![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands (E.G., C.L., C.T., K.S., J.A.R., G.J.P.); Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy (E.G., S.N., S.R., R.D.); and Medical Oncology Branch, National Cancer Institute, Bethesda, Maryland (G.G.)
Received October 3, 2007; accepted January 10, 2008
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
erlotinib (48 h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of these markers.
). Many patients who failed first-line treatment are in a sufficient performance status to receive second-line treatment. There are three agents presently registered for this indication: docetaxel, pemetrexed, and erlotinib. Pemetrexed and erlotinib are distinctly less toxic than docetaxel, and their use has rapidly increased (Hanna et al., 2004; Shepherd et al., 2005). Both patient characteristics (never-smoking status, female gender, adenocarcinoma histology, East Asian race) and molecular markers are predictive of benefit to EGFR tyrosine-kinase inhibitors (TKIs), such as erlotinib and gefitinib. In particular, higher response rates to these targeted agents have been associated with the occurrence of EGFR-activating mutations (Lynch et al., 2004; Paez et al., 2004) and absence of k-Ras mutations (Eberhard et al., 2005).
Clinical benefit observed with erlotinib is not restricted to patients carrying EGFR-activating mutations (Shepherd et al., 2005), and the clinical response to EGFR-TKIs may be associated with EGFR copy number or expression (Dziadziuszko et al., 2006) or with molecular changes affecting several downstream EGFR intracellular signal transducers, such as the Ras/Raf/mitogen-activated protein kinase and the phosphatidylinositol-3'-kinase (PI3K)/Akt (Cappuzzo et al., 2004).
A key issue in the novel treatment modalities against NSCLC is the integration of EGFR-TKIs with chemotherapy. Targeted therapies should have limited overlapping toxicities in combination with conventional chemotherapy, and preclinical studies have shown that coadministration with EGFR-TKIs enhanced the effect of different cytotoxic agents against various tumor models (Magné et al., 2003; Morelli et al., 2005; Bianco et al., 2006), including NSCLC cells (Van Schaeybroeck et al., 2006; Li et al., 2007).
However, the combined treatment of patients with advanced NSCLC with either gefitinib or erlotinib and standard two-drug (gemcitabine/cisplatin and carboplatin/paclitaxel) chemotherapy regimens (Giaccone et al., 2004; Herbst et al., 2004, 2005; Gatzemeier et al., 2007) failed to improve survival. The lack of the expected positive outcome may have been caused by several factors: 1) predictive markers for response to EGFR-TKIs in combination with chemotherapy have not yet been identified, and therefore patient selection is still lacking, and 2) the choice of chemotherapeutic agents, doses, and schedules has been determined empirically or from single-agent studies (Eberhard et al., 2005). Therefore, further studies are needed to investigate new therapies combining erlotinib with cytotoxic chemotherapy and to describe molecular mechanisms and potential markers of drug combination activity.
Antimetabolites are widely used in combination regimens because of their ability to biochemically modulate the cytotoxicity of other drugs (Peters et al., 2000). Pemetrexed is an inhibitor of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) (Shih et al., 1997). Resistance to pemetrexed may be related mainly to an increased TS expression (Sigmond et al., 2003), although limited uptake by the reduced folate carrier (RFC) or decreased polyglutamylation may be other important parameters (Mauritz et al., 2002). Preclinical studies demonstrated that its combination with cisplatin, taxanes, and gemcitabine produced additive or synergistic cytotoxicity (Tonkinson et al., 1999; Teicher et al., 2000; Giovannetti et al., 2005).
The ability of pemetrexed to deplete cellular ribonucleotide pools, modulate cell cycle, and induce apoptosis (Tonkinson et al., 1999) makes this drug an attractive agent for polychemotherapy regimens. In particular, pemetrexed acts on several pathways involved in DNA synthesis and cell death control, including the Akt pathway (Giovannetti et al., 2005), whereas recent studies showed that the TS inhibitor 5-fluorouracil (5-FU) increased EGFR phosphorylation, thus potentially favoring EGFR-TKI activity (Van Schaeybroeck et al., 2005). Moreover, in vitro studies demonstrated that EGFR-TKIs decreased TS expression and activity, determining synergistic interaction with 5'-deoxy-5-fluorouridine (Magné et al., 2003; Budman et al., 2006).
The present work was aimed at evaluating molecular mechanisms underlying the synergistic cytotoxicity between erlotinib and the antimetabolite pemetrexed through the in vitro assessment of their pharmacological interaction. We observed a strong synergistic interaction between these agents against a panel of six NSCLC cell lines, regardless of their genetic signature, related to either erlotinib or pemetrexed activity. Furthermore, we characterized several factors, including apoptosis induction, modulation of EGFR and Akt phosphorylation, and expression of critical genes involved in pemetrexed activity, which may contribute to this synergistic interaction.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Lines. Cell lines were maintained as monolayer cultures in RPMI 1640 medium or DMEM (containing 2 mM L-glutamine) supplemented with 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin. The human NSCLC cell lines NCI-H460 (H460), NCI-H1703 (H1703), NCI-H292 (H292), and NCI-H1650 (H1650) were cultured in RPMI 1640 medium, whereas A549 and SW1573 cells were cultured in DMEM. Cells were grown in 75-cm2 flasks (Costar, Cambridge, MA) at 37°C in 5% CO2/95% air and harvested with trypsin-EDTA when they were in exponential growth.
Analysis of Mutations and Polymorphisms in Determinants of Drug Activity. The NSCLC cells used in this study have been characterized previously for EGFR and k-Ras mutations (Janmaat et al., 2006). Furthermore, we evaluated genetic variations that may influence pemetrexed sensitivity, such as the polymorphic tandem repeat sequence of the enhancer region of the thymidylate synthase (TS) promoter (TSER) and the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism.
