Anaesthetic drugs can influence the function of the immune system in a negative fashion [1-3], e.g. by impairing the chemotactic activity of macrophages , monocytes , eosinophils  and neutrophil granulocytes , by reducing the oxidative ability and phagocytic activity in macrophages  and by down-regulating natural killer cell function . Additionally, anaesthetic drugs impair leukocyte recruitment to inflamed tissues by reducing their transmigration through endothelial cell monolayers [8,9] and by decreasing the secretion of chemokines such as interleukin-8 . Thus anaesthetic drugs inhibit immune function not only by inhibiting cell migration, but also impair the recruitment of immune competent cells during inflammation. Both processes are prerequisites for efficient immune supervision.
Because of the immune suppressive characteristics of anaesthetic drugs, there is an ongoing discussion whether these compounds might have an adverse influence on wound healing processes and parasitic diseases , whether they would predispose postoperative and intensive care patients to infection  or whether they might promote tumour progression in patients . However, the question whether anaesthetic drugs might also promote tumour progression by influencing the migration of cancer cells is not yet clear. It is well known that the primary cause of death in cancer disease is not attributed to the formation of a primary tumour but rather to the growth of metastases at distant organ sites for which the induction of cell migration is a prerequisite .
A relationship between the gamma aminobutyric acid (GABA)-ergic system and oncogenesis has been suggested for a variety of tumours  including breast cancer [13,14]. This concurs with our recent finding that clinically relevant doses of the GABA-A receptor agonist propofol resulted in an increased migration of the human breast carcinoma cell line MDA-MB-468 . However, the observation that propofol can activate the migration of the MDA-MB-468 breast cancer cell line is in contrast to other studies showing that propofol impairs the migration of immune competent cells [2, 4-6] and even cancer cells .
In order to clarify how the migration of MDA-MB-468 breast carcinoma cells is altered by propofol we investigated for GABA-A receptor expression on this cell line. Propofol is a specific GABA-A receptor agonist that acts on this receptor by both enhancing the action of GABA on the GABA-A receptor  as well as directly activating GABA-A receptors . Here we show that the MDA-MB-468 breast carcinoma cells express a functional GABA-A receptor. Activation of this receptor resulted in the permanent engagement of voltage-gated L-type calcium channels leading to reorganization of the actin cytoskeleton and induction of cell migration.
Solutions and drugs application
The anaesthetic drug long-chain triglyceride (LCT)-propofol (Abbot, Wiesbaden, Germany) was diluted in Leibovitz's-15 (L-15; PAA Laboratories GmbH, Linz, Austria) medium and used in a final concentration of 6 μg mL−1. Control experiments were performed with a dilution of the solvent (LCT 20; Lipovenoes, Fresenius Kabi, Bad Homburg, Germany). The GABA-A receptor antagonist bicuculline (Sigma, Munich, Germany) was dissolved in dimethyl sulfoxide and diluted in L-15 medium containing a dilution of LCT solvent. Bicuculline was used in a final concentration of 50 μmol. Verapamil (Merck BioSciences GmbH, Schwalbach, Germany) was dissolved in phosphate buffer saline (PBS), diluted in L-15 containing a dilution of LCT and used in a final concentration of 5 μmol. Verapamil was selected from a spectrum of calcium channel blockers since it selectively blocks plasma membrane bound voltage-sensitive L-type calcium channels, which are activated as a consequence of GABA-A receptor engagement.
The human breast carcinoma cell line MDA-MB-468 (HTB-132) was purchased from ATCC (Rockville, USA) and maintained in L-15 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin. Cells were cultivated in a humidified atmosphere at 37°C.
Western blot analysis of GABA-A receptor expression of MDA-MB-468 and U373 cells (obtained from C. Kaltschmidt, Institute for Neurobiochemistry, University of Witten/Herdecke) was performed as described previously . Detection of the GABA-A receptor was performed with the primary polyclonal goat anti-GABAA Rγ3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the secondary mouse-anti-goat horse-radish-peroxidase conjugated antibody (Dianova, Hamburg, Germany). Antibodies were used at a concentration of 0.1 μg mL−1. Bands were visualized using the highly sensitive enhanced chemiluminescence system (Amersham/Pharmacia Biotech, Freiburg, Germany).
