Sequence-dependent antiproliferative effects of cytotoxic drugs and epidermal growth factor receptor inhibitors

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1 Annals of Oncology 16 (Supplement 4): iv61 iv68, 2005 doi: /annonc/mdi910 Sequence-dependent antiproliferative effects of cytotoxic drugs and epidermal growth factor receptor inhibitors M. P. Morelli 1, T. Cascone 1, T. Troiani 1,2, F. De Vita 1, M. Orditura 1, G. Laus 3, S. G. Eckhardt 2, S. Pepe 3, G. Tortora 3 & F. Ciardiello 1 * 1 Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale F. Magrassi e A. Lanzara, Seconda Università degli Studi di Napoli, Napoli, Italy; 2 University of Colorado Health Sciences Center, Denver, CO, USA; 3 Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Università degli Studi di Napoli Federico II, Napoli, Italy Introduction Background: Epidermal growth factor receptor (EGFR) inhibitors are in clinical development in cancer treatment. Preclinical studies have shown potential antitumor efficacy of these agents in combination with chemotherapy or with radiotherapy. However, controversial results have been obtained in different clinical trials. Materials and methods: The effects on proliferation, cell cycle distribution and induction of apoptosis of three different anti-egfr agents (gefitinib, ZD6474, cetuximab) were evaluated in different sequences of combination with either a platinum derivative (cisplatin, carboplatin, oxaliplatin) or a taxane (docetaxel, paclitaxel) in KYSE30 cells, a model of a human cancer cell line with a functional EGFR autocrine pathway. Results: The combination of a cytotoxic drug with an EGFR inhibitor caused different antiproliferative effects on KYSE30 cancer cells depending on the treatment schedule. An antagonistic effect was observed when treatment with each EGFR inhibitor was done before chemotherapy. In contrast, a synergistic antiproliferative activity was obtained when chemotherapy was followed by treatment with EGFR antagonists. This effect was accompanied by potentiation of apoptosis and arrest of the surviving cancer cells in the G 2 /M phases of the cell cycle. Conclusions: This study provides a rationale for the evaluation of a potentially synergistic sequence of cytotoxic drugs and EGFR inhibitors in a clinical setting. Key words: Cytotoxic drugs, combination therapy, epidermal growth factor receptor inhibitors, monoclonal antibodies, small molecule tyrosine kinase inhibitors Experimental and clinical work supports the view that the epidermal growth factor receptor (EGFR) is a relevant target for cancer therapy [1]. Different modalities have been used to block EGFR activation and/or function in cancer cells [2 4]. Two therapeutic approaches have shown clinical activity in advanced human cancers: monoclonal antibodies and small molecule inhibitors of the EGFR tyrosine kinase enzymatic activity [2 4]. Monoclonal antibodies (MAbs) are generally directed at the external domain of the EGFR to block ligand binding and receptor activation. Tyrosine kinase inhibitors (TKI) block the autophosphorylation of the tyrosine kinase domain of the EGFR. Three EGFR inhibitors are currently *Correspondence to: Dr Fortunato Ciardiello, Cattedra di Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale F. Magrassi e A. Lanzara, Seconda Università degli Studi di Napoli, Via S. Pansini, Naples, Italy. Tel: ; Fax: ; fortunato.ciardiello@unina2.it licensed in USA and several other countries worldwide for chemotherapy-refractory cancers. The blocking anti-egfr MAb cetuximab has been approved for the treatment of irinotecan-resistant colorectal cancer; whereas two EGFR-TKIs, erlotinib and gefitinib, are available for the second and the third line treatment, respectively, of advanced non-small cell lung cancer. Overexpression of EGFR is associated with resistance to hormonotherapy, cytotoxic drugs or radiotherapy [1, 2 4]. In this respect, it has been postulated that EGFR overexpression would serve as a cell survival response to counteract apoptotic signaling in cancer cells exposed to a cytotoxic damage [5]. Preclinical studies with cetuximab, gefitinib, erlotinib and, more recently, with the dual EGFR- and vascular endothelial growth factor receptor (VEGFR)-TKI, ZD6474, demonstrate that these agents potentiate the antitumor activity of standard cytotoxics and radiotherapy [2 4, 6 10]. Taken together, these studies support the hypothesis that cellular damage induced by chemotherapy or by ionizing radiations can convert EGFR ligands from growth factors into survival factors q 2005 European Society for Medical Oncology

