Elsevier

Biochemical Pharmacology

Volume 72, Issue 8, 16 October 2006, Pages 941-948
Biochemical Pharmacology

Receptor tyrosine kinase (RTK) inhibition is effective in chemosensitising EGFR-expressing drug resistant human ovarian cancer cell lines when used in combination with cytotoxic agents

https://doi.org/10.1016/j.bcp.2006.07.022Get rights and content

Abstract

This study has focused on the use of RTK inhibitors in the treatment of ovarian cancer. We have used the human ovarian cancer cell line PEO1 alongside two in-house derived drug resistant variants: PEO1CarboR (8-fold acquired resistance to carboplatin and cisplatin) and the Pgp expressing PEO1TaxR (15-fold acquired resistance to paclitaxel). These variant cell lines were shown to have a higher expression of EGFR 1.6- and 2.0-fold increase, respectively, compared with the parental cell line.

We have shown that the RTK inhibitor GW282974A (an analogue of GW2016; lapatinib) is effective in chemosensitisation of drug resistant EGFR over-expressing cells giving rise to a synergistic effect when used in combination with either cisplatin or paclitaxel in chemosensitivity assays. These effects were also seen at the level of apoptosis using the Annexin V assay and expression levels of the IAP Survivin. A reduction in the downstream signalling effector phosphorylated ERK was seen in both resistant cell lines when GW282974A was used in combination with either cisplatin or paclitaxel. This reduction was not so apparent in cells treated with the single agent GW282974A or cytotoxic agent. Interestingly, we did not show evidence for an enhanced sensitivity to the RTK inhibitor in our EGFR expressing resistant lines versus parental PEO1 cells. However, the paclitaxel resistant cell line appeared more sensitive to the chemosensitising effects of GW282974A, in line with its increased EGFR expression. Our data suggest that RTK inhibition is effective in circumvention of tumour cell drug resistance that occurs in conjunction with EGFR overexpression.

Introduction

Over the last decade, many advances have been made in the understanding of the molecular biology of cancer cells. Growth factor receptor overexpression is a common feature of malignant cells and this is often accompanied by an elevated production of growth factors produced in either a paracrine or autocrine fashion, driving the proliferation of the abnormal cells. Aberrant cell cycle regulation and signal transduction pathways can mediate many important events including growth-factor induced proliferation and can also influence the apoptotic sensitivity of tumour cells. Ovarian cancer frequently responds to first line therapy using cytotoxic drugs such as the taxanes and platinum agents. However, drug resistance is a frequent problem and 5 years survival rates are between 20% and 30% at best. Normal ovarian epithelial cells express relatively low levels of epidermal growth factor (EGFR; HER1) whereas, approximately 70% of ovarian cancers express elevated levels of EGFR [1]. Moreover, a number of studies have indicated that overexpression of EGFR is associated with a poor prognosis in a variety of human malignancies, including ovarian cancer [2], [3]. There is also evidence of an association of EGFR expression with VEGF expression in ovarian cancers [4].

The idea of using chemotherapeutic agents combined with agents designed to target tyrosine kinase receptor phosphorylation has been explored in a number of laboratory-based studies. For example, Pegram et al. [5] used a c-ERBb2-directed monoclonal antibody with a variety of chemotherapeutic agents including doxorubicin, VP-16, paclitaxel and 5-FU in human tumour xenograft models of a c-ERBb2-transfected human breast carcinoma. The effects of the antibody therapy with VP-16 were synergistic and with doxorubicin an additive effect was demonstrated. The dual EGFR and c-ERBb2 directed small molecule tyrosine kinase inhibitor CI1033 used in combination with the topoisomerase-I directed compound SN-38 was shown to be synergistic in T98G glioblastoma cells [6]. Importantly, these effects have translated to clinical trials where the response rate of paclitaxel was significantly enhanced by the use of trastuzumab (Herceptin®), which targets HER2/c-ERBb2 in breast cancer patients (reviewed in [7]).

The small molecule inhibitor lapatinib (GW2016), a specific dual tyrosine kinase inhibitor of EGFR and ErbB2 has shown activity against human tumour xenografts expressing these receptors and also to AKT-overexpressing human tumour xenografts [8]. In addition, a report by Sewell et al. [9] showed a TGFα stimulated growth of PEO1 cells which was reversed by gefitinib (Iressa®), with reduced phospho-ERK1/2 and reduced phospho-AKT expression.

In the present study we have looked at the effects of using a small molecule inhibitor against EGFR in terms of its possible sensitisation effects on drug resistant PEO1 ovarian cancer cells. This has involved use of PEO1CarboR platinum-resistant and PEO1TaxR paclitaxel-resistant ovarian cancer cells treated with combinations of the small molecule inhibitor GW282974A (an analogue of GW2016) and cytotoxic agents. Experiments were designed to assess possible synergistic effects of drug combinations by looking at their effects on chemosensitivity and apoptosis.

Section snippets

Cell culture

All cell culture reagents were obtained from Sigma (Poole, UK) unless stated otherwise. The ovarian carcinoma parental cell line PEO1 (obtained from Prof. Fran Balkwill, formerly ICRF, London, UK) were cultured as monolayers in RPMI-1640 medium and supplemented with 10% foetal calf serum (FCS, heat inactivated; Invitrogen, Paisley, UK). The drug resistant variants PEO1TaxR and PEO1CarboR were derived by step-wise incubation of the inducing agent over a number of months until a stable resistance

Levels of EGFR measured in PEO1 cell lines

The parental PEO1 cell line showed detectable EGFR levels (in line with the findings of others [12]). Both the PEO1CarboR and PEO1TaxR cells showed increased levels of EGFR expression relative to the parental cells, as indicated in Table 1. We were also able to confirm these results using the same antibody in western immunoblotting (data not shown).

Effects of combination treatment of GW2974A and cytotoxic drugs in PEO1 cell lines

Fig. 1 shows a selection of typical isobologram graphs obtained showing CI versus fractional effect. For the drug resistant cells, all data points

Discussion

The importance of EGFR in ovarian cancer is highlighted by the fact that up to 70% of cases may show overexpression and it has been implicated in the progression of this disease. Evidence of autocrine and paracrine regulation via TGFα/EGFR activation in ovarian cancer cell lines has also been demonstrated [14]. The present study and work of others [15] suggest that acquired resistance to cytotoxic agents may be associated in part with an upregulation in EGFR expression. Whilst we saw no

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Current address: Novartis, Horsham Research Centre, Wimblehurst Road, Horsham West Sussex RH12 5AB, UK.

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