In vitro comparative evaluation of trastuzumab (Herceptin®) combined with paclitaxel (Taxol®) or docetaxel (Taxotere®) in HER2-expressing human breast cancer cell lines

J.-L. Merlin+, M. Barberi-Heyob and N. Bachmann

Centre Alexis Vautrin, Laboratoire de Recherche en Oncologie, Vandoeuvre-les-Nancy, France

Received 26 October 2001; revised 4 April 2002; accepted 17 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background:

Trastuzumab (Herceptin®) has clinical indication in association with paclitaxel (Taxol®) for the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer. Synergistic interactions have been reported with taxane derivatives in HER2-expressing breast cancer cells. However, no direct comparison of the potential interest in combining trastuzumab with either paclitaxel or docetaxel (Taxotere®) has been reported.

Materials and methods:

The present study was designed to evaluate in a comparative way the interaction of trastuzumab with paclitaxel or docetaxel in HER2-overexpressing human breast cancer cell lines. HER2 expression was documented in MCF-7, MDA-MB453 and SK-BR3 cell lines using immunocytochemistry with purified mouse anti-human monoclonal antibody. Cytotoxicity assays were performed using the sulforhodamine B assay and in vitro interactions between trastuzumab and taxanes were analyzed using the median-effect principle.

Results:

Trastuzumab cytotoxicity was confirmed to be directly related to HER2 expression level. At the IC50, the combination of trastuzumab with either paclitaxel or docetaxel led to synergism in all cell lines. However, considering mean values calculated in the IC30–IC70 range of concentrations, trastuzumab interacted additively with docetaxel in SK-BR3 and MDA-MB453 cell lines while additive and synergistic interactions were achieved with paclitaxel in SK-BR3 and MDA-MB453, respectively. On the same basis, trastuzumab yielded synergistic interaction with both taxanes in the MCF-7 cell line.

Conclusions:

The present study shows that at least additive interactions are observed when trastuzumab is combined with either paclitaxel or docetaxel in weak to moderate or more than moderate HER2-expressing cells. Some interesting results were achieved in cells displaying weak HER2 expression which could suggest some further potential interest in trastuzumab.

Key words: docetaxel, HER2, paclitaxel, trastuzumab


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human epidermal growth factor 2 (HER2) proto-oncogene, also known as c-erbB-2, belongs to a gene family encoding a growth factor protein family. The protein encoded by this gene is a 185 kDa transmembrane tyrosine kinase receptor (p185HER2/neu) homologous to epidermal growth factor (EGF) receptor [1].

As a consequence of HER2 gene amplification, p185HER2/neu overexpression occurs in 25–30% of breast carcinomas and is associated with a poor prognosis [2, 3]. The prognostic significance of HER2 overexpression has also been reported in other tumor types such as those of bladder, colon, stomach, testis, lung [4] and pancreas [5] and male breast cancers [6].

Preclinical studies demonstrated that HER2 overexpression is associated with chemoresistance to anticancer drugs, including the taxane derivatives paclitaxel and docetaxel [7]. Down-regulation of HER2 overexpression [8] has been reported to circumvent the resistance to paclitaxel in ovarian cancer cells.

4D5 mouse monoclonal antibody was reported to be directed against an extracellular epitope of p185HER2/neu protein [9] and exerts an antiproliferative effect against murine and human cells overexpressing p185HER2/neu [10]. Trastuzumab is a humanized form of 4D5 [11], which showed higher affinity for p185HER2/neu and similar antiproliferative activity against HER2-overexpressing cells.

Preclinical studies performed in vitro [12] as well as in vivo [13] in breast carcinoma overexpressing HER2 reported additive effects to synergistic interactions between trastuzumab and anticancer drugs including alkylating agents, platinum derivatives, topoisomerase 2 inhibitors and taxanes.

