Targeting the insulin-like growth factor-1 receptor by picropodophyllin as a treatment option for glioblastoma

Abstract

Glioblastoma (GB) is the most common malignant brain tumor in adults. It has limited treatment opportunities and is almost exclusively fatal. Owing to the central role the insulin-like growth factor-1 receptor (IGF-1R) plays in malignant cells, it has been suggested as a target for anticancer therapy including GB. The cyclolignan picropodophyllin (PPP) inhibits IGF-1R without affecting the highly homologous insulin receptor. Here, we show that PPP inhibits growth of human GB cell lines along with reduced phosphorylation of IGF-1R and AKT. In vivo, PPP-treatment causes dramatic tumor regression not only in subcutaneous xenografts but also in intracerebral xenografts, indicating passage of PPP across the blood-brain barrier.

Figures

Fig. 1.

Effect of PPP on growth of GB cell lines. Twelve glioma cell lines were treated with 0.05, 0.2, 0.5, 1.0, or 2.5 µM PPP for 48 hours followed by analysis of relative number of viable cells using the resazurin assay. Three independent experiments were performed for each cell line. The results were pooled followed by the calculation of mean values, which are depicted. Standard deviations were always less than 10%.

Fig. 2.

Effect of PPP on IGF-1R and AKT phosphorylation. U-343MGa Cl 2:3, U-563MG, and U-1242MG Cl 4 cells were serum-starved overnight and then treated for 1 hour with 0.5 or 2.5 µM PPP before 5 minutes of stimulation with 50 ng/mL IGF-1. (A) Lysates were subjected to IGF-1Rβ immunoprecipitation followed by Western blotting using p-Tyr or IGF-1Rβ antibodies. (B) The expression of p-AKT (Ser473) and AKT were analyzed by Western blotting. Mean ratios and SDs of triplicates are shown.

Fig. 3.

Effect of PPP on GB xenografts. (A) U-563MG cells were injected subcutaneously into SCID mice. When the tumors reached sizes of 150–250 mm3, treatment with i.p. injections twice daily with 20 mg/kg PPP or DMSO was initiated and continued for 6 days. Tumor volumes were determined every second day. At the end of the experiment, tumors were dissected, homogenized, and proteins extracted followed by analysis of the expression levels of p-AKT and AKT using Western blotting. (B) U-87MG cells were implanted into the right hemisphere of nude rats. After 7 days, treatment with i.p. injections twice daily with 20 mg/kg PPP or DMSO was initiated and continued for 10 days. Following anesthesia, the animals were sacrificed, the brains dissected, and the tumor sizes determined. Mean tumor volumes and SDs of triplicates are shown. As for the subcutaneous xenografts, the tumor tissues from the intracranial xenografts were analyzed for the expression levels of p-AKT and AKT using Western blotting. (C) Sections of fresh-frozen tissue from the intracerebral xenografts were stained with hematoxylin and eosin. (D) Fresh-frozen tumor tissue from 8 human primary gliomas as indicated was analyzed for the expression of the IGF-1R by Western blotting.