Mesenchymal stem cells (MSC) are self-renewing clonal progenitor cells of nonhematopoietic tissues that exhibit a marked tropism to wounds and tumors. The authors' studies aimed at exploring how local anesthetics would affect MSC biology.
Proliferation, colony formation, in vitro wound healing, and bone differentiation assays of culture-expanded bone-marrow-derived murine MSC were performed in the presence of increasing concentrations of lidocaine, ropivacaine, and bupivacaine. Cytotoxicity was monitored by measuring lactate dehydrogenase activity and phosphatidylserine exposure/propidium iodide staining (early apoptotic cells/necrotic cells). Measurements of mitochondrial function in intact and permeabilized cells, transcriptional changes, and changes in nuclear factor κ-light-chain-enhancer of activated B cells signaling in MSC treated with ropivacaine were used to further characterize the biologic effects of local anesthetics on MSC.
All local anesthetics reduced MSC proliferation at 100 μM, consistent with cell cycle delay or arrest at the G0/1-S phase transition. They increased lactate dehydrogenase release and the number of annexin V-positive MSC but not necrotic MSC. Colony formation was decreased, differentiation into osteoblasts impaired, and in vitro wound healing delayed. Mitochondrial respiration and adenosine 5'-triphosphate concentrations were reduced. Microarray analysis revealed significant expression changes in lysosomal genes and genes controlling sterol metabolism, indicating an impaired phospholipid metabolism in the lysosome. Multiple transcriptional programs related to cell differentiation, tumorigenesis, and metastasis were negatively affected by ropivacaine.
The authors' studies demonstrate that local anesthetics significantly affect important aspects of MSC biology. These experiments provide novel rationales for the perioperative use of local anesthetics in patients with cancer but also highlight the potentially detrimental effects of local anesthetics on wound healing.
What We Already Know about This Topic
Mesenchymal stem cells are implicated in wound healing and tumor growth
Local anesthetics have antiproliferative effects on many cell types, possibly including tumor cells, but their effects on mesenchymal stem cells are unknown
What This Article Tells Us That Is New
Local anesthetics impaired proliferation, differentiation, and respiration and were cytotoxic to murine mesenchymal stem cells in vitro
The possibility of beneficial antitumor effects and detrimental effects on wound healing in vivo requires additional study
MESENCHYMAL stem cells (MSC) are self-renewing clonal progenitor cells of nonhematopoietic tissues that exhibit a marked tropism to wounds and tumors.1A tumor is often regarded as a “nonhealing wound,” and vice versa , a wound can be regarded as “a healing tumor” because of the many similarities between tumor growth and tissue repair.2MSC are recruited from the bloodstream to tumors, healing wounds, or sites of tissue injury by multiple growth factors and chemokines, where they differentiate into fibroblasts, pericytes, endothelial cells, and even terminally differentiated cells, such as osteoblasts, chondrocytes, astrocytes, neurons, and myocytes (“multilineage differentiation”).3Engrafted at the sites of tissue damage, they secrete growth factors and cytokines (e.g. , vascular endothelial growth factor, platelet-derived growth factor) that facilitate vasculogenesis and the healing process.4Conversely, most experimental studies also show that MSC promote tumor growth. Coinjection of bone-marrow–derived MSC with green fluorescent protein-labeled breast cancer cells into immune-incompetent mice accelerates tumor growth.5Likewise, coinjection of adult- and fetal-derived MSC with colon cancer cells into a mouse xenograft model leads to increased formation of highly vascularized tumors.6Although some studies report proinflammatory and antiproliferative effects of MSC on tumor growth, probably by increased mobilization of macrophages and granulocytes, MSC are known to secrete proangiogenic factors, such as vascular endothelial growth factor, fibroblast-derived growth factor, platelet-derived growth factor, and stromal cell-derived factor-1, which potentially facilitate endothelial and smooth muscle cell proliferation in tumors.7MSC also secrete chemokine (C-C motif) ligand 5, which was shown to enhance the metastatic potential of breast cancer cells.5
Local anesthetics are commonly used in the perioperative setting for pain treatment to reversibly block the conductance in neurons (regional anesthetics, nerve blocks, wound infiltration). If overdosed, they exert detrimental toxic effects mainly on neural and cardiac tissues resulting in life-threatening seizures and respiratory and cardiac arrest. Reports on their cytotoxicity also revealed adverse effects on mitochondrial respiration resulting in marked oxidative stress.8Other studies indicate a dose-dependent inhibition of proliferation of fibroblasts and tenocytes at concentrations as low as 10 μM.9These observations raise concerns that direct administration (e.g. , using wound catheters) of local anesthetics at even low concentrations could weaken the wound and delay its closure. Conversely, there is emerging evidence that local anesthetics applied in the perioperative setting are capable of preventing tumor spreading during cancer surgery.10So far, it remains elusive whether local anesthetics affect the biology of MSC, key players in tissue repair and tumor growth. Therefore, we hypothesized that lidocaine, bupivacaine, and ropivacaine would inhibit the proliferation of MSC in a dose-dependent manner and set out to unravel the underlying mechanisms. The expected inhibition of MSC by local anesthetics could indeed represent an important mechanism by which local anesthetics applied in the perioperative period might help prevent perioperative metastasis and improve long-term survival of patients undergoing cancer surgery. Conversely, inhibition of MSC proliferation by prolonged application of local anesthetics potentially could delay wound closure and promote wound dehiscence.
