Antiparasitic mebendazole shows survival benefit in 2 preclinical models of glioblastoma multiforme

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer, and despite treatment advances, patient prognosis remains poor. During routine animal studies, we serendipitously observed that fenbendazole, a benzimidazole antihelminthic used to treat pinworm infection, inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines, mebendazole displayed cytotoxicity, with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells, and in vitro activity was correlated with reduced tubulin polymerization. Subsequently, we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections, has a long track-record of safe human use, and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials.

Figures

Fig. 1.

Mebendazole (MBZ) inhibited intracranial tumor growth in the syngeneic GL261 mouse model. (A) Inhibition of GL261 mouse glioma cells by MBZ and albendazole (ABZ) showing the half-maximal inhibitory concentration (IC50) of MBZ at 0.24 μM and the IC50 of ABZ at 0.30 μM. (B) Hematoxylin and eosin staining of a coronal section of a C57BL/6 mouse brain implanted with GL261 glioma cells in the frontal lobe. (C) Mice implanted with GL261 cells expressing firefly luciferase were injected with 100 mg/kg luciferin and measured by Xenogen after 20 days of MBZ treatment. Four animals per group were randomly selected and shown. The total photon counts are displayed in the bar graph to the right. (D) Kaplan-Meier survival curve of C57BL/6 mice implanted with GL261 glioma cells and treated with MBZ. MBZ was given orally beginning 5 days after tumor implantation at a daily dose of 50 mg/kg for the first 20 days of treatment then changed to 50 mg/kg for 5 days, with 2 days off, each week. MBZ treatment increased the mean survival to 49 days compared with the 30 days of control (P < .0001). m, Mean survival days; n, number of animals. (E) Kaplan-Meier survival curve of C57BL/6 mice implanted with GL261 glioma cells treated with ABZ. MBZ was given orally beginning 5 days after tumor implantation at either 50 mg/kg or 150 mg/kg, as indicated, every day for the first 20 days of treatment and subsequently changed to 5 days a week. P < .001 for ABZ, 150 mg/kg, versus control; P = .015 for ABZ, 50 mg/kg, versus control; P = .38 (not significant) for ABZ, 150 mg/kg, versus 50 mg/kg

Fig. 2.

Mebendazole (MBZ) improved the survival in the 060919 human glioblastoma multiforme (GBM) xenograft mouse model. (A) The half-maximal inhibitory concentration (IC50) levels of albendazole (ABZ), MBZ, and temozolomide (TMZ) in the 060919 human GBM neurosphere line are shown. (B) Hematoxylin and eosin staining of a coronal section of a nude mouse brain implanted with 060919 cells in the frontal lobe (10x). White arrows point to the invasive tumor cells. (C) Kaplan-Meier survival curve of 060919 xenografts treated with oral MBZ (50 mg/kg), ABZ (150 mg/kg), or TMZ (15 mg/kg) started 5 days after tumor implantation. Mice were treated daily for 20 days, followed by the same daily dose for 5 days per week. MBZ treatment increased the mean survival to 65 days compared with the 48 days of control. M, mean survival in days; n, number of animals. Significance values were as follows: MBZ versus control, P = .0016; ABZ versus control, P = .45; TMZ versus control, P = .30. (D) Mice implanted with 060919 cells expressing firefly luciferase were injected with 100 mg/kg luciferin and measured by Xenogen before and after 20 days of MBZ treatment. Three animals in each group were randomly selected and shown in the picture on the left side. Different color bars were applied on day 0 and day 20 of treatment. (E) The total photon counts of animals displayed in D were shown in the graph.

Fig. 3.

Mebendazole (MBZ) disrupted microtubule polymerization in 060919 glioblastoma multiforme (GBM) cells. (A) 060919 GBM neurosphere cells were incubated with MBZ, colchicine (Col), or paclitaxol (PTX) at indicated concentrations for 24 h. After lysing cells with hypotonic buffer, the lysates were separated by centrifugation. The pellets (P) containing polymerized tubulin were resuspended in equal amount of lysis buffer and loaded along with the supernatant (S) containing depolymerized tubulin on SDS-PAGE for anti-α-tubulin (αTub) Western blot. The anti-β-Actin (βAct) blot served as a loading control. (B) The signals on the anti-α-tubulin blot were quantified and the percentage of polymerized tubulin (% P) induced by individual treatments was calculated with the formula: % P = P/(P + S) × 100. Mean and standard deviation (SD) are indicated. (C) MBZ disrupted the microtubule structure of 060919 cells. 060919 cells were cultured in adherent fashion with serum-containing media, treated for 24 h with 1 μM of MBZ or 10 nM cholchicine, and stained with anti-αTub antibody, Texas Red secondary antibody, and DAPI.

Fig. 4.

Mebendazole (MBZ) plus temozolomide (TMZ) extends survival further than TMZ alone in the GL261 mouse model. (A) The half-maximal inhibitory concentration (IC50) levels of MBZ and TMZ with various glioblastoma multiforme (GBM) cell lines and normal mouse astrocytes. (B) Inhibition of GBM cell growth by MBZ, TMZ, or MBZ and TMZ combined. (C) Kaplan–Meier survival curve of C57BL/6 mice implanted with GL261 glioma cells and treated with TMZ (15 mg/kg) or TMZ plus MBZ (50 mg/kg). MBZ and TMZ were administered daily starting 5 days post tumor implantation for 20 days, followed by dosing 5 days per week. The TMZ + MBZ treatment increased the mean survival to 50 days, compared to the 30 days for controls (P < .0001) and 41 days for TMZ alone. The P = .0015 for TMZ plus MBZ versus TMZ. M, mean survival in days; n, number of animals. (D) Mice implanted with GL261 cells expressing firefly luciferase were injected with 100 mg/kg luciferin and measured by Xenogen after 25 days of MBZ treatment. Four animals in the TMZ and TMZ plus MBZ groups were randomly selected and are shown in the top picture, whereas the total photon counts are displayed on the lower graph. The color bars were set as follows: min = 2e + 5; max = 2e + 7.