2 iv62 for cancer cells that express functional EGFR [5]. In this situation, the blockade of EGFR signaling in combination with cytotoxic drugs or with radiotherapy could cause irreparable cancer cell damage leading to increased programmed cell death. Several studies have been conducted to translate these preclinical results in a clinical setting [3 4]. Clinical trials have generally been performed with conventional chemotherapy regimens in combinations with anti-egfr drugs given continuously in the case of small molecule, orally bioavailable EGFR-TKIs, or mostly on a weekly basis for the anti-egfr MAbs which are usually administered intravenously to the patient. However, conflicting results have been obtained. For example, cetuximab seems to increase the antitumor activity of standard chemotherapy regimens in metastatic colorectal cancer patients [11 12], whereas no effect on antitumor activity and no survival advantage has been obtained by the combined treatment of advanced non-small cell lung cancer patients with either gefitinib or erlotinib and standard 2-drug chemotherapy regimens [13 16]. Relevant clinical issues are still open for the optimal use of EGFR antagonists in combination with cytotoxic drugs. Clinical and pathologic criteria for the selection of potentially responding patients are not yet well understood. Although EGFR expression is necessary for the activity of these drugs, it is not sufficient to define a cancer as EGFR-dependent. Novel clinical pharmacology studies are necessary to better understand the pharmacokinetic and pharmacodynamic interactions of EGFR inhibitors and chemotherapy with the aim of designing rationale schedules of treatment. In this respect, some preclinical studies suggest that the antitumor effects of combining EGFR inhibitors and cytotoxic drugs could be schedule-dependent [17]. In the present study, we have used a human epithelial cancer cell line with a functional EGFR-dependent autocrine pathway as an in vitro model for defining the differential effects of various schedules of combination of anti-egfr agents and cytotoxic drugs on cell growth, cell cycle distribution and induction of apoptosis. For this purpose, we have tested three EGFR inhibitors (gefitinib, ZD6474, cetuximab) and five chemotherapeutic agents belonging to two classes of drugs: platinum-derivatives (cisplatin, carboplatin, oxaliplatin) and taxanes (docetaxel, paclitaxel). Materials and methods Drugs Gefitinib and ZD6474 were kindly provided by Dr Anderson Ryan, Astra- Zeneca Pharmaceuticals (Macclesfield, UK). Cetuximab was a gift of Merck Pharma Italy (Milan, Italy). Paclitaxel, cisplatin and carboplatin were purchased from Sigma Chemicals Co., St. Louis, MO. Docetaxel and oxaliplatin were obtained from Sanofi Aventis Italy (Milan, Italy). Cell line Human KYSE30 esophageal squamous epithelial cancer cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI medium supplemented with 20% heat-inactivated fetal bovine serum (FBS), 20 mm 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), ph 7.4, penicillin (100 UI/ml), streptomycin (100 mg/ml) and 4 mm glutamine (ICN, Irvine, UK) in a humidified atmosphere of 95% air and 5% CO 2 at 378C. Figure 1. (A) IC 50 values for each drug were calculated by performing dose-response experiments with: gefitinib (range, mm) each day for 3 days of treatment; ZD6474 (range, mm) each day for 3 days; cetuximab (range, mg/ml) each day for 3 days; cisplatin (range, mm) for 24 hours; carboplatin (range, mm) for 24 hours; oxaliplatin (range, mm) for 24 hours; docetaxel (range, ng/ml) for 24 hours; paclitaxel (range, nm) for 24 hours. Each experiment was performed in triplicate. (B) Western blot analysis of protein expression following EGFR inhibitor treatment in KYSE30 cancer cells. Cells were treated with: lane 2, gefitinib (1 mm); lane 3, ZD6474 (1 mm); or lane 4, cetuximab (2.5 mg/ml) for 24 h. Lane 1, control untreated cells. For the evaluation of EGFR and P-EGFR expression, total cell protein extracts were immunoprecipitated with the MAb 528 anti-egfr MAb, resolved by a 7.5% SDS PAGE and probed with either the PY20 anti-p-tyr MAb (P-EGFR) or an anti-human EGFR Mab (EGFR). For the evaluation of MAPK, P-MAPK, AKT, P-AKT expression, total cell protein extracts were fractionated through 4 20% SDS PAGE, transferred to nitrocellulose filters, and incubated with the appropriate antibodies as described in Materials and methods. Immunoreactive proteins were visualized by enhanced chemiluminescence.