Materials and Methods
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (Publication No. 85-23, revised 1996), and the experimental protocol used in this investigation was approved by the University of Alberta Animal Policy and Welfare Committee (Edmonton, Alberta, Canada).
Supplies and Chemicals
All chemicals were purchased from Sigma (Oakville, Ontario, Canada) unless otherwise stated. Dulbecco modified Eagle's medium, fetal bovine serum, penicillin-streptomycin, trypsin-EDTA solution, and Dulbecco phosphate buffered saline were obtained from Invitrogen (Burlington, Ontario, Canada).
MSC Isolation and Expansion
Mesenchymal stem cells were isolated from femurs and tibias of C57BL6/J mice 8–10 weeks old. Marrow was extruded by inserting a 26.5-gauge needle into the shaft of the bone and flushing it with complete cell culture media (Dulbecco modified Eagle's medium supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin). The aspirate was resuspended in complete media and distributed into T75 cell culture flasks. The flasks were incubated at 37°C in a humidified atmosphere of 5% CO2-95% air in a cell culture incubator. After 24 h, nonadherent cells were removed, fresh media was added, and the adherent cells were allowed to reach 80% confluence, before splitting. Passage 3 cells were used for immunophenotypical characterization and for the osteogenic differentiation assay. Passage 7–15 (P7–P15) cells were used for all other experiments.
Characterization of MSC
Fluorescence-activated cell sorting (FACS) of cell surface antigens was performed to characterize the immunophenotype of MSC, in accordance with the minimal criteria for definition of MSC.11MSC were collected, counted on a hemocytometer, and adjusted to a final density of 6 × 105cells/ml. Cells were washed twice with FACS buffer (sodium azide [0.05%] and bovine serum albumin [0.1%] in phosphate buffered saline), then incubated with fluorescein isothiocyanate or phycoerythrin-coupled monoclonal antibodies against stem cell antigen-1 (clone E13–161.7; Biolegend, Burlington, Ontario, Canada), CD105 (clone MJ7/18; Biolegend), c-kit (CD117; clone 2B8), CD44 (clone IM7), CD45 (clone 30-F11), and CD34 (clone RAM34) (BD Biosciences, Mississauga, Ontario, Canada) or isotype-matched control immunoglobulin G at the manufacturers' recommended concentrations in the dark at + 4°C for 30 min.12Fluorescent-labeled cells were washed twice with FACS buffer and fluorescence signals of scatter-gated cells were measured using FACSCanto II flow cytometer and FACSDiva software (BD Biosciences).
Osteogenic Differentiation Assay
The osteogenic differentiation assay was performed in accordance with the minimal criteria for definition of MSC.11MSC (P3) were plated on 13-mm Thermanox plastic cover slips (Nunc, Rochester, NY) in 24-well plates and cultured to confluence. To induce osteogenesis, adherent cultures were treated every other day with osteogenic media consisting of Dulbecco modified Eagle's medium supplemented with fetal bovine serum (10%), penicillin-streptomycin (1%), 10 mM glycerol phosphate, 0.2 mM ascorbate-2-phosphate, and 0.5 μM dexamethasone. Increasing concentrations (to 250 μM) of lidocaine, bupivacaine, or ropivacaine were added. Negative control consisted of cells cultured in complete media. After 3 weeks, the cells were fixed in 4% formalin. Mineralization of the extracellular matrix was visualized by staining with alizarin red S (2.5%, pH 4.2) for 30 min at room temperature.
Cell Proliferation and Colony-forming Unit Assays
Mesenchymal stem cells were plated in six-well plates at a density of 6 × 104cells/well and cultured for 72 h in complete media in the presence or absence of increasing concentrations (10, 100, 500 μM) of the local anesthetics lidocaine, bupivacaine, and ropivacaine. After 24, 48, and 72 h, MSC were collected, stained with 0.4% trypan blue, and counted using a hemocytometer. In some experiments, MSC were treated with the respiratory chain inhibitor antimycin A (0.1 and 0.2 μM) or with the antioxidant N -acetylcysteine (4 mM and 10 mM), concomitantly added to ropivacaine. Colony-forming unit assays were performed as described previously.13Briefly, MSC were seeded at the low density of 100 cells/64 cm2(in 9-cm Petri dishes) in the presence or absence of increasing concentrations of local anesthetics. Media were changed every 96 h for 2 weeks. Cultures were washed with phosphate buffered saline, fixed with formalin (4%), and stained with crystal violet (3%). Colonies with ≥40 cells were counted.
