Local anesthetics have a broad spectrum of pharmacological actions that go beyond the familiar pain relief and antiarrhythmic effects.1,2 The most commonly used anesthetic, lidocaine, effectively inhibits the invasiveness of cancer cells at concentrations used in surgical procedures.3 This antiinvasive effect seems unrelated to its anesthetic activity (sodium channel blockade). Other local anesthetics have been shown to trigger apoptosis in a variety of human cells.4,5 The mechanisms underlying these effects are not yet fully understood.
Recent evidence indicates that wound infiltration with local anesthetics reduces postoperative pain after breast surgery.6,7 The apoptosis-inducing activity of local anesthetics may thus provide additional benefits to their use that could have substantial clinical implications. In the present study, we sought to examine the effects of 2 common local anesthetics, lidocaine and bupivacaine, on breast tumor cells in vitro and in a murine xenograft model.
Lidocaine and bupivacaine were purchased from Sigma (St. Louis, MO). Specific pan-caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-9 inhibitor (z-LEHD-fmk) were obtained from BD Biosciences (San Jose, CA).
Cell Lines and Culture Conditions
Human breast tumor cell lines MCF-7 and MCF-10A were purchased from the American Type Culture Collection. The MCF-7 cell line is derived from malignant pleural effusions of breast adenocarcinoma. MCF-10A is a spontaneously immortalized but nontransformed human mammary epithelial cell line derived from breast tissue with fibrocystic changes. MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (SAFC Biosciences, Brooklyn, Victoria, Australia), 2 mM·L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. MCF-10A cells were cultured in Dulbecco’s modified Eagle’s medium/F12 supplemented with 5% horse serum (Invitrogen/Gibco, Carlsbad, CA), 20 ng/mL epidermal growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 µg/mL insulin, 100 U/mL penicillin, and 100 µg/mL streptomycin. Both cell lines were incubated in an atmosphere of humidified 5% CO2 at 37°C.
Cells were plated 5 × 103/well in 96-well tissue culture plates and treated with lidocaine or bupivacaine (individually or in combinations) at indicated concentrations for 6, 24, or 48 hours. To determine the cell viability, 200 µL MTS reagent (0.2 mg/mL, Promega, Madison, WI) was added to the cells, which were then incubated in the dark at 37°C for 1.5 hours. The absorbance was measured at 490 nm using Varioskan Flash (Thermo Fisher Scientific, Waltham, MA). The median 50% effective dose (ED50) values were calculated using the probit method of Miller and Tainter.8
DNA Fragmentation Analysis
DNA was extracted using QIAamp DNA mini kit (51306; Qiagen, Valencia, CA) according to the instructions of the manufacturer. After DNA quality was verified with spectrophotometric measurements, 1 µg extracted DNA was electrophoresed on a 1.5% agarose gel, visualized by EZ-vision dye staining (Amresco, Solon, OH) under ultraviolet illumination, and photographed using Digital Image Stocker (DS-30, FAS III, Toyobo, Osaka, Japan). Apoptotic DNA fragmentation of samples corresponding to <1500 bp DNA was densitometrically determined.
Annexin V Apoptosis Assay
The annexin V-fluorescein isothiocyanate (FITC) apoptosis kit (BioVision) was used to determine whether cell death was due to apoptosis. Cells were plated on coverslips in 24-well culture plates. After treatment with lidocaine or bupivacaine at indicated concentrations for 4 hours, cells were stained with 2.5 μg/mL annexin V-FITC and 4 μg/mL Hoechst 33285 (Sigma) in binding buffer (10 mM HEPES/NaOH [pH 7.4], 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) for 20 minutes at room temperature in the dark. Cells were then fixed with 2% formaldehyde in binding buffer for 5 minutes. After washing with binding buffer, cells were mounted and observed with a fluorescence microscope (Axio Imager A1, Carl Zeiss AG, Oberkochen, Germany). For quantitative analysis, the fraction of FITC-positive cells (apoptotic cells) in each group was determined based on calculations from 10 randomly selected fields under the fluorescence microscope.
Western Blot Assay
Total proteins were extracted using M-PER lysis buffer (Thermo). Lysates were centrifuged, and proteins were heat denatured. Protein concentration was determined using the Bradford assay kit (Bio-Rad, Hercules, CA). Total proteins (30 µg) were separated by 10% to 15% SDS-PAGE and then transferred onto a nitrocellulase membrane (Amersham Biosciences/GE Healthcare, Piscataway, NJ). Membranes were blocked in 5% (w/v) nonfat milk and immunoblotted with primary antibodies as indicated. Antibodies were diluted 1:1000 for anti-caspase 8 (ALX-804-429; Enzo, Farmingdale, NY), anti-caspase 9 (9508; Cell Signaling, Danvers, MA), anti-caspase 7 (9494; Cell Signaling), and anti–poly ADP-ribose polymerase (PARP) (P248; Sigma) and 1:10 000 for anti-β-actin (A5441; Sigma) and α-tubulin (T5168; Sigma). Immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and the enhanced chemiluminescence system (ECL plus; Merck Millipore, Darmstadt, Germany).
In Vivo Xenograft Model
The xenograft model was established and modified as previously described.9 In brief, 17-estradiol was implanted into 30 female BALB/c nude mice. Twenty-four hours later, 1 × 107 MCF-7 cells in 100 μL mixture of phosphate buffered saline and growth factor–reduced matrigel were injected subcutaneously into the trunks of mice. Tumor growth was determined by caliper measurements. Tumor volume was calculated as ½ × length × width2 to approximate an ellipsoid volume. When the tumor volume reached 100 mm3, mice were treated with peritumoral injections of lidocaine, bupivacaine, or saline (N = 10 per group, killed at 2 time points). To ensure precise delivery of local anesthetic solutions or saline, we used a short-needle, 30-gauge, 0.3-mL insulin syringe. A total of 0.1 mL of 0.5% (w/v) lidocaine (21.3 mM), 0.125% (w/v) bupivacaine (4.3 mM), or 0.9% (w/v) saline were infiltrated to the normal tissue, abutting the tumor by 5 separate injections (0.02 mL × 5). After 24 and 48 hours, 5 mice from each group were killed, and the tumor tissues were harvested. Proteins were extracted from the tumor tissues and subjected to Western blot analysis. Tissue samples were also fixed in 10% buffered formalin, embedded