3 iv63 Table 1. Effects on KYSE30 cancer cell growth of the combination of EGFR inhibitors and cytotoxic drugs Sequence Cisplatin Carboplatin Oxaliplatin Docetaxel Paclitaxel Gefitinib followed by chemotherapy (CI at IC 50 ) Chemotherapy followed by gefitinib (CI at IC 50 ) ZD6474 followed by chemotherapy (CI at IC 50 ) Chemotherapy followed by ZD6474 (CI at IC 50 ) Cetuximab followed by chemotherapy (CI at IC 50 ) Chemotherapy followed by cetuximab (CI at IC 50 ) CI values were calculated according to the Chou and Talalay mathematical model for drug interactions using the Calcusyn software. As a measure of interaction, CI values at IC 50 are indicated for the two different sequences of combination therapy: 1, EGFR inhibitor (gefitinib, ZD6474 or cetuximab) treatment for three consecutive days followed by 24 hours of treatment with either of each cytotoxic drug (cisplatin, carboplatin, oxaliplatin, docetaxel, paclitaxel) or 2, treatment with a cytotoxic drug (cisplatin, carboplatin, oxaliplatin, docetaxel, paclitaxel) for 24 hours followed by EGFR inhibitor (gefitinib, ZD6474 or cetuximab) treatment for three consecutive days. KYSE30 cell viability was determined 7 days after plating the cells using the MTT assay. KYSE30 cancer cells were treated with 7 different concentrations of gefitinib (range, mm), ZD6474 (range, mm), cetuximab (range, mg/ml), cisplatin ( mm), carboplatin ( mm), oxaliplatin ( mm), docetaxel ( ng/ml), paclitaxel ( nm). Each experiment was performed in triplicate. CI is a quantitative measure of the degree of interaction between different drugs. When CI is equal to 1, it denotes additivity; if CI is greater than 1, antagonism; CI values between 1 and 0.7 indicate slight synergism; CI values of 0.7 to 0.3, synergism; CI values less than 0.3, strong synergism. Figure 2. Chou and Talalay method-derived CI plots for the growth inhibitory effects on KYSE30 cancer cells of the sequence of treatment with: carboplatin followed by ZD6474 (A); cisplatin followed by ZD6474 (B); oxaliplatin followed by ZD6474 (C); docetaxel followed by ZD6474 (D); paclitaxel followed by ZD6474 (E). CI is a quantitative measure of the degree of interaction between different drugs. When CI is equal to 1, it denotes additivity; if CI is greater than 1, antagonism; CI values between 1 and 0.7 indicate slight synergism; CI values of 0.7 to 0.3, synergism; CI values less than 0.3, strong synergism.

