Colorectal cancer is a major public health concern. Although chemotherapy appears to be of very limited value in advanced colorectal cancer, there have been many efforts to apply combination chemotherapy in patients with primary disease.1, 2, 3
The combination of fluorouracil and leucovorin used to be recognized as standard therapy for colorectal cancer, but the topoisomerase I inhibitor, irinotecan (CPT-11), has recently been demonstrated to be active against colorectal cancer that was resistant to prior therapy.4, 5 Moreover, the CPT-11/5-FU/LV combination has been approved as standard chemotherapy by the US FDA for metastatic colorectal cancer.6 However, patients treated with CPT-11 plus bolus 5-FU/leucovorin have been found to have a 3-fold higher rate of treatment-induced or treatment-exacerbated death than patients treated with other arms of the respective studies.7 We have therefore been seeking a new combination regimen containing CPT-11 and target-based drugs.
The development of target-based drugs, including receptor tyrosine kinase inhibitors (TKI), is one of the promising strategies for cancer chemotherapy.8, 9 Colorectal cancers express receptors of the type 1 tyrosine kinase family, including epidermal growth factor receptor (EGFR) and c-erbB-2,10, 11, 12 and the EGFR has emerged as a central molecular target for modulation in cancer therapeutics. The correlation between high expression of EGFR and clinically aggressive malignant disease has made EGFR a promising target of therapy for many epithelial tumors, which represent approximately 2/3 of all human cancers. In solid cancers, including colorectal cancers, high EGFR expression correlates with poor prognosis.11 Gefitinib (“Iressa”) is an orally active, selective EGFR-TKI that blocks signal transduction pathways involved in the proliferation and survival of cancer cells and in other host-dependent processes promoting cancer growth.13, 14 In EGFR tyrosine kinase assays, gefitinib has an IC50 of 0.033 μM. Inhibition of c-erbB-2 and KDR occurs at doses 100-fold higher than for EGFR inhibition.15 We have previously demonstrated that gefitinib exerts high growth-inhibitory activity against EGFR-positive tumors in a xenograft model,16 and gefitinib is therefore expected to be a potent therapeutic agent against EGFR-positive colorectal cancers. In recent years, it has been shown that the combined treatment of established human colorectal cancer xenograft with anti-EGFR drug (cetuximab or gefitinib) and with topoisomerase I inhibitor, topotecan, increase the antitumor activity of these drugs.17, 18 The aim of the present study was to investigate the combination effect of gefitinib and CPT-11 and to elucidate the biochemical mechanism of synergistic interaction in colorectal cancers.
MATERIAL AND METHODS
Drugs and chemicals
Gefitinib (N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(morpholin-4-yl)propoxy] quinazolin-4-amine) was provided by AstraZeneca (Cheshire, UK). Gefitinib was dissolved in dimethyl sulfoxide (DMSO) for the in vitro study and suspended in 5% glucose, pH 6, for the in vivo study. CPT-11 was obtained from Yakult Honsha (Tokyo, Japan). CPT-11 was dissolved in 45 mg/ml solvitol (pH 3-4) for both the in vivo and in vitro studies.
Female BALB/c nude mice, 6-weeks-old, were purchased from Japan Charles River Co., Ltd. (Atsugi, Japan). All mice were maintained in our laboratory under specific-pathogen-free conditions.
Cells and culture
Human colorectal cancer cell lines WiDR, LS-174T, COLO320DM, COLO320HSR, Lovo, SW480 and HCT116 were obtained from ATCC (Lockville, MD). Lovo cells, SW480 and HCT116 cells were maintained in HAM's F12 medium (GIBCO BRL, Grand Island, NY), Leibovitz's L-15 medium and McCoy's 5A medium (GIBCO BRL), respectively, all supplemented with 10% heat-inactivated fetal bovine serum (FBS). Other cell lines were maintained in RPMI1640 (Nikken Bio Med. Lab., Kyoto, Japan) supplemented with 10% FBS.