Confocal laser scanning microscopy
Visualization of the effects of propofol and verapamil on the calcium influx and reorganization of the actin cytoskeleton was performed using an inverse confocal laser scanning microscope (Leica TCS 4D; Leica, Bensheim, Germany) as previously described [11,19]. In brief, for calcium influx analysis, MDA-MB-468 cells (2 × 105) were seeded onto coverslips and incubated with phosphate saline solution (PSS) buffer (145 mmol NaCl, 5.6 mmol KCl, 1 mmol MgSO4, 1 mmol CaCl2, 20 mmol HEPES, 10 mmol glucose, pH 7.4 (all chemicals from Sigma)) containing 10 μmol Fluo-3 for 30 min at 37°C. Cells were washed twice with PSS followed by addition of fresh L-15 media and incubated for 30 min at 37°C. Coverslips were placed under a self-constructed flow chamber. During calcium measurement, cells were exposed to a continuous flow of L-15 media or L-15 media supplemented with 6 μg mL−1 propofol, 5 μmol verapamil or a combination of both. A time series of 50-115 confocal images was recorded for each experiment, each series containing images recorded at 6 s intervals. The calcium signalling of individual cells was analysed using the histogram function of Corel Photopaint software. The grey-scale value of the first histogram of each cell was normalized to 100%, and the detected grey-scale values were normalized with respect to this 100% figure. For studying actin reorganization, MDA-MB-468 cells (2 × 104) were seeded in chamberslides (Nunc, Wiesbaden, Germany) and treated for 20 min with 6 μg mL−1 propofol, 5 μmol verapamil or a combination of both. Subsequently, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature, washed three times with PSS and treated with 0.5% (v/v in PSS) Triton X-100 (Sigma) for 10 min at room temperature. Cells were washed again three times with PSS, and the F-actin cytoskeleton was stained for 2 h at room temperature with TexasRed-X-Phalloidin (Molecular Probes, Leiden, The Netherlands).
Cell migration assay and statistical analysis of cell migration data
The migratory behaviour of MDA-MB-468 breast carcinoma cells was analysed using the three-dimensional collagen matrix assay combined with time-lapse video microscopy and subsequent computer-assisted cell tracking as described previously [11,19]. In brief, liquid collagen solution (Vitrogen 100; Nutacon, Leimuiden, The Netherlands) was mixed with 10 × MEM (Sigma), sodium bicarbonate (Sigma), MDA-MB-468 cells (6 × 104) and the appropriate substances to be tested. Cell migration within the three-dimensional collagen lattice was recorded for at least 8 h by time-lapse video microscopy and subsequently analysed by computer-assisted cell tracking. For data analysis, 30 cells of each sample were randomly selected and two-dimensional projections of the paths were digitized as x/y coordinates in 15 min intervals. Statistical significance was calculated by t-test. Levels of significance <0.1% (P < 0.001) were considered significant.
GABA-A receptor expression of MDA-MB-468 cells was determined by Western blot analysis (Fig. 1). Compared to the human astrocytoma cell line U373, which was used as a positive control, MDA-MB-468 breast carcinoma cells showed a weaker expression of the γ subunit of the GABA-A receptor. Long-term stimulation of MDA-MB-468 cells with 6 μg mL−1 propofol did not alter the expression of the γ subunit of the GABA-A receptor (Fig. 1).
The mode of calcium signalling in MDA-MB-468 cells treated with propofol (6 μg mL−1), verapamil (5 μmol) and a combination of both was determined on a single cell level by confocal laser scanning microscopy. Untreated MDA-MB-468 cells displayed baseline transient calcium oscillations (Fig. 2a). Treatment of MDA-MB-468 cells with 6 μg mL−1 propofol resulted in a permanent opening of calcium channels indicated by elevated intracellular calcium levels. The propofol-mediated influx of calcium started approximately 200 s after addition of this anaesthetic drug (Fig. 2b). In order to prove whether the propofol driven calcium influx was attributed to the opening of plasma membrane bound calcium channels, the voltage-sensitive L-type calcium channel blocker verapamil was added to the experimental setup. Control experiments with verapamil alone showed no alterations in the autologous calcium signalling of MDA-MB-468 cells. Verapamil (5 μmol) treated MDA-MB-468 cells still displayed baseline transient calcium oscillations (Fig. 2c). A similar result was obtained for MDA-MB-468 cells co-treated with propofol (6 μg mL−1) and verapamil (5 μmol). MDA-MB-468 cells, co-incubated with both substances, still showed baseline transient calcium oscillations (Fig. 2d).
We next examined the actin distribution within MDA-MB-468 cells before and after (20 min) treatment with propofol (6 μg mL−1), verapamil (5 μmol) and a combination of both. Untreated MDA-MB-468 cells showed the typical distribution of the actin cytoskeleton of a quiescent cell. Actin fibres are bundled in parallel on the bottom of the cell as well as beneath the plasma membrane (Fig. 3a). Treatment of MDA-MB-468 cells with propofol (6 μg mL−1) resulted in the reorganization of the actin cytoskeleton. Newly polymerized actin fibres were found to be located beneath the plasma membrane and in lamellipodia formation (Fig. 3b). MDA-MB-468 cells solely treated with verapamil (5 μmol) showed no alterations in the distribution of the actin cytoskeleton (Fig. 3c) as compared to control cells, which is congruent to the obtained calcium influx data showing that verapamil had no influence on the autologous calcium signalling (Fig. 2c). As expected, co-treatment of MDA-MB-468 cells with propofol (6 μg mL−1) and verapamil (5 μmol) blocked the propofol-induced actin reorganization (Fig. 3d) due to the verapamil-mediated inhibition of plasma membrane bound voltage-sensitive L-type calcium channels. Actin fibre bundles located at the bottom of a cell are still visible.