Cytotoxicity Assays. To evaluate the cytotoxic activity of single drugs, the cell growth inhibitory effect of erlotinib (0.001-50 µM, 72-h exposure) and pemetrexed (0.001-100 µM, 24- and 72-h treatment) was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. For this purpose, cells were plated at 104 cells/well, and growth inhibition was expressed as the percentage of control (vehicle-treated cells) absorbance (corrected for absorbance before drug addition). The IC50 value was calculated by nonlinear least-squares curve-fitting (Prism; GraphPad Software Inc., San Diego, CA).
Drug Combination Studies. Several studies have shown a more efficient interaction when cytotoxic chemotherapeutic agents are given before EGFR-TKIs (Morelli et al., 2005; Van Schaeybroeck et al., 2005). Therefore, combination studies were focused on simultaneous treatment and on treatment with an initial 24-h exposure to both pemetrexed and erlotinib, followed by a washout and an additional 48-h treatment with erlotinib alone. According to the expected biological effects and pharmacokinetics data available, pemetrexed was used for 24 or 72 h, whereas erlotinib was always used for 72 h in a continuous or a pulsatile administration. Each combination was tested in at least six different concentrations, using a constant ratio calculated with respect to drug IC50 values. The cytotoxicity of the combination was compared with the cytotoxicity of each drug alone using the combination index (CI), where CI < 0.9, CI = 0.9 to 1.1, and CI > 1.1 indicated synergistic, additive, and antagonistic effects, respectively. Data analysis was carried out using CalcuSyn software (Biosoft, Oxford, UK). Because we considered growth inhibition lower than 50% as not relevant, CI values at fraction affected (FA) of 0.5, 0.75, and 0.9 were averaged for each experiment, and this value was used to calculate the mean between experiments (Peters et al., 2000).
Cell Cycle Analysis. Cell cycle modulation induced by treatments at IC50 values for 72 h was studied by propidium iodide staining and flow cytometry analysis using a FACScan (BD Biosciences, San Jose, CA). Data analysis was carried out with CELLQuest (BD Biosciences), whereas cell cycle distribution was determined using Modfit software (Verity Software House, Topsham, ME).
Evaluation of Apoptosis. Erlotinib, pemetrexed, and their combination were also characterized for their ability to induce apoptosis, which was detected after 72-h drug exposure. Apoptosis was measured by evaluating the sub-G1 region of the previous FACS analysis and by fluorescence microscopy analysis with bisbenzimide staining (Giovannetti et al., 2005). Two hundred cells from randomly chosen microscopic fields were counted, and the apoptotic index (AI) was calculated as the percentage ratio between the number of cells displaying apoptotic features and the number of counted cells.
EGFR, ERK1/2, and Akt Phosphorylation Assays. To study the effect of drug treatments on the activation of EGFR and of ERK1/2 and Akt, cells were exposed to IC50 values of erlotinib, pemetrexed, and erlotinib-pemetrexed combination and stimulated with EGF (10 ng/ml), as described previously (Janmaat et al., 2003). After protein extraction from cell pellets, EGFR phosphorylation at the tyrosine residue at position 1173 [EGFR (pY1173)], dual phosphorylation of ERK2 at threonine 185 and tyrosine 187 [ERK2 (pTpY185/187)] and ERK1 at threonine 202 and tyrosine 204 [ERK1 (pTpY202/204)] and Akt phosphorylation at serine residue 473 [Akt (pS473)] were evaluated with specific enzyme-linked immunosorbent assays (BioSource International, Camarillo, CA), and normalized, respectively, to the total EGFR, ERK1/2 and Akt, and protein content (Bianco et al., 2006).
Pharmacological Interaction Study with the PI3K Inhibitor LY294002. To evaluate whether inhibition of the PI3K/Akt pathway may modulate the effects of the erlotinib-pemetrexed combination, we studied the pharmacological interaction of LY294002 with erlotinib, pemetrexed, and their combination. The cell growth-inhibitory effect was detected in cells treated with erlotinib (0.001-50 µM), pemetrexed (0.001-100 µM), LY294002 (30 µM), and their combinations for 72 h, whereas drug interaction with LY294002 was assessed at a nonconstant concentration ratio using the CI method.
Real-Time RT-PCR. To compare the possible influence of gene expression profile on drug sensitivity, we selected cells characterized by heterogeneous patterns of EGFR and TS expression (Janmaat et al., 2003). However, the basal expression of EGFR and TS was also assessed in this study to evaluate the effect of 72-h treatment with IC50 levels of erlotinib, pemetrexed, and their combination in all cell lines. Because previous studies have shown a strong correlation between expression levels of TS and its upstream transcriptional regulator E2F-1 (Li et al., 1995; Huang et al., 2007), and because other studies showed that EGFR-TKIs affected E2F-1 (Kobayashi et al., 2006; Suenaga et al., 2006), we also evaluated the expression of E2F-1 mRNA. Moreover we studied the expression levels of DHFR, GARFT, RFC, 
-glutamyl hydrolase (GH), and folyl-polyglutamate synthetase (FPGS). RNA was extracted by the QiaAmp RNA mini-Kit (QIAGEN, San Diego, CA) and reverse-transcribed (Bianco et al., 2006). Forward and reverse primers and probes were designed with Primer Express (Applied Biosystems, Foster City, CA) on the basis of TS, DHFR, FPGS, and GARFT gene sequence (Giovannetti et al., 2005), whereas primers and probes for E2F-1, RFC, and -glutamyl hydrolase were obtained from Applied Biosystems Assay-on-Demand Gene expression products (Hs001572991_m1, Hs00228858_m1, and Hs00914167_m1). Finally, EGFR basal expression was detected as reported previously (Dziadziuszko et al., 2006). Amplification data were normalized to β-actin, and quantification of gene expression was performed using standard curves obtained with dilutions of cDNA from Quantitative-PCR Human-Reference Total-RNA (Stratagene, La Jolla, CA).