We used the tetrazolium dye [3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT] assay to evaluate the cytotoxicity of various drug concentrations. A 200 ml volume of an exponentially growing cell suspension (5 × 103–1.5 × 104 cells/ml) was seeded into a 96-well microtiter plate and 20 μl of each drug at various concentrations was added. After incubation for 72 hr at 37°C, 20 μl of MTT solution (5 mg/ml in phosphate buffered saline, PBS) was added to each well and the plates were incubated for a further 4 hr at 37°C. After centrifuging the plates at 200g for 5 min, the medium was aspirated from each well, and 180 μl of DMSO was added to each well to dissolve the formazan. Optical density was measured at 562 and 630 nm with a Delta Soft ELISA analysis program interfaced with a Bio-Tek Microplate Reader (EL-340, Bio-Metallics, Princeton, NJ). Each experiment was performed in 6 replicate wells for each drug concentration and carried out independently 3 or 4 times. The IC50 value was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the survival curves. Percent survival was calculated as follows: (mean absorbance of 6 replicate wells containing drugs − mean absorbance of 6 replicate background wells)/(mean absorbance of 6 replicate drug-free wells − mean absorbance of 6 replicate background wells) × 100.
Specific primers designed for EGFR CDS were used for detection of EGFR mRNA as described elsewhere.16 First-strand cDNA was synthesized from the cells' RNA with an RNA PCR Kit (TaKaRa Biomedicals, Ohtsu, Japan). After reverse transcription of 1 μg of total RNA with Oligo(dT)-M4 adaptor primer, the whole mixture was used for PCR with 2 oligonucleotide primers (5′-AATGTGAGCAGAGGCAGGGA-3′,5′GGCTTGGTTTGGAGCTTCTC-3′). PCR was performed with initial denaturation at 94°C for 2 min, 25 cycles of amplification (denaturation at 94°C for 30 sec, annealing at 55°C for 60 sec and extension at 72°C for 105 sec).
Immunoprecipitation and immunoblotting
The cultured cells were washed twice with ice-cold PBS, lysed in EBC buffer (50 mM Tris-HCI, pH 8.0, 120 mM NaCI, 0.5% Nonidet P-40, 100 mM NaF, 200 mM Na orthovanadate and 10 mg/ml each of leupeptin, aprotinin and phenylmethylsulfonyl fluoride). The lysate was cleared by centrifugation at 20,000g for 5 min, and the protein concentration of the supernatant was measured by BCA protein assay (Pierce, Rockford, IL). For Immunoblotting, 20 μg samples of protein were electrophoretically separated on a 7.5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membrane was probed with rabbit polyclonal antibody against EGFR (1005; Santa Cruz Biotech, Santa Cruz, CA), HER2/neu (c-18; Santa Cruz), phospho-EGFR specific for Tyr 845, Tyr 1045, and Tyr 1068 (numbers 2231, 2235 and 2234; Cell Signaling, Beverly, MA) and cleaved PARP (number 9544; Cell Signaling) as the first antibody, followed by horseradish peroxidase-conjugated secondary antibody. The bands were visualized by electrochemiluminescence (ECL, Amersham, Piscataway, NJ). For immunoprecipitation, 5 × 106 cells were washed, lysed in EBC buffer, and centrifuged. The resultant supernatants (1,500 μg) were incubated with the anti-EGFR antibody (1005) at 4°C overnight. The immunocomplex were absorbed onto protein A/G-Sepharose beads, washed 5 times with lysate buffer, denatured and subjected to electrophoresis on a 7.5% polyacrylamide gel followed by immunostaining probed with antiphosphotyrosine antibody (PY-20, BD Bioscience Clontech, Tokyo, Japan).
Combined effect of gefitinib and CPT-11 in vitro
The combined effect of gefitinib and CPT-11 on colorectal cancer cell growth was evaluated by the combination index (CI) analysis method.6 For any given drug combination, CI represents the degree of synergy, additivity or antagonism. CI was expressed in terms of fraction-affected (Fa) values, which represents the percentage of cells killed or inhibited by the drug. Using the mutually exclusive (α=0) or mutually nonexclusive (α=1) isobologram equation, the Fa/CI plots for each cell line was constructed by computer analysis of the data generated from the median effect analysis. CI values were interpreted as follows: <1.0 = synergism; 1.0 = additive and >1.0 = antagonism.
Using the median-effect method, developed by Chou and Talalay, the dose-response curve was plotted for each drug and for multiple doses of a fixed-ratio combination by using the equation:
where, D is the dose-administered, Dm is the dose required for 50% inhibition of growth, fa is the fraction affected by dose D, fu is the unaffected fraction and m is a coefffect curve. The dose-response curve was plotted by logarithmic conversion of the equation to determine the m and Dm values, and the dose Dx required for x percent effect (fa)x was then calculated as
Thus, Cl can be defined by the isobologram equation
where (Dx)1 is the dose of Drug-1 required to produce x percent effect alone, and (D)1 is the dose of Drug 1 required to produce the same x percent effect in combination with Drug 2; similarly, (Dx)2 is the dose of Drug 2 required to produce x percent effect alone and (D)2 is the dose of Drug 2 required to produce the same x percent effect in combination with Drug 1. Theoretically, Cl is the ratio of the combined dose to the sum of the single-drug doses at an isoeffective level. Consequently, Cl values <1 indicate synergism, values >1 indicate antagonism and a value of 1 indicates additive effects. The Cl values obtained from both the classical nonconservative (α=0) and conservative (α=1) isobologram equations are presented in this report.
