Abstract
Background: It has been hypothesized that anaesthesia during primary breast cancer surgery may influence recurrence or metastasis. Effects of anaesthetic drugs on breast cancer cell function are unclear. The Neuroepithelial Cell Transforming Gene 1 (NET1) gene is associated with promoting migration in adenocarcinoma in vitro. Therefore, we investigated the role of NET1 in the effect of anaesthetic drugs propofol and bupivacaine on breast cancer cell function in vitro. Materials and Methods: Estrogen receptor-negative (ER-negative) MDA-MB-231 and ER-positive MCF7 breast cancer cells were incubated with propofol (1-10 μg/ml) and bupivacaine (0.5-100 μg/ml) or control medium. Cell functions were determined with the CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Chemotaxis 96-well Cell Migration and Invasion Assay. NET1 gene expression was determined by real-time PCR with gene silencing using siRNA and stimulation by lysophosphatidic acid (LPA). Results: Propofol did not reduce breast cancer cell proliferation of MCF7 or MDA-MB-231 cells. However, it did reduce migration of both MCF7 and MDA-MB-231 cells which was reversed by silencing NET1. Propofol reduced invasion of MCF7 but not of MDA-MB-231 cells, which was unaffected by siRNA. Propofol reduced expression of NET1 by 42-88% in MCF7 and by 49-79% in MDA-MB-231 cells. Bupivacaine had no significant effect on breast cancer cell function or NET1 expression. Conclusion: Propofol reduced NET1 expression and cell migration in both ER-positive and -negative cells, which was reversed by silencing NET1, implying a role for NET1 in mediating the effect of propofol on breast cancer cell function in vitro.
Breast cancer is the most common type of cancer in women and the second leading cause of cancer death, usually caused by recurrence and metastases (1). Although surgery is usually the key initial treatment, it is well-established that it inadvertently releases tumour cells to the circulation (2). Whether a micrometastasis becomes an established recurrence depends on the balance between pro-tumour and tumour-resisting influences in the perioperative period (3). It has been hypothesized that anaesthetic drugs and techniques, and postoperative pain and analgesic techniques may affect this balance, and potentially influence breast cancer recurrence or metastasis (4, 5). Increasingly, investigation has focused on potential direct effects of anaesthetic and analgesic drugs on cancer cell functions which may be essential to metastatic potential, including proliferation, migration and invasion. Studies of propofol using in vitro cell culture models of cancer cells have yielded conflicting results (6, 7).
More promising results have been materialised from experimental investigation of local anaesthetics, particularly amides, which suggest that they have direct signalling effects on the migratory function of cancer cells, either via voltage-gated sodium channels or Src intracellular signaling (8, 9).
Separately, single-gene activation and single-gene nucleotide gene polymorphisms (SNP) that affect processes essential for cancer cell function may affect cancer metastasis, both in vitro (10) and clinically: An SNP of the A118G mu-opioid receptor (MOR) is associated with resistance to the analgesic effect of morphine and to breast cancer progression (11).
Single-gene activation of the NET1 gene, which has a role in gastric cancer cell migration, is also associated with lymph node-positive breast cancer in patients at high risk for metastasis (12). Furthermore, we have shown that the stimulatory effect of morphine on the migration of estrogen receptor-positive breast cancer cells is attenuated by silencing of the NET1 gene, implying a role for this gene in breast cancer cell function (13).
However, no study to our knowledge has evaluated the influence of the NET1 gene on the effect of propofol or bupivacaine, two agents commonly used in anaesthesia and analgesia for patients undergoing breast cancer surgery, on breast cancer cell functions crucial to metastasizing potential. Therefore, we investigated the effect of these two drugs on estrogen receptor-positive and estrogen receptor-negative breast cancer cells in vitro, and on the role of the NET1 gene in these processes.