Western Blot. To evaluate the possible role of breast cancer resistance protein (BCRP) and multidrug-related proteins (MRPs) expression on pemetrexed sensitivity, as suggested in a study on freshly explanted human tumor specimens (Hanauske et al., 2007), we performed Western blot analysis of BCRP, MRP1, MRP2, MRP3, MRP4, and MRP5 expression in the panel of NSCLC cells. Total lysates were prepared in buffer containing 50 mM Tris, pH 7.6, 20% (v/v) glycerol, 5 mM dithiothreitol, 0.5% (v/v) Nonidet P-40, and 4.0% (v/v) of a protease inhibitor cocktail. Lysates were sonicated and centrifuged. The protein-containing supernatant was collected, and protein content was determined using a Bradford assay. In each lane of a minigel system (Bio-Rad Laboratories, Hertfordshire, UK), 30 µg of proteins was loaded. The following monoclonal antibodies (kindly provided by Dr. G. L. Scheffer, VU University Medical Centre, Amsterdam, The Netherlands) were used: rat anti-MRP1 (MRP-r1; 1:500), -MRP4 (M4I-10; 1:200), -MRP5 (M5I-10; 1:250), and -BCRP (BXP-53; 1:200), as well as mouse anti-MRP2 (M2III-6; 1:500) and -MRP3 (M3II-21; 1:500). As secondary antibodies, horseradish peroxidase-conjugated rabbit anti-rat or anti-mouse (1:2000; Dako Denmark A/S, Glostrup, Denmark) were used. As a loading control, expression of β-actin was determined using an antibody against β-actin (1:10,000; Millipore Bioscience Research Agents, Temecula, CA).
Erlotinib, pemetrexed, and their combination, at IC50 values, were also studied for their ability to modulate protein expression of possible targets or surrogate markers of drug activity, such as TS and E2F-1. After 72-h exposure, cell pellets were collected and lysed, and 20 µg of proteins was loaded and separated on a 10% SDS-PAGE gel, followed by blotting onto a nitrocellulose membrane. The membrane was preincubated in blocking buffer [0.5% milk powder, 0.5% BSA in Tris-buffered saline/Tween 20 (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween 20)] for 1 h, whereas the primary polyclonal TS (1:1000, kindly provided by Dr. G. W. Aheme, Sutton, UK) and monoclonal E2F-1 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were added overnight at 4°C. After washing in Tris-buffered saline/Tween 20, the blots were incubated for 1 h with anti-rabbit (for TS) and anti-mouse (for E2F-1) horseradish peroxidase-labeled secondary antibodies (1:2000; Dako Denmark A/S). Antibody binding was detected using enhanced chemoluminescence. Densitometric analysis of the images captured on the VersaDoc3000 instrument (Bio-Rad) was performed with the Kontron Analysis Image software (Kontron-Electronik, Munich, Germany).
Evaluation of TS Catalytic and in Situ Activity. To evaluate the possible modulation of TS activity, pellets of A549 and SW1573 cells treated as described above in the gene expression analyses were suspended in an ice-cold 200 mM Tris-HCl buffer, pH 7.4, containing 20 mM β-mercaptoethanol, 100 mM NaF, and 15 mM CMP. After sonification and centrifugation, 50 µl of the enzyme-containing suspension was used for determination of the protein content according to Bradford assay, whereas the remaining volume was used for the TS catalytic assay. This assay determines the catalytic activity of TS by means of the 3H release during the TS-catalyzed conversion of 1 µM[3H]dUMP to TMP. Pemetrexed is only an effective TS inhibitor in its polyglutamylated form. Therefore, to be able to evaluate the long-term effect of pemetrexed, which is mostly mediated by polyglutamylation, we also determined the potential inhibition of TS in intact cells after 72-h drug exposure at IC50 values. This TS in situ activity was measured in all cell lines by exposure to [5-3H]deoxycytidine (0.3 µM final concentration; specific activity, 1.6 mCi/mol) during the last 2 h of the assay and by processing the medium of each sample as described above for the catalytic assay (van Triest et al., 1997).
Statistical Analysis. All experiments were performed in triplicate and repeated at least twice. Data were expressed as mean values ± S.E. and analyzed by Student's t test or analysis of variance followed by the Tukey's multiple comparison test. The Pearson/Spearman correlation and regression analysis were used to demonstrate the relationship between gene expression profile and chemosensitivity, and between erlotinib and gefitinib cytotoxic activity, TS mRNA and protein expression, and modulation of E2F-1 and TS protein expression; the level of significance was P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Cytotoxicity of Pemetrexed and Erlotinib and Correlation with Genetic Background. Erlotinib caused a concentration-dependent inhibition of proliferation in all cell lines, with IC50 values ranging from 0.20 to 18.17 µM, in H1650 and H460 cells, respectively (Table 1). A concentration-dependent inhibition of cell growth was also observed after 72-h exposure to pemetrexed, with IC50 values ranging from 0.08 (SW1573) to 1.05 µM (H460). Higher IC50 values were obtained after 24-h exposure to pemetrexed followed by 48-h incubation in drug-free medium. In contrast, erlotinib exposure for 24 h, followed by washout and addition of new medium with erlotinib for the ensuing 48 h, slightly decreased IC50 values observed after continuous 72-h incubation. H1650 cells, carrying the EGFR-activating mutation Del746-750, were the most sensitive cells, whereas H460 cells, harboring the G61H mutation in k-Ras, were the least sensitive cells to both erlotinib and pemetrexed, after all of the different exposures. In contrast, no relationship was found between tandem repeat sequence of the enhancer region of the TSER promoter or MTHFR polymorphisms and pemetrexed chemosensitivity. In this panel of NSCLC cells, a significant correlation was found between erlotinib and gefitinib sensitivity (R2 = 0.92, P = 0.017).
