Elsevier

Biochemical Pharmacology

Volume 67, Issue 9, 1 May 2004, Pages 1689-1700
Biochemical Pharmacology

Combination treatment of glioblastoma multiforme cell lines with the anti-malarial artesunate and the epidermal growth factor receptor tyrosine kinase inhibitor OSI-774

https://doi.org/10.1016/j.bcp.2003.12.035Get rights and content

Abstract

New drugs and combination modalities for otherwise non-responsive brain tumors are urgently required. The anti-malarial artesunate (ART) and the EGFR tyrosine kinase inhibitor OSI-774 reveal profound cytotoxic activity. The effectiveness of a combination treatment and the underlying molecular determinants of cellular response are unknown. In the present investigation, we studied ART and OSI-774 in glioblastoma multiforme (GBM) cell lines. Supra-additive inhibition of cell growth was observed in U-87MG.ΔEGFR cells transduced with a deletion-mutant constitutively active EGFR gene, while additive effects were present in cells transduced with wild-type EGFR (U-87MG.WT-2N), kinase-deficient EGFR (U-87MG.DK-2N), mock vector controls (U-87MG.LUX), or non-transduced parental U-87MG cells. Among nine other non-transduced GBM cell lines, supra-additive effects were found in two cell lines (G-210GM, G-599GM), while ART and OSI-774 acted in an additive manner in the other seven cell lines (G-211GM, G-750GM, G-1163GM, G-1187GM, G-1265GM, G-1301GM, and G-1408GM). Sub-additive or antagonistic effects were not observed. Genomic gains and losses of genetic material in the non-transduced cell lines as assessed by comparative genomic hybridization were correlated with the IC50 values for ART and OSI-774 and subsequently subjected to hierarchical cluster analysis and cluster image mapping. A genomic profile of imbalances was detected that predicted cellular response to ART and OSI-774. The genes located at the genomic imbalances of interest may serve as candidate resistance genes of GBM cells towards ART and OSI-774. In conclusion, the combination treatment of ART and OSI-774 resulted in an increased growth inhibition of GBM cell lines as compared to each drug alone.

Introduction

Current standard chemotherapies for cancer are still inadequate regarding drug resistance and toxicity. As a consequence of dose-limiting toxicity, concentrations necessary to kill resistant subpopulations of tumor cells can frequently not be achieved, leading to the ultimate failure of chemotherapy. Faced with these major limitations of current cancer chemotherapy, there is an urgent requirement for new drugs and combination treatment modalities with acceptable toxicity profiles and activity against otherwise non-responsive tumors.

The human epidermal growth factor receptor (EGFR, HER1) represents a valuable target for the development of novel therapeutic strategies. A specific deletion-mutation in the EGFR gene increases cellular tumorigenicity [1], and amplification and/or over-expression of the EGFR is of prognostic relevance for many types of solid tumors including glioblastoma multiforme (GBM) [2]. Upon ligand binding and homo- or heterodimerization with HER2, another member of the EGFR gene family, the intracellular tyrosine kinase domain is activated. EGFR activation can stimulate the Raf/Mek/Erk, the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/Akt, and the phospholipase Cγ (PLCγ)/protein kinase C (PKC) signal transduction pathways [3]. The activation of EGFR-coupled signaling routes drive mitogenic and other cancer-promoting processes, e.g., proliferation, invasion, angiogenesis, cell motility, cell adhesion, inhibition of apoptosis, and the development of drug resistance. The deregulated activation of the receptor’s tyrosine kinase activity, e.g., by gene amplification or deletions throughout the ligand-binding domain in exons 2–7, is critical for the malignant transformation of cells.

Since these functional and structural alterations have been strongly implicated in increased cellular proliferation and tumorigenesis, decreased apoptosis, and poor prognosis, several strategies, including monoclonal antibodies, immunotoxin conjugates, anti-sense oligonucleotides, siRNA, and ribozymes, have attempted to ultimately intercept cellular effects mediated by quantitatively and qualitatively aberrant EGFR [4]. Small molecules, e.g., ZD1839, OSI-774, PKI-166, PD158780, AG1478, CGP59326, and CI-1033, either interfere with the signal-transducing tyrosine phosphorylation activity or enhance ubiquitination and endocytotic degradation of EGFR. A salient feature of EGFR inhibitors is that they improve tumor cell killing if applied in combination with standard antineoplastic agents [5], [6]. Hence, such combination treatment approaches may provide novel solutions for more effective treatments of human cancers.