Materials and Methods
Cell cultures. Both cell lines were obtained from the European Collection of Cell Cultures (Salisbury, UK). MDA-MB-231 is an oestrogen-receptor-negative human breast adenocarcinoma cell line. MDA-MB-231 cells were cultured in L-Liebowitz 15 medium supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin solution. MCF7 is an oestrogen and progesterone-receptor-positive cell. MCF7 cells were grown in minimum essential medium with Earle's salts and sodium bicarbonate, supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin solution. Both cell lines were incubated at 37°C, in humidified atmospheric air with 5% CO2. Cells were grown as monolayers in 75 ml standard tissue culture plastic ware (Sarstedt Ltd, Dublin, Ireland). The media were changed every three days, and part of the culture was passaged every week after trypsinization. For experiments, cells were harvested from 70% subconfluent cultures by trypsinization, resuspended in media and added to assay plates as per protocol.
Drug exposure. Propofol (diisopropylphenol) was obtained in its generic chemical form from Sigma (Saint Louis, MO, USA). It was further diluted in dimethyl sulphoxide (DMSO) to 500 μg/ml and stored. Immediately before the experiment, it was again diluted with cell media to 1, 2, 4, 6, 8 and 10 μg/ml. These concentrations correlated with reported serum levels of propofol in patients after administration of clinically relevant doses intravenously (14). Similarly, bupivacaine was prepared at concentrations of 0.5, 1.0, 10, 20 and 100 μg/ml. Corresponding concentrations of Dimethyl Sulphoxide (DMSO) in media were prepared as controls. Lysophosphatidic acid (LPA) is an established driver of RHO A activation and has been shown to stimulate RHO A-mediated cytoskeletal re-arrangement events (15). LPA was obtained from Sigma Aldrich in powder form and dissolved in DMSO to obtain a concentration of 10 μM. Cells were exposed to LPA by adding it to cell media at a concentration of 10 μM. LPA was used before exposure to the anaesthetic drugs. The duration of exposure was 1, 2, and 4 h. Following exposure, the cells were harvested and used in cell migration assay or RNA extraction and PCR as described below.
Cell proliferation assay. Cell proliferation was determined using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Inc, Madison, USA) according to the manufacturer's protocol. Six parallel sets of assays were performed simultaneously and the average data were used for analysis. Cells were added to 96-well plates at a density of 50,000 cells/well. The plates were first incubated for 24 h in medium supplemented with FBS to allow cell attachment, followed by 24 hours' incubation in serum-free medium. Propofol and bupivacaine at concentrations listed above were added to appropriate wells. DMSO at corresponding concentrations was used as control. The plates were incubated for pre-determined time intervals (6, 12, 24 and 36 hours). Proliferation is defined as an increase in the number of cells as a result of cell growth and division and was determined by measuring the change in absorbance, realtive to thta of the control, resulting from changes in the proportion of living cells on a plate. Absorbance was measured with a spectrophotometric plate reader using 490 nm filter set.
Cell migration assay. Cancer cell migration across microporous membrane was assessed using the Chemotaxis 96-well Cell Migration Assay (Chemicon International, Billerica, USA) according to the manufacturer's protocol. Six parallel sets of assays were carried out simultaneously and the averaged data was used for analysis. Cell migration was quantified by the number of cells that migrated directionally through an 8 μm pore-size membrane into a lower chamber containing chemoattractant (150 μl of media with 20% FBS). A total of 50,000 cells were seeded to each upper chamber of the 96-well plate. Propofol, bupivacaine and DMSO (control) diluted in media with 10% FBS were added to both upper and lower chambers of the appropriate wells. Samples without cells, but containing Cell Detachment Buffer, Lysis Buffer and CyQuant Dye were used as control for background fluorescence. Assay culture plates were incubated for 24 h at 37°C in 5% CO2. Migrated cells were recovered from the lower chamber and transferred into a 96-well flat-bottomed plate (Costa). Total cell migration was determined by measuring the optical density of the collected migrated cells with a spectrophotometric fluorescence plate reader using a 480/520 nm filter set. Final results for migration were corrected for any change in proliferation that may have occured. Results are given relative to control.
