Targeting Bcl-xL with Navitoclax Effectively Eliminates Senescent Tumor Cells That Appear Following CEP-1347-Induced Differentiation of Glioma Stem Cells

Abstract

Cellular senescence is a state of the durable cell cycle arrest of dysfunctional cells, which has been associated with the promotion of tumor cell reprogramming into a stem cell state. We previously reported that the mixed lineage kinase (MLK) inhibitor CEP-1347 promotes the differentiation of glioma stem cells (GSCs)-key contributors to glioblastoma recurrence and therapy resistance-into non-stem tumor cells. However, we also noted that CEP-1347-treated GSCs exhibited a morphological change suggestive of senescence. Therefore, we herein investigated whether CEP-1347 induces senescence in GSCs and, consequently, if senescent GSCs may be eliminated using senolytics. Cell death induced by CEP-1347 in combination with senolytic agents or with the knockdown of anti-apoptotic BCL2 family genes, as well as the effects of CEP-1347 on the expression of senescence markers and anti-apoptotic Bcl-2 family proteins, were examined. The results obtained showed that CEP-1347 induced senescence in GSCs accompanied by the increased expression of Bcl-xL. Among the panel of senolytic agents tested, navitoclax, a BH3 mimetic, efficiently induced cell death in GSCs when combined with CEP-1347 at concentrations clinically achievable in the brain. The knockdown of Bcl-xL resulted in more pronounced GSC death in combination with CEP-1347 than that of Bcl-2. These results suggest that combining CEP-1347 with the targeting of Bcl-xL, the expression of which increases with CEP-1347-induced senescence, is a rational approach to ensure the elimination of GSCs, thereby improving the outcomes of glioblastoma treatment.

Keywords: drug repositioning; glioblastoma multiforme; stem cell capacity; tumor-initiating cells.

Figure 1

CEP-1347 induces senescence in GSCs. (a) GSCs (GS-Y01 and GS-Y03) treated with 250 nM CEP-1347 for three days were subjected to a Western blot analysis. (b) Cells were treated as in (a), and the area occupied by the cells was measured (right graphs). Representative phase contrast images are shown in the left panels, with multinucleated cells indicated by arrows. (c) Cells treated with CEP-1347 at the indicated concentrations for three days were stained with SPiDER-β-gal and analyzed by flow cytometry. The percentage of SPiDER-β-gal-positive cells (upper graphs) and representative flow cytometric histograms (lower panels) are shown. (d) Cells treated as in (a) were subjected to an RT-PCR analysis. * p < 0.05 vs. control. Bars: 100 µm.

Figure 2

The combination of CEP-1347 and senolytics potently induces cell death in GSCs. GSCs (GS-Y01 and GS-Y03) treated as indicated with 250 nM CEP-1347, 2 μM OTX015, 100 nM dasatinib, 10 μM quercetin, and/or 500 nM navitoclax for three days were subjected to the PI uptake assay. The percentage of PI-positive cells (a) and representative images (b) are shown. * p < 0.05, † p < 0.05 vs. cells treated with 250 nM CEP-1347 in combination with 500 nM navitoclax. ns: not significant. Bars: 50 μm.

Figure 3

The combination of CEP-1347 and Bcl-xL targeting BH3 mimetics potently induces apoptosis in GSCs. (a) GSCs (GS-Y01 and GS-Y03) treated with 250 nM CEP-1347 for three days were subjected to a Western blot analysis. (b,c) GSCs treated as indicated with 250 nM CEP-1347, 500 nM navitoclax, 500 nM venetoclax, and/or 125 nM A-1331852 for three days were subjected to the PI uptake assay. The percentage of PI-positive cells (b) and representative images (c) are shown. (d) GSCs were treated as described in (b) and subjected to a Western blot analysis. * p < 0.05, Bars; 50 μm.

Figure 4

The combination of CEP-1347 and Bcl-xL depletion potently induces apoptosis in GSCs. GSCs (GS-Y01 and GS-Y03) were transiently transfected with siRNA against BCL2 or BCL2L1 (encoding Bcl-xL) or with control siRNA (siControl). One day after transfection, cells were treated with 250 nM CEP-1347 for three days and then subjected to the PI uptake assay (a,b) or a Western blot analysis (c). * p < 0.05, Bars: 50 μm.

Figure 5

Navitoclax exhibits potent GSC-killing activity in combination with CEP-1347 at concentrations clinically achievable in the brain. GSCs (GS-Y01 and GS-Y03) treated with 250 nM CEP-1347 and/or navitoclax at the indicated concentrations for three days were subjected to the PI uptake assay. The percentage of PI-positive cells (a) and representative images (b) are shown. (c) GSCs were treated as described in (a) and subjected to a Western blot analysis. * p < 0.05 vs. cells treated with CEP-1347 alone. Bars: 50 μm.