Anesth Analg 2006;102:1103-1107
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000198330.84341.35
ANESTHETIC PHARMACOLOGY
The Antiproliferative Effect of Lidocaine on Human Tongue Cancer Cells with Inhibition of the Activity of Epidermal Growth Factor Receptor
Masahiro Sakaguchi, MD*,
Yoshihiro Kuroda, PhD
, and
Munetaka Hirose, MD*
*Department of Anesthesiology, Kyoto Prefectural University of Medicine; and Graduate School of Pharmaceutical Sciences, Kyoto University, Japan
Address correspondence and reprint requests to Munetaka Hirose, MD, Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kamigyoku, Kyoto 602-8566, Japan. Address e-mail to hirose{at}koto.kpu-m.ac.jp.
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Abstract
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Local anesthetics suppress proliferation in several cancer cells. The mechanism of the suppression, however, is unknown. Our previous study shows that lidocaine, at the level of tissue concentration under topical or local administration, has a direct inhibitory effect on the activity of epidermal growth factor receptor (EGFR), which is a potential target for antiproliferation in cancer cells. Therefore, we hypothesized that lidocaine would suppress the proliferation of cancer cells through the inhibition of EGFR activity. We investigated the effects of lidocaine (40–4000 µM) on proliferation of a human tongue cancer cell line, CAL27, which has a high level of EGFR expression, and also examined the effect of lidocaine on epidermal growth factor (EGF)-stimulated autophosphorylation of EGFR in CAL27 cells. A clinical concentration of lidocaine (400 µM) suppressed both serum-induced and EGF-induced proliferation of CAL27 cells and inhibited EGF-stimulated tyrosine kinase activity of EGFR without cytotoxicity. A larger concentration of lidocaine (4000 µM) showed cytotoxicity with an antiproliferative effect. We suggest that the inhibition of EGF-stimulated EGFR activity is one of the mechanisms of the antiproliferative effect of lidocaine on CAL27 cells. Lidocaine administered topically within the oral cavity for cancer pain relief may suppress the proliferation of human tongue cancer cells.
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Introduction
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Topical lidocaine and other local anesthetics can be used for relief of pain induced by oral or rectal cancer (1–3). However, local anesthetics reportedly showed antiproliferative or cytotoxic effects on cancer cells (4–7) and prevented the proliferation of colon cancer in patients with ulcerative colitis (4). Therefore, it is likely that local anesthetics, administered topically within the oral cavity for cancer pain relief, would suppress proliferation of oral cancer cells.
The epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, plays a crucial role in regulating cellular proliferation or differentiation of epithelial cells and tumors of epithelial cell origin. Binding of the extracellular domain of EGFR to its ligands, mainly epidermal growth factor (EGF), leads to their dimerization followed by autophosphorylation of tyrosine kinase residue and subsequent activation of signal transduction pathways involved in cell growth and proliferation. The EGFR is a potential anticancer target for head and neck cancer, breast cancer, colorectal cancer, and lung cancer (8,9). In the present study, we hypothesized that lidocaine would suppress proliferation of head and neck cancer cells with inhibition of tyrosine kinase activity of the EGFR, because our previous study showed that lidocaine directly inhibits tyrosine kinase activity of the EGFR (10). We used human tongue squamous cell carcinoma CAL27 cells, which have a high level of EGFR expression and are highly sensitive to a selective EGFR-tyrosine kinase inhibitor (11).
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Methods
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Antiphosphotyrosine antibody (4G10) and anti-EGFR rabbit polyclonal antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-rabbit immunoglobulin (Ig)G antibody and chemiluminescence luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). EGF was obtained from Gibco (Grand Island, NY). Nonidet P-40 was from Nacalai tesque (Kyoto, Japan), protein A-Sepharose CL-4B was from Pharmacia Biotech (Piscataway, NJ), and nitrocellulose membrane and Laemmli sample buffer were from Bio-Rad Laboratories (Hercules, CA). Other chemicals were from Sigma Chemical Co. (St. Louis, MO).
CAL27 cell was purchased from American Type Culture Collection (Manassas, VA). Cells were routinely cultured with Dulbecco modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS) (ICN Biomedicals, Aurora, OH) and penicillin-streptomycin (Nacalai tesque) in 25-cm2 culture flasks in a 37°C under an atmosphere of 5% CO2 and 95% air.