4 iv64 Immunoprecipitation and Western blot analysis Total cell protein extracts were obtained from KYSE30 cells, which were cultured for 24 h in the presence or absence of gefitinib, 1 mm, ZD6474, 1 mm, or cetuximab, 2.5 mg/ml. For the assessment of EGFR expression and evaluation of EGFR phosphorylation, proteins were first immunoprecipitated with the anti-egfr MAb 528, kindly provided by Dr John Mendelsohn, University of Texas MD Anderson Cancer Center, Houston, TX, USA. Subsequently, immunoprecipitates were resolved by a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and probed with either an anti-human EGFR MAb (Transduction Laboratories, Lexington, KY, USA) or the PY20 anti-ptyrosine MAb (Transduction Laboratories). Furthermore, total cell protein extracts from KYSE30 cells were resolved by a 4 20% SDS PAGE and probed with one of the following antibodies: anti-akt mouse MAb (Cell Signaling), anti-phosho (ser 473)-akt mouse MAb (Cell Signaling); anti-erk 1 2 mouse MAb (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-phospho-erk1/2 mouse MAb (Santa Cruz Biotechnology). Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham International, UK). Evaluation of antiproliferative effects The antiproliferative activity of single agent treatment was assessed in monolayer culture conditions by plating KYSE30 cancer cells in 48 multiwell cluster dishes (Becton Dickinson). After 24 h, cells were treated with different concentrations of: gefitinib each day for 3 days; ZD6474 each day for 3 days; or cetuximab each day for 3 days; cisplatin for 24 h; carboplatin for 24 h; oxaliplatin for 24 h; docetaxel for 24 h; paclitaxel for 24 h. After 7 days from the beginning of the experiment the proportion of surviving cells was determined using the 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. All the experiments were performed in triplicate. For the evaluation of the antiproliferative effects of the combined treatment with a cytotoxic drug and an EGFR antagonist, two treatment schedules were used: 1, EGFR inhibitor (gefitinib, ZD6474 or cetuximab) treatment for three consecutive days followed by 24 h of treatment with either of each cytotoxic drug (cisplatin, carboplatin, oxaliplatin, docetaxel, paclitaxel); or 2, treatment with a cytotoxic drug (cisplatin, carboplatin, oxaliplatin, docetaxel, paclitaxel) for 24 h followed by EGFR inhibitor (gefitinib, ZD6474 or cetuximab) treatment for three consecutive days. KYSE30 cell viability was determined 7 days after plating the cells using the MTT assay. Each experiment was performed in triplicate. The results of the sequential treatment with cytotoxic drugs and EGFR inhibitors were analyzed according to the method of Chou and Talalay [18 19], by using the Calcusyn software program (Biosoft, Cambridge, UK). The resulting Combination Index (CI) is a quantitative measure of the degree of interaction between different drugs. When CI is equal to 1, it denotes additivity; if CI is greater than 1, antagonism; CI values between 1 and 0.7 indicate slight synergism; CI values of 0.7 to 0.3, synergism; CI values less than 0.3, strong synergism. To evaluate the antiproliferative efficacy of repeated weekly chemotherapy dosing along with EGFR inhibitor intermittent or continous treatment, KYSE30 cells were plated in 12 multiwell cluster dishes (Becton Dickinson) on day 1. On day 2 and day 9, cells were treated with either docetaxel (1 ng/ml) or oxaliplatin (5 mm) for 24 h, washed and treated with either gefitinib (1 mm), ZD6474 (1 mm), or cetuximab (2.5 mg/ml), each day for 3 days (days 3 to 5 and 10 to 12), or alternatively treated continuously with an EGFR inhibitor at the same doses on days 3 to 12. KYSE30 cell survival was measured on day 15 after plating the cells by the MTT assay. Each experiment was performed in triplicate. Flow cytometric analysis of cell cycle distribution and cell death To evaluate the effects on cell cycle and the induction of apoptosis, KYSE30 cells were plated in 100 mm tissue culture dishes (Becton Dickinson) and treated with the indicated concentrations of either gefitinib, ZD6474, cetuximab, docetaxel, or oxaliplatin alone or in the sequence of a cytotoxic drug for 24 h followed by an EGFR antagonist for 48 h. After additional 24 h, both adherent and detached cells were harvested. Flow cytometric analysis of apoptotic cell death was performed on cell pellet fixed in 70% ethanol, washed in PBS and mixed with ribonuclease (Sigma) and propidium iodide (Sigma) solution as previously reported [20]. DNA content was analyzed by a FACScan flow-cytometer (Becton Dickinson, San Jose, CA) coupled with a Hewlett Packard computer, and the percent of apoptotic cells was calculated by gating the hypodiploid region on the DNA content histogram using the LYSYS software (Becton Dickinson) as previously reported [20]. Cell cycle data analysis was performed using the CELL-FIT software (Becton Dickinson) as previously reported [20]. Results We have selected KYSE30, a human esophageal squamous epithelial cancer cell line, which possesses an amplified EGFR gene and has high EGFR protein expression with a functional EGFR-dependent autocrine growth. We evaluated the effects on KYSE30 cell growth of three different EGFR inhibitors, two small molecule EGFR-TKIs, such as gefitinib and ZD6474, and a blocking anti-egfr MAb, cetuximab. Similarly, the cytotoxic effects of three platinum derivatives, cisplatin, carboplatin and oxaliplatin, and of two taxanes, docetaxel and paclitaxel were determined. A dose-response Table 2. Cell cycle distribution of KYSE30 cells after treatment with cytotoxic drugs followed by EGFR inhibitors G 0 /G 1 (%) S (%) G 2 /M (%) Control Gefitinib ZD Cetuximab Docetaxel Docetaxel followed by gefitinib Docetaxel followed by ZD Docetaxel followed by cetuximab Oxaliplatin Oxaliplatin followed by gefitinib Oxaliplatin followed by ZD Oxaliplatin followed by cetuximab KYSE30 cells were plated in 100 mm tissue culture dishes as described in Materials and Methods. After 24 hours, the cells were treated for 24 hours with docetaxel, 1 ng/ml, oxaliplatin, 5 mg/ml, or for 48 hours with gefitinib, 1 mm, ZD6474, 1 mm, cetuximab, 2.5 mg/ml. Alternatively, KYSE30 cells were treated first with the same dose of cytotoxic drug for 24 hours and subsequentely with the EGFR inhibitor for 48 hours. Cell cycle analysis of surviving cells was done 96 hours after plating the cells. Each experiment was performed in duplicate.

5 iv65 Figure 3. KYSE30 cancer cells were treated for 24 hours with docetaxel, 1 ng/ml (B), oxaliplatin, 5 mg/ml (C), or for 48 hours with gefitinib, 1 mm (D), cetuximab, 2.5 mg/ml (E), ZD6474, 1 mm (F). A, Control, untreated cells. Both adherent and detached cells were harvested and flow cytometric analysis of cell death was performed. Each experiment was done in duplicate. growth inhibitory effect was observed. Figure 1A summarizes the 50% inhibitory concentration (IC 50 ) of all drugs which were tested. We next evaluated the effects of EGFR inhibitor treatment on EGFR activation and on two key intracellular mediators of EGFR-driven mitogenic activity, such as MAPK and AKT. As shown in Figure 1B after 24 h of treatment with each EGFR inhibitor an almost complete inhibition of the activated, phosphorylated forms of EGFR, AKT and MAPK was observed. To determine whether a potentiation of the antiproliferative activity could be obtained by the combination of chemotherapy and EGFR inhibitors, a series of experiments were performed on KYSE30 cells which were treated with different doses of each chemotherapy agent and each EGFR inhibitor with two opposite sequences of treatment. To determine any interaction in cytotoxicity between EGFR inhibitors and chemotherapy, cell growth experiments were performed according to the Chou and Talalay method [18 19]. As illustrated in Table 1, a clear synergistic growth inhibitory effect was found with every drug used only for the sequence in which chemotherapy was followed by EGFR inhibitor treatment. As an example, combination