Correlation between Gene Expression and Chemosensitivity. The expression of genes involved in the action of erlotinib and pemetrexed showed a large heterogeneity, suggesting that the different sensitivity to drug treatment may be related to variable cellular gene expression profiles (Table 1). No correlation was found between EGFR mRNA gene expression and erlotinib sensitivity in this panel of NSCLC cells. However, a clear correlation was found between the IC50 values of pemetrexed and the expression of its target enzymes TS (R2 = 0.72, P = 0.030) and DHFR (R2 = 0.97, P = 0.003); the lower chemosensitivity of the H460 cells seemed mostly dependent on the higher expression levels of the genes encoding TS and DHFR with respect to the other cells. In addition, this cell line has a low expression of RFC, which may be associated with a low uptake of pemetrexed despite a high expression of FPGS. In contrast, the sensitive H1650 cells had a high RFC expression.
Correlation between Protein Expression and Chemosensitivity. TS protein expression was measured with Western blot, and the analysis of band density demonstrated a significant link between TS mRNA and protein expression in control cells (R2 = 0.92, P = 0.002). A significant correlation was also found between TS protein expression and pemetrexed sensitivity (R2 = 0.73, P = 0.03; data not shown). In contrast, no correlation was found between the protein expression of BCRP and MRPs and pemetrexed sensitivity (Fig. 1).
|
50%). In all of our experiments, we obtained growth-inhibition curves still decreasing at the highest concentration values toward the d = 0 absorption values, which for the combination were well below those for the single drugs. Therefore, the computer-generated CI values at FA = 0.9 are correct, and we included this value for calculation of the mean of CI values. The average CI values for erlotinib-pemetrexed combinations in all of the NSCLC cell lines are summarized in Fig. 2, D and E. Simultaneous drug administration resulted in a synergistic-additive effect in A549 cells (mean CI, 0.92), whereas a synergistic interaction was observed in all of the other NSCLC cell lines, with CI values ranging from 0.46 (H460 cells) to 0.66 (H292) (Fig. 2D). The pemetrexed + erlotinib (24 h) erlotinib (48 h) schedule proved to be the most effective combination in all cell lines (Fig. 2E), displaying the strongest synergistic effect in H460 (mean CI, 0.09). To evaluate the mechanisms underlying the additive-synergistic (in A549 cells) and the synergistic interaction (in all of the other NSCLC cells), several biochemical analyses were performed with the simultaneous combination, as detailed below.
|
Cell Cycle Modulation and Induction of Apoptosis. DNA flow cytometry studies were performed to evaluate the effect of erlotinib, pemetrexed, and their combination on the cell cycle distribution and to determine whether their cell cycle-modulating activity might provide clues to optimize drug scheduling. Both agents were able to affect the cell cycle of NSCLC cells (Table 2). Erlotinib caused a 1.2- to 1.4-fold increase in the population of cells in the G1 phase. In contrast, a 72-h treatment with pemetrexed resulted in a 1.3- to 2.0-fold increase in the percentage of cells in the S phase. The increment in the S phase cell population was most pronounced in A549 and H1650 cells (1.7- and 2-fold, respectively), whereas H292 cells showed only a 1.3-fold enhancement. A marked increase in the S phase population with respect to controls was also observed for the simultaneous combination in all NSCLC cells. In the 24-h pemetrexed + erlotinib followed by 48-h erlotinib schedule, however, the increase in the S phase population was lower, whereas more cells accumulated in the G2/M phase.
|
All treatments induced cell death, as shown by the presence of a cell population with sub-G1 DNA content in the FACS analysis, which was confirmed by fluorescence microscopy analysis after bisbenzimide staining to determine the AI values (Table 2). Cells exposed to erlotinib, pemetrexed, and their combination, at the IC50 values, presented typical apoptotic morphology with cell shrinkage, nuclear condensation, and fragmentation, and rupture into apoptotic bodies. Erlotinib slightly increased AI with respect to controls in A549, SW1573, H460, and H1703 cells, whereas higher AI values were observed in the more sensitive cell lines (8.0 and 6.5% in H1650 and H292 cells, respectively). Pemetrexed induced more apoptosis than erlotinib, with AI values ranging from 6.2 to 13.7% in H460 and H292 cells, respectively. The combination of the two drugs additionally increased the AI, up to 21.2% in H292 and H1650 cells, and the 24-h pemetrexed + erlotinib followed by 48-h erlotinib schedule was slightly more active and caused a significant induction of apoptosis compared with both controls and pemetrexed-treated cells in all cell lines.
Modulation of EGFR, ERK1/2, and Akt Phosphorylation. As expected, erlotinib induced a significant suppression of EGF-induced phosphorylation of EGFR at the tyrosine residue pY1173 in all of the NSCLC cell lines, with percentages of reduction of EGFR-phosphorylated protein ranging from -39.6 to -65.5% with respect to controls in A549 and H1650 cells, respectively. Conversely, pemetrexed significantly enhanced pY1173-EGFR levels, varying from +33.3 to +74.4% in all cell lines, whereas drug combinations reduced the phosphorylation status of EGFR but generally to a lower extent than erlotinib alone (Fig. 3A). All of these molecular changes, which occurred before 24 h, might influence the activity of erlotinib when this agent is added to the cells for 48 h after 24-h exposure to pemetrexed + erlotinib.