Since actin reorganization is a prerequisite in the process of cell migration we next investigated the migratory activity of MDA-MB-468 cells. Cell migration was recorded for 8 h by time-lapse video microscopy and subsequently analysed by computer-assisted cell tracking. Untreated MDA-MB-468 cells showed a mean spontaneous migration rate of about 64.4 ± 7.0%, which was significantly increased by propofol (6 μg mL−1) to 85.0 ± 5.0% (Fig. 4a; P < 0.001). In accordance with both the calcium influx as well as the actin reorganization data, verapamil (5 μmol) alone had no influence on the spontaneous migration of MDA-MB-468 cells (mean migratory activity: 66.5 ± 7.8%). In contrast to this, verapamil blocked the propofol-induced migration of MDA-MB-468 cells (Fig. 4a). In the presence of both substances we determined a mean migratory activity of about 63.0 ± 2.0%, which is equal to the migratory activity of both untreated and solely verapamil treated cells. Similar results were achieved using the GABA-A receptor inhibitor bicuculline (Fig. 4b). Bicuculline (50 μmol) alone had no effect on the spontaneous migration of MDA-MB-468 cells (control: 56.3 ± 8.5% vs. bicuculline (50 μmol): 60.3 ± 12.5%; P = 0.044) but blocked significantly the propofol-induced migration of MDA-MB-468 cells (propofol (6 μg mL−1): 80.5 ± 7.1% vs. propofol (6 μg mL−1) + bicuculline (50 μmol): 52.5 ± 8.6%; P < 0.001). Concomitant with verapamil, bicuculline only inhibited propofol-induced migration but had no effect on the spontaneous migration of MDA-MB-468 cells.
We have investigated the relationship between GABA-A receptor modulators, the mode of calcium oscillation, actin reorganization and the induction of migration of the human breast carcinoma cell line MDA-MB-468. From our previous findings that different anaesthetic drugs have the ability to activate GABA-A receptors, thereby modulating the migratory activity of the MDA-MB-468 breast carcinoma cell line in vitro  we concluded that this cell line must express a functional GABA-A receptor. It is well recognized that the GABA-ergic system is involved in the hormonal regulation and the pathogenesis of breast cancer . In addition to this, a close relationship between the expression of peripheral-type benzodiazepine receptors and breast cancer progression has been described . Western blot analysis revealed that MDA-MB-468 breast carcinoma cells express the γ subunit of the GABA-A receptor. This result is in accordance with recent findings of Jiang and colleagues, who have shown that the π subunit of the GABA-A receptor could be detected in breast tumour tissue . In addition to this, GABA receptor expression was also detected in gastrointestinal epithelium, pancreas and ovary tissue .
Control MDA-MB-468 cells (treated with the solvent LCT 20) showed autologous baseline transient oscillations, which is in accordance with previously published data . These autologous baseline transient calcium oscillations are not attributed to the lipid emulsion itself, but rather to the fact that an autocrine epidermal growth factor and transforming growth factor-α loop exist in this epidermal growth factor receptor over-expressing cell line . Treatment of MDA-MB-468 cells with propofol resulted in a permanent opening of calcium channels concomitantly with the reorganization of the actin cytoskeleton. These findings are consistent with the data of Labrakakis and colleagues and Oscarsson and colleagues [24,25]. Labrakakis and co-workers reported that activation of the GABA-A receptor complex resulted in an increase in intracellular calcium levels in human glioma cells due to activation of calcium channels. Additionally, Oscarsson and colleagues demonstrated that treatment of cortical neurons with clinically relevant doses of propofol resulted in an increase in intracellular calcium levels and reorganization of the actin cytoskeleton. In summary, our data indicate that MDA-MB-468 cells must express a functional GABA-A receptor, which is activated by propofol.
Co-treatment of propofol with verapamil blocked the propofol-induced sustained calcium influx in MDA-MB-468 cells clearly indicating that GABA-A receptor activation by propofol results in the activation of voltage-gated L-type calcium channels since verapamil is a well-known voltage-gated L-type calcium channel blocker . Interestingly, baseline transient calcium oscillations are still detectable in propofol/verapamil co-treated and verapamil mono-treated cells. As mentioned above these baseline transient calcium oscillations are attributed to the autocrine epidermal growth factor and transforming growth factor-α loop that exists in this cell line . Thus neither propofol nor verapamil interfere with this autologous signalling.