index (CI) plots of KYSE30 cell growth inhibition are shown for the sequence of chemotherapy followed by ZD6474 treatment (Figure 2). In contrast, the reverse sequence, i.e. EGFR inhibitor followed by chemotherapy, resulted for all drug tested in an interaction which was of antagonistic type (CI > 1.0) (Table 1). We also evaluated the effects of chemotherapy and/or of EGFR inhibitor treatment on KYSE30 cell cycle distribution and on the induction of programmed cell death. For these experiments, we have chosen oxaliplatin and docetaxel as prototypes of a platinum derivative and of a taxane, respectively. A flow cytometric analysis of cell cycle distribution revealed that 48 hours of treatment with either gefitinib, ZD6474, or cetuximab caused a marked increase in the proportion of cells in the G 0 /G 1 phases (Table 2). Docetaxel treatment for 24 hours determined an accumulation of cells in G 2 /M, whereas oxaliplatin treatment induced mostly an S phase arrest. These effects on the cell cycle distribution of surviving KYSE30 cells were accompanied by a relatively modest induction of apoptosis by single agent treatment (Figure 3). However, the sequence of either docetaxel or oxaliplatin for

6 iv66 Figure 4. (A), Oxaliplatin followed by ZD6474; (B), oxaliplatin followed by gefitinib; (C), oxaliplatin followed by cetuximab; (D), docetaxel followed by ZD6474; (E), docetaxel followed by gefitinib; (F), docetaxel followed by cetuximab. KYSE30 cancer cells were treated with either oxaliplatin, 5 mg/ml, or docetaxel, 1 ng/ml, for 24 hours and subsequently with gefitinib, 1 mm, cetuximab, 2.5 mg/ml, or ZD6474, 1 mm, for 48 hours. Both adherent and detached cells were harvested and flow cytometric analysis of cell death was performed. Each experiment was done in duplicate. 24 hours followed by each EGFR inhibitor for 48 hours caused a marked 3- to 6-fold induction of apoptosis in KYSE30 cells with a marked accumulation of the surviving cells in the G 2 /M phases of the cell cycle (Figure 4 and Table 2). In contrast, the sequence of each EGFR inhibitor for 48 hours followed by either docetaxel or oxaliplatin for 24 hours determined a marked KYSE30 cell growth arrest in G 0 /G 1 without induction of apoptosis (data not shown). To characterize further the optimal in vitro combination of chemotherapy and EGFR inhibitor, KYSE30 cells were treated for 24 hours with either docetaxel or oxaliplatin for two times every 7 days, or with the same chemotherapy schedule followed by continuous dosing of each EGFR inhibitor for up to 12 days or by intermittent dosing of each EGFR inhibitor only for the three days after each chemotherapy treatment. As shown in Figure 5, the most effective antiproliferative results were found when chemotherapy was given with the sequential intermittent administration of the EGFR inhibitor, whereas the schedule of prolonged EGFR inhibitor treatment in combination with chemotherapy did not result in any increase of KYSE30 cell growth inhibition as compared to single agent treatment. Discussion The possibility of combining conventional cytotoxic drugs with novel agents that specifically interfere with key pathways controlling cancer cell survival, proliferation, invasion and/or metastatic spreading has generated a wide interest [21]. This could be a promising therapeutic approach for several reasons. First, since the cellular targets for these agents and their mechanism(s) of action are different from those of cytotoxic drugs, it is possible their combination with chemotherapy without cross-resistance. Second, alterations in the expression and/or the activity of genes that regulate mitogenic signals not only can directly cause perturbation of cell growth, but also may affect the sensitivity of cancer cells to conventional chemotherapy and radiotherapy [5, 21]. The EGFR autocrine pathway is one of the more frequently growth factor-driven mechanisms in the development and progression of human epithelial cancers. For this reason, in the past few years several preclinical and clinical studies have been conducted to test the antitumor activity of selective EGFR antagonists and cytotoxic therapies. In the present study, we have demonstrated in an in vitro model of an EGFR-positive human cancer epithelial cell line