Growth-inhibition assay in vivo
Experiments were performed in accordance with the United Kingdom Coordinating Committee on Cancer Research Guidelines for the welfare of animals in experimental neoplasia (second edition).
In vivo experiments were scheduled to evaluate the combined therapeutic effect on preexisting tumors of oral or intraperitoneal administration of gefitinib and intravenous injection of CPT-11. The dose of each drug was set based on the results of a preliminary experiment involving administration of each drug alone. Ten days before administration, 1 × 107 WiDR and COLO320DM or 2 × 106 Lovo cells were injected subcutaneously into the back of mice. Five or 6 mice per group were injected with tumor cells. Tumor bearing mice were either given gefitinib, 40 mg/kg/day p.o. on days 1–10, or CPT-11, 40 mg/kg/day i.v. on days 1, 5 and 9, or both, or placebo (5%(w/v) glucose solution). Alternatively, gefitinib, 30 or 60 mg/kg, i.p. days 1–14, and i.v. CPT-11, 16.7 or 33.3 mg/kg, i.v. on days 1, 5 and 9, were administered to the mice. Tumor diameters were measured with calipers on days 1, 4, 7, 10, 14, 18 and 22 to evaluate the effects of treatment, and tumor volume was determined by using the following equation: tumor volume = ab2/2 (mm3) (where a is the largest diameter of the tumor and b is the shortest diameter). Day “x” denotes the day on which the effect of the drugs was estimated, and day “0” denotes the first day of treatment. All mice were sacrificed on day 22 after measuring their tumors.
Differences between the test groups were analyzed by 1-factor ANOVA followed by Fisher's protected least significant difference (PLSD). A value of p<0.05 was considered statistically significant.
EGFR and HER2 expression and EGFR autophsophorylation in colorectal cancer cells
We examined EGFR mRNA expression by RT-PCR analysis using 2 specific primers. Approximately 570 bp-long PCR products were amplified in all cell lines that exhibited expression of EGFR mRNA (Fig. 1a). Comparison of the protein expression levels of EGFR in colorectal cancer cells by immunoblotting (Fig. 1b) revealed high expression in Lovo and WiDR cells. EGFR protein was also detected in LS-174T, COLO320DM, COLO320HSR, HCT116 and SW480 cells, although the expression levels in COLO320DM and COLO320HSR are subtle. The highest expression level of phosphrylated EGFR measured by phospho-specific EGFR antibody (Tyr845, Tyr1045 and Tyr1068) was observed in Lovo cells (Fig. 1b). Because the function of EGFR is closely related to that of other HER families including HER2/neu, we also examined the protein level of HER2/neu. High expression of HER2/neu were observed in LS-174T, HCT-116 and SW480 (Fig. 1b).
Cellular sensitivity of colorectal cancer cells to gefitinib and CPT-11
The growth inhibitory effect of gefitinib and CPT-11 on colorectal cancer cells was examined by MTT assay. The IC50 values of gefitinib for the cell lines ranged from 1.2 μM (Lovo cells) to 160 μM (HCT116 cells) (Table I). No significant relationship was observed between EGFR expression levels and IC50 values among these cell lines. However, Lovo cells, which exhibited the highest EGFR expression and its phosphorylation, were the most sensitive to gefitinib. On the other hand, the IC50 values of CPT-11 for the cell lines ranged from 5.2 μM (Lovo) to 35 μM (SW480). The range of sensitivity to gefitinib was wider than to CPT-11.
Table I. In Vitro Growth-Inhibitory Activity of Gefitinib and CPT-11 in Human Colorectal Cancer Cells (MTT Assay)1
Concentration range (μM)
Concentration range (μM)
The IC50 value (μM) of each drug was measured by MTT assay, as described in the Materials and Methods. Each value is a mean ± SD of 3 or 4 independent experiments–N.D., not determined.
