Repurposing the antihelmintic mebendazole as a hedgehog inhibitor

Abstract

The hedgehog (Hh) signaling pathway is activated in many types of cancer and therefore presents an attractive target for new anticancer agents. Here, we show that mebendazole, a benzamidazole with a long history of safe use against nematode infestations and hydatid disease, potently inhibited Hh signaling and slowed the growth of Hh-driven human medulloblastoma cells at clinically attainable concentrations. As an antiparasitic, mebendazole avidly binds nematode tubulin and causes inhibition of intestinal microtubule synthesis. In human cells, mebendazole suppressed the formation of the primary cilium, a microtubule-based organelle that functions as a signaling hub for Hh pathway activation. The inhibition of Hh signaling by mebendazole was unaffected by mutants in the gene that encodes human Smoothened (SMO), which are selectively propagated in cell clones that survive treatment with the Hh inhibitor vismodegib. Combination of vismodegib and mebendazole resulted in additive Hh signaling inhibition. Because mebendazole can be safely administered to adults and children at high doses over extended time periods, we propose that mebendazole could be rapidly repurposed and clinically tested as a prospective therapeutic agent for many tumors that are dependent on Hh signaling.

Conflict of interest statement

Potential conflicts of interest: None.

©2014 American Association for Cancer Research.

Figures

Figure 1

MBZ inhibits Hh signaling. (A) Shh-Light II cells maintained in low serum conditions were incubated in ShhN-conditioned medium or control medium, in the presence of MBZ at the indicated concentrations. The activity of the stably integrated Gli-luc reporter was measured after 48 h of treatment. The effect on this assay of 0.2 μM vismodegib (Vis) is indicated by the dashed line. (B) C3H10T1/2 mouse fibroblasts were co-transfected with the Gli-luc and renilla luciferase reporters. After 24 h, MBZ was added for an additional 48h in low serum media prior to cell lysis and measurement of luciferase activity. The effect of 0.2 μM vismodegib (Vis) is indicated by the dashed line. (C) The effect of MBZ on Gli-luc reporter activity in Shh-Light2 cells was compared with that of the structurally related benzamidizoles albendazole, fenbendazole, and tiabendazole. Treatment times and conditions were as in (A). (D) Endogenous levels of Gli1 transcripts in syngeneic, GL261 gliomas and in normal brain tissue from the contralateral region were measured by qRT-PCR. (E) Gli1 expression was measured by qRT-PCR in untreated and MBZ-treated GL261 tumors. Each measurement was standardized to a parallel measurement from a contralateral brain section that did not contain tumor tissue. (F) Relative protein levels of Gli1 in three untreated and three MBZ-treated GL261 tumors were assessed by immunoblot. (G) Immortalized hTERT-RPE1 cells or DAOY medulloblastoma cells (H) growing in low serum were treated with ShhN-conditioned or control medium for 48 h. MBZ was included during this treatment period at the concentrations indicated. GLI1 and PTCH1 transcript levels were assessed by qRT-PCR.

Figure 2

Effect of MBZ on Hh-signaling, growth and survival of Hh-dependent medulloblastoma cells. Subconfluent DAOY cultures maintained under low serum conditions were treated for 48 h with MBZ at varying concentrations. (A) GLI1 expression was assayed by qRT-PCR. (B) Cell proliferation was assessed by measuring the incorporation of BrdU over 2 h. (C) Cell survival was quantified by a clonogenic assay. (D) The effect of MBZ on cell viability was comparatively assessed by CellTiter-Blue in hTERT-RPE1 (blue) and DAOY (red). (E) The expression of GLI1 protein and cleavage of caspase-3 were assessed by immunoblot in DAOY and hTERT-RPE1 cells treated with MBZ for 12 h, under low serum conditions. α-tubulin was probed as a loading control. (F) Representative nuclei from MBZ-treated DAOY cells and untreated controls, stained with Hoechst 33258. Scale bar, 20 μm. (G) Annexin V stained cells were quantified by flow cytometry, after 24 h of MBZ treatment (1 μM) under low serum conditions. (H) The proportion of Annexin V positive cells after treatment with various concentrations of MBZ, as in (G).

