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Anesth Analg 2008; 107:1592-1597
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e3181844cef
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ANESTHETIC PHARMACOLOGY

Lidocaine Inhibits NIH-3T3 Cell Multiplication by Increasing the Expression of Cyclin-Dependent Kinase Inhibitor 1A (p21)

Sukumar P. Desai, MD, Koji Kojima, MD, PhD, Charles A. Vacanti, MD, and Shohta Kodama, MD, PhD

From the Department of Anesthesiology, Perioperative and Pain Medicine, Laboratory for Tissue Engineering and Regenerative Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.

Address correspondence and reprint requests to Shohta Kodama, MD, PhD, Department of Anesthesiology, Perioperative and Pain Medicine, Laboratory for Tissue Engineering and Regenerative Medicine, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Address e-mail to skodama{at}zeus.bwh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: We explored molecular mechanisms by which lidocaine inhibits growth in the murine embryonic fibroblast cell line NIH-3T3. Local anesthetics can adversely affect cell growth in vitro. Their effects on wound healing are controversial. We examined the effects and novel mechanisms by which lidocaine affects in vitro multiplication of the murine fibroblast cell line NIH-3T3.

METHODS: NIH-3T3 cells were grown in culture with lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM]. Cell multiplication was assessed by determining cell counts on subsequent days, while mechanisms by which inhibition occurred were evaluated by bromodeoxyuridine uptake, gene expression using polymerase chain reaction array, and Western blot analysis to verify increased levels of affected proteins.

RESULTS: Lidocaine caused dose-dependent inhibition of multiplication of NIH-3T3 cells. Effects ranged from no inhibition [0.05 and 0.5 mM] and mild inhibition [1 mM], to severe inhibition [2 and 5 mM] [P = 0.006]. Lidocaine 2 mM inhibited bromodeoxyuridine uptake at day 3.5 [P = 0.02 versus control, and P = 0.0495 vs 1 mM lidocaine]. On day 1.5, lidocaine upregulated expression of cyclin-D1 and cyclin-dependent kinase inhibitor 1A [p21]. On day 2.5, lidocaine increased the levels of p21 protein.

CONCLUSIONS: Low concentrations of lidocaine, as would be seen in plasma after spinal, epidural, or plexus anesthesia, do not significantly affect multiplication of fibroblasts. Higher doses of lidocaine arrest cell multiplication at the S-phase of the growth cycle by upregulation of p21, an extremely potent inhibitor of cell multiplication. Higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms by which local anesthetics (LA) cause toxicity are not well understood, although it is known that LAs exert pharmacological actions primarily by inhibiting ionic flux through Na+ channels of excitable tissues.1 LAs produce adverse effects on the heart,2,3 blood vessels,4 and immune system.5 Their effects on neutrophils include decreased chemotaxis,5–7 and a delayed onset of the inflammatory response.6 Reversible and irreversible local toxicity, including cell necrosis, has been observed when these drugs are injected into skeletal muscle.8 LA toxicity has been described on chondrocytes in vitro, in vivo, and in clinical practice.9–12

The depressant effects of LAs on multiplication of culture cells have been demonstrated in human fibroblasts,13 human endothelial cells,13 human keratinocytes,13 type II pneumocytes,14 lung fibroblasts,14 and corneal epithelial cells.15 The mechanisms by which LAs inhibit growth include mitochondrial damage,16,17 caspase activation,18,19 inhibition of tyrosine kinase20 and signaling by lysophosphatidate,21 and activation of p38 mitogen-activated protein kinase.20,22 The adverse effects of lidocaine and other LAs on wound healing remain controversial.23–28 Wound healing may be retarded due to reduction of mucopolysaccharide synthesis.27,29 This effect may be explained by changes in the stability of the cellular membrane and of sodium and calcium conductance.28 Although some studies have shown that wound healing was impaired by LA,23,25,27,28 others have not been able to verify deleterious effects.7