10 ± 1.1
33 ± 7.5
100.4 ± 10.1
11 ± 3.8
11 ± 0.6
177.0 ± 12.2
23 ± 0.6
35 ± 5.5
1.2 ± 0.59
5.2 ± 0.82
In vitro combined effect of gefitinib and CPT-11 on colorectal cancer cell lines
Based on the results of the evaluation of in vitro growth-inhibition, 4 cell lines (WiDR, COLO320DM, Lovo, and SW480 cells) were selected for the in vitro combination study. Cells were treated with gefitinib or CPT-11 alone or in concomitant combination at fixed molar ratio for 72 hr. The ratios of gefitinib and CPT-11 were set based on the IC50 values of each cell line. Growth rate values are averages of data from at least 3 independent experiments. The effects of combinations of gefitinib and CPT-11 on cell growth are shown in Figure 2. CI values of <1, >1 and 1 indicate a supra-additive effect (synergism), antagonistic effect and additive effect, respectively. A low CI index was observed in WiDR, COLO320DM and Lovo cells over a wide range of inhibition levels. Synergistic effects were also observed in the relatively high Fa values in SW480 cells. These results suggest that gefitinib and CPT-11 had a synergistic effect on most of the colorectal cancer cell lines in vitro.
In vivo combination effects of gefitinib and CPT-11
In order to determine whether the combination of these 2 drugs is also synergistic against colorectal cancer in vivo, the growth-inhibitory effect of the combination was evaluated against the colorectal cancer cells in tumor xenografts. The growth inhibitory effect of gefitinib, 30 mg/kg, i.p. days 1–10, and CPT-11, 40 mg/kg, i.v. days 1, 5 and 9, on WiDR cells was evaluated (Fig. 3a,b). Administration of gefitinib or CPT-11 alone suppressed the tumor volume of WiDR cells with a T/C value of 73.9% and 69.2%, respectively, at day 22, (Fig. 3c), whereas gefitinib+CPT-11 suppressed WiDR tumors with T/C value of 51.8% at day 22, but this was not statistically significant (Fig. 3d, p=0.164 by 1-factor ANOVA). A 10% body weight loss was observed until day 15 in mice given the combination, but body weight recovered by day 22 (Fig. 3e). No growth inhibitory effect of single or combined therapy of CPT-11 and gefitinib in COLO320DM cells were observed (data not shown). In mice transplanted with Lovo cells, with a high EGFR expression level, marked tumor growth inhibition was achieved with gefitinib+CPT-11 (Fig. 3f). The T/C of the combination schedule at day 11 was 22.8% and significantly lower than in the control (p<0.0012 by Fisher's PSLD, Fig. 3g). A 10% maximum body weight loss until day 15 was also observed in mice treated with the combination (Fig. 3j).
Alternatively, the combined effect of oral administration of gefitinib and intravenous administration of CPT-11 was evaluated in mice transplanted with Lovo cells. Gefitinib, 30 or 60 mg/kg p.o. days 1–14, and CPT-11, 16.7 or 33.3 mg/kg i.v. days 1, 5 and 9, were administered (schedule 2, Fig. 4a), and greater growth inhibition was observed in mice treated with this combination, compared to the controls (Fig. 4b) A more marked growth-inhibitory effect was observed at a higher dose of CPT-11 (16.7 vs. 33.3 mg/kg), but there was no difference between 30 mg/kg and 60 mg/kg of gefitinib in the combination. The combination of gefitinib (30 and 60 mg/kg) and CPT-11 (33.3 mg/kg/i.v.) resulted in tumor reduction during treatment that was significant at day 15 (Fig. 4c). The T/C values immediately after the completion of treatment (at day 15) and at day 22 are summarized in Fig. 4d. More severe body weight loss was observed, ∼20% at day 15, in mice treated with 60 mg/kg of gefitinib alone or with CPT-11, suggesting that CPT-11 does not enhance the body weight loss induced by gefitinib. Body weight recovered by day 22 (Fig. 4e). No deaths of were observed during the treatment or observation period.
Induction of EGFR phosphorylation and enhanced gefitinib-induced PARP activation by CPT-11
To elucidate the synergistic effects of CPT-11 and gefitinib, we examined the effect of exposure of CPT-11 on EGFR phosphorylation in Lovo and WiDr cells. Phosphorylated EGFR was detected with anti-phosphotyrosine antibody (PY-20) against immunoprecipitated EGFR and increased phosphorylation of EGFR was observed after exposure to CPT-11 in Lovo cells in dose- and time- dependent manner (3–24 hr) (Fig. 5a). The dose-dependent activation of EGFR by CPT-11 was also obtained in WiDR cells (Fig. 5b). CPT-11-induced phosphorylation of EGFR was observed without ligand-stimulation. The EGFR activation was completely inhibited by 24 hd exposure of 5 μM gefitinib. gefitinib-induced apoptosis measured by PARP activation was enhanced by combination with CPT-11, although no PARP activation was induced by CPT-11 alone (Fig. 5c). These results suggest that the modification of EGFR by CPT-11 increases the cellular sensitivity to gefitinib, resulting the synergistic effect of CPT-11 and gefitinib. We also observed the effect of gefitinib on the expression and the activity of topoisomerase I by immunoblotting and decatenatnion assay. No modification of topoisomerase I by gefitinib was observed (data not shown).
Evidence has suggested that the new EGFR-targeting drug gefitinib is active against gastrointestinal malignancies as well as non-small cell lung cancer. Combination of gefitinib with cytotoxic drugs has been evaluated in the U.S. and Europe,19, 20 but combination with CPT-11 has not been evaluated. CPT-11 is a potent DNA-targeting drug in patients with colorectal cancer that is refractory to treatment with fluorouracil and leucovorin,4, 5 although a higher rate of treatment-induced toxicity was suspected in a retrospective analysis.7 In preclinical study, Ciadiello et al.17, 18 reported that supra-additive combination effect of EGFR-targeting drug (cetuximab or gefitinib) and topoisomerase I inhibitor, topotecan was observed in human colorectal cancer GEO xenograft. We have therefore studied the synergistic potential for a new combination regimen containing CPT-11 and gefitinib. The synergistic potential of CPT-11 combined with gefitinib demonstrated in our study suggests that the gefitinib/CPT-11 combination is a promising regimen for colorectal cancer patients. Schedule 2, administration of oral gefitinib and intravenous CPT-11 designed in a xenograft model, was based on possible clinical administration of the drugs, and thus a treatment schedule consisting of intermittent i.v. CPT-11 and continuous gefitinib p.o. may be applicable to colorectal cancer in humans.
In xenograft models, body weight loss was observed when administered in combination as well as when each drug was administered alone. However, body weight loss rapidly recovered immediately after the completion of administration, and no deaths were observed. Diarrhea is the dose-limiting toxicity of CPT-11 in humans,7 and it is also observed in patients treated with gefitinib.21, 22 However, no diarrhea or related phenomena were observed in the mouse model treated with combinations of these drugs. These results suggest that this regimen is intensive but can be tolerated, at least in mice.
The in vitro and in vivo experiments in our study demonstrated the synergistic potential of gefitinib − CPT-11 combination. We previously reported that topoisomerase I up-regulation by counter-part drugs was a possible mechanism for the synergy in an CPT-11 containing regimen.23 On the other hand, the synergistic potential of gefitinib with topotecan, cisplatin, paclitaxel or radiation has been reported.18, 24, 25, 26, 27, 28 To elucidate the biochemical mechanism underlying the synergistic interaction between the gefitinib and CPT-11, the effect of CPT-11 on EGFR-phosphorylation was examined (Fig. 5). Increased phosphorylation of EGFR was observed after exposure to CPT-11 in dose and time-dependent manner in WiDR and Lovo cells. Since EGFR expression and phosphorylation were the major determining factors for sensitivity of the cells to gefitinib-induced growth-inhibition,14 biochemical modulation of EGFR by CPT-11 might be responsible for the synergistic interaction between gefitinib and CPT-11. EGFR is induced and activated by cellular stress, such as oxidative stress and UV irradiation.29, 30, 31, 32, 33, 34 Ohmori et al.22 demonstrated that increased autophosphorylation of EGFR was obtained in cisplatin-exposure in human lung cancer cells. A number of reports suggest that EGFR promotes cell survival through the activation of the ERK or the AKT pathway.31, 32 EGFR activation induced by these cellular stress may play a survival response against apoptosis.31, 32 In the present study, PARP activation by gefitinib was markedly enhanced by combination with CPT-11 at 5 μM exposure, which is comparable with IC50 value of CPT-11 in Lovo cells, although no PARP activation was observed by monotherapy of CPT-11. On the other hand, gefitinib does not modify the expression and the activation of topoisoerase I (data not shown). These result suggest that the inhibitory effect of gefitinib on the activated survival signal transduction induced by CPT-11 lead to synergistic effect. The findings of the present study suggest that biological modulation by various anticancer agents including DNA damaging agents will contribute to the synergistic effects of these anticancer agents and gefitinib in EGFR expressing tumor and support clinical evaluation of gefitinib in combination with DNA-targeting agents, especially CPT-11, in the treatment of colorectal cancers.