OSI-774 is a quinazoline that inhibits the tyrosine kinase activity of EGFR and induces apoptosis and cell cycle arrest [7], [8]. In a recent study, we have shown that the ability of GBM cell lines to induce EGFR mRNA expression after exposure to OSI-774 was associated with decreased susceptibility to the anti-tumorigenic effect of OSI-774 as a cellular effort to counteract functional EGFR inhibition by this drug [9].

Another novel compound with profound cytotoxicity against tumor cells is ART. This is a semisynthetic derivative of artemisinin, the active principle of Artemisia annua L. Though initially described as an anti-malarial drug, ART proved to be active in 55 cell lines of the National Cancer Institute (N.C.I.), USA [10]. Mining the N.C.I.’s database for the mRNA expression microarray data for 465 genes showed that EGFR gene expression correlated inversely with the cellular sensitivity to ART, indicating that EGFR is a determinant of cellular response to ART [11].

While both drugs are well described concerning their activity against tumor cells, nothing is known about the effectiveness of a combination treatment and the underlying molecular determinants of cellular response. In the present investigation, we have analyzed the small molecule EGFR tyrosine kinase inhibitor OSI-774 in combination with ART in a panel of 14 GBM cell lines, in order to identify molecular factors that may determine the cellular response to this combination treatment. Therefore, ART was tested in combination with OSI-774 in GBM cell lines transduced with expression vectors carrying either wild-type or deletion-mutant EGFR cDNAs as well as in a panel of GBM cell lines with varying degrees of inherent EGFR expression. Furthermore, the comparative genomic hybridization technique was used to identify genomic imbalances that correlated with the IC50 values of GBM cell lines for ART and OSI-774. Finally, a profile of genomic aberrations has been identified by hierarchical cluster analysis that predicted sensitivity or resistance of GBM cell lines to the combination of ART and OSI-774.

Section snippets

Drugs

OSI-774 ([6,7-bis(2-methoxy-ethoxy)quinazoline-4-yl]-(3-ethylphenyl)amine) was kindly provided by OSI Pharmaceuticals. ART was obtained from Saokim Ltd.

Cell lines

The establishment of the parental human GBM cell line U-87MG and its derivatives which over-express exogenous wild-type epidermal growth factor receptor (U-87MG.WT-2N), tyrosine kinase-deficient EGFR (U-87MG.DK-2N), constitutively active EGFR with a genomic deletion of exons 2–7 (U-87MG.ΔEGFR), or control expression vector (U-87MG.LUX),

Growth inhibition assays

As a starting point, growth inhibition after exposure to OSI-774 has been determined in all glioblastoma cell lines investigated. OSI-774 has been applied in a concentration range from 0.1 to 30 μM to U-87MG.ΔEGFR cells transduced with a deletion-mutant EGFR and U-87MG control cells (Fig. 1a). The IC50 values for OSI-774 calculated from the dose-response curves were 1.6 μM for U-87MG.ΔEGFR and 8.6 μM U-87MG cells. Hence, U-87MG.ΔEGFR cells were 5.5-fold more sensitive to OSI-774 than U-87MG cells.

Additive and supra-additive growth inhibition by ART and OSI-774

In the present investigation, we analyzed the combination treatment of ART and OSI-774 in a panel of 14 GBM cell lines. OSI-774 was applied in a concentration of 1 μM in combination with ART. In a recent phase I study on patients with advanced solid malignancies [22], the average minimal steady state plasma concentration of OSI-774 was 1.2 μg/ml (3 μM). Hence, the concentration of 1 μM that was active in the present investigation is safely within the range of clinically achievable plasma

Acknowledgements

T.R. was funded by the German-Mexican Exchange Program of the Deutsche Forschungsgemeinschaft (given to E.G.) and Conacyt, Mexico. The project was additionally supported by an intramural grant of the University of Göttingen (given to M.-E.H.). We thank Karen Thoma for skillful technical assistance.

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