Cell invasion assay. Cell invasion was investigated by the Biocoat Matrigel Invasion Chambers (BD Biosciences, Bedford, MA, USA) according to the manufacturer's protocol. Six parallel sets of assays were carried out and average data were used for analysis. 24-Well invasion chambers were removed from −20°C storage and allowed to come to room temperature. The inserts were rehydrated for 2 h by adding 250 μl of serum-free medium to each chamber. After that, the medium was replaced with 500 μl of a 500,000 cells/ml cell suspension in serum-free medium with or without propofol or bupivacaine as appropriate; 750 μl of medium with 20% FBS with or without propofol or bupivacaine in corresponding concentrations was added to the outer chamber as chemo-attractant. Plates with and without propofol or bupivacaine were incubated for 6 hours at 37°C with 5% CO2. Following incubation, non-invasive cells were removed from the upper chamber using cotton swabs soaked with PBS. Cells that had invaded through the Matrigel membrane were fixed with methanol and stained with haematoxylin. Inserts were then de-hydrated by soaking in solutions with increasing ethanol concentration; the membrane was then removed from the insert and mounted on a slide with DPX mounting medium. Cells were visualized at ×10 magnification; the number of cells in five fields per slide was counted and averaged. Invasion was expressed as a ratio of invading cells incubated with drugs compared with controls.
RNA extraction and polymerase chain reaction. RNA was isolated from cells when they were 80% confluent. The growth medium was removed and cells were washed twice with PBS. Once all PBS was removed, 1 ml of TRIzol (Sigma-Aldrich, Ireland) was added to one 75 ml flask of cells and left for five minutes at room temperature with occasional shaking. Once cells were lysed, as judged microscopically, the suspension formed was removed to a clean 1.5 ml microfuge tube, 200 μl chloroform was added and the mixture was shaken, left at room temperature for 15 minutes and centrifuged at 13,000 ×g at 4°C for 15 min. The upper aqueous layer was transferred to a fresh 1.5 ml tube. Care was taken not to transfer the DNA and protein-containing lower and inter phase. Ice-cold isopropanol (0.5 ml) was added to the aqueous phase, the tube shaken and left to stand on ice for 10 minutes before it was centrifuged at 13,000 ×g at 4°C for 10 min. The supernatant was then removed and 1 ml of sterile 75% ethanol was added to wash the pellet by gentle vortexing and centrifugation at 7500 ×g for 5 min. The ethanol was removed and the pellet was allowed to air-dry for five minutes. Pellets were resuspended in 50 μl 0.1% DEPC treated water by heating at 60°C for 15 min. All RNA was stored at −80°C.
An aliquot of 2 μg of total RNA was treated with DNAse I and reverse transcribed using random hexamers and SuperScript II reverse transcriptase (Invitrogen Ltd. UK). Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and synthesised by Sigma-Aldrich. The sequences of primers used for PCR were: GAPDH forward: 5’-TGC ACC ACC AAC TGC TTA GC-3’, GAPDH reverse: 5’-GGC ATG GAC TGT GGT CAT GAG-3’, NET1 forward: 5’-CTG TTC ACC TCG GGA CAT TT-3’, NET1 reverse: 5’-TGG AGC TGT CAG ACG TTT TG-3’. Polymerase chain reaction (PCR) was carried out in 50 ml containing 0.5 ml of Taq polymerase (Invitrogen, UK) and 1 ml of cDNA. Polymerase chain reaction products were run on a 2% agarose gel with a parallel 100 bp DNA ladder (Promega, UK). Real-time PCR was carried out according to the manufacturer's instructions using the LightCycler RNA SYBR Green 1 Amplification Kit (Roche Applied Science) together with the Light Cycler instrument. All measurements were independently repeated six times. The maximum concentration of total RNA template used was 0.5 mg/ml. The following components were added to 1 ml of total RNA in a 20 ml capillary tube: 10.2 ml of PCR-grade water, 4 ml of SYBR Green 1 reaction mix, 2.4 ml of 25 mM MgCl2, 0.4 ml of RT–PCR enzyme mix and 1 ml each of forward and reverse primers. The reaction conditions were as follows: reverse transcription: 55°C for 10 min followed by denaturation at 95°C for 30 s. Forty-five PCR cycles were then run at denaturation at 95°C for 10 s; annealing (GAPDH: 54°C, NET1: 52°C) for 10 s and extension: 72°C for 13 s. Data analysis using the delta delta Ct method was performed using the LightCycler version 4.0 software. GAPDH expression levels were used to normalise NET1 expression.
