TRAIL and paclitaxel synergize to kill U87 cells and U87-derived stem-like cells in vitro

Abstract

U87-derived stem-like cells (U87-SLCs) were cultured using serum-free stem cell media and identified by both biological behaviors and markers. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and paclitaxel (PX), in combination or alone, was used to treat U87-MG human glioma cells (U87 cells) or U87-SLCs. The results showed that TRAIL/PX cannot only synergistically inhibit U87 cells but also U87-SLCs. We observed a significantly higher apoptotic rate in U87 cells simultaneously treated with TRAIL/PX for 24 h compared to cells treated with either drug alone. Furthermore, there was a remarkably higher apoptosis rate in U87-SLCs induced by the TRAIL/PX combination compared with either drug alone. Unlike the simultaneous treatment in U87 cells, U87-SLCs were pretreated for 24 h with 1 μmol/L of PX followed by 1000 ng/mL of TRAIL. Protein assays revealed that TRAIL/PX synergy was related to DR4, cleaved caspase-8 and cleaved caspase-3 upregulation, whereas the mitochondrial pathway was not involved in TRAIL-induced apoptosis. The present study indicates that PX can sensitize U87 cells and U87-SLCs to TRAIL treatment through an extrinsic pathway of cell apoptosis. The combined treatment of TRAIL and PX may be a promising glioma chemotherapy because of its successful inhibition of U87-SLCs, which are hypothesized to influence chemotherapeutic outcomes of gliomas.

Keywords: U87-derived stem-like cells (U87-SLCs); apoptosis; glioma; glioma stem cells; paclitaxel; tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).

Figure 1

(A) Tumor spheres cultured in serum-free stem cell media (magnification, 200×); (B) Tumor sphere cells were CD133-positive (magnification, 200×); (C) and (D) Tumor spheres differentiated to express the glial cell marker, glial fibrillary acidic protein (GFAP) (red) and neuronal marker, Tu-20 (green). Cell nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; blue, magnification, 200×).

Figure 2

The proportion of cells in G0/G1 phase. (A) U87 cells; (B) U87-SLCs.

Figure 3

(A) and (B) In an MTT assay, TRAIL or PX alone inhibited U87 cells in vitro in a concentration-dependent manner; (C) The synergic effect of TRAIL/PX on U87 cells (# CDI = 0.83; * CDI = 0.59); (D) Flow cytometric apoptosis assay.

Figure 3

(A) and (B) In an MTT assay, TRAIL or PX alone inhibited U87 cells in vitro in a concentration-dependent manner; (C) The synergic effect of TRAIL/PX on U87 cells (# CDI = 0.83; * CDI = 0.59); (D) Flow cytometric apoptosis assay.

Figure 4

(A) TRAIL weakly inhibited U87-SLCs; (B) A high concentration of PX showed a relatively high inhibitory effect on U87-SLCs; (C) No synergy occurred when U87-SLCs were treated simultaneously with TRAIL and PX after 24 h. However, a modified administration scheme was synergic (# CDI = 0.80); (D) Flow cytometric apoptosis assay.

Figure 5

(A) and (B) In the TRAIL/PX combination treatment with synergy, significantly higher expression levels of DR4, cleaved caspase-8 and cleaved caspase-3, but not DR5, were observed by quantitative analysis (# p < 0.05; * p < 0.01) either in U87 cells or U87-SLCs; (C) Upregulation of DR4, cleaved caspase-8 and cleaved caspase-3 in the TRAIL/PX combination treatment with synergy was visualized by Western blot.