A phase III clinical study [14] was performed in 469 patients with metastatic breast carcinoma overexpressing HER2. First-line chemotherapy consisted of either paclitaxel (alone or combined with trastuzumab), or doxorubicin and cyclophosphamide (alone or combined with trastuzumab). A significant increase in response rate and time to progression were observed in patients receiving trastuzumab with a good safety profile. Docetaxel was also evaluated [15, 16] in combination with trastuzumab in phase II trials as first- or second-line chemotherapy for women with metastatic breast cancer whose tumors overexpressed HER2 and appeared to be a well-tolerated regimen. In non-small-cell lung carcinoma, combination of trastuzumab with either paclitaxel or docetaxel was recently evaluated in a randomized phase II trial [17]; no difference in response rate and toxicity was observed between the two taxanes.

The present study was designed to evaluate the combination of trastuzumab with either paclitaxel or docetaxel in human breast carcinoma cell lines expressing different levels of HER2.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Drugs
Paclitaxel was purchased from Sigma (Saint Quentin Fallavier, France), docetaxel (Taxotere®) from Aventis Pharma (Montrouge, France), trastuzumab (Herceptin®) from Roche Pharma AG (Reinach, Switzerland). Paclitaxel was solubilized in ethanol and diluted in sterile water. Docetaxel and trastuzumab were solubilized according to the manufacturer’s procedure.

Cell lines and culture conditions
MCF-7, MDA-MB453 and SK-BR3 human breast cancer cell lines (kindly given by Jean-Philippe Peyrat, Centre Oscar Lambret, Lille, France) were maintained in RPMI-1640 (Life Technologies, Cergy-Pontoise, France) without phenol red supplemented with 9% heat-inactivated fetal calf serum (Dutscher SA, Brumath, France), 100 U–100 µg/ml penicillin–streptomycin (Life Technologies), 2 mmol/l l-glutamine (Life Technologies) in 75 cm2 plastic flasks (Costar; Dutscher SA) at 37°C in a humidified atmosphere with 5% CO2, and passaged weekly by trypsinization (trypsin–EDTA; Life Technologies).

HER2 expression analysis
Immunocytochemistry
Cytospun cells were fixed in Preservcyt® (Cytyc, Montrouge, France) according to the manufacturer’s recommendations, then placed in 10 mmol/l citrate buffer, pH 6, in a pressure cooker for 4 min. The cells were then stained with A0485 anti-HER2 monoclonal antibody (Dako, Trappes, France) at 1:1000 dilution in phosphate-buffered saline (PBS) for 30 min at room temperature with a basic diaminobenzidine detection kit (Ventana, Illkirch, France) using biotinylated immunoglobulin secondary antibody according to the manufacturer’s recommendations. Briefly, the cells were treated with avidin-horseradish peroxidase conjugate, then by hydrogen peroxide and diaminobenzidine as chromogen substrate with light hematoxylin counterstaining.

Flow cytometry
Cell suspensions were prepared according to Stal et al. [18]. Briefly 106 cells/ml were fixed in 1% paraformaldehyde for 3 min then centrifuged and resuspended in 0.5% bovine serum albumin in PBS. The cell suspensions were then exposed to the anti-HER2 monoclonal antibody (9G6; Pharmingen). The negative control was IgG1 immunoglobulin. Both were then processed in the same way, with addition of the secondary fluorescein isothiocyanate (FITC)-conjugated antibody after washing and resuspension in 1 ml 0.5% bovine serum albumin in PBS. The samples were analyzed using flow cytometry (FACScalibur®; Becton Dickinson, Meylan, France) with 488 nm argon laser excitation and 530 nm detection of the FITC signal; 10 000 cellular events were analyzed per sample.