Gene silencing by RNA interference. Two siRNA duplexes were designed and synthesised for silencing NET1 gene, as previously described17 (Qiagen Inc. CA, USA). The duplexes had the following sequences: NET1(1) sense, 5’-GGA GGA UGC UAU AUU GAU A-3’; NET1(1) antisense, 5’-UAU CAA UAU AGC AUC CUC C-3’; NET1(2) sense, 50-GGU GUG GAU UGA UUG GAA A-30; NET1(2) antisense 5’-UUU CCA AUC AAU CCA CAC C-3’. A chemically synthesised non-silencing siRNA duplex with the following sequence: sense, 5’-UUC UCC GAA CGU GUC ACG U-3’; antisense, 5’-ACG UGA CAC GUU CGG AGA A-3’ that had no known homology with any mammalian gene was used to control for nonspecific silencing events. A total of 300,000 cells were added to each well of a 6-well plate in 3 ml growth media and incubated under the standard conditions for 24 h. A volume of 2.4 ml growth medium was mixed with 5 μg siRNA, 500 μl buffer and 30 μl RNAifect (ratio siRNA:RNAifect=1:6) (Qiagen). Following incubation, the medium was removed from the cells and this mixture was added dropwise. The cells were incubated for 48 hours under standard conditions either before being assayed or before RNA was extracted as described above.
Statistical analysis. Values of absorbance as a measure of proliferation and of fluorescence as a measure of migration of each breast cancer cell line at each concentration of study drug were obtained from six separate experiments, each performed in triplicate, and compared with corresponding values for cells observed in control wells. Data from six separate runs of PCR were analysed using the delta delta Ct method with the LightCycler version 4.0 software. Having confirmed that recorded data in all groups were normally-distributed using the Kolmogorov-Smirnov test, differences between paired groups (i.e. before versus after an experimental manipulation) and between different groups were evaluated using within group and independent group analysis of variance, respectively, as appropriate. Post-hoc Dunnet's test was used to correct for repeated measures. p-Values less than 0.05 were indicated statistical significance.
Results
NET1 expression in breast cancer cell lines. Both MCF7 and MDA-MB-231 breast cancer cell lines were found to express NET1 gene. Expression of NET1 gene is described in terms of multiples of baseline level observed, or fold changes vs. the baseline or untreated value. Incubation of cells for two hours with 10 μM LPA significantly stimulated expression of NET1 gene in MCF7 cells by 2.9-fold and in MDA-MB-231 cell line by 78-fold (Figure 1A and B). After four hours incubation, however, NET1 expression had returned to the baseline value, indicating that the NET1 response is time-limited. Incubation with 10 μM LPA also increased migration by 60% in MCF7 cells (p=0.045) and by 299% in MDA-MB-231 (p=0.005) compared to the control (Figure 1C and D).
Propofol inhibited expression of NET1 in both cell lines compared to baseline (indicated by a value of 1.0), but not in a dose-dependent manner, (Figure 2A and B), by 32-88% in MCF7 and by 49-79% in MDA-MB-231 cells (p<0.05).
Propofol did not significantly effect proliferation of ER-positive or ER-negative cells (Figure 3A and B).
Figure 4A demonstrates the efficacy of siRNA in silencing NET1 expression by 96% in both cell lines. Silencing NET1 reduced LPA-stimulated migration by 15%, p=0.04 in ER positive MCF7 cells, but no significant difference was observed in ER-negative MDA-MB-231 cells.