Cytotoxicity Assay and Annexin V-Propidium Iodide Staining
Mesenchymal stem cells were plated in six-well plates at a density of 6 × 104cells/well, allowed to attach overnight, and cultured for 24 h in complete media in the presence or absence of increasing concentrations (10, 100, 500 μM) of the local anesthetics lidocaine, bupivacaine, and ropivacaine. Cytotoxicity was assessed using the Cytotoxicity Detection Kit (lactate dehydrogenase) (Roche Diagnostics Canada, Laval, Quebec, Canada) according to the manufacturer's instructions. Phosphatidylserine exposure on the plasma membrane surface was determined using the annexin V-fluorescein isothiocyanate apoptosis kit (Sigma) according to the manufacturer's instructions. Fluorescence signals of scatter-gated cells were measured using FACSCanto II flow cytometer and FACSDiva software.
Cell Cycle Analysis
Mesenchymal stem cells were collected and fixed in ice-cold ethanol (70%) overnight. Fixed cells were resuspended in 0.5 ml phosphate buffered saline and incubated in a solution containing Triton X-100 (0.1%), 1.0 mg/ml DNase-free RNase, and 1.0 mg/ml propidium iodide for 30 min at room temperature.14Fluorescence signals were measured using FACSCanto II flow cytometer, and the proportion in G0/G1, S, and G2/M phases was estimated using FlowJo cell cycle analysis program (Tree Star Inc., Ashland, OR).
In Vitro Wound Healing Assay
Mesenchymal stem cells were seeded into 24-well cell culture plates at a density of 6 × 105cells/well and cultured until 90% confluent.15To more closely reproduce the conditions in a real wound, the monolayers were treated with 20 ng/ml tumor necrosis factor α (TNFα; R&D Systems, Minneapolis, MN) in the presence or absence of 100 μM ropivacaine and incubated for 24 h. Cell monolayers were scraped in a straight line using the tip of a 1,000-μl pipette to create a “wound.” Debris was removed by rinsing the cells with culture media, and fresh culture media supplemented by TNFα and local anesthetics were added. Time-lapse images of the wound surface area were obtained at 0, 3, and 6 h after scratching using an inverted microscope (Leica Microsystems Inc., Richmond Hill, Ontario, Canada) and analyzed with OpenLab software (Quorum Technologies Inc., Guelph, Ontario, Canada).
Expression of Intercellular Adhesion Molecule 1 (ICAM-1)
Mesenchymal stem cells were treated with TNFα (20 ng/ml) and exposed to increasing concentrations of local anesthetics for 60 min. Cells were collected, washed, resuspended in FACS buffer, and incubated for 30 min with a R-phycoerythrin–conjugated monoclonal ICAM-1 antibody (clone 3E2) or with isotype-matched control antibody (BD Biosciences) in the dark at 4°C. Fluorescence signals of scatter-gated cells were measured using FACSCanto II flow cytometer and FACSDiva software.
Expression of Cell Cycle-related Proteins
The expression of the cell cycle regulatory components p16INK4a, p27Kip1, and proliferating cell nuclear antigen was investigated by Western blotting in MSC exposed to ropivacaine for 24 h. Cells were collected, washed twice with ice-cold phosphate buffered saline, and centrifuged at 6,000g for 10 min, and the pellets were snap-frozen in liquid N2. For Western blotting, cell pellets were thawed in lysis buffer (20 mM Tris, pH 7.4; 150 mM NaCl2, 1 mM EDTA, 1 mM EGTA, Triton X-100 (1%), 2.5 mM sodium pyrophosphate, protease, and phosphatase inhibitor cocktails). Cell lysates were homogenized and centrifuged at 12,000g for 15 min at 4°C, and protein concentrations were determined with the Bradford assay (Bio-Rad, Hercules, CA). Proteins were separated on SDS-polyacrylamide (12%) gels and electrophoretically transferred onto a nitrocellulose membrane (Bio-Rad, Mississauga, Ontario, Canada). Membranes were probed overnight at 4°C with the following primary antibodies: rabbit anti-p16 INK (SAB4500072; Sigma), rabbit anti-p27 KIP1 (SAB4500068; Sigma), antiproliferating cell nuclear antigen (clone PC10, 2586 Cell Signaling Technology; distributed by New England Biolabs Ltd., Pickering, Ontario, Canada), α-tubulin (loading control; T6074; Sigma). After incubation with antiimmunoglobulin G-horseradish-peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1.5 h at room temperature, protein bands were quantified using ImageJ software.§§
Immunoblotting and Electrophoretic Mobility Shift Assay in TNFα-treated Cells
Mesenchymal stem cells were treated for 60 min with 20 ng/ml TNFα in the presence or absence of ropivacaine 100 μM and collected and processed for Western blotting as described. Primary antibodies against nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) were rabbit anti-IκB-α (ab7217; Abcam, Cambridge, MA) and rabbit antiphospho-IκB-α (ab12135; Abcam). Electrophoretic mobility shift assays were performed using the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) p65 LightShift Chemiluminescent electrophoretic mobility shift assay kit (Product No. 89859; Thermo Scientific, Rockford, IL). Nuclear extracts were prepared from frozen cell pellets using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Product No. 78833; Thermo Scientific).