We examined the effect of low and high concentrations of lidocaine on the in vitro growth rate of murine embryonic fibroblasts. We sought to verify whether or not lidocaine affected cell multiplication in vitro. The next logical step was to explore the mechanisms by which lidocaine exerted any inhibitory effects on DNA synthesis as evidenced by reduced uptake of bromodeoxyuridine (BrdU). Exploring the molecular mechanisms further, we examined gene expression using polymerase chain reaction (PCR) array. Lastly, we verified translation of these effects by measuring cellular levels of the regulatory protein affected by these genes using Western blot analysis.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
The murine fibroblast cell line NIH-3T3 (ATCC, Manassas, VA) was cultured in Dulbecco’s modified Eagle medium (High Glucose, Invitrogen, Carlsbad, CA), with 10% Fetal Bovine Serum (Invitrogen) and 1% penicillin/streptomycin (Sigma, St. Louis, MO). Lidocaine hydrochloride (Sigma) was prepared freshly before each use. Cell counts as well as exposure to medium containing lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM] began after cells became adherent to the culture dish. Cell counts were performed on days 0, 1, 2, 3, 4, and 5; and 0.4% Trypan Blue (Invitrogen) was used to exclude dead cells.

BrdU Uptake
Drug effects on the phase of the cell cycle were assessed by examining nuclear uptake of BrdU (BD Biosciences, San Diego, CA) by NIH-3T3 cells grown in one-well culture slides. After growth with medium alone for 1 day, lidocaine was applied to culture slides at the concentrations of 0, 1, and 2 mM at day 0. BrdU was added to culture slides at a concentration of 1 mM and incubated for 45 min at day 3.5. The slides were then fixed and stained with biotinylated anti-BrdU antibody and streptavidine indocarbocyanine (Cy3; Jackson ImmunoResearch, West Grove, PA) was used as the secondary antibody. 4', 6-diamidino-2-phenyindole dilactate (Sigma, St. Louis, MO), was applied as a nuclear counterstain. Cell counts were performed using fluorescent microscopy (NIKON, Melville, NY), a Spot CCD camera, and analyzing software (Diagnostic Instruments, Sterling Heights, MI).

PCR Array Analysis
NIH-3T3 cells were exposed to medium containing lidocaine at concentrations of 0, 1, and 2 mM for 1.5 days at which time their mRNA was purified using an RNeasy Mini kit (Qiagen, Valencia, CA).30 Purified mRNA was subjected to PCR array analysis (SuperArray Bioscience, Frederick, MD). The expression of genes was evaluated by the [Delta][Delta]Ct method.31 Final results were reported by SuperArray Bioscience [reference number P2914]. Ninety-six genes involved in the regulation of the cell cycle were studied in the PCR array. Of these, 20 that showed changes are included in Table 1. We narrowed our search further to genes that control the transition between the G1 to S phase of the cell cycle.


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  		Table 1. Messenger RNA Expression of Molecules Known to Regulate Cell Multiplication

 

Western Blot Analysis
Cultured NIH-3T3 cells exposed to lidocaine for 2.5 days were subjected to protein extraction. Extracted protein was analyzed using a standard BCA Protein Assay kit (Pierce Biotechnology). Monoclonal antibody to p21 (Cell Signaling Technology, Beverly, MA) and IRDy 800 CW conjugated antimouse IgG (Rockland Immunochemical, Gilbertsville, PA) were used to detect target molecules. Direct infrared fluorescence was used to detect proteins, and also for densitometric analysis (Odyssey, LI-COR, Lincoln, NE).

Statistical Analysis
The effects of time and lidocaine concentration on cell growth were evaluated by two-way analysis of variance. The Mann-Whitney U-test was used to determine significant differences in BrdU uptake. The level of statistical significance chosen was P = 0.05.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cells were exposed to lidocaine upon becoming adherent to the bottom of the culture plate. Although we began with a cell density of 1 x 104 cell/mL before adhesion, cell counts were performed a day later (day 0) [day of cell adhesion, and exposure to drug]. This count was used as the starting count, and subsequent counts were standardized to it [day 0 value] as a reference [100%] Figure 1A. Each data point represents the mean of five independent experimental observations, and bars represent sem. In the control group, cell growth resulted in near confluence at the conclusion of the study.


