ABSTRACT
Tumor Treating Fields (TTFields) are an FDA-approved anticancer treatment using alternating electric fields. Here, we present a protocol to perform live-cell imaging (LCI) of cells during TTFields treatment with the Inovitro LiveTM system. The setup we describe dissipates TTFields-related heat production and can be used in conjunction with any LCI-compatible microscope setup. This approach will enable further elucidation of TTFields' mechanism of action at the molecular level and facilitate the development of promising combination strategies.
Subject(s)
Electric Stimulation Therapy , Neoplasms , Combined Modality Therapy , Electric Stimulation Therapy/methods , Humans , Neoplasms/diagnostic imagingABSTRACT
Background: Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves maximal surgical resection of the tumor followed by radiation and chemotherapy (temozolomide [TMZ]). The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status in the tumor. Methods: A drug screening aimed at evaluating the potential recycling and repurposing of known drugs was conducted in TMZ-resistant GBM cell lines and primary cultures of newly diagnosed GBM with different MGMT promoter methylation status, phenotypic/genotypic background and subtype, and validated with sphere formation, cell migration assays, and quantitative invasive orthotopic in vivo models. Results: We identified hydroxyurea (HU) to synergize with TMZ in GBM cells in culture and in vivo, irrespective of MGMT promoter methylation status, subtype, and/or stemness. HU acts specifically on the S-phase of the cell cycle by inhibiting the M2 unit of enzyme ribonucleotide reductase. Knockdown of this enzyme using RNA interference and other known chemical inhibitors exerted a similar effect to HU in combination with TMZ both in culture and in vivo. Conclusions: We demonstrate preclinical efficacy of repurposing hydroxyurea in combination with TMZ for adjuvant GBM therapy. This combination benefit is of direct clinical interest given the extensive use of TMZ and the associated problems with TMZ-related resistance and treatment failure.
Subject(s)
Brain Neoplasms/drug therapy , DNA Replication/drug effects , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/drug effects , Glioblastoma/drug therapy , Hydroxyurea/pharmacology , Temozolomide/pharmacology , Animals , Antineoplastic Agents, Alkylating/pharmacology , Apoptosis , Brain Neoplasms/classification , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Proliferation , Drug Repositioning , Glioblastoma/classification , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Mice , Nucleic Acid Synthesis Inhibitors/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Enhancer of Zeste Homolog 2 (EZH2) has emerged as a promising therapeutic target for treatment of a broad spectrum of tumors including gliomas. We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcb1 and/or Abcg2 knockout mice. All EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by very low membrane permeability. Both P-gp and Bcrp1 restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcb1;Abcg2 knockout mice. These results are likely due to poor membrane permeability and question the clinical usefulness of GSK126. Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series.