Cytotoxicity assays
Cytotoxicity was analyzed using a sulforhodamine-B (SRB) (Aldrich-Chimie, St Quentin Fallavier, France) assay as previously reported [19]. Briefly, cell suspensions containing 104 viable cells/ml were plated into 96-well dishes and allowed to attach for 24 h at 37°C in a 5% CO2 atmosphere. The cells were then exposed to trastuzumab (0.7–338 nmol/l) from day 1 to day 5. When combined, taxanes (0.1 nmol/l to 1 µmol/l for paclitaxel and 0.01 nmol/l to 0.1 µmol/l for docetaxel) were added at day 3 for 48 h. Control experiments including trastuzumab alone and taxanes alone were performed using the same time schedule. The ratio of trastuzumab to paclitaxel was fixed at 0.14 according to previous results [12]. The trastuzumab to docetaxel molar ratio was adjusted to 0.28, according to the protocols used in clinical trials in which the trastuzumab dose is kept constant at a 4 mg/kg loading dose then 2 mg/kg while paclitaxel and docetaxel doses differ (175–200 mg/m2 and 75–100 mg/m2, respectively), according to the maximal tolerated dose of each compound [1416]. In every case, blank control experiments were performed to evaluate the effect of excipients used to solubilize each drug. After being exposed to drugs, the cells were washed with PBS and fixed by means of protein precipitation with trichloroacetic acid at 4°C for 1 h. After washing with water, cells were stained with SRB. Protein-bound stain was solubilized with unbuffered Tris base [tris(hydroxymethyl)aminomethane] (Merck, Darmstadt, Germany). The absorbance of each well was then measured at 540 nm using a Multiskan MCC/340 microplate reader (Flow Laboratories, Les Ulis, France). Each concentration was assayed in sextuplicate and each experiment was repeated at least three times. Results were expressed as relative absorbance compared with untreated controls. Drug concentrations inhibiting 50% of cell growth (IC50) were calculated using the median-effect principle [20] based on the equation fa/fu = (D/Dm)m, with fa being the dead cell fraction; fu, the surviving cell fraction (fu = 1-fa); D, the concentration tested; Dm, the IC50 and m a Hill-type coefficient illustrating the sigmoidicity of the dose-effect plot. Dm is extrapolated using linear regression analysis of the median-effect plot representing log(fa/fu) as a function of log(D).

Combination analysis
Drug combination analysis was performed according to Chou and Talalay multiple drug interaction analysis [20] based on the calculation of combination indices (CIs) using the following equation: CI = (DHER/DHERx) + (DTAX /DTAXx) + (DHER x DTAX/DHERx x x DTAXx), with DHER, DTAX being each drug (trastuzumab, taxane) concentration in the mixture, required to induce x% cytotoxicity and DHERx, DTAXx the concentration of each drug required to induce the same cytotoxicity when used alone. The interaction is considered as synergistic if CI <1, additive if CI = 1, antagonistic if CI >1.

Statistical analysis
All results were analyzed for statistical significance using a non-parametric Mann–Whitney U test with a limit set to P <0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HER2 expression
Immunocytochemistry.
According to the consensus used for classification of HER2 expression in tumor specimens [3], MCF-7, MDA-MB453 and SK-BR3 cell lines were found to express HER2 at weak (1+), weak to moderate (2+) and more than moderate levels (3+) in more than 10% of the cells, respectively (Table 1).


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Table 1. HER2 expression using immunocytochemistry
 
Flow cytometry.
Flow cytometry data allowed further characterization of HER2 expression in the three cell lines. HER2 expression was detected in nearly all cells from all three cell lines (>97%). As observed using immunocytochemistry, significant differences in fluorescence intensity were detected among the three cell lines and HER2 expression was found to be respectively 3- and 18-fold higher in MDA-MB453 and SK-BR3 than in MCF-7 cells (Table 2).


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Table 2. HER2 expression using flow cytometry
 
Cytotoxicity assays
Trastuzumab alone.
Under the experimental conditions used, the IC50 of trastuzumab was not reached in any cell line in the tested concentration range (0.7–338 nmol/l). Trastuzumab cytotoxicity increased with concentration from 2, 3 and 19% cell kill at 0.7 nmol/l, up to 10, 16 and 30% of cell kill at the maximal tested concentration (338 nmol/l), respectively, in MCF-7, MDA-MB453 and SK-BR3 cell lines. In SK-BR3 cell line, the IC50 [standard deviation (SD)] for trastuzumab was extrapolated to 820 (183) nmol/l.