High-resolution Respirometry in Intact and Permeabilized Cells
The respiratory capacity of the mitochondrial electron transport chain in control and ropivacaine (100 μM)-treated MSC after 24 h of incubation was measured with a high-resolution respirometry (Oxygraph-2K; Oroboros, Innsbruck, Austria) at 37°C. Analysis of the coupling states in intact cells was performed with membrane-permeant inhibitors or uncouplers (see figure, Supplemental Digital Content 1, https://links.lww.com/ALN/A820, for a detailed protocol). To evaluate the site(s) of inhibition by ropivacaine, we measured phosphorylation rates in digitonin-permeabilized cells in the presence of various mitochondrial complex-specific substrates and/or inhibitors (see figure, Supplemental Digital Content 2, for a detailed protocol, https://links.lww.com/ALN/A821). At the end of every run, cell suspensions were collected and stored at −80°C for citrate synthase activity measurements.
Determination of Adenosine 5′-triphosphate (ATP) Concentrations
Adenosine 5′-triphosphate concentrations were measured in control and ropivacaine (100 μM)-treated MSC after 24 h of incubation using the ATP bioluminescent somatic cell assay kit (Sigma). A subset of samples was also treated with either iodoacetate (200 μM; inhibitor of glycolysis) or antimycin A (10 μM, complex III inhibitor) during the last hour of the incubation period. These additional experiments were performed to assess the contribution of glycolytically produced ATP to total ATP in MSC. Data were normalized to citrate synthase activity.
Citrate Synthase Activity
To relate the observed respiration rates to mitochondrial content, the activity of the mitochondrial matrix marker enzyme citrate synthase was measured at 412 nm by monitoring the formation of thionitrobenzoate, the product of reaction between 5,5′dithiobis-2-nitrobenzoate (a chromogen) with the thiol group of free coenzyme A that is produced in the formation of citrate.16The reaction was initiated by the addition of 0.5 mM oxaloacetate in the presence of 0.3 mM acetyl-coenzyme A, and the rate of absorbance change was monitored for 2 min.
Measurements of Reactive Oxygen Species (ROS)
Cellular production of hydrogen peroxide in response to ropivacaine was measured by loading cells with 2′,7′-dichlorodihydrofluorescein diacetate (20 μM; diluted in the medium from a 10-mM stock solution in dimethyl sulfoxide) for 45 min in the dark. During the last 30 min, ropivacaine (100, 250, and 500 μM) or antimycin A (2.5 μM, positive control) was added to the plates. Cells not loaded with the fluorescent dye served as negative controls. Cells were harvested, resuspended in FACS buffer, and their fluorescence signals collected using FACSCanto II flow cytometer and FACSDiva software.
Control and ropivacaine (100 μM)-treated MSC were collected after a 24 h-incubation and processed for total RNA isolation using the Qiagen RNeasy MiniKit (QIAGEN Inc., Toronto, Ontario, Canada) according to the manufacturer's instructions. RNA samples were processed for microarray analysis (Affymetrix Mouse Exon 1.0 ST Arrays; Affymetrix, Santa Clara, CA) in accordance with the minimum information about a microarray experiment (MIAME) guidelines.17Data are available at the Gene Expression Omnibus database under the series number GSE31827. Gene set enrichment analysis was performed to assess alterations in global gene expression in response to ropivacaine.18,19Microarray results were confirmed by real-time polymerase chain reaction assays (the primers used and the validation data are presented in the table, Supplemental Digital Content 3, https://links.lww.com/ALN/A822).
Values are given as mean ± SD for the indicated number of independent observations (n). The significance of differences in variables among groups was determined by Student t test (two groups) or by analysis-of-variance (ANOVA) followed by the Holm-Sidak method for post hoc analysis or by nonparametric methods (Kruskal-Wallis test) depending on the underlying data distribution. Proliferation data were analyzed using two-way ANOVA followed by the Holm-Sidak method for post hoc multiple comparisons. Wound healing assay data were analyzed using two-way repeated measures ANOVA followed by the Student–Newman–Keuls test. Differences are considered significant if P < 0.05. SigmaStat (version 3.5; Systat Software, Inc., Chicago, IL) was used for the analyses.
The immunophenotypical characterization by flow cytometry revealed that MSC were uniformly positive for stem cell antigen-1 (97.4%) and CD105 (endoglin; 96.7%) but negative for the hematopoietic-endothelial antigens CD34 (0.4%), CD45 (0.3%), and c-kit (CD117; 0.2%). MSC also expressed the hyaluronan receptor CD44 (8.5%).
Local Anesthetics Dose-dependently Exert Antiproliferative Effects, Increase Markers of Cellular Injury, Delay In Vitro Wound Healing, and Impair Osteogenic Differentiation in MSC
For clarity, ropivacaine data are presented. Comparative results obtained with lidocaine, bupivacaine, and ropivacaine are depicted in figures 1,–,4of Supplemental Digital Content 4 (https://links.lww.com/ALN/A823). Ropivacaine inhibited cell proliferation at concentrations ≥100 μM (fig. 1A). The population doubling time in untreated cultures was 39.4 h but increased to 92.7 h in cultures treated with 100 μM ropivacaine. Inhibition of cell growth was accompanied with lactate dehydrogenase release (fig. 1B), a marker of cytotoxicity and increased plasma membrane permeability. A dose-dependent impairment of colony formation (fig. 1C) and an increase in annexin V binding to phosphatidylserine (marker of early apoptosis) (fig. 2A) were observed.
Cell cycle analysis after 24 h of exposure to ropivacaine revealed a higher percentage of MSC in G0/1phase and a lower percentage in the S phase (fig. 2B), consistent with cell cycle arrest. Ropivacaine up-regulated the expression of the cell cycle-regulatory proteins p16, p27, and proliferating cell nuclear antigen (fig. 2C). Growth inhibition was reversible after cessation of ropivacaine treatment (100 μM), indicating a reversible pharmacologic, rather than an irreversible toxic, effect (see figure, Supplemental Digital Content 5, https://links.lww.com/ALN/A824). Wound healing assays, which measure the migration of MSC in response to mechanical damage of a confluent cell layer, revealed that ropivacaine at a concentration of 100 μM has a pronounced inhibitory effect on cell migration (fig. 3). To test whether local anesthetics also affect cell differentiation, osteogenic differentiation assays were performed in the presence and absence of increasing concentrations of the drugs. All anesthetics dose-dependently reduced the deposition of mineralized matrix, with bupivacaine being more potent, abolishing osteogenesis at a concentration of 250 μM (see figure, Supplemental Digital Content 6, https://links.lww.com/ALN/A825).
Ropivacaine Reduces the Expression of ICAM-1, a Key Surface Receptor in MSC Migration and Differentiation, via the IκB–NF-κB Signaling Pathway
Because previous studies demonstrated the importance of ICAM-1 in MSC migration and differentiation,20,21we hypothesized that local anesthetics would decrease ICAM-1 expression in the presence of TNFα. Ropivacaine markedly decreased TNFα-induced ICAM-1 expression in MSC in a concentration-dependent manner (fig. 4A). IκB phosphorylation and NF-κB translocation to nuclei subsequently were determined in MSC exposed to ropivacaine. Ropivacaine inhibited TNFα-induced IκB phosphorylation (fig. 4B) and abolished translocation of transcriptional factor NF-κB to nuclei (fig. 4C).