|
Synergistic Cytotoxic Effects of Erlotinib and Pemetrexed with LY294002. To determine whether the effects of erlotinib and pemetrexed on EGFR phosphorylation and the activity of downstream pathways are responsible for their synergistic interaction, we investigated the effect of the specific PI3K inhibitor LY294002 at an effective concentration (Janmaat et al., 2006). LY294002 enhanced the growth inhibition of erlotinib in all NSCLC cell lines tested, with IC50 values ranging from 0.05 to 8.01 µM in H1650 and H460 cells, respectively. Similar effects were observed in the combination with pemetrexed and with the erlotinib-pemetrexed simultaneous combination. The mean CI values showed an additive-synergistic interaction in all of these combinations (Fig. 4), indicating that additional inhibition of PI3K by LY294002 led to a somewhat enhanced effect.
|
|
Modulation of E2F-1 mRNA and Protein Expression by Erlotinib, Pemetrexed, and Their Combination. To further investigate the causes of the decrease in TS and DHFR mRNA after erlotinib exposure, we also evaluated E2F-1 mRNA. Both erlotinib and the drug combination significantly reduced E2F-1 mRNA levels in SW1573, H292, H460, H1650, and H1703 cells, as shown in Fig. 5B. Similar results were observed in the blots of E2F-1, showing a slight increase in E2F-1 protein expression in pemetrexed-treated cells, whereas a reduction was observed after both erlotinib and erlotinib-pemetrexed exposure in most NSCLC cells. Finally, the modifications in TS and E2F-1 expression levels after erlotinib treatment were positively correlated (data not shown).
Modulation of TS Catalytic Activity in Cell Extracts and in Situ Activity. Given the changes in TS levels described above, we measured the activity of this enzyme using both a catalytic assay in cell extracts and an in situ assay in intact cells. The catalytic assay demonstrated a significant increase in TS activity in pellets from cells treated with pemetrexed for 72 h, in line with the results of TS mRNA and protein expression analysis, whereas erlotinib significantly decreased TS activity (by 35 and 60% in A549 and SW1573 cells, respectively). No clear differences with respect to controls were detected in samples exposed to the erlotinib-pemetrexed simultaneous combination (Fig. 5D).
Because in the extracts the drug is washed away, we also determined the activity of the enzyme in intact cells. In contrast to the results of the catalytic assay, we found that pemetrexed significantly inhibited TS in all cell lines when measured using the in situ assay. In addition, erlotinib inhibited the TS in situ activity (Fig. 5E). It is most interesting that the combination almost completely inhibited the TS in situ, and statistical analysis revealed significant reductions with respect to those observed after pemetrexed exposure.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
In a recent article, schedule-dependent synergism of pemetrexed and erlotinib was found, associated with pemetrexed-induced EGFR phosphorylation (Li et al., 2007). Similar effects were observed for other cytotoxic drugs in NSCLC cells displaying a synergistic interaction with gefitinib (Van Schaeybroeck et al., 2006) and with 5-FU in colorectal cancer cells (Van Schaeybroeck et al., 2005). However, our findings are novel because they show that the synergistic interaction seems to be mediated by several mechanisms, which enhanced both the sensitivity to erlotinib and to pemetrexed (Table 3). In addition to the effect of pemetrexed on EGFR phosphorylation, downstream mediators of the EGFR signal transduction pathway were affected not only by erlotinib but also by pemetrexed, which reduced Akt phosphorylation.
|
Previous work has shown controversial results regarding the modulation of phospho-Akt by pemetrexed (Giovannetti et al., 2005; Li et al., 2007), which may be related to the discrepancy between drug exposure conditions and different sensitivity of experimental methods. Our findings are in agreement with several studies demonstrating a reduced Akt phosphorylation after exposure to the antimetabolite gemcitabine (Giovannetti et al., 2005; Bianco et al., 2006; Chun et al., 2006; Mey et al., 2006; Feng et al., 2007). In particular, Feng et al. (2007) proposed a model of gemcitabine-induced apoptosis via EGFR degradation, explaining the possible mechanism by which a cytotoxic compound may affect EGFR and Akt signaling pathways. Drug treatment, through c-Src activation, leads to EGFR phosphorylation, which promotes ubiquitination of the receptor. EGFR is then targeted to proteosomes and lysosomes for degradation, resulting in phospho-Akt down-regulation and induction of apoptosis (Feng et al., 2007).
These data also fit with the concept that an additional inhibition may be required to prevent the inhibition of one pathway by an EGFR antagonist and may lead to optimal use of alternative signaling pathways (Janmaat et al., 2006). Furthermore, the combination of pemetrexed and erlotinib with the PI3K inhibitor LY294002 resulted in a synergistic interaction, suggesting that combinations of specific signal transduction inhibitors targeting different steps of EGFR-PI3K pathways may be a successful strategy against NSCLC. Because phospho-Akt regulates antiapoptotic mechanisms and previous in vitro studies showed that its down-regulation by pemetrexed correlated with the enhancement of gemcitabine-induced apoptosis and antitumor activity in lung and bladder cancer cells (Giovannetti et al., 2005; Mey et al., 2006), the reduction of pS-473Akt may explain the increased apoptosis found in the pemetrexed-erlotinib combination.
This increased induction in apoptosis may be related to cell cycle modulation, which was reported to be important for the efficacy of the combination of EGFR-TKIs with cytotoxic compounds (Morelli et al., 2005; Bianco et al., 2006). Cellular damage induced by chemotherapy can convert EGFR ligands from growth factors into survival factors for cancer cells that express functional EGFR (Mendelsohn and Baselga, 2000). In this context, the blockade of EGFR signaling after the exposure to cytotoxic drugs could cause irreparable cell damage leading to apoptosis. Therefore, the 24-h pemetrexed + erlotinib combination followed by 48-h erlotinib exposure may favor this process, because treatment with erlotinib after pemetrexed may prevent repair of cell damage and induce apoptosis.
In addition to the effects of pemetrexed on signaling pathways, the present study also shows that erlotinib interfered with the cytotoxic activity of pemetrexed. Indeed, erlotinib significantly inhibited the activity of TS, whose expression is significantly correlated with pemetrexed sensitivity both in the preclinical and in the clinical setting (Giovannetti et al., 2005; Gomez et al., 2006). These data are in agreement with previous observations that not only antifolates and 5-FU modulate TS. Vinorelbine suppressed TS expression in the PC14 NSCLC cells, favoring the activity of 5-FU (Matsumoto et al., 2004), whereas other drugs such as platinum derivatives and irinotecan can down-regulate TS levels as well (van der Wilt et al., 1992; Guichard et al., 1998).
For instance, TS, as an RNA binding protein, also regulates its own synthesis by impairing the translation of its mRNA, and the binding to a specific inhibitor leads to up-regulation of TS protein (Chu et al., 1991). In agreement with this hypothesis and with the observed increase in TS mRNA expression, as detected previously with pemetrexed and 5-FU (Peters et al., 2002; Mauritz et al., 2007), TS protein expression and catalytic activity in cell extracts were enhanced after pemetrexed exposure.
Recent studies reported that EGFR-TKIs decreased TS expression and activity in breast and colorectal cancer cells, showing synergistic interaction with 5'-deoxy-5-fluorouridine (Magné et al., 2003; Budman et al., 2006). In the latter study, it was postulated that TS down-regulation was related to a decrease in S phase and increase of the G1 phase. However, the marginal increase in G1 phase observed in the present study does not fit with this hypothesis, and the effects on mRNA and protein expression suggest that TS modulations are caused by mechanisms involving transcriptional control.
In particular, our data show a reduction of E2F-1 mRNA and protein expression after both erlotinib and erlotinib-pemetrexed exposure. These results may be related to the nuclear effects of EGFR, which influence the activity of some cell cycle proteins and transcription factors, including E2F-1 (Lo and Hung, 2006). High levels of free E2F-1 up-regulate the transcription of several genes, including TS and DHFR (Li et al., 1995). EGFR-TKIs may affect E2F-1 directly or via down-regulation of cyclin D1 (Kobayashi et al., 2006; Suenaga et al., 2006). After nuclear translocation, EGFR interacts with DNA-binding transcription factors, E2F-1 and STAT3 and up-regulate expression of cyclin D1, inducible nitric-oxide synthase, and B-Myb (Lo and Hung, 2006). In addition, the Ras/mitogen-activated protein kinase/ERK-dependent pathway is also implicated in the modulation of the expression of the cyclin D1 gene, and cyclin D1 down-regulation results in E2F-1 inhibition (Kobayashi et al., 2006). In this regard, transfection of gastric cells with a cyclin D1 antisense oligodeoxynucleotide reduced TS and DHFR mRNA and significantly increased 5-FU and methotrexate cytotoxicity (Shuai et al., 2006). Therefore, the down-regulation of TS is most likely related to decreased transcription, which also explains the synergistic interaction and the reduction of TS activity after combination treatment, as detected using the in situ assay.
In conclusion, the present study characterizes several molecular mechanisms and determinants involved in the synergistic effect between erlotinib and pemetrexed against NSCLC cells, regardless of their genetic signature. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination, and favored apoptosis induction. Erlotinib significantly reduced TS expression and activity, whereas the combination additionally decreased TS in situ activity, possibly via down-regulation of E2F-1 expression. The modulation of all of these determinants influences the cytotoxic activity of this combination. Although the extrapolation of in vitro data to the clinical setting should be considered with caution, these results may have implications for the rational development of chemotherapeutic regimens, including erlotinib and pemetrexed for the treatment of NSCLC.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
This work was presented previously in abstract form: Giovannetti E, Smid K, Mey V, Tekle C, Nannizzi S, Del Tacca M, Rodriguez JA, Danesi R, Giaccone G, and Peters GJ (2007) Synergistic interaction between erlotinib and pemetrexed in non-small cell lung cancer (NSCLC) cells. 97th Annual Meeting American of the Association for Cancer Research; 2007 Apr 14-18; Los Angeles, CA. Abstract 4078, American Association for Cancer Research, Philadelphia, PA.
ABBREVIATIONS: NSCLC, non-small-cell lung cancer; 5-FU, 5-fluorouracil; AI, apoptotic index; BCRP, breast cancer resistance protein; CI, combination index; DHFR, dihydrofolate reductase; EGFR, epidermal growth factor receptor; FA, fraction affected; FPGS, folyl-polyglutamate synthetase; GARFT, glycinamide ribonucleotide formyltransferase; MRPs, multidrug-related protein; PI3K, phosphatidylinositide 3-kinase; RFC, reduced folate carrier; TKI, tyrosine-kinase inhibitor; TS, thymidylate synthase; LY294002, (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride); ERK, extracellular signal-regulated kinase; RT-PCR, reverse-transcriptase polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; FACS, fluorescence-activated cell sorting; TSER, tandem repeat sequence of the enhancer region of the thymidylate synthase promoter; MTHFR, methylenetetrahydrofolate reductase; GH, -glutamyl hydrolase.
Address correspondence to: Dr. Elisa Giovannetti, Division of Pharmacology and Chemotherapy, Department of Internal Medicine, 55, Via Roma, 56126 Pisa, Italy. E-mail: elisa.giovannetti{at}gmail.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Budman DR, Soong R, Calabro A, Tai J, and Diasio R (2006) Identification of potentially useful combinations of epidermal growth factor receptor tyrosine kinase antagonists with conventional cytotoxic agents using median effect analysis. Anticancer Drugs 17: 921-928.[CrossRef][Medline]
Cappuzzo F, Magrini E, Ceresoli GL, Bartolini S, Rossi E, Ludovini V, Gregorc V, Ligorio C, Cancellieri A, Damiani S, et al. (2004) Akt phosphorylation and gefitinib efficacy in patients with advanced non-small-cell lung cancer. J Natl Cancer Inst 96: 1133-1141.
Chu E, Koeller DM, Casey JL, Drake JC, Chabner BA, Elwood PC, Zinn S, and Allegra CJ (1991) Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad Sci U S A 88: 8977-8981.
Chun PY, Feng FY, Scheurer AM, Davis MA, Lawrence TS, and Nyati MK (2006) Synergistic effects of gemcitabine and gefitinib in the treatment of head and neck carcinoma. Cancer Res 66: 981-988.
Dziadziuszko R, Witta SE, Cappuzzo F, Park S, Tanaka K, Danenberg PV, Barón AE, Crino L, Franklin WA, Bunn PA Jr, et al. (2006) Epidermal growth factor receptor messenger RNA expression, gene dosage, and gefitinib sensitivity in non-small cell lung cancer. Clin Cancer Res 12: 3078-3084.
Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens SL, Herbst RS, Ince WL, Jänne PA, Januario T, Johnson DH, et al. (2005) Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 23: 5900-5909.
Feng FY, Varambally S, Tomlins SA, Chun PY, Lopez CA, Li X, Davis MA, Chinnaiyan AM, Lawrence TS, and Nyati MK (2007) Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity. Oncogene 26: 3431-3439.[CrossRef][Medline]
Gatzemeier U, Pluzanska A, Szczesna A, Kaukel E, Roubec J, De Rosa F, Milanowski J, Karnicka-Mlodkowski H, Pesek M, Serwatowski P, et al. (2007) Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the Tarceva Lung Cancer Investigation Trial. J Clin Oncol 25: 1545-1552.
Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, Natale RB, Schiller JH, Von Pawel J, Pluzanska A, et al. (2004) Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial-INTACT 1. J Clin Oncol 22: 777-784.
Giovannetti E, Mey V, Nannizzi S, Pasqualetti G, Marini L, Del Tacca M, and Danesi R (2005) Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells. Mol Pharmacol 68: 110-118.
Gomez HL, Santillana SL, Vallejos CS, Velarde R, Sanchez J, Wang X, Bauer NL, Hockett RD, Chen VJ, Niyikiza C, et al. (2006) A phase II trial of pemetrexed in advanced breast cancer: clinical response and association with molecular target expression. Clin Cancer Res 12: 832-838.
Guichard S, Hennebelle I, Bugat R, and Canal P (1998) Cellular interactions of 5-fluorouracil and the camptothecin analogue CPT-11 (irinotecan) in a human colorectal carcinoma cell line. Biochem Pharmacol 55: 667-676.[CrossRef][Medline]
Hanauske AR, Eismann U, Oberschmidt O, Pospisil H, Hoffmann S, Hanauske-Abel H, Ma D, Chen V, Paoletti P, and Niyikiza C (2007) In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression. Invest New Drugs 25: 417-423.[CrossRef][Medline]
Hanna N, Shepherd FA, Fossella FV, Pereira JR, De Marinis F, von Pawel J, Gatzemeier U, Tsao TC, Pless M, Muller T, et al. (2004) Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small cell lung cancer previously treated with chemotherapy. J Clin Oncol 22: 1589-1597.
Herbst RS, Giaccone G, Schiller JH, Natale RB, Miller V, Manegold C, Scagliotti G, Rosell R, Oliff I, Reeves JA, et al. (2004) Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial-INTACT 2. J Clin Oncol 22: 785-794.
Herbst RS, Prager D, Hermann R, Fehrenbacher L, Johnson BE, Sandler A, Kris MG, Tran HT, Klein P, Li X, et al. (2005) TRIBUTE-a phase III trial of erlotinib HCl (OSI-774) combined with carboplatin and paclitaxel (CP) chemotherapy in advanced non-small cell lung cancer (NSCLC). J Clin Oncol 23: 5892-5899.
Huang CL, Liu D, Nakano J, Yokomise H, Ueno M, Kadota K, and Wada H (2007) E2F1 Overexpression correlates with thymidylate synthase and survivin gene expressions and tumor proliferation in non-small-cell lung cancer. Clin Cancer Res 13: 6938-6946.
Janmaat ML, Kruyt FA, Rodriguez JA, and Giaccone G (2003) Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited anti-proliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways. Clin Cancer Res 9: 2316-2326.
Janmaat ML, Rodriguez JA, Gallegos-Ruiz M, Kruyt FA, and Giaccone G (2006) Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells. Int J Cancer 118: 209-214.[CrossRef][Medline]
Kobayashi S, Shimamura T, Monti S, Steidl U, Hetherington CJ, Lowell AM, Golub T, Meyerson M, Tenen DG, Shapiro GI, et al. (2006) Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signaling. Cancer Res 66: 11389-11398.
Li T, Ling YH, Goldman ID, and Perez-Soler R (2007) Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in human non-small cell lung cancer cells. Clin Cancer Res 13: 3413-3422.
Li W, Fan J, Hochhauser D, Banerjee D, Zielinski Z, Almasan A, Yin Y, Kelly R, Wahl GM, and Bertino JR (1995) Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines. Proc Natl Acad Sci U S A 92: 10436-10440.
Lo HW and Hung MC (2006) Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br J Cancer 94: 184-188.[CrossRef][Medline]
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350: 2129-2139.