Taxanes alone.
Each drug was evaluated alone in the three cell lines. The mean IC50 (SD) for paclitaxel in MCF-7, MDA-MB453 and SK-BR3 was found to be 28.2 (16), 22.3 (7) and 101.4 (26) nmol/l, respectively, while for docetaxel the mean IC50 was 9.1 (1.8), 11.9 (2.2) and 20.2 (2.3) nmol/l, respectively. No significant difference in IC50 was observed between MCF-7 and MDA-MB453 cell lines with either paclitaxel or docetaxel. HER2 overexpression in SK-BR3 was associated with a significant increase in the IC50 of taxanes (P <0.01) as compared with the two other cell lines. This increase was significantly more pronounced for paclitaxel (4.6-fold) than for docetaxel (1.7-fold).

Drug combinations.
Combinations of trastuzumab with paclitaxel or docetaxel were evaluated in the three cell lines. Analysis of drug interaction was based on CI values.

At the IC50, all combinations of trastuzumab with either paclitaxel or docetaxel were found to be synergistic (Figure 1) with CIs being significantly lower than 1 (P <0.05).



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Figure 1. Median-effect plot of paclitaxel alone (plain black line) or combined with trastuzumab (dotted black line) and docetaxel alone (plain gray line) or combined with trastuzumab (dotted gray line) in MCF-7 (A), MDA-MB435 (B) and SK-BR3 (C) cells.

 
Mean CIs were calculated from CI values achieved at IC30, IC40, IC50, IC60 and IC70 as reported previously by Pegram et al. [12]. When mean CI values were considered (Table 3), significant differences appeared between the cell lines. In MCF-7 cells, both taxanes showed synergistic interaction with trastuzumab. In MDA-MB453 cells, only paclitaxel yielded synergistic interactions with trastuzumab while docetaxel yielded additive interaction. In SK-BR3 cells, both taxanes showed additive interaction.


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Table 3. Taxane–trastuzumab interaction analysis
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Trastuzumab is an innovative monoclonal antibody therapy indicated in patients with advanced breast carcinoma whose tumors overexpress HER2, thus representing ~30% of primary breast carcinomas. At present, trastuzumab is registered in metastatic breast carcinoma as monotherapy in recurrent disease and in association with paclitaxel as first-line regimen. Recent clinical trials [15, 16] reported that docetaxel could also be combined safely with trastuzumab in breast carcinoma.

The rational design of such clinical trials is based on preclinical results evaluating the association of taxanes derivative with trastuzumab. Results achieved with paclitaxel in cell lines and/or human tumor xenografted mice revealed additive or synergistic interactions [12, 13]. Until now, no rational study has been reported either in vitro or in vivo comparing the potency of trastuzumab combination with paclitaxel or docetaxel, although this could be of great interest for designing clinical trials. Moreover, some clinical trials have been initiated [15] claiming that docetaxel interacts synergistically with trastuzumab, although no preclinical data have been published with docetaxel. Only ternary combinations of taxanes (paclitaxel or docetaxel) with carboplatin and trastuzumab have been reported to yield therapeutic advantage in HER2-overexpressing cell lines [21], with equal potency of both taxanes. Considering this controversy, the present study was designed to evaluate, in a direct comparative way, paclitaxel versus docetaxel in combination with trastuzumab in human breast carcinoma cell lines.

