AIM: To analyze the biological effects of prolonged in vitro exposure of HT-29 and LoVo colon cancer cell lines to gefitinib (Iressa™), an inhibitor of epidermal growth factor receptor (EGFR) activity, and ZD6474, an inhibitor of both KDR and EGFR activities.

METHODS: Cells were treated with each drug for up to 2 wk using either a continuous or an intermittent (4 d of drug exposure followed by 3 d of washout each week) schedule.

RESULTS: In both cell types, prolonged exposure (up to 14 d) to gefitinib or ZD6474 produced a similar inhibition of cell growth that was persistent and independent of the treatment schedule. The effects on cell growth were associated with a pronounced inhibition of p-EGFR and/or p-KDR expression. Treatment with gefitinib or ZD6474 also inhibited the expression of EGFR downstream signal molecules, p-Erk1/2 and p-Akt, although the magnitude of these effects varied between treatments and cell lines. Furthermore, expression of the drug resistance-related protein ABCG2 was shown to significantly increase after 14 d of continuous exposure to the two drugs.

CONCLUSION: We conclude that long-term exposure of colon cancer cells to gefitinib and ZD6474 does not modify their cytotoxic effects but it might have an effect on sensitivity to classical cytotoxic drugs.

Recently, a greater understanding of the molecular basis of cancer has fostered the development of rationally designed molecular-targeted therapies with agents that have been shown to prevent cell proliferation, differentiation and survival through the inhibition of receptor tyrosine kinases (TKs), such as epidermal growth factor receptor-1 (EGFR) and vascular endothelial growth factor receptors (VEGFRs)[1,2].

Drugs that specifically target these receptors act via inactivation of the tyrosine kinase function of EGFR receptors resulting in lack of recruitment and phosphorylation of several intracellular substrates. A major downstream signalling route involved in this process is the Ras-Raf-MAPK pathway finally leading to ERK1 and 2 inactivation[3,4]. Another important target in EGFR signalling is PI3K and the downstream AKT protein transducing signals to the cascade of survival and motility[5,6]. Recently, the relevant role of the oncosuppressor gene PTEN in uncoupling some of these signalling pathways and thus generating gefitinib resistance has also been stressed[7].

Gefitinib (Iressa) ia a well-known oral EGFR inhibitor that is able to reduce tumour growth and the formation of metastases in a range of human cancer cell lines and human tumour xenografts[8,9]. In the clinical setting, gefitinib monotherapy has demonstrated antitumour activity in patients with recurrent or refractory non-small-cell lung cancer[10,11] and it has been approved for clinical cancer treatment in several countries. ZD6474 is a novel, orally available antiangiogenic agent that selectively targets two key tumour growth pathways by inhibiting VEGFR and EGFR tyrosine kinase activities. Preclinical studies have shown ZD6474 to be a potent inhibitor of VEGF-induced endothelial cell proliferation, tumour-induced angiogenesis and tumour growth[12].

Combining gefitinib or ZD6474 with other biological or cytotoxic agents has resulted in enhanced antitumour effects in vitro and in vivo[13-17]. Several ongoing clinical trials are therefore investigating the clinical efficacy of these targeting molecules when administered (1-21 or 1-28 d) in combination with agents such as taxols, gemcitabine, cisplatin, oxaliplatin, 5-FU and irinotecan [http://www.clinicaltrials.gov]. However, we have recently demonstrated that the effect of gefitinib used in combination with some cytotoxic drugs, can be schedule-dependent and have concluded that only extensive analysis of their main pathways of action could help in the design of an optimal multi-drug therapy[13,15,16]. The great influence that schedules can have in combination treatments with TK-inhibitors and other cytotoxic drugs is also evinced by the fact that EGFR inhibitors can variously interact with proteins involved in resistance to some conventional cytotoxic drugs[18,19]. In particular, ABCG2 and mdr-P glycoprotein (P-gP), belonging to ATP-binding cassette (ABC) transporters, are involved in sensitivity to topoisomerase I inhibitors[20] and anthracyclines[21].

In vitro data analyzing the activity of TK inhibitors on cell growth , apoptosis induction or cell cycle and target modulation are mainly limited to very short term cell exposures to drugs, generally lasting no longer than 5 d[22-24]. Although such exposure times are useful to clarify the molecular mechanisms of action of this class of drugs, they do not account for some major cell mechanisms controlling the expression and function of EGFR receptors[25]. Some such mechanisms that are known to produce EGFR down-regulation are endocytosis, pH-sensitive dissociation, dephosphorilation by PTP1B, trafficking to the lysosome, etc.[26-29].

Data from in vivo studies on the effects of prolonged exposure to TK inhibitors are scant. An in vivo study on long-term exposure confirmed that TK inhibitors are able to reduce tumour growth when utilized alone or in association with other drugs[9,30] but at least one other study has demonstrated that tumour cell lines can develop resistance to gefitinib[31]. The only in vitro report analyzing the pharmacological and biological effects of prolonged exposure to an EGFR inhibitor supports the hypothesis that duration of cell exposure to such a drug is important in modulating its antitumour effect and synergism with other drugs[32].

The aim of this study was to investigate the cytotoxic and biomolecular effects of different gefitinib and ZD6474 long-term exposure modalities on colon cancer cell lines and, whether phosphorylation of their specific targets (EGFR and/or KDR), activity of downstream signalling molecules and multidrug resistance proteins were modified in an exposure time-dependent manner.

