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Research Articles: Therapeutics, Targets, and Development

Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of Integrin-linked kinase (ILK)

Departments of ¹ Advanced Therapeutics, ² Medical Biophysics, ³ Medical Oncology, and ⁴ Cancer Genetics, BC Cancer Agency; Departments of ⁵ Pathology and Laboratory Medicine and ⁶ Biochemistry and Molecular Biology and ⁷ Faculty of Pharmaceutical Sciences, University of British Columbia; ⁸ Department of Medicinal Chemistry, QLT, Inc., Vancouver, British Columbia, Canada; and ⁹ Department of Pharmacy, National University of Singapore, Republic of Singapore

Requests for reprints: Dawn N. Waterhouse, Department of Advanced Therapeutics, BC Cancer Agency, Vancouver, British Columbia, Canada. E-mail: dwater{at}bccrc.ca

Abstract

Integrin-linked kinase (ILK) was assesed as a therapeutic target in glioblastoma xenograft models through multiple endpoints including treatment related changes in the tumor microenvironment. Glioblastoma cell lines were tested in vitro for sensitivity toward the small-molecule inhibitors QLT0254 and QLT0267. Cell viability, cell cycle, and apoptosis were evaluated using MTT assay, flow cytometry, caspase activation, and DAPI staining. Western blotting and ELISA were used for protein analysis (ILK, PKB/Akt, VEGF, and HIF-1). In vivo assessment of growth rate, cell proliferation, BrdUrd, blood vessel mass (CD31 labeling), vessel perfusion (Hoechst 33342), and hypoxia (EF-5) was done using U87MG glioblastoma xenografts in RAG2-M mice treated orally with QLT0267 (200 mg/kg q.d.). ILK inhibition in vitro with QLT0254 and QLT0267 resulted in decreased levels of phospho-PKB/Akt (Ser⁴⁷³), secreted VEGF, G₂-M block, and apoptosis induction. Mice treated with QLT0267 exhibited significant delays in tumor growth (treated 213 mm³ versus control 549 mm³). In situ analysis of U87MG tumor cell proliferation from QLT0267-treated mice was significantly lower relative to untreated mice. Importantly, VEGF and HIF-1 expression decreased in QLT0267-treated tumors as did the percentage of blood vessel mass and numbers of Hoechst 33342 perfused tumor vessels compared with control tumors (35% versus 83%). ILK inhibition with novel small-molecule inhibitors leads to treatment-associated delays in tumor growth, decreased tumor angiogenesis, and functionality of tumor vasculature. The therapeutic effects of a selected ILK inhibitor (QLT0267) should be determined in the clinic in cancers that exhibit dysregulated ILK, such as PTEN-null glioblastomas. [Mol Cancer Ther 2008;7(1):59–70]

Grant support: National Cancer Institute of Canada (including support from the Canadian Breast Cancer Research Alliance) and QLT, Inc. S. Dedhar was supported by grants from the National Cancer Institute of Canada with funds raised by the Canadian Cancer Society.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹⁰ http://rsb.info.nih.gov/nih-image/

¹¹ Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 5/11/07; revised 9/19/07; accepted 12/ 4/07.