APO010, a synthetic hexameric CD95 ligand, induces human glioma cell death in vitro and in vivo

Abstract

Death receptor targeting has emerged as one of the promising novel approaches of cancer therapy. The activation of one such prototypic death receptor, CD95 (Fas/APO-1), has remained controversial because CD95 agonistic molecules have exhibited either too strong toxicity or too little activity. The natural CD95 ligand (CD95L) is a cytokine, which needs to trimerize to mediate a cell death signal. Mega-Fas-Ligand, now referred to as APO010, is a synthetic hexameric CD95 agonist that exhibits strong antitumor activity in various tumor models. Here, we studied the effects of APO010 in human glioma models in vitro and in vivo. Compared with a cross-linked soluble CD95L or a CD95-agonistic antibody, APO010 exhibited superior activity in glioma cell lines expressing CD95 and triggered caspase-dependent cell death. APO010 reduced glioma cell viability in synergy when combined with temozolomide. The locoregional administration of APO010 induced glioma cell death in vivo and prolonged the survival of tumor-bearing mice. A further exploration of APO010 as a novel antiglioma agent is warranted.

Fig. 1.

APO010 induces DEVD-amc–cleaving caspase activity and cell death in human glioma cell lines. (A–C) U87MG, LNT-229, or LN-308 cells were exposed to increasing concentrations of APO010, sCD95L, or CH11 for 20 hours in triplicates. Cell density was assessed by crystal violet staining. Data are expressed as mean and standard deviation (n = 3); 1 representative out of 3 independent experiments with similar results is shown. (D and E) U87MG or LNT-229 cells were treated as in (A) and (B) in triplicates and assessed at 3 (U87MG) or 6 hours (LNT-229) for DEVD-cleaving caspase activity. Data are expressed in relation to untreated controls as mean and standard deviation (n = 3); 1 representative out of the 3 independent experiments with similar results is shown. The level of statistical significance for a superior effect of APO010 over sCD95L and CH11 was assessed in (A)–(E) by one-way ANOVA and Bonferroni post hoc testing (*P < .05; **P < .01; ***P < .001; ⁺⁺P < .01 for APO010 and sCD95L vs CH11). (F) LNT-229 cells were treated as indicated for 20 hours. Cellular lysates were examined for cleavage of caspases 8 and 3 and for PARP and GAPDH by immunoblot.

Fig. 2.

Caspase-dependent induction of annexin V/PI labeling by APO010. The cells were treated with CD95 agonists as indicated and characterized by annexin V/PI labeling 6 (U87MG) or 20 hours (LNT-229) later. Quantitative data for U87MG, LNT-229, LN-308, and SV-FHAS cells are provided in (A)–(D). Coexposure to zVAD-fmk (50 μM) was included to assess the caspase dependency of cell death induction. Data are expressed as the mean percentage of annexin V–positive cells ± SEM from the 3 independent experiments. The level of statistical significance was assessed by one-way ANOVA and Bonferroni post hoc testing (**P < .01).

Fig. 3.

Targeted alterations in CD95 expression at the cell surface result in altered sensitivity toward APO010. U87MG or LNT-229 cells were transfected with siRNA targeting CD95 (CD95si) or scrambled control siRNA (scr). (A) Seventy-two hours after transfection, the cells were assessed for the expression of CD95 by flow cytometry. (B) Seventy-two hours after transfection the cells were treated with APO010 (200 ng/mL) and characterized by annexin V/PI labeling 6 (U87MG) or 20 hours (LNT-229) later. Data are expressed as the mean percentages of annexin V–positive cells ± SEM from the 3 independent experiments. (C) LN-308 cells were stably transfected to overexpress CD95. Subclones LN-308.CD95 K2 or K3 and LN-308.neo controls were exposed to increasing concentrations of APO010 for 20 hours in triplicates in a 96-well format. Cell density was assessed by crystal violet staining. Data are expressed in relation to untreated controls as mean and SEM (n = 3). One of the 3 representative independent experiments with similar results is shown. The level of statistical significance was assessed by one-way ANOVA and Bonferroni post hoc testing (*P < .05; **P < .01).

Fig. 4.

Synergistic induction of cell death by APO010 and TMZ. LNT-229 cells were treated with TMZ for 96 hours followed by APO010 for another 20 hours. The graphs show the results of treatment with either agent alone, the predicted effect assuming independent (additive) effects, and the truly observed effect. The bars express the percentage of living cells as assessed by flow cytometry after annexin V and PI staining. The data are expressed as mean and SEM from 3 independent experiments. We studied LNT-229.neo control cells (TMZ 90 μM) (A), MGMT-transfected cells (TMZ 90 μM) (B), MGMT-transfected cells additionally treated with O6BG (50 µM; TMZ 90 μM) (C), and LN-308 cells (TMZ 600 μM) (D). (E) LNT-229 cells were pretreated with TMZ (90 μM) as indicated above followed by addition of APO010 and assessed at 6 hours for DEVD-amc–cleaving caspase activity. Data are expressed relative to untreated controls as mean and standard deviation (n = 3). One representative out of the 3 independent experiments with similar results is shown.

Fig. 5.

Locoregional APO010 administration induces glioma cell death in vivo and prolongs the survival of tumor-bearing mice. (A) The mice were treated i.c. with PBS or APO010 (40 ng) on days 7 and 14 after the intracranial implantation of 10⁵ U87MG cells and monitored for survival. (B) The mice were treated with PBS or APO010 (0.015 mg/kg body weight) i.p. 3 times per week and monitored for survival. (C) The animals were treated as in (A) and tumors were removed on day 15 after tumor inoculation and stained with hematoxylin-eosin (a and b) or for apoptotic cell death by TUNEL (c and d) or immunohistochemistry for activated caspase 3 (e and f). Size bar in the lower right panel: 40 µm. Analysis of statistical significance was performed by Student 2-tailed t-test (**P < .01; n.s., not significant). (D) Mice were treated as described in (A) and (B) with a single injection. After perfusion of the mice with PBS, the tumors were removed 4 (upper panel) or 24 hours (lower panel) after treatment. Pooled tumor lysates were subjected to immunoblot, and APO010 was assessed using an anti-ACRP30 antibody (clone ne.na). Purified APO010 served as a positive control.