Propofol reduced migration compared with the control in both cell lines, to a greater extent in ER positive MCF7 cells (by 60-72%, p<0.01 for each comparison between 4-10 μg/ml, Figure 5A), but also in ER-negative MDA-MB-231 cells (by 40%, p=0.04 for 6-10 μg/ml; Figure 5B). When breast cancer cells with NET1 expression silenced (using siRNA), were incubated with propofol or control medium, the inhibitory effect of propofol on breast cancer cell migration was abrogated in both cell lines (Figure 5C and D).
Effect of propofol on invasion. Propofol inhibited invasion of ER-positive MCF7 cells at concentrations of 4-10 μg/ml by 40-85% compared with the control, (p<0.05 at each concentration), but not in ER-negative MDA-MB-231 cells, (Figure 6A and B). Silencing the NET1 gene had no significant influence on the effect of propofol on invasion in either cell line (Figure 6C and D).
Effect of bupivacaine. In pilot experiemnts, we observed that bupivacaine had no significant effect on NET1 expression. Moreover, bupivacaine had no effect on migration of either MCF7 or MDA-MB-231 cells compared with the control medium (Figure 7A and B). Therefore, it was not appropriate to undertake silencing NET1 studies with bupivacaine.
Discussion
As far as we are aware of, this is the first study to investigate on direct effects of propofol and bupivacaine on breast adenocarcinoma cell migration in vitro, using two different cells lines of the same histological type but different metastatic potential, and to evaluate the role of the NET1 gene in mediating these effects. Propofol reduced migration in both breast cancer cell lines and invasion in ER-positive but not ER-negative cells. The migration, but not invasion effects were abrogated after silencing NET1 gene expression, suggesting that the inhibitory effect of propofol on migration may be mediated at least in part, by NET1 gene expression. Bupivacaine on the other hand, had no appreciable effect on breast cancer cell migration. The implication of these findings is that propofol, but not bupivacaine, affects the expression of the NET1 gene, which has independently been linked with metastatic potential in cancer cells, including breast cancer.
The findings regarding propofol are consistent with a study of HeLa, HT1080, HOS and RPMI-7951 human cancer cells where it inhibited migration by modulating RHO A proteins, thus inhibiting formation of actin stress fibres. RHO A proteins are known to regulate the actin cystoskeleton and thus migration and invasion abilities of cancer cells (16). They are in turn regulated by the NET1 gene. In addition to NET1 being associated with higher a risk of metastasis in lymph node-positive breast cancer (12), knockdown of NET1 gene expression has been shown to reduce the intracelullar levels of RhoA and to reduce the gastric cancer cell migration and invasion (17). In our present study, when NET1 gene expression was stimulated with LPA, an established driver of RHO A activation, we found enhanced migration in both breast adenocarcinoma cell lines, but to a greater extent in the ER-negative cells. Conversely, when NET1 expression was silenced, there was no significant decrease in cell migration, suggesting that NET1 gene does not mediate the inhibitory effect of propofol on breast cancer cell migration. Propofol can cause changes in the immune system, specifically in the cell-mediated immune response to tumour. It has been shown to stimulate the cellular immune response and affect production of proinflammatory cytokines (6), However, it may also inhibit migration of macrophages and neutrophils in vitro (18, 19).
However, propofol may also have direct effects on the ability of cancer cells to proliferate, migrate, and invade. Available data have yielded conflicting results. Propofol has been shown to prevent apoptosis of osteoblasts exposed to oxidative stress (20), reduce cell proliferation of cardiac fibroblasts in rats (21) and stimulate ER negative breast cancer cell migration in vitro (22). This effect on migration was shown to be mediated by stimulation of the GABAA receptor and a subsequent change in calcium signalling and actin re-organisation within neurons (23). It is, therefore, plausible that our observation of an effect of NET1 on propofol in cancer cell migration may be attributable to the recognised activity of propofol at the GABAA receptor. The effect of propofol on cancer cell function may also be mediated by intracellular actin re-organization, which has also been shown in neurons (24). Propofol also has inhibitory effects on in vitro migration and invasion of several types of cancer (cervical cancer, malignant melanoma, osteosarcoma, and fibrosarcoma cells) (25), by modulation of RHO A, known to regulate actin cystoskeleton and thus migration and invasion abilities of cancer cells (16).