Figure 121
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  		Figure 1. The effects of lidocaine on cell number and BrdU uptake. (A) Shows cell number compared with day 0 for the first 5 days in culture in the presence of lidocaine. Two-way analysis of variance shows significant effects of lidocaine concentration and duration in culture on the rate of multiplication of cultured cells. (B) Shows nuclear staining and BrdU uptake with and without lidocaine. (C) Shows significant inhibition of BrdU uptake with 1 mM [P = 0.0495] and 2 mM lidocaine [P = 0.02]. Each data point represents the mean of five independent experimental observations, and bars represent sem.

 

Two-way analysis of variance indicated that cell numbers were significantly affected both by time [P = 0.0002] and drug concentration [P = 0.006]. Evaluation of the effect of drug concentration showed that lidocaine at concentrations of 2 mM and 5 mM completely inhibited cell growth. Cell counts with 0 mM [control], 0.05 mM, and 0.5 mM lidocaine were similar, and higher than those with 1 mM lidocaine. Cell counts with 1 mM lidocaine were higher than those with 2 mM and 5 mM lidocaine, which were similar to one another. Evaluation of the effect of time [duration in cell culture] showed that cell counts on days 0, 1, 2, and 3 were similar. These counts differed from counts on day 4, which in turn, differed from counts on day 5.

The effect of lidocaine on the S-phase of the cell cycle was evaluated by determining BrdU uptake in cells cultured on chamber slides [Figure 1B]. Nuclear staining is shown in blue, whereas BrdU uptake is shown in red. Cells that display dual staining indicate the presence of DNA replication and cell multiplication, whereas those staining blue indicate nuclei without DNA synthesis. The effects of 2 mM lidocaine clearly show not only a decrease in cell density, but also, a decrease in cells in the multiplication phase. Figure 1C shows the effects of lidocaine on BrdU uptake on day 3: 2 mM lidocaine inhibited BrdU uptake when compared to controls [9 ± 8 vs 33 ± 7% percent cells in growth phase, mean ± sd, P = 0.02], and also when compared to 1 mM lidocaine [9 ± 8 vs 30 ± 13% percent cells in growth phase, mean ± sd, P = 0.0495].

We examined 96 genes involved in the regulation of the cell cycle. Partial results from this analysis are included in Table 1. Of the 96 genes studied, 20 showed changes and these results are included in the table. Among genes with known effects on the G1 to S phase, cyclin-dependent kinase inhibitor 1A [also known as p21] shows the highest ratio when expression at 2 mM lidocaine is compared to control [ratio = 3.32]. It is for this reason that we examined cellular levels of this protein to confirm that upregulation of the gene resulted in higher levels of protein within the cell.

Figure 2A shows black and white as well as color displays of the protein p21 obtained from cells exposed to lidocaine [0 mM, 1 mM, and 2 mM] using Western blot analysis. The color display includes additional semidensitometric information that is described on the scale to the right of the panel. Red represents signals that exceed 1900 fluorescence/pixel, yellow those below 1750 fluorescence/pixel, and green those below 1600 fluorescence/pixel. The red signal confirms upregulation of the protein being studied — p21.


Figure 221
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  		Figure 2. (A) shows increased levels of p21 protein in cells exposed to lidocaine 1 mM and 2 mM, using Western blot analysis. (B) Shows the site of action of regulatory molecules in the cell cycle.35

 

Even a brief representation or discussion of the various controllers involved in the cell cycle is beyond the scope of this article. However, the segment of the cell cycle control affected by lidocaine is shown in Figure 2B. Upregulation of the potent inhibitor p21 explains the negative effects on cell multiplication.