To investigate the effect of lidocaine on serum-induced cell proliferation, CAL27 cells were detached with trypsin/EDTA and were seeded in 96-well plates at 1 x 103 cells per well. The cells were exposed to the culture media containing DMEM and10% FBS with or without lidocaine (40, 400, or 4000 µM) for 1, 3, and 5 days. We also investigated the effect of lidocaine on EGF-induced cell proliferation. CAL27 cells were exposed overnight to a serum-free medium and then exposed to the FBS-free culture media with or without EGF (10 ng/mL) and lidocaine (40, 400, or 4000 µM) for 1, 3, and 5 days. The proliferation of cells was examined using CellTiter96® Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI). After cells were incubated with CellTiter96® Aqueous One Solution for 1 h, the absorbance at 492 nm was measured using a 96-well plate reader (multiscan BICHROMATIC; Labsystems, Helsinki, Finland).
To investigate the effect of lidocaine on EGF-stimulated autophosphorylation of EGFR, CAL27 cells were detached with trypsin/EDTA and cultured to be confluent in 100-mm dishes with DMEM containing 10% FBS. They were exposed overnight to serum-free medium, after which EGF (100 ng/mL) and lidocaine (40, 400, or 4000 µM) were added. After incubation in a 37°C, 5% CO2 environment for 5 min, the compounds were replaced with 5 mL of phosphate-buffered saline. The cells were harvested and suspended in lysis buffer (50 mM of HEPES, pH value of 7.5, 150 mM of NaCl, 2 mM of EDTA, 1% [vol/vol] Nonidet P-40, 10% [vol/vol] glycerol, 10 mM of sodium fluoride, 2 mM of sodium vanadate, 1 mM of phenylmethylsulfonyl fluoride, 10 mM of sodium pyrophosphate, 5 µg/mL of aprotinin, and 0.5 µg/mL of pepstatin) and laid on ice for 30 min. Insoluble material was removed by centrifugation at 15,000 rpm for 15 min. Aliquots of the supernatants containing equal amounts of protein, as determined using the Bradford protein assay with Bradford reagent, were subjected to immunoprecipitation for 1 h with the anti-EGFR antibody. After the addition of protein A-Sepharose CL-4B, the immunoprecipitates were washed 3 times in a wash buffer (50 mM of HEPES, pH value of 7.5, 150 mM of NaCl, 2 mM of EDTA, 0.1% [vol/vol] Nonidet P-40, 10% [vol/vol] glycerol, 10 mM of sodium fluoride, 2 mM of sodium vanadate, 1 mM of phenylmethylsulfonyl fluoride, 10 mM of sodium pyrophosphate, 5 µg/mL of aprotinin, and 0.5 µg/mL of pepstatin). The samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis by adding Laemmli sample buffer and boiling for 5 min. The immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 7.5% (vol/vol) acrylamide solving gels and transferred electrophoretically to nitrocellulose membrane. The membranes were then blocked in 5% (wt/vol) dried milk in phosphate-buffered saline containing 0.1% (vol/vol) polyoxyethylene sorbitan monolaurate (Tween 20) for 1 h at room temperature and were then immunoblotted with the appropriate antibody. The antibody complexes were visualized by chemiluminescence luminol reagent. Bands of interest were scanned and quantified by using LightCapture AE-6960 (ATTO Corporation, Tokyo, Japan).
Cell death was quantified both by double staining with Hoechst 33342 and propidium iodide (PI) and by measuring lactate dehydrogenase (LDH) released from dead cells. CAL27 cells were exposed overnight to serum-free medium, and then EGF (10 ng/mL), lidocaine (40, 400, or 4000 µM), or both were added and incubated for 3 days. Cells were stained with Hoechst 33342 (1 µg/mL) and PI (1 µg/mL) for 10 min and analyzed under a fluorescence microscope. Because Hoechst 33342 stains all nuclei blue and PI stains nuclei of cells with a disrupted plasma membrane red, viable cells were observed as blue intact nuclei, necrotic cells as red intact nuclei, early apoptotic cells as fragmented blue nuclei, and terminal apoptotic cells as red fragmented or condensed nuclei. LDH released from dead cells was measured using the LDH-Cytotoxic Test (Wako, Osaka, Japan) after incubation for 3 days with EGF, lidocaine, or both.
