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Keywords:

  • combination;
  • gefitinib;
  • “Iressa”;
  • colorectal cancer;
  • irinotecan

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib (“Iressa”) in combination with the DNA topoisomerase I inhibitor CPT-11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT-PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC50 range of 1.2–160 μM by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT-11 induced supra-additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT-11 had a supra-additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT-11-exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT-11 increased phosphorylation of EGFR in Lovo and WiDR cells in time- and dose-dependent manners. This EGFR activation was completely inhibited by 5 μM gefitinib and gefitinib-induced apoptosis was enhanced by combination with CPT-11, measured by PARP activation although no PARP activation was induced by 5 μM CPT-11 alone. These results suggested that these modification of EGFR by CPT-11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT-11 and gefitinib. These findings imply that the EGFR-TKI gefitinib and CPT-11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT-11, in the treatment of colorectal cancers. © 2003 Wiley-Liss, Inc.

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

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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.

Animals

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.

Growth-inhibition assay

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.

RT-PCR

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:

  • equation image

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

  • equation image

Thus, Cl can be defined by the isobologram equation

  • equation image

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.

Statistical analysis

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.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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).

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Figure 1. EGFR expression in colorectal cancer cells. (a) Expression of EGFR mRNA in each cell line was detected by RT-PCR using specific primers designed for EGFR CDS. Expression of G3PDH mRNA was detected. Twenty-five cycles of PCR amplification were performed for each PCR product. Lanes 1–7 represent LS-174T, WiDR, COLO320DM, COLO320HSR, HCT116, Lovo and SW480 cells, respectively. (b) A 20 μg sample of total cell lysates was separated by 7.5% SDS-PAGE, transferred to PVDF membrane, and incubated with a specific anti-human EGFR, HER2/neu and phospho-EGFR (Tyr845, Tyr992 and Tyr1068).

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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
Cell linegefitinibCPT-11
IC50 (μM)Concentration range (μM)IC50 (μM)Concentration range (μM)
  • 1

    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.

WiDR10 ± 1.10.83–5333 ± 7.51.6–160
LS-174T100.4 ± 10.1N.D.13N.D.
COLO320DM11 ± 3.80.63–10011 ± 0.61.6–160
COLO320HSR22N.D.5.5N.D.
HCT116177.0 ± 12.2N.D.11N.D.
SW48023 ± 0.61.6–1035 ± 5.51.6–50
Lovo1.2 ± 0.590.31–255.2 ± 0.820.16–10

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.

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Figure 2. Combination index (CI) plots of interactions between gefitinib and CPT-11. Cells were treated with gefitinib and CPT-11 alone and in combination at fixed molar ratios (molar ratios of gefitinib to CPT-11 of 3:1 and 1:1 [(a) WiDR], 4:1 and 1:1 [(b) COLO320DM], 1:2 and 1:5 [(c) Lovo], 1:1 [(d) SW480]. Using the mutually exclusive (CI) or mutually nonexclusive (CI′) isobologram equation, the affected fraction (Fa)-CI plot for each cell was constructed by computer analysis of the data generated from the median effect analysis. CI values <1 occurred over a wide range of inhibition levels, indicating synergy.

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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).

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Figure 3. In vivo combined effect of gefitinib and CPT-11 on WiDR and Lovo tumor xenografts. (a) Treatment schedule. (b) (WiDR) and F (Lovo), Tumor growth curves. Female nude mice bearing WiDR or Lovo xenografts were randomly allocated to treatment with 5% (w/v) glucose solution (diamond), gefitinib (square), CPT-11 (triangle), or the combination (×). Tumor volume was calculated as described in Material and Methods. Each data point represents the mean tumor volume of 5 mice. E (WiDR) and J (Lovo) Percent change in body weight in the gefitinib (hatched square) and combination (×) group. C (WiDR) and G (Lovo) Ratio of tumor volume in the control (C) to tumor volume in the treatment group (T) at day 22 and day 15. D (WiDR), H and I (Lovo) Histogram of mean tumor volume at day 11 and day 22 bars, S.D. Statistical analysis was performed by 1-factor ANOVA, followed by Fisher's PLSD between 2 groups, as described in the Material and Methods section. *Significant difference (p<0.05; Fisher's PLSD) compared to the control.

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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.

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Figure 4. The dose-dependent effect of combination therapy on Lovo cells in vivo. (a) Treatment schedule. (b) Significant growth-inhibition was observed in mice treated with the combination. Mice were allocated to 9 groups (6 mice/group) [closed diamond, 5%(W/V) glucose solution; ×, CPT-11 16.7 mg/kg; + CPT-11 33.3 mg/kg; square, gefitinib 30 mg/kg; star, gefitinib 30 mg/kg + CPT-11 16.7 mg/kg; blue line, gefitinib 30 mg/kg + CPT-11 33.3 mg/kg; open triangle, gefitinib 60 mg/kg; circle, gefitinib 60 mg/kg + CPT-11 16.7 mg/kg; light blue line, filled square, gefitinib 60 mg/kg + CPT-11 33.3 mg/kg]. (c) Mean tumor volumes and results of the statistical analysis at days 15 and 22, bars, S.D. *Significant difference (p<0.05) compared to the control. (d) T/C(%) at day 15 and 22. (e) Treatment-related body weight loss occurred in mice treated with gefitinib 60 mg/kg (triangle, circle, and light blue line).

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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).

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Figure 5. The effect of CPT-11 on EGFR phosphorylation in WiDR cells. Lovo (a) and WiDR (b) cells (5×106) were treated with 5 or 50 μM CPT-11 for 6 hr. Additionally Lovo cells were treated with 5 μM CPT-11 for 24 hr. The 1,500 μg of total cell lysate was immunoprecipitated with an anti-EGFR antibody. Tyrosine-phosphorylated EGFR was determined with an anti-phosphotyrosine antibody and the membranes were reblotted by anti-EGFR antibody. (c) Lovo cells were treated with gefitinib or CPT-11 alone (lane 2 and 3) and in combination (lane 4) for 24 hr. A 20 μg of protein of each sample was analyzed by Western blotting using antiphospo-EGFR (Tyr 1068) and cleaved PARP antibody.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

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.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Blijham G, Wagener T, Wils J, de Greve J, Buset M, Bleiberg H, Lacave A, Dalmark M, Selleslag J, Collette L, Sahmoud T. Modulation of high-dose infusional fluorouracil by low-dose methotrexate in patients with advanced or metastatic colorectal cancer: final results of a randomized European Organization for Research and Treatment of Cancer Study. J Clin Oncol 1996; 14: 226673.
  • 2
    O'Connell MJ, Klaassen DJ, Everson LK, Cullinan S, Wieand HS. Clinical studies of biochemical modulation of 5-fluorouracil by leucovorin in patients with advanced colorectal cancer by the North Central Cancer Treatment Group and Mayo Clinic. NCI Monogr 1987; 1858.
  • 3
    Focan C, Kreutz F, Focan-Henrard D, Moeneclaey N. Chronotherapy with 5-fluorouracil, folinic acid and carboplatin for metastatic colorectal cancer; an interesting therapeutic index in a phase II trial. Eur J Cancer 2000; 36: 3417.
  • 4
    Shimada Y, Yoshino M, Wakui A, Nakao I, Futatsuki K, Sakata Y, Kambe M, Taguchi T, Ogawa N. Phase II study of CPT-11, a new camptothecin derivative, in metastatic colorectal cancer. CPT-11 Gastrointestinal Cancer Study Group. J Clin Oncol 1993; 11: 90913.
  • 5
    Cunningham D, Pyrhonen S, James RD, Punt CJ, Hickish TF, Heikkila R, Johannesen TB, Starkhammar H, Topham CA, Awad L, Jacques C, Herait P. Randomised trial of irinotecan plus supportive care versus supportive care alone after fluorouracil failure for patients with metastatic colorectal cancer. Lancet 1998; 352: 14138.
  • 6
    Saltz LB, Cox JV, Blanke C, Rosen LS, Fehrenbacher L, Moore MJ, Maroun JA, Ackland SP, Locker PK, Pirotta N, Elfring GL, Miller LL. Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer: Irinotecan Study Group. N Engl J Med 2000; 343: 90514.
  • 7
    Rothenberg ML, Meropol NJ, Poplin EA, Van Cutsem E, Wadler S. Mortality associated with irinotecan plus bolus fluorouracil/leucovorin: summary findings of an independent panel. J Clin Oncol 2001; 19: 38017.
  • 8
    Modi S. Seidman AD. An update on epidermal growth factor receptor inhibitors. Curr Oncol Rep 2002; 4: 4755.
  • 9
    Saijo N, Tamura T, Nishio K. Problems in the development of target-based drugs. Cancer Chemother Pharmacol 2000; 46: S435.
  • 10
    Baselga J. The EGFR as a target for anticancer therapy: focus on cetuximab. Eur J Cancer 2001; 37: S1622.
  • 11
    Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001; 37: S915.
  • 12
    Speer G, Cseh K, Winkler G, Takacs I, Barna I, Nagy Z, Lakatos P. Oestrogen and vitamin D receptor (VDR) genotypes and the expression of ErbB-2 and EGF receptor in human rectal cancers. Eur J Cancer 2001; 37: 14638.
  • 13
    Albanell J, Rojo F, Baselga J. Pharmacodynamic studies with the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839. Semin Oncol 2001; 5: 5666.
  • 14
    Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000; 19: 655065.
  • 15
    Woodburn JR, Morris CQ, Kelly H. EGF receptor tyrosine kinase inhibitors as anti-cancer agents-preclinical and early clinical profile of ZD1839. Cell Mol Biol Lett 1998; 3: 3489.
  • 16