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show that lidocaine inhibits growth of cultured murine embryonic fibroblasts [NIH-3T3 cells] in a dose-dependent and time-dependent manner. Decreased BrdU uptake confirms that lidocaine may decrease DNA synthesis and inhibit the cell cycle in transitioning from the G1 to S phase. Gene expression showed increased expression of a protein known to be a potent inhibitor of the cell cycle. We propose that the observed negative effects on cell multiplication are caused by upregulation of cyclin-dependent kinase inhibitor 1A [also known as p21]. Western blot analysis has shown that lidocaine increases cellular levels of p21. Our results are not at variance with other published reports of the cellular effects of LA, and this report provides one more mechanism by which lidocaine and other LAs exert may cellular actions.

LA drugs are unusual in that they require an enormous range of effective tissue concentrations depending on the actions desired [Table 2]. When used as topical anesthetics on mucosal surfaces, lidocaine is dispensed as a 4% solution [185 mM], whereas therapeutic plasma concentrations during treatment of cardiac arrhythmia are 1.5–2.0 µg/mL [0.006–0.007 µM]. Our results suggest that higher concentrations of lidocaine, as would occur at the site of injection, are associated with inhibition of cell growth. High tissue concentrations of LA for prolonged periods can occur when postoperative infusions are administered into the wound, in a joint, around a nerve plexus, or the epidural space. Lower concentrations as would occur in plasma after epidural or spinal anesthesia are not associated with any inhibition in fibroblast growth. In fact, lidocaine concentrations higher than plasma concentrations associated with seizures did not affect growth of fibroblasts, which suggests that lidocaine administered as part of neuraxial or plexus block would not inhibit fibroblast growth or affect wound healing.


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  		Table 2. Usual Concentration of Lidocaine When Used Clinically During Various Applications

 

An interesting effect of decreasing cell proliferation, as seen with higher doses of lidocaine, might be the potential beneficial effect in recovery from ischemia and reperfusion injury. Excessive cell growth and liberation of inflammatory mediators may be detrimental to the survival of injured tissue. The antiproliferative effect of drugs such as lidocaine may thus be beneficial in this setting, as well as in scenarios where scar formation needs to be suppressed. Two specific surgical instances where such actions might be especially useful postoperatively would be after correction of strictures, and excision of keloids.

Limitations of our study include the fact that it was performed in vitro and that the cell line used in our model has an embryonic murine origin. NIH-3T3 cells grow easily and rapidly in the laboratory, and thus provide a convenient model for the study of drug effects. Primary cell lines derived from humans show extreme variability in growth behavior, but we plan on using commercially available human fibroblast cell lines such as human dermal fibroblast-fetal, or human cortical neuron during future trials. In addition, we plan on enhancing our current murine data with genetic analysis using knock-out mouse or siRNA approaches. In addition, during clinical use with single-injection techniques, tissue concentrations of lidocaine would decrease within hours, while our study was extended for 5 days. Such long-term exposure would be seen with the use of LA infusion via catheters [epidural, intraarticular, near a nerve plexus, or within the wound] during the postoperative period. We did not specifically study the effects of short-term exposure to lidocaine [i.e., expose the cells only for 1–2 h to lidocaine, after which cells are grown in a lidocaine-free medium]. Since these studies were not performed, we are unable to speculate on the effects of short-term exposure to lidocaine. Moreover, our studies evaluated fibroblast number and other quantitative descriptors without evaluating the effects of lidocaine on fibroblast function. Lastly, other effects of LA such as enzyme inhibition, ionic flux, connective tissue synthesis, apoptosis, and necrosis were not studied in this trial on cell proliferation.

In conclusion, we have shown that lidocaine affects multiplication of murine embryonic fibroblasts in a dose-dependent and time-dependent manner. At low doses, little or no effect is seen on cell multiplication, whereas substantial toxicity appears as dosage is increased. These effects are associated with decreased DNA synthesis, increased gene expression for p21, and increase cellular levels of p21. Further studies need to verify whether clinically significant impairment of wound healing occurs. In addition, the effects of suppressing fibroblast proliferation could be studied after repair of strictures or keloid excision.


   Footnotes
 
Accepted for publication April 29, 2008.

Supported by Intramural Funds and done at the Laboratory for Tissue Engineering and Regenerative Medicine, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2008 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press