Mebendazole and radiation in combination increase survival through anticancer mechanisms in an intracranial rodent model of malignant meningioma

Abstract

Purpose: Meningiomas are a frequent tumor of the central nervous system. Although mostly benign, approximately 5% present as atypical or malignant tumors. Treatments for atypical meningiomas include gross total resection and radiotherapy, but about 33% of patients have recurrent tumors, sometimes as a higher grade. Recently, the brain penetrant anthelmintic drug, mebendazole, has shown promise as an anticancer agent in rodent models of glioblastoma and medulloblastoma.

Methods: The half maximal inhibitory concentration (IC₅₀) effect on colony formation, cell proliferation, and caspase-3/7 markers of apoptosis of mebendazole with and without radiation was measured in vitro. Mice intracranially implanted with KT21MG1 human meningioma were administered mebendazole alone or in combination with radiation. Survival benefit was evaluated, while tumors were investigated by immunohistochemical staining for apoptosis, cell proliferation, and vascular density.

Results: In vitro experiments on meningioma cell lines showed the IC₅₀ for mebendazole in the range of 0.26-0.42 µM. Mebendazole alone induced cytotoxicity, however the combination had a greater reduction in colony formation and resulted in higher levels of cleaved caspase-3. The in vivo study showed both, mebendazole alone and the combination, to have a survival benefit with an increase in apoptosis, and decreases in tumor cell and vascular proliferation.

Conclusion: These preclinical findings indicate that mebendazole alone or in combination with radiation can be considered for the treatment of malignant meningioma. The mechanism of action for this combination may include an increase in apoptosis, a reduction in proliferation and angiogenesis, or a combination of these effects.

Keywords: Brain; Mebendazole; Meningioma; Preclinical; Radiation.

Fig. 1

Mebendazole Alone and in Combination with Radiation Increases the Median Survival of the KT21MG1 Intracranial Model of Malignant Meningioma by Decreasing Xenograft Tumor Growth. Mebendazole alone and in combination with radiation significantly extended the life of the intracranial mouse model of malignant meningioma in two combined experiments. a.) The KT21MG1 cell line was implanted intracranially into female athymic nude mice. The combination significantly increased median survival in mice compared to all groups. b.) IVIS Xenogen imaging of the KT21MG1 xenograft expressing luciferase illustrates that the combination significant reduced the growth of the tumor in comparison to the control and mebendazole groups (p≤0.05) twelve days after implantation. c.) Average luminescence based on IVIS data was quantified for each treatment group

Fig. 2

Mebendazole Alone and in Combination with Radiation Decreases Xenograft Tumor Growth by Altering Pathways Involved in Apoptosis, Cell Proliferation, and Angiogenesis in vivo. Representative brain tissue sections were submitted to hematoxylin and eosin for each treatment group. Brain tissue sections were subjected to immunohistochemical analysis for cleaved caspase-3(10x), Ki67 (10x), and CD31 (10x) and are illustrated above for all treatment groups. The average protein expression for each treatment group was quantified using ImageJ deconvolution.

Fig. 3

The Effects of Mebendazole Alone and in Combination with Radiation on Cell Viability and Colony Formation of Meningioma Cell Lines. Meningioma cells lines were treated with different concentrations (0.001μM-100μM) of mebendazole (MBZ) alone or in combination with radiation (12 Gy) by CIXD and accessed for cell viability by CCK-8 assay. a.) The IC₅₀ values represent 72 hours of treatment with mebendazole alone and in combination with radiation. Data represents three experiments with four replicates each. b.) KT21MG1 and c.) IOMM LEE cells, both malignant meningioma cell lines, were treated with mebendazole alone and in combination with radiation (2 Gy) to evaluate the effects of treatment on colony formation. Data is represented by three replicates. Double asterisks indicate that treatment is significant (p≤0.05) to DMSO treatment alone, while single asterisks indicate significance (p≤0.05) of a particular treatment to a particular treatment group

Fig. 4

Mebendazole Alone and in Combination with Radiation Decreases Cell Proliferation and Increases Apoptosis in Cell Lines of Malignant Meningioma. The KT21MG1 a.) and IOMM LEE b.) cell lines were treated with different concentrations of mebendazole (0.001μM-100μM) alone for 72 hours and with a single dose of radiation(12 Gy) 24 hours after mebendazole treatment.. Proliferation activity in response to treatment was evaluated by a chemiluminescent BRDU-ELISA measuring DNA synthesis. Data represents four replicates per treatment group. The KT21MG1 c.) and IOMM LEE d.) cell lines were treated with different concentrations of mebendazole (0.001μM-100μM) alone for 72 hours and with a single dose of radiation(12 Gy) 24 hours after mebendazole treatment. Apoptotic activity in response to treatment was evaluated by a chemiluminescent ELISA measuring caspase-3/7 pathway activity. Data represents four replicates per treatment group

Fig. 5

Mebendazole Alone and in Combination with Radiation Induces Apoptosis through the Caspase-3/7 Pathway in Cell Lines of Malignant Meningioma. Malignant cell lines were treated, collected, lysed, ran on an SDS-PAGE, transferred to PVDF membrane and probed for proteins markers of apoptosis. a-b.) KT21MG1 and c-d.) IOMM LEE cells were analyzed by western blot for PARP, Cleaved PARP, Caspase-3, Cleaved Caspase-3, Caspase-7, Cleaved Caspase-7, and GAPDH following treatment with mebendazole alone and in combination with radiation (12 Gy)