Magné N, Fischel JL, Dubreuil A, Formento P, Ciccolini J, Formento JL, Tiffon C, Renee N, Marchetti S, Etienne MC, et al. (2003) ZD1839 (Iressa) modifies the activity of key enzymes linked to fluoropyrimidine activity: rational basis for a new combination therapy with capecitabine. Clin Cancer Res 9: 4735-4742.
Matsumoto S, Igishi T, Hashimoto K, Kodani M, Shigeoka Y, Nakanishi H, Touge H, Kurai J, Makino H, Takeda K, et al. (2004) Schedule-dependent synergism of vinorelbine and 5-fluorouracil/UFT against non-small cell lung cancer. Int J Oncol 25: 1311-1318.[Medline]
Mauritz R, Peters GJ, Priest DG, Assaraf YG, Drori S, Kathmann I, Noordhuis P, Bunni MA, Rosowsky A, Schornagel JH, et al. (2002) Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis. Biochem Pharmacol 63: 105-115.[CrossRef][Medline]
Mauritz R, van Groeningen CJ, Smid K, Jansen G, Pinedo HM, and Peters GJ (2007) Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expression after administration of 5-fluorouracil to patients with colorectal cancer. Int J Cancer 120: 2609-2612.[CrossRef][Medline]
Mendelsohn J and Baselga J (2000) The EGF receptor family as targets for cancer therapy. Oncogene 19: 6550-6565.[CrossRef][Medline]
Mey V, Giovannetti E, De Braud F, Nannizzi S, Curigliano G, Verweij F, De Cobelli O, Pece S, Del Tacca M, and Danesi R (2006) In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients. Br J Cancer 95: 289-297.[CrossRef][Medline]
Morelli MP, Cascone T, Troiani T, De Vita F, Orditura M, Laus G, Eckhardt SG, Pepe S, Tortora G, and Ciardiello F (2005) Sequence-dependent antiproliferative effects of cytotoxic drugs and epidermal growth factor receptor inhibitors. Ann Oncol 16 (Suppl 4): iv61-iv68.
Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304: 1497-1500.
Peters GJ, Backus HH, Freemantle S, van Triest B, Codacci-Pisanelli G, van der Wilt CL, Smid K, Lunec J, Calvert AH, Marsh S, et al. (2002) Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism. Biochim Biophys Acta 1587: 194-205.[Medline]
Peters GJ, van der Wilt CL, van Moorsel CJ, Kroep JR, Bergman AM, and Ackland SP (2000) Basis for effective combination cancer chemotherapy with antimetabolites. Pharmacol Ther 87: 227-253.[CrossRef][Medline]
Pfister DG, Johnson DH, Azzoli CG, Sause W, Smith TJ, Baker S Jr, Olak J, Stover D, Strawn JR, Turrisi AT, et al. (2004) American Society of Clinical Oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol 22: 330-353.
Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, Campos D, Maoleekoonpiroj S, Smylie M, Martins R, et al. (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353: 123-132.
Shih C, Chen VJ, Gossett LS, Gates SB, MacKellar WC, Habeck LL, Shackelford KA, Mendelsohn LG, Soose DJ, Patel VF, et al. (1997) LY231514, a pyrrolo [2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 57: 1116-1123.
Shuai XM, Han GX, Wang GB, and Chen JH (2006) Cyclin D1 antisense oligodexoy-neucleotides inhibits growth and enhances chemosensitivity in gastric carcinoma cells. World J Gastroenterol 12: 1766-1769.[Medline]
Sigmond J, Backus HH, Wouters D, Temmink OH, Jansen G, and Peters GJ (2003) Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression. Biochem Pharmacol 66: 431-438.[CrossRef][Medline]
Suenaga M, Yamaguchi A, Soda H, Orihara K, Tokito Y, Sakaki Y, Umehara M, Terashi K, Kawamata N, Oka M, et al. (2006) Antiproliferative effects of gefitinib are associated with suppression of E2F-1 expression and telomerase activity. Anticancer Res 26: 3387-3391.
Teicher BA, Chen V, Shih C, Menon K, Forler PA, Phares VG, and Amsrud T (2000) Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts. Clin Cancer Res 6: 1016-1023.
Tonkinson JL, Worzalla JF, Teng CH, and Mendelsohn LG (1999) Cell cycle modulation by a multitargeted antifolate, LY231514, increases the cytotoxicity and antitumor activity of gemcitabine in HT29 colon carcinoma. Cancer Res 59: 3671-3676.
van der Wilt CL, van Laar JA, Gyergyay F, Smid K, and Peters J (1992) Biochemical modification of the toxicity and the anti-tumour effect of 5-fluorouracil and cisplatinum by WR-2721 in mice. Eur J Cancer 28A: 2017-2024.[CrossRef]
Van Schaeybroeck S, Karaiskou-McCaul A, Kelly D, Longley D, Galligan L, Van Cutsem E, and Johnston P (2005) Epidermal growth factor receptor activity determines response of colorectal cancer cells to gefitinib alone and in combination with chemotherapy. Clin Cancer Res 11: 7480-7489.
Van Schaeybroeck S, Kyula J, Kelly DM, Karaiskou-McCaul A, Stokesberry SA, Van Cutsem E, Longley DB, and Johnston PG (2006) Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells. Mol Cancer Ther 5: 1154-1165.
van Triest B, Pinedo HM, Telleman F, van der Wilt CL, Jansen G, and Peters GJ (1997) Cross-resistance to antifolates in multidrug resistant cell lines with P-glycoprotein or multidrug resistance protein expression. Biochem Pharmacol 53: 1855-1866.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  J.-C. Ko, S.-C. Ciou, C.-M. Cheng, L.-H. Wang, J.-H. Hong, M.-Y. Jheng, S.-T. Ling, and Y.-W. Lin Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells Carcinogenesis, July 1, 2008; 29(7): 1448 - 1458. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