Analysis of the biomolecular effects that the TK inhibitors, gefitinib and ZD6474, can have on tumour cells after prolonged drug exposure is instrumental in leading to optimization of their use in monotherapies and in combination with other biological or conventional cytotoxic drugs[10,11,33,34]. Several cell effects have been demonstrated to depend directly on the modality and duration of cell exposure to these drugs; these effects include receptor expression, function of signal transduction, modulation of drug resistance proteins, etc. Nonetheless, and in spite of the clinical modalities of administration of these drugs, in vitro studies describing the inhibitory effect of gefitinib or ZD6474 on cell growth have considered only short drug exposures of 1-5 d[22-24].

In our study, we assessed the effects of prolonged exposure to the TK inhibitors, gefitinib and ZD6474, on cell viability and on their specific molecular targets by directly monitoring the modulation of the phosphorylated form of EGFR and of the two effectors, Akt and Erk1/2, that are important in the cell survival and proliferation pathways, respectively.

Two weeks exposure to gefitinib resulted in up to 70% cell growth inhibition and no apparent differences between the continuous and the intermittent treatment schedules. Moreover, as already reported for erbitux[32], gefitinib inhibited the phosphorylated forms of the receptor p-EGFR and of the downstream effector p-Erk1/2 involved in the proliferation pathway. The effects of gefitinib on p-Akt were less dramatic and appeared to be cell line-specific.

Comparison of these results with those obtained after short-term drug exposure[13] highlighted differences in the molecular effects produced by different drug schedules. Although a high rate of cells died after 2 wk of drug exposure in our investigation, cells tried to escape the attack by an exogenous agent by further modulating the survival and proliferation pathways. These findings suggest that a combination of Gefitinib and other PI3K/Akt pathway inhibitors, such as mTor inhibitor, may produce a more powerful synergistic effect.

ZD6474 proved to be able to inhibit the growth of our two colon cancer cell lines, thus confirming similar effects previously observed in GEO cells[23]. In both cell lines, the ZD6474-mediated inhibition of growth was associated with almost complete inhibition of p-KDR and p-EGFR, as well as a slight inhibition of p-Akt. In contrast, the modulation of p-Erk1/2 by ZD6474 was evident in HT-29 cells only.

Another aspect we considered was the effect on the expression of the tumour suppressor PTEN. Unlike Nagata, who reported PTEN modulation after 1 hour of exposure to the anti-ErbB2 antibody, trastuzumab[35], no appreciable modulation of this protein was observed in our study after short or long, continuous or alternate drug exposures. Our conclusion was that 14 d of drug exposure may be long enough for recovery of the possibly transient and short-term modulation of this gene potentially occurring in the first few hours after treatment with TK inhibitors.

Tyrosine kinase inhibitors have shown the potential to modulate cytotoxic drug resistance, through an interaction with ABCG2 and PgP[18,19]. In our study, ABCG2, which is involved in camptothecin resistance[20], was detectable in both HT-29 and LoVo cells while PgP, which is involved in anthracycline resistance, was not detectable in either cell line. The increased ABCG2 expression, following exposure to gefitinib or ZD6474, was cell line-specific. The HT-29 cells showed a progressive exposure-related increase of 100%-150%, while expression in the LoVo cells was unaffected. The increased expression of ABCG2 in HT-29 cells was schedule-dependent, and it was higher with continuous than with intermittent incubation. Our results seem to be in contrast with those obtained by Nakamura[19] but the experimental conditions used were completely different. Nakamura used a short time (15 min) to show a gefitinib-dependent increase in topotecan accumulation in transfectant cells, overexpressing ABCG2, and 4 d of gefitinib plus cytotoxic drugs exposure to led to reversal of drug resistance. In our experiments the ability of gefitinib to enhance ABCG2 expression proved to be a late effect of the drug and may account for the antagonism between Topoisomerase-I inhibitors and TK-inhibitors in HT-29 cells induced by a pre-exposure to gefitinib for 5 d[13]. Moreover, the cell-line-specific effects of gefitinib and ZD6474 on drug-related proteins may provide an explanation for the different results. Wakeling and Ciardiello obtained[30,31] when analyzing the onset of drug resistance in vivo after long-term drug intake.

In conclusion, our investigation studied, in an in vitro model of colon cancer, some crucial points related to the clinical use of these TK inhibitor drugs. Evidence has emerged that long term use (maximum 14 d) of these drugs does not lead to a loss of cell activity and intermittent or continuous exposures to these drugs do not produce significantly different toxic effects. The main signal transduction steps for TK receptor pathways were also studied and the findings demonstrated that the effects of these drugs may be highly cell-line-specific (HT-29 vs Lovo), drug-dependent (gefitinib vs ZD6474) and schedule-related (continuous vs intermittent), thus indicating that a predictive factor for these drugs cannot be easily identified or broadly applicable. The data concerning the modulation of drug resistance related proteins in tumour cells treated with long-term drug exposure are even more interesting. ABCG2 expression was shown to be induced by these drugs in a cell-line- specific and schedule-dependent manner. This evidence should suggest caution in choosing exposure times when combining these drugs with Topoisomerase-I inhibitors in particular. The findings of this study can be instrumental in the implementation of future in vitro and clinical studies on these drugs.