Three human breast carcinoma cell lines were selected with different levels of expression of HER2. Using immunocytochemistry, the three cell lines were found to fit within the three classes used for pathological classification of human tumors with regard to HER2 expression. Using flow cytometry, expression of HER2 in the SK-BR3 and MDA-MB453 cell lines was 3- and 18-fold higher, respectively, than in the MCF-7 cell line. However, in the three cell lines, HER2 was found to be expressed in a large majority (>95%) of the cells. These data are fully consistent with those from original work on the same cell lines [22, 23]. In this panel of cell lines, when used alone, trastuzumab cytotoxicity was found to correlate with HER2 expression level, with a cytotoxicity profile being consistent with those reported by Pegram et al. [12] in the SK-BR3 cell line. The results presented in the present paper show that the cell sensitivity to either paclitaxel or docetaxel appears to be related to HER2 expression level, as already reported with these drugs in breast cancer cell lines [24] and with paclitaxel in ovarian cell lines [8].

The present study was performed using different trastuzumab:taxane ratios for paclitaxel and docetaxel. This experimental point is consistent with standard clinical protocols used in breast cancer in which paclitaxel is administered at between 175 and 225 mg/m2 and docetaxel between 75 and 100 mg/m2, while the trastuzumab dose is kept constant at 4 mg/kg starting dose then 2 mg/kg whatever the taxane [1416]. It is also consistent with the mean ratio of the IC50 observed in vitro between the two taxanes in a large panel of cell lines [25].

The results presented in the present paper confirmed data reported previously showing that in SK-BR3 HER/neu-overexpressing cells, paclitaxel yielded additive interaction with trastuzumab at the same trastuzumab:paclitaxel molar ratio as previously evaluated [12]. However, in the same cell line, no synergism but a clear additive interaction was found with docetaxel. Only in the moderately HER2-expressing MDA-MB453 cell line was a difference observed between the two taxanes. In this cell line, paclitaxel yielded synergistic while docetaxel yielded additive interaction. In the estrogen receptor-negative SK-BR3 and MDA-MB453 cell lines [26], the sensitization to taxanes could be mediated by a positive interaction with the pro-apoptotic caspase-3 pathway [8]. In MCF-7 cells, which we found to express HER2 at a lower level than SK-BR3 and MDA-MB453 cells and which are known to be deficient in caspase-3 activation [27], the combination of either paclitaxel or docetaxel with trastuzumab still produced synergistic interaction. As opposed to MDA-MB453 and SK-BR3 cell lines, MCF-7 cells express estrogen receptors, EGF and insulin-like growth factor-I (IGF-I) [28]. In such a case, the interaction with trastuzumab observed could be mediated by its inhibitory effect on the phosphatidylinositol 3-kinase-dependent pathway [29], whose interaction with estrogen receptor [30] or EGF and IGF-I-mediated growth stimulation [28], as well as its critical role in anticancer drug sensitivity including paclitaxel [31] and gemcitabine [32], were recently described. The raf-1 kinase pathway was also implicated. Indeed, some recent results in human ovarian and cervical cell lines [33] suggest that raf-1 kinase activity could explain the difference in sensitivity toward paclitaxel and docetaxel, confirming data reported in breast cancer cells and therefore, that phosphorylation of raf-1 could be an important signaling event in paclitaxel- but not docetaxel-induced cell death [34].

In conclusion, the present study shows that both taxanes are equally efficient when combined with trastuzumab in more than moderate HER2-expressing cells since, at least, additive interactions are observed when trastuzumab is combined with either paclitaxel or docetaxel. Some interesting results were achieved in cells displaying weak to moderate, and weak HER2 expression and suggest further potential interest in trastuzumab.


    Acknowledgements
 
The authors are grateful to Dr R. Michel Parache for the cytological analysis. This study was presented at the 92nd Annual Meeting of the American Association for Cancer Research.


    Footnotes
 
+ Correspondence to: Dr J.-L. Merlin, Centre Alexis Vautrin, Laboratoire de Recherche en Oncologie, Avenue de Bourgogne, 54511 Vandoeuvre-les-Nancy cedex, France. Tel: +33-383-59-83-07; Fax: +33-383-44-78-51; E-mail: jl.merlin{at}nancy.fnclcc.fr Back


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 Materials and methods
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 Discussion
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