Data were analyzed by one-way analysis of variance with Scheffe post hoc analysis. The statistical significance was established at the P < 0.05 level. All values are reported as mean ± sd.
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Results
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Lidocaine (400 µM) significantly suppressed serum-induced proliferation of CAL27 on 3 and 5 days (Fig. 1). A larger concentration of lidocaine (4000 µM) abolished cell viability in CAL27 cells on 3 and 5 days. In the serum-free medium, EGF augmented the proliferation of CAL27 cells, and lidocaine showed dose-dependent suppression of the EGF-stimulated cell proliferation of CAL27 on 3 and 5 days (Fig. 2).
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Figure 1. The effect of lidocaine on serum-induced cell proliferation of CAL27 cells. The cells were incubated for 1, 3, and 5 days in 10% fetal bovine serum (FBS) with or without lidocaine. The absorbance at 492 nm corresponds to the cell proliferation. *P < 0.05 compared with the absorbance at lidocaine 0 µM on each day. The results presented are the mean values for four separate experiments (±sd).
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Figure 2. The effect of lidocaine on epidermal growth factor (EGF)-induced cell proliferation of CAL27 cell line. The cells were incubated for 1, 3, and 5 days in a serum-free medium with EGF, lidocaine, or both. The absorbance at 492 nm corresponds to the cell proliferation. *P < 0.05 compared with the absorbance at EGF alone (solid bar) on each day. The results presented are the mean values of four separate experiments (±sd).
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To assess tyrosine phosphorylation of EGFR in CAL27 cells, the equal amounts of protein from CAL27 were subjected to immunoprecipitation with anti-EGFR antibody followed by immunoblotting with a antiphosphotyrosine antibody or anti-EGFR antibody. EGF stimulation resulted in a marked increase in tyrosine phosphorylation of EGFR. EGF-stimulated responses of EGFR were taken as 100%. Both 400 µM and 4000 µM of lidocaine significantly attenuated EGF-stimulated tyrosine phosphorylation of EGFR (Fig. 3). EGFR protein levels did not differ among every line.
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Figure 3. Levels of tyrosine phosphorylation of epidermal growth factor receptor (EGFR) in CAL27 cells. Cell lysates were generated from CAL27 cells stimulated with or without epidermal growth factor (EGF), lidocaine (40, 400, or 4000 µM), or both for 5 min. The EGFR protein was immunoprecipitated (IP) using an anti-EGFR antibody. The tyrosine phosphorylation of EGFR was detected by immunoblotting (IB) using an antiphosphotyrosine antibody (pY). The immunoblot was reprobed with anti-EGFR antibody to verify protein expression levels in the cells. Results displayed in the upper panels represent typical immunoblots of autophosphorylation of EGFR and immunoblots of protein content. *P < 0.05 compared with the EGFR-stimulated autophosphorylation without lidocaine (100%). The results presented are the mean values for four separate experiments (±sd).
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In the cell death assay, apoptotic cells increased significantly in the serum-free medium without EGF. EGF, however, suppressed the increased level of apoptotic cells without EGF (Fig. 4). This effect of EGF might have been due to an antiapoptotic effect of EGF. However, 4000 µM of lidocaine significantly increased PI positive cells and showed fragmented blue nuclei and condensed red nuclei, which correspond to early and late apoptotic cells. The LDH assay also showed that 4000 µM of lidocaine significantly increased cell death (Fig. 4).
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Figure 4. Effect of lidocaine on cell death was assessed by double staining with Hoechst 33342 and propidium iodide (PI) and also done by lactate dehydrogenase (LDH) release. After incubation for 3 days with or without epidermal growth factor (EGF) and lidocaine (40 µM, 400 µM, and 4000 µM), PI (red)-positive cells were counted as necrotic cells, and fragmented blue and condensed red nuclei were counted as early and late apoptotic cells in at least 100 cells for each experiments. LDH activity released in the medium was also assayed. *P < 0.01 compared with CAL27 cells stimulated with EGF alone. The results presented are the mean values for four separate experiments (±sd).