A previous study of Belouchi and colleagues has shown that the effect of propofol strongly depends on the presence of extracellular albumin . Thereby, increasing levels of extracellular albumin concentration correlates with a decreased inhibitory effect of propofol because of albumin-propofol interaction. It is not clear to what extent the effect of propofol was influenced by albumins in our study. Since in each experiment MDA-MB-468 cells were incubated in medium containing 10% fetal calf serum it can be assumed that a certain amount of propofol is removed due to binding to albumin proteins. However, in our previous study we have tested three different propofol concentrations (3, 6 and 9 μg mL−1) and the observed increase in the migratory activity of MDA-MB-468 cells was similar for all three propofol concentrations. As there was no difference between the lowest (3 μg mL−1) and the moderate (6 μg mL−1) propofol concentration we conclude that the potential inhibitory effect of extracellular albumins can be neglected.
Increased levels of intracellular calcium trigger cell motility dependent signalling cascades  by regulating proteins, which interact with the actin cytoskeleton . In our study, actin reorganization occurred in MDA-MB-468 cells within 20 min of propofol treatment. Newly polymerized F-actin fibres were found to be located beneath the plasma membrane and in lamellipodia formation. The latter one is a prerequisite in the induction of cell migration . Blockade of voltage-gated L-type calcium channels with verapamil prevented the propofol-induced actin reorganization in MDA-MB-468 cells. Actin fibre bundles located at the bottom of a cell are still visible in propofol/verapamil co-treated MDA-MB-468 cells. These results indicate that the propofol-mediated sustained opening of voltage-gated L-type calcium channels is responsible for the rearrangement of the actin cytoskeleton. In accordance with these data propofol induced migration in MDA-MB-468 breast carcinoma cells, which is congruent with previously published data , whereas co-treatment with verapamil blocked the propofol-induced migration of this cell line. Verapamil alone did not affect MDA-MB-468 cell migration and the propofol-induced migration of MDA-MB-468 cells was solely blocked to control levels by verapamil, which is in accordance with the calcium influx and the actin reorganization data.
In contrast to our results a recent study of Mammoto and colleagues revealed that propofol could inhibit the invasion of human cancer cells . In this study the authors demonstrated that the invasion of several human cancer cell lines (HT1080, HOS, HeLa, RPMI-7951) was dose-dependently blocked by clinically relevant doses of propofol. Additionally, animal trials showed that the pulmonary metastasis rate of LM-8 mouse osteosarcoma cells was significantly reduced in mice receiving 20 or 40 mg kg−1 propofol per day. As a consequence the authors concluded from their results that propofol might be an ideal anaesthetic for cancer surgery . However, the question has to be addressed whether the human cancer cell lines used by Mammoto and colleagues do express a functional GABA-A receptor or not? For instance, HeLa cells treated with 5 μg mL−1 propofol for 60 min showed no redistribution of the actin cytoskeleton . In contrast, our results clearly show that the actin cytoskeleton of MDA-MB-468 breast carcinoma cells is rearranged within 20 min of propofol (6 μg mL−1) treatment. Additionally, Oscarsson and colleagues demonstrated that treatment of cortical neurons with clinically relevant doses of propofol resulted in an increase in intracellular calcium levels and reorganization of the actin cytoskeleton . Thus it can be assumed that above-mentioned human carcinoma cell lines used by Mammoto and colleagues are negative for GABA-A receptor expression and therefore respond to propofol treatment in a different manner as compared to the GABA-A receptor positive cell line MDA-MB-468.
In summary, our data show that MDA-MB-468 breast cancer cells express a functional GABA-A receptor. Activation of this receptor with propofol resulted in sustained increased intracellular calcium concentrations leading to actin reorganization and induction of migration in this human breast cancer cell line. However, it is difficult to hypothesize that propofol may act as a general promotor of GABA-A receptor positive cancer cell migration since only one cell line was investigated. Nonetheless, our data show that the migration of cancer cells expressing a functional GABA-A receptor can be triggered by propofol. GABA-A receptor expression is found in various tissues including gastrointestinal epithelium, pancreas and ovary  and a relationship between the GABA- ergic system and oncogenesis has been suggested . It might therefore be speculated that propofol might not be the appropriate anaesthetic drug in the surgery of GABA-A receptor positive tumours such as breast cancer. However, this should be verified in further in vitro and in vivo studies, including animal trials and prospective clinical studies.
This work was supported by the Fritz-Bender-Foundation, Munich, Germany.
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