    Naruse I, Ohmori T, Ao Y, Fukumoto H, Kuroki T, Mori M, Saijo N, Nishio K. Antitumor activity of the selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) Iressa™ (ZD1839) in a EGFR-expressing multidrug resistant cell line in vitro and in vivo. Int J Cancer 2002; 98: 3105.
  • 17
    Ciardiello F, Bianco R, Damiano V, De Lorenzo S, Pepe S, De Placido S, Fan Z, Mendelsohn J, Bianco AR, Tortora G. Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225. Clin Cancer Res 1999; 5: 90916.
  • 18
    Ciardiello F, Caputo R, Bianco R, Damiano V, Pomatico G, De Placido S, Bianco A.R, Tortora G. Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin Cancer Res 2000; 6: 205363.
  • 19
    Sirotnak FM, Zakowski MF, Miller VA, Scher HI, Kris MG. Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 2000; 6: 488592.
  • 20
    Slichenmyer WJ, Fry DW. Anticancer therapy targeting the erbB family of receptor tyrosine kinases. Semin Oncol 2001; 28: 6779.
  • 21
    Kusaba H, Tamura T, Nakagawa K, Yamamoto N, Kudoh S, Negoro S, Takeda K, Tanigawara Y, Fukuoka M. A phase I intermittent dose-escalation trial of ZD1839 (‘IRESSA’) in Japanese patients with solid malignant tumors. Clin Cancer Res 2000; 6: abs381.
  • 22
    Kris M, Ranson M, Ferry D, Hammond L, Averbuch S, Ochs J, Rowinsky E. Phase I study of oral ZD1839(‘IRESSA’): A novel inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TKI)—Evidence of good tolerability and activity. Clin Cancer Res 1999; 5: abs 99
  • 23
    Kanzawa F, Koizumi F, Koh Y, Nakamura T, Tatsumi Y, Fukumoto H, Saijo N, Yoshioka T, Nishio K. In vitro synergistic interactions between the cisplatin analogue nedaplatin and the DNA topoisomerase I inhibitor irinotecan and the mechanism of this interaction. Clin Cancer Res 2001; 7: 2029.
  • 24
    Ohmori T, Ao Y, Nishio K, Saijo N, Arteaga CL, Kuroki T. Low dose cisplatin can modulate the sensitivity of human non-small cell lung carcinoma cells to EGFR tyrosine kinase inhibitor (ZD1839; ‘Iressa’) in vivo. Proc Am Assoc Cancer Res 2000; 41: abs 3072.
  • 25
    Magne N, Fischel JL, Dubreuil A, Formento P, Marcie S, Lagrange JL, Milano G. Sequence-dependent effects of ZD1839 (‘Iressa’) in combination with cytotoxic treatment in human head and neck cancer. Br J Cancer 2002; 86: 81927.
  • 26
    Raben D, Helfrich BA, Chan D, Johnson G, Bunn PA Jr. ZD1839, a selective epidermal growth factor receptor tyrosine kinase inhibitor, alone and in combination with radiation and chemotherapy as a new therapeutic strategy in non-small cell lung cancer. Semin Oncol 2002; 29: 3746.
  • 27
    Sirotnak FM, Zakowski MF, Miller VA, Scher HI, Kris MG. Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD1839 (Iressa), an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 2000; 6: 488592.
  • 28
    Williams KJ, Telfer BA, Stratford IJ, Wedge SR. ZD1839 (‘Iressa’), a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor, potentiates radiotherapy in a human colorectal cancer xenograft model. Br J Cancer 2002; 86: 115761.
  • 29
    Goldshmit Y, Erlich S, Pinkas-Kramarski R. Neuregulin rescues PC12-ErbB4 cells from cell death induced by H2O2. Regulation of reactive oxygen species levels by phosphatidylinositol 3-kinase. J Biol Chem 2001; 276: 4637985.
  • 30
    Meves A, Stock SN, Beyerle A, Pittelkow MR, Peus D. H2O2 mediates oxidative stress-induced epidermal growth factor receptor phosphorylation. Toxicol Lett 2001; 122: 20514.
  • 31
    Miyazaki Y, Hiraoka S, Tsutsui S, Kitamura S, Shinomura Y, Matsuzawa Y. Epidermal growth factor receptor mediates stress-induced expression of its ligands in rat gastric epithelial cells. Gastroenterology 2001; 120: 10816.
  • 32
    Wang X, McCullough KD, Franke TF, Holbrook NJ. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 2000; 275: 1462431.
  • 33
    Benhar M, Engelberg D, Levitzki A. Cisplatin-induced activation of the EGF receptor. Oncogene 2002; 21: 872331.
  • 34
    Kitagawa D, Tanemura S, Ohata S, Shimizu N, Seo J, Nishitai G, Watanabe T, Nakagawa K, Kishimoto H, Wada T, Tezuka T, Yamamoto T, et al. Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation: its implication in an anti-apoptotic function. J Biol Chem 2002; 277: 36671.