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Discussion
The present study showed that 400 µM of lidocaine causes an antiproliferative effect on CAL27 cells without cytotoxicity, and 4000 µM of lidocaine induces cytotoxicity. Both concentrations of lidocaine inhibit the tyrosine kinase activity of EGFR in CAL27 cells.
Topical administration of lidocaine, usually as a combination analgesic, is often used for relief of pain induced by oral or rectal cancer at the concentration of 0.5%–2.5% (almost equivalent to 19–93 mM) (1–3). Although the precise concentration of lidocaine in the tissue is unclear, because lidocaine diffuses and is absorbed after topical administration, lidocaine likely reaches to the concentration of approximately 400 µM in the tissue, which is about a hundredth of the original concentration (10). When lidocaine is injected into the tissue for nerve block, it is thought to be diluted to around a hundredth or less of the concentration at the site of peripheral nerve (12), and approximately 600 µM of lidocaine is required to block afferent nociceptive fibers (13). The present study suggests that this level of tissue concentration after topical lidocaine administered orally causes an antiproliferative effects on human tongue cancer cells with the inhibition of tyrosine kinase activity of the EGFR.
Topical local anesthetics administered rectally also have beneficial clinical effects in patients with ulcerative colitis (4,14). Martinsson (4) studied human colon adenocarcinoma cell lines (HT29) with local anesthetics and suggested that the antiproliferative effect of ropivacaine or lidocaine on colon cancer cells is useful for treatment of ulcerative colitis, which has an increased incidence of colorectal cancer. However, gefitinib, a selective EGFR-tyrosine kinase inhibitor, inhibits EGF-stimulated autophosphorylation of EGFR in HT29 with suppression of the proliferation of HT29 (15). Therefore, mechanisms of the antiproliferative effect of topical local anesthetics on colorectal cancer cells may also involve the inhibition of tyrosine kinase activity of the EGFR.
In our previous study, lidocaine (400 µM) suppressed EGF-stimulated autophosphorylation of the purified EGFR (10). Because lidocaine can interact with the aromatic ring of phenylalanine (F) and tyrosine (Y), with the negatively charged amino acids aspartic acid (D) and glutamic acid (E), and with the basic amino acids lysine (K) and arginine (R) (16), it is likely that lidocaine directly inhibits its tyrosine kinase activity of the EGFR by binding to the autophosphorylation sites of the EGFR, which are surrounded by these amino acids (such as DADEY 988-992, DNPDYQQDFF 1144-1153, ENAEYLR 1169-1175) (10). In the serum-free medium in the present study, EGF augmented the proliferation of CAL27 cells, and lidocaine suppressed the EGF-stimulated proliferation. These results suggest that the inhibitory effect of lidocaine on the activity of EGFR is one of the mechanisms of antiproliferative effect of lidocaine on CAL27 cells.
The cytotoxic effect of lidocaine is reportedly observed at a large concentrations (2000–4000 µM or more) in both cancer and noncancer cells (5–7,17,18). Several mechanisms of the cytotoxic effect of lidocaine on these cells have been proposed, such as a reduction in glycolysis and adenosine triphosphate levels (5), an increase in the concentration of intracellular calcium ions (17), and mitochondrial dysfunction (18). In addition to the inhibition of EGFR activity, many mechanisms are involved in the cytotoxic effect of lidocaine (4000 µM) in CAL27 cells. Because this concentration of lidocaine is apparently larger than the clinical concentration in the tissue, the cytotoxic effect of local anesthetics on cancer cells cannot be expected in clinical settings, even with topical or local administration.
The limitation of the present study is that we used only CAL27 cells possessing a high level of EGFR expression. There are various types of human tongue cancer cell lines with a wide range of EGFR expression representative of that found in human tumors (11). Further study is required to investigate the effect of lidocaine on the proliferation of cancer cells with a low level of EGFR expression and also is required to investigate the effect of topical lidocaine on the growth of tumor tissue in an animal study.
In conclusion, a clinically relevant concentration of lidocaine in the tissue, which can be achieved by administration topically within the oral cavity for cancer pain relief, suppresses the proliferation of human tongue squamous cell carcinoma with inhibition of the tyrosine kinase activity of the EGFR.
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Footnotes
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Supported, in part, by Grants-in-Aids for Scientific Research (#16591554 to M.H.) from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan.
Accepted for publication October 31, 2005.
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Abstract
Introduction