Figure 3

MBZ suppresses the growth of medulloblastoma cells and Hh signaling in vivo. DAOY cells expressing firefly luciferase were grown as orthotopic xenograft tumors following injection into the cerebella of nude mice. After 5 d, mice were treated with 50 mg/kg MBZ (n=6) or mock-treated (Vehicle; n=8). (A) Survival of mice with DAOY cell-derived orthotopic tumors. Median survival was 75 d in the control (Vehicle) group and 113 d in the MBZ-treated group (p=0.001). One MBZ-treated mouse was euthanized after surviving six months without presenting any symptoms of lethal tumor growth. (B) Total RNA was harvested from representative tumors at the time of death. GLI1, PTCH1 and PTCH2 transcripts were quantified by qRT-PCR. (C) Tumor growth in live animals was quantified by bioluminescence imaging 5 d after implantation and again following 60 d of treatment. Representative images from the 60 d time point are shown at right.

Figure 4

MBZ inhibits activation of SMO. (A) NIH3T3 fibroblasts co-transfected with Gli-luc and renilla reporter plasmids were maintained in low serum conditions for 48 h in the presence of ShhN-conditioned or control media. During this period, MBZ or vismodegib were added at the indicated concentrations. (B) A direct comparison of Gli-luc activation in NIH3T3 cells 48 h after treatment with ShhN ligand, or 72 h after cotransfection with plasmids that drive exogenous expression of the Ptch1-resistant Smo mutant SmoM2, GLI1 or GLI2 in low serum media. (C) MBZ or vismodegib was added to SmoM2-, GLI1-, or GLI2-transfected NIH3T3 cells during 48 h of incubation in low serum conditions. The activation of a co-transfected Gli-luc reporter by each overexpressed gene, in the absence of drug treatment, was normalized to 100.

Figure 5

MBZ inhibits formation of primary cilia. (A) A Smo-FLAG fusion protein was expressed in hTERT-RPE1 cells by transient transfection. After 48 h incubation in low serum and treatment with 1 uM MBZ or vehicle, cells were fixed, permeabilized and stained with antibodies directed against acetyl-α-tubulin (green) and FLAG (red). Nuclei were counterstained with DAPI. Scale bar, 10 μm. (B) Cilia were numerically assessed by acetyl-α-tubulin staining in hTERT-RPE1 cells maintained in low serum conditions and treated with MBZ. Primary cilia (indicated by arrows) could be visualized on individual cells (inset). Scale bar, 20 μm. (C) The effects of MBZ or 0.2 μM vismodegib (red line) on the proportion of ciliated cells. (D) hTERT-RPE1 cells were treated with MBZ at the indicated concentrations or with 10 nM paclitaxel (red line) for 48 h under low serum conditions. Polymerized and unpolymerized tubulin fractions were quantified by immunobloting, normalized to the loading control β-actin, and expressed as the proportion of polymerized tubulin compared to the combined polymerized and unpolymerized tubulin. (E) GLI1 expression was assessed in DAOY cells that were incubated with MBZ for 48 h under the low serum conditions that allow formation of the primary cilium (“Treat in low serum”), or that were first maintained in low serum for 20 h prior to before adding MBZ for an additional 48 h (“Pre-incubate in low serum”). (F) Cell viability was assessed by CellTiter-Blue after the treatments described in (E).

Figure 6

Additive effects of MBZ and vismodegib against SMO signaling. Wild type Smo or the Smo D477G mutant were expressed with the Gli-luc and renilla luciferase reporters by co-transfection into Smo−/− MEFs. After 24 h, cells were treated with (A) vismodegib or (B) MBZ at the indicated concentrations, in the presence of ShhN-conditioned medium. (C) The effects of 1 μM MBZ and 0.2 μM vismodegib on Smo-dependent activation of the Gli-luc reporter were assessed against an expanded panel of Smo mutants. WT, wild type Smo. (D) The combined effects of vismodegib and MBZ on relative Gli-luc activity were tested in Shh Light2 cells under low serum conditions with supplemental ShhN-media. The relative luciferase readout was normalized to 100 for each MBZ concentration, so that the curves could be superimposed. The IC50 of vismodegib was unchanged by addition of MBZ. (E) A modification of the experiment shown in D, in which MBZ was titrated into the Shh Light2 Gli-luc assay along with fixed concentrations of vismodegib.