl (middle) and bisacodyl (right) on proliferating TG1 cells (─), quiescent TG1 cells (▲), human primary astrocytes (О) and human fetal neural stem cells (◻). Curves were fitted as in A (n = 3). (C-D) Cell viability measurements (trypan blue staining) on proliferating (P) or quiescent (Q) TG1 cells (C) and HA cells (D) treated with bisacodyl (compound 1 in Fig 9).
Fig 7.
Activity of Prestwick Library hit compounds on GSCs and control cell types.
Graphical presentation of the activity of the high-throughput screen hit compounds (expressed as 1/EC50) on proliferating (P) and quiescent (Q) GSCs derived from three patients (TG1, TG16 and OB1), human primary astrocytes (HA cells), human fetal neural stem cells (f-NSC) and a human embryonic kidney cell line (HEK 293). Upper, middle and low panels: group 1, group 2 and group 3 molecules as defined in Table 1, respectively. EC50 values correspond to mean values calculated from fitted dose-response curves to ATP-Glo assay readings of three independent experiments (n = 3). *: Given that the maximum effect of bisacodyl on HA cells is a 30% reduction of ATP levels which is not due to cell death (see Fig 6D), the corresponding EC50 value was not taken into account. **: EC50 values observed >100 μM. ***: EC50 values not determined.
Fig 8.
Bisacodyl stability in GSCs’ culture media.
Bisacodyl was dissolved in freshly prepared culture medium used for the culture of proliferating cells (A) or in conditioned culture medium of quiescent TG1 GSCs (B) at a final concentration of 10 μM in 1% DMSO. The presence of bisacodyl (diamonds) and its deacetylated derivatives (mono- (squares) and di- (triangles) deacetylated forms), was followed as a function of time. Ordinate represents area under the HPLC elution peaks of the different molecular species present.
Fig 9.
Tables representing structure-activity data of bisacodyl (compound 1) and bisacodyl derivatives (compounds 2–19) on proliferating and quiescent TG1 glioblastoma stem-like cells (GSCs). The ATP-Glo cell survival assay was used to measure compound activity. Compound structures are shown. Compounds were tested in triplicate in each experiment.