Ropivacaine Inhibits Mitochondrial Respiration Depleting Cellular ATP Content and Imposes Oxidative Stress on MSC
Mitochondrial oxygen consumption was measured in intact and permeabilized MSC, and all data were normalized to citrate synthase activity. No difference in citrate synthase activity between the control and ropivacaine-treated cells was observed (data not shown). Intact MSC treated with 100 μM ropivacaine exhibited a significant reduction (∼28%) in oxygen consumption while respiring on endogenous substrates (fig. 5A). The treated MSC also exhibited a significant reduction (∼25%) in maximal mitochondrial oxidative capacity as assessed by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone-induced oxygen consumption. However, the leak respiration rate (oligomycin-insensitive fraction of respiration) was unchanged. To determine the site(s) of inhibition by ropivacaine (i.e. , to isolate the flux going through specific respiratory complexes), we used a multiple substrate-inhibitor combination protocol in permeabilized MSC. Our experiments show marked reductions in all examined complexes (i.e. , complex I, complex II, and complex IV; fig. 5B). Glutamate-malate–driven flux through complex I was decreased by 34% in ropivacaine-treated MSC compared with untreated cells. Complex II-dependent respiration was even more impaired (40% reduction), whereas complex IV-driven flux was reduced by 30%. Ropivacaine induced a small but significant dose-dependent increase in the production of ROS (fig. 5C). Subsequent experiments showed that 100 μM ropivacaine exposure for 24 h also reduced cellular ATP content by ∼20% compared with untreated MSC (fig. 5D). To determine whether the cellular ATP depletion was entirely attributable to inhibition of mitochondrial respiration, ATP content of MSC was measured after ropivacaine exposure in the presence of iodoacetate, an inhibitor of glycolysis, or antimycin A, a complex III inhibitor (fig. 5D). These experiments demonstrate that MSC can produce ATP through glycolysis in the presence of oxygen (Warburg effect), and the reduction in ATP concentrations in ropivacaine-treated cells is indeed attributable to dysfunctional mitochondria. To test whether inhibition of mitochondrial respiration by ropivacaine would be causally related to their antiproliferative action, MSC were concomitantly exposed to ropivacaine and the antioxidant N -acetylcysteine. Antimycin A, an inhibitor of the respiratory chain, dose-dependently delayed MSC proliferation in a manner similar to that of ropivacaine and served as positive control. Treatment with the antioxidant N -acetylcysteine did not reverse the effect of ropivacaine, implying that mechanisms other than ROS alone are mediating the antiproliferative action of ropivacaine (fig. 5E).
Transcriptional Profiling Uncovers a Lysosomal Storage Disorder but also Reveals the Anticancer Potential of Ropivacaine Elicited in MSC
Ropivacaine treatment (100 μM, 24 h) induced significant transcriptional changes in MSC compared with untreated cells. Among the top up-regulated transcripts are genes related to cholesterol metabolism (e.g. , lanosterol synthase, mevalonate [diphospho]decarboxylase), the lysosome (e.g. , α-galactosidase, lysosomal ATPase), cell cycle control (e.g. , dipeptidyl-peptidase 2, G0/G1switch gene 2), and stress response (e.g. , metallothioneins). However, ropivacaine treatment repressed genes related to differentiation processes (e.g. , pleiotrophin, asporin, transcription factor Sp7/osterix, and osteoglycin; see figure, Supplemental Digital Content 7, https://links.lww.com/ALN/A826). Gene set enrichment analysis clearly confirmed that amphiphilic local anesthetics have detrimental effects on membranes. Ropivacaine significantly increases the metabolism of lipids and cholesterol, essential components of mammalian cell membranes, but also up-regulates lysosomal processes, consistent with an increase phospholipid turnover (table 1; figure, Supplemental Digital Content 8, https://links.lww.com/ALN/A827). Ropivacaine reduced the expression of chemokines (figure, Supplemental Digital Content 8, https://links.lww.com/ALN/A827) and of pathways related to angiogenesis and metastasis formation (fig. 6, table 1, and table 2). The analysis also uncovered important perturbations of transcriptional developmental programs caused by ropivacaine treatment (table 2).
Our experiments were motivated by recent findings that the perioperative use of local anesthetics appears to improve long-term survival in cancer patients, which could be caused by antiproliferative cytostatic effects of local anesthetics on tumor cells.10In a retrospective analysis of patients undergoing surgery for breast cancer, the use of a paravertebral nerve block combined with general anesthesia was associated with a better cancer-free survival.22A number of additional studies could confirm these promising findings in patients with prostate23and colon24cancer. In a study with melanoma patients undergoing surgery, patients receiving local anesthetics as opposed to general anesthesia showed a decrease in tumor recurrence.25Taken together, there is increasing evidence that the antiproliferative effects of local anesthetics could mitigate perioperative tumor growth and metastasis formation.