Recent molecular analysis has shown that activation of a single gene, affecting a process essential for metastasis, can be sufficient to induce metastasis in vitro (26). One such gene is the NET1 gene, which has been identified through the Serial Analysis of Gene Expression (SAGE) database as being overexpressed in breast and gastric adenocarcinoma (28, 29). Our knowledge of the effects of anaesthetic drugs cells (27). NET1 gene has a key role in organization of actin cytoskeleton and thus in the ability of cancer cells to migrate on intracellular series of molecular events is still fragmentary, but a change in gene expression may be one of these events. Propofol has been shown to affect gene expression in vital organs, including the brain, in animal models and it has also been shown that anaesthesia with propofol increases gene expression of pro-inflammatory cytokines in alveolar macrophages (30, 31).
However, our results showing an inhibitory effect of propofol on migration of breast cancer cells are contradictory to similar studies (22). This may be attributable to our use of different breast cancer cell lines. Moreover, propofol has several mechanisms of action, including activation of GABAA receptors, and modulation of calcium influx through slow calcium-ion channels (32). Many studies showing an inhibitory effect of propofol on the migration of cells (cancer cells, macrophages, neutrophils) did not include specific investigations of the receptors involved (32). A previous cell culture model suggested that propofol-mediated reduction in breast cancer cell migration was through histone de-acetylation inhibition, but this work was limited to conjugated molecules of propofol (33).
This work has some limitations on the extent to which our data can be interpreted. Proliferation, migration and invasion could also have been analysed by labelling cells and counting via microscopy or by flow cytometry, whereas we elected to measure these functions by absorbance, which does not distinguish between living and dead cells. In the migration assay, the need to transfer the cells to a different vessel for the absorbance measurement introduces a potential source of error. It is noteworthy that our observed effects of propofol and NET1 on breast cancer cell functions are not dose-related. This implies that these effects might not be mediated by any specific receptor pathway, but rather by exerting inhibitory effects on the cellular microenvironment.
Bupivacaine is an amide local anaesthetic, commonly used in regional anaesthesia. While it has been hypothesized that the potential benefit of regional anaesthesia in cancer surgery lies in inhibition of the stress response (34), recent experimental data suggest that local anaesthetics, particularly amides have direct effects on cancer cells, including disruption of DNA replication (35), inhibition of mesenchymal cells (36) and direct anticancer cell effects not mediated by sodium channel blockade by modulation of the SRC cell signalling system (8). Further investigation suggests that local anaesthetic effects on certain types of sodium channels specifically inhibit colonic cancer cell activity (9). However, we found no significant effect of bupivacaine on either ER-positive or ER-negative breast cancer cells, which was not influenced by silencing of the NET1 gene. Perhaps this discrepancy may be attributable to breast cancer cells having different tumour biology compared with other cancer cells where these apparent benefits of amide local anaesthetics were demonstrable.
In conclusion, NET1 gene is expressed in breast adenocarcinoma cell lines in vitro and drives excessive migration of these cells. Its expression is reduced by exposure of cells to propofol which results in a corresponding decrease in in vitro migration. These results were more evident in the ER-negative cell line, which represents a more aggressive breast cancer type. Further studies are needed to clarify in detail the pathway of the direct effect of propofol on cell migration. A potential role of estrogen receptors in this pathway also merits further investigation.
Footnotes
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Funding
This study was supported by the Sisk Mater Anaesthesia Fellowship, a National Institute for Academic Anaesthesia (NIAA), UK project grant and The Eccles Breast Cancer Research Fund.
- Received September 6, 2013.
- Revision received October 12, 2013.
- Accepted October 15, 2013.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved