Antiangiogenic effects of noscapine enhance radioresponse for GL261 tumors

Abstract

Purpose: To assess the effects of noscapine, a tubulin-binding drug, in combination with radiation in a murine glioma model.

Methods and materials: The human T98G and murine GL261 glioma cell lines treated with noscapine, radiation, or both were assayed for clonogenic survival. Mice with established GL261 hind limb tumors were treated with noscapine, radiation, or both to evaluate the effect of noscapine on radioresponse. In a separate experiment with the same treatment groups, 7 days after radiation, tumors were resected and immunostained to measure proliferation rate, apoptosis, and angiogenic activity.

Results: Noscapine reduced clonogenic survival without enhancement of radiosensitivity in vitro. Noscapine combined with radiation significantly increased tumor growth delay: 5, 8, 13, and 18 days for control, noscapine alone, radiation alone, and the combination treatment, respectively (p < 0.001). To assess the effect of the combination of noscapine plus radiation on the tumor vasculature, tubule formation by the murine endothelial 2H11 cells was tested. Noscapine with radiation significantly inhibited tubule formation compared with radiation alone. By immunohistochemistry, tumors treated with the combination of noscapine plus radiation showed a decrease in BrdU incorporation, an increase in apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, and a decrease in tumor vessel density compared with tumors treated with radiation alone.

Conclusion: Noscapine enhanced the sensitivity of GL261 glioma tumors to radiation, resulting in a significant tumor growth delay. An antiangiogenic mechanism contributed to the effect. These findings are clinically relevant, particularly in view of the mild toxicity profile of this drug.

Fig. 1. In vitro effect of noscapine and radiation

(a), Survival fraction of T98G and GL261 cell lines at escalating doses of noscapine. (b), Survival curve for T98G and GL261 cell lines after radiation (single dose = 2.5, 5.0 and 7.5 Gy) or radiation and noscapine (10 μM for T98G and 50 μM for GL261).

Fig. 2. In vivo effect of noscapine: enhancement of radioresponse

(a), Tumor growth delay assay. (b), Time in days for tumors to grow four-fold. Tumor volumes are shown for each treatment group at the indicated time points. *A statistically significant difference was found between the control and noscapine treated groups (p < 0.001).

Fig. 3. Effect of noscapine and radiation on tumor cell proliferation and apoptosis

(a), Left panel: Representative images of BrdU staining from each group (X200). Right panel: Percentage of BrdU positive cells per microscopic. Columns and error bars: mean ± SD. *A statistically significant difference was found between radiation treatment alone and the combined treatment group (p < 0.005). (b), Left panel: Representative images of TUNEL staining from each group (X200). Average number of TUNEL positive cells per microscopic field. Columns and error bars: mean ± SD. *A statistically significant difference was found between radiation treatment alone and the combined treatment group (p < 0.04).

Fig. 4. Effect of noscapine alone and combined with radiation on blood vessel formation in vitro and in vivo

(a), Left panel: Representative images of tubule formation at 6 h (X40). Middle panel: Number of branch points per well. Columns and error bars: mean ± SD. Right panel: Representative histograms for cells analyzed by flow cytometry for the number of Annexin V positive cells. *A statistically significant difference was found between the control and noscapine treated groups (p < 0.0001). (b), Left panel: Representative images of tubule formation at 6 h (X40). Middle panel: Number of branch points per well. Columns and error bars: mean ± SD. Number of branch points per well (middle). Columns and error bars: mean ± SD. Right panel: Number of colonies in the clonogenic survival assay per treatment group as percent of control. Columns and error bars: mean ± SD. *A statistically significant difference was found between the control and noscapine treated groups (p < 0.001). (c), Left panel: Representative images of sections stained for CD31 (X100). Right panel: Number of blood vessels per field. Columns and error bars: mean ± SD. *A statistically significant difference was found between radiation treatment alone and the combined treatment group (p < 0.001).