Most previous studies focused on the direct toxicity of local anesthetics on various cancer cells,26but there is no information on how local anesthetics affect MSC, key players of tumor growth and metastasis formation.27,28Although some controversies exist with respect to the definite role of MSC in tumor growth and propagation, most recent in vivo studies clearly demonstrate their high potential to boost tumor growth and metastasis by the release of growth factors, enhanced angiogenesis, immunomodulation, and a phenomenon called “epithelial-to-mesenchymal transition,” a process whereby MSC transform into “carcinoma-associated fibroblasts” and promote the dedifferentiation and spreading of tumor cells into the body.29On the other hand, MSC are essential elements in tissue repair30and thus are currently used in numerous investigational regenerative therapies for otherwise incurable diseases.31
Our experiments show the following salient findings. First, MSC are sensitive to the antiproliferative effects of local anesthetics at concentrations of 10–100 μM that previously were measured in skeletal muscle tissue after the establishment of femoral blocks.32Of note, these concentrations are significantly lower than the ED50(550 μM) for ropivacaine to inhibit growth of HT-29 colon adenocarcinoma cells in vitro in the presence of 10% serum.26Second, at these concentrations local anesthetics do not induce cell death but inhibit cell growth by arresting cells in the G0/1phase. Third, our experiments suggest that multiple mechanisms may be involved in the observed antiproliferative actions, including inhibition of the IκB–NF-κB–ICAM-1 signaling pathway, which enables efficient cell-to-cell communication and thus is critical in proliferation and migration,20,21inhibition of mitochondrial respiration with subsequent depletion of cellular ATP and formation of ROS, and profound changes in the transcriptome. Because many previous studies reported marked and rapid deterioration in bioenergetics of mitochondria exposed to local anesthetics,33,34it appears that the observed transcriptional changes and the down-regulation of the cell surface receptor ICAM-1 are a consequence, rather than a cause, of the antiproliferative effects of local anesthetics in response to energy loss and oxidative stress. In fact, ROS-mediated attenuation of ICAM-1 expression was shown in paclitaxel-treated breast cancer cells.35Inhibition of complex II previously was implicated in cellular senescence and aging causing cell cycle arrest.36Byun et al. 36reported that inhibition of complex II of the respiratory chain induces a delay in cell cycle progression without cell death. This is consistent with our findings that ropivacaine increased the expression of the cell cycle inhibitors p16INK4and p27KIP1. The increase in proliferating cell nuclear antigen, which plays an unprecedented role in controlling DNA synthesis, DNA repair, and cell cycle progression,37indicates a block between G1and S phase.38Byun et al. 36also showed that the delay of cell cycle progression was attributable to formation of ROS, rather than ATP depletion. Although ropivacaine increased the formation of ROS, our experiments do not support ROS formation as the single cause of the antiproliferative actions. Rather, our transcriptional analysis points to a number of additional mechanisms perhaps underlying ropivacaine-induced cell cycle inhibition. Our microarray analysis also showed up-regulation of genes controlled by the transcription factor sterol regulatory element-binding protein-1a, which is known to cause G1cell cycle arrest through accumulation of cyclin-dependent kinase inhibitors p16, p21, and p27,39and up-regulation of the cell cycle inhibitory G0G1switch gene 2.40In our experiments, ropivacaine-treated cells did not show uncoupling, but uncoupling was shown previously for bupivacaine.34Local anesthetics also were reported to directly inhibit F1F0ATP synthase and to decrease and depolarize the mitochondrial potential. Although mitochondrial dysfunction appears to be the predominant cause of cell cycle arrest and antiproliferative action in our experiments, we cannot entirely rule out that changes in lipid composition of mitochondrial and other membranes, as evidenced in our transcriptional analysis, may have contributed, at least on the longer-term (i.e. , after 24 h) to mitochondrial dysfunction. In fact, local anesthetics similar to other cationic amphiphilic drugs, such as tetracyclines and amiodarone, induce steatosis and phospholipidosis,41characterized by intracellular phospholipid and cholesterol-triglyceride accumulation interfering with vital cellular functions.42Up-regulation of cholesterol and lysosomal-peroxisomal pathways can be regarded as a cellular defense mechanism against the membrane-disrupting effects of local anesthetics. Collectively, our results provide evidence that local anesthetics markedly inhibit growth of MSC by multiple mechanisms.
In our experiments, most of the biologic effects of local anesthetics on MSC were observed at a concentration of 100 μM, which is clearly above the toxic serum concentrations previously reported for local anesthetics. Although these high concentrations cannot be used systemically (i.e. , “at a distance” in tumor patients), it is still possible to use high concentrations locally (i.e. , in situ for the benefit of the patient). Relatively high concentrations of local anesthetics are reached by placing catheters close to the site of surgery,32where tumor cells are likely to be spread by surgical manipulations. Complete surgical resections of tumors often are impossible, and under these conditions local anesthetics may inhibit tumor cells to access the vascular system and metastasize. In accordance with this hypothesis, infiltration of local anesthetics into wounds was reported to be associated with lower cancer recurrence after melanoma excision.25In addition, because tumor cells prefer sites of injury and healing for colonization, in situ treatment may still have an important impact on disease progression.43Nouette-Gaulain et al. 32report a ropivacaine concentration in muscular tissue of 30 μM when applying a femoral nerve block with only seven bupivacaine injections (1 mg/kg) in a rat model. However, a continuous infusion of local anesthetics over the postoperative course is likely to cause additional accumulation of local anesthetics and increase peak local concentrations, inhibiting metastasizing tumor cells in growth and proliferation. Interestingly, it was shown that even very low subanesthetic concentrations of bupivacaine can become cytotoxic if applied over an extended time.44Moreover, in our in vitro experiments, we used 20% serum to create optimal conditions for MSC to grow and proliferate. However, because proliferation is a balance between promoting and inhibiting growth stimuli, it is possible that under in vivo conditions with less favorable conditions and a functional tumor-inhibiting immune system, much lower concentrations of local anesthetics (i.e. , in the nanomole range, may be sufficient to inhibit or kill tumor cells. Accordingly, Martinsson reports that a reduction in serum concentration from 10 to 1% increases the sensitivity of cultured HT-29 colon adenocarcinoma cells to ropivacaine-induced inhibition of proliferation by 50%.26Nevertheless, it is possible that the putative improved outcome in cancer patients receiving local anesthetics and/or regional anesthesia is attributable to the concomitant pain relief leading to a reduced consumption of morphine, which has been reported to be proangiogenic.45Clearly, our studies need additional in vivo validation.
Considerations on the Use of Local Anesthetics against Tumor Growth and Metastasis Formation in Surgical Patients
The role of MSC in tumor growth may be particularly important in the context of surgery, where tissue damage caused by surgery evokes a massive surge of these cells from the bone marrow.46Most recent in vivo studies strongly support the tumor growth-promoting actions of MSC, although not all bone marrow-derived MSC may promote tumor progression equally.47MSC promote tumor growth and metastasis formation in multiple ways, including antiapoptotic proliferative effects, immunosuppression, drug resistance, paracrine secretion of growth factors and chemokines, and “epithelial-to-mesenchymal transition.”28In addition, MSC have a propensity to homing toward tumor cells and are prone to malignant self-transformation because of chromosomal instability.48Our unbiased microarray screen now reveals for the first time that ropivacaine markedly down-regulates transcripts related to G-protein coupled receptors, chemokines, and growth factor signaling in MSC, consistent with antiproliferative antiinflammatory and cytostatic actions. Ropivacaine up-regulates transcripts such as RB1, a negative regulator of the cell cycle, known to be suppressed in retinoblastoma, bladder cancer, and sarcoma. Our comprehensive analysis also shows that ropivacaine suppresses multiple gene sets with promoter regions containing transcription factor consensus sequences associated with stemness and/or cell differentiation. The identification of large groups of genes harboring common transcription factor binding sites (table 2) is essential for understanding the regulatory modules that control stem cell processes such as differentiation or metabolism. However, the false-discovery rates are rather high. Likely reasons for this may be the limited number of samples per group (n = 4) and/or the noise inherent to the expression data of these particular gene sets. Irrespective of the underlying reasons, the results clearly point to the necessity to further validate the findings. Among the most prominent consensus sequences were -CAGGTA- matching with transcription factor 8, also called ZEB1 (zinc finger E-box-binding homeobox 1), and -NTGGNNNNNNGCCAANN- matching with neurofibromin 1. Transcription factor 8 or ZEB1 inactivity promotes tumorigenicity and metastasis by angiogenesis,49whereas lack of neurofibromin 1 enhances cell growth by enhancing signal transducer and activator of transcription-3.50Interestingly, an MSC-like phenotype is the hallmark of tumor aggressiveness in human primary glioblastomas.51In this study, genes that were down-regulated by ropivacaine, such as collagen type III α1, transforming growth factor-β induced, or tenascin C (see figure, Supplemental Digital Content 8, https://links.lww.com/ALN/A827), were highly overexpressed in aggressive glioblastoma tumors and strong predictors of survival.
What Do Our Results Suggest with Respect to the Direct Application of Local Anesthetics to Wounds?
Mesenchymal stem cells form an essential component of the complex wound healing process, which consists of cell migration and proliferation, deposition of extracellular matrix, vasculogenesis, and matrix metalloprotease-mediated tissue remodeling. Using an excisional wound splitting model in mice, Wu et al. 30showed that injection of bone marrow derived MSC around the wound markedly accelerated would healing and closing in normal and diabetic mice. These authors also observed increased formation of angiopoietin-l and endothelial cell tube formation after MSC injection, indicating that MSC participate in revascularization, a critical step in tissue repair. Reduced wound breaking strength and impaired healing were reported in rat models of acute wound repair after exposure to local anesthetics.52A recent study using a mouse model of cutaneous wound healing was unable to demonstrate adverse effects of lidocaine and bupivacaine, but the local anesthetics were applied only once over 3 days as bolus and not as continuous infusion.53
The local anesthetics used in our study are widely administered in perioperative medicine. The question of whether ester local, as opposed to amide local, anesthetics or general anesthetics (namely volatile anesthetics and propofol) would have similar or opposite effects on MSC is important and should be tested in future experiments. We recognize that the results of our experiments were obtained through in vitro primary cell cultures of MSC. Although it is inappropriate to use our data for extrapolation to the clinical environment, they provide valuable novel information about mechanisms underlying the putative anticancer and tissue repair inhibiting effects of local anesthetics. Clearly, future in vivo experimental and clinical studies will be necessary to clarify the roles of local anesthetics and MSC in perioperative tumor spreading and tissue repair.