Abstract 1727: Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin
Authors: Bouzinab Kaouthar , Thomas Neil R. , Turyanska Lyudmila , Gershkovich Pavel , Weston Nicola , Ashford Marianne B. , Bradshaw Tracey D. 
Published: September 2020

Background: Temozolomide (TMZ) is a DNA alkylating prodrug used for the treatment of malignant, glioblastoma multiforme (GBM) tumors. Yet, TMZ therapy is blighted by resistance systems in GBM that thwart eradication of all cancer cells remaining after surgery and radiotherapy. Resistance mechanisms include drug efflux by P-glycoprotein 1 (Pgp), present on the blood brain barrier (BBB) and on GBM cells, overexpression of O6-methylguanine DNA-methyltransferase (MGMT), which removes cytotoxic O6-methylated guanine (O6-MeG) lesions caused by TMZ, and deficiency in mismatch repair (MMR), which leads to tolerance of O6-MeG lesions. Nanoscale delivery systems are proposed as a strategy to enhance drug targeting and accumulation under systemically tolerable conditions. Apoferritin (AFt), a hollow protein capsule, has been used to deliver anticancer agents. Transferrin receptor 1 (TfR1) binding sites on the AFt cage has reportedly been shown to allow AFt to cross the BBB and accumulate in GBM cells, as both BBB endothelial and GBM (but not glial) cells express TfR1. Thus, we use AFt for targeted delivery, specific uptake and controlled release of TMZ in GBM.

Methods: TMZ was encapsulated into AFt via molecular diffusion through the channels in the AFt cage. Encapsulation efficiency and stability for this formulation were assessed by dynamic light scattering (DLS) and UV-vis spectroscopy. In vitro assays were conducted (MTT, live cell count, clonogenic, flow cytometry and ELISA) against isogenic GBM cell lines, U373V (vector control) and U373M (MGMT overexpressing), as well as MMR deficient and Pgp overexpressing HCT116 (colorectal carcinoma) and healthy MRC-5 (lung fibroblasts) cells. Environmental scanning electron microscopy (ESEM) and confocal microscopy were further employed to assess morphological changes to the cells after treatment.

Results: Encapsulation of &gt; 500 molecules of TMZ per AFt cage was achieved, with encapsulation efficiency &gt; 70%. MTT assays demonstrated significantly increased activity of AFt-TMZ compared to TMZ alone in all cancer cell lines, with 50% growth inhibition (GI50) in resistant cancer cell lines at &lt; 15 μM, compared to &gt; 200 μM for TMZ alone. Clonogenic and flow cytometric cell cycle and DNA damage analyses further corroborate these findings. Additionally, ELISA quantification demonstrated significantly (P &lt; 0.001) elevated O6-MeG levels following treatment of U373V and U373M cells with AFt-TMZ compared to naked TMZ, correlating with enhanced γ-H2AX burden in cells following exposure to AFt-TMZ. Against MRC-5, lower activity was observed, with GI50 for AFt-TMZ &gt; 70 μM. Furthermore, cell shrinkage and blebbing were observed in GBM cells following 24 h treatment with AFt-TMZ.

Conclusions: TMZ delivered by AFt demonstrated enhanced potency. Overcoming resistance by enhancing TMZ intracellular accumulation, may prove to be a turning point in enhancing drug efficacy in GBM.

Citation Format: Kaouthar Bouzinab, Neil R. Thomas, Lyudmila Turyanska, Pavel Gershkovich, Nicola Weston, Marianne B. Ashford, Tracey D. Bradshaw. Challenging resistance to temozolomide in glioblastoma by drug encapsulation in apoferritin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1727.