7 iv67 Figure 5. (A), Oxaliplatin in combination with ZD6474; (B), oxaliplatin in combination with gefitinib; (C), oxaliplatin in combination with cetuximab; (D), docetaxel in combination with ZD6474; (E), docetaxel in combination with gefitinib; (F), docetaxel in combination with cetuximab. KYSE30 cancer cells were plated in 12 multiwell cluster dishes on day 1 as described in Materials and Methods. Cells were treated as follows: with either docetaxel (1 ng/ml) or oxaliplatin (5 mg/ml) for 24 h on days 2 and 9 (lane 1); with either gefitinib (1 mm), ZD6474 (1 mm), or cetuximab (2.5 mg/ml), each day for 10 days from days 3 to 12 (lane 2); with either gefitinib (1 mm), ZD6474 (1 mm), or cetuximab (2.5 mg/ml), each day from days 3 to 5 and 10 to 12 (lane 3); with either docetaxel (1 ng/ml) or oxaliplatin (5 mg/ml) for 24 h on days 2 and 9, washed and treated with either gefitinib (1 mm), ZD6474 (1 mm), or cetuximab (2.5 mg/ml), each day for 10 days from days 3 to 12 (lane 4); with either docetaxel (1 ng/ml) or oxaliplatin (5 mg/ml) for 24 h on days 2 and 9, washed and treated with either gefitinib (1 m), ZD6474(1 mm), or cetuximab (2.5 mg/ml), each day from days 3 to 5 and 10 to 12 (lane 5). KYSE30 cell survival was measured on day 15 after plating the cells by the MTT assay. Each experiment was performed in triplicate. with a functional EGFR-dependent autocrine growth pathway that the potentiation of the antiproliferative activity of selected cytotoxic drugs, such as platinum derivatives and taxanes, by combined treatment with EGFR inhibitors is schedule and sequence dependent. In fact, for all drugs tested, only when KYSE30 cancer cells were first exposed to a cytotoxic drug and then treated with an EGFR inhibitor a frankly synergistic growth inhibitory effect was observed. This was accompanied by a significant increase in cancer cell death and in a profound effect on cell cycle distribution of the surviving cancer cells which were accumulating in the G 2 /M phases of the cell cycle. To our knowledge, this study represents the most extensive in vitro evaluation of the antitumor activity of combining structurally and functionally different EGFR inhibitors, such as two small molecule EGFR-TKIs and an anti-egfr blocking Mab, with selected cytotoxics, such as platinum derivatives and taxanes. Similary to our study, Xu et al. Have recently shown a sequence-dependent antiproliferative synergy between gefitinib and oxaliplatin in HT-29 and LoVo human colon cancer cell lines [22 23]. The same authors have extended these observations to the combination of gefitinib and the topoisomerase I inhibitor irinotecan [24]. Collectively, our and other studies suggest that a more effective approach to combine chemotherapy with EGFR targeted agents is a sequence in which cytotoxic drug treatment is followed by EGFR inhibitor administration. The results of our study also suggest that, when multiple cycle of therapy are given, this sequence should be recycled with a relatively short-term and intermittent exposure to the EGFR antagonist after chemotherapy in order to avoid negative cell kinetic interactions. Phase I and II clinical trials which explore the clinical activity of this biologic and pharmacologic rationale are currently ongoing. Acknowledgements This study was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC). We thank Dr Anderson Ryan, AstraZeneca Pharmaceuticals, Macclesfield, UK, for the generous gift of gefitinib and of ZD6474 and for helpful discussions. References 1. Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995; 19: Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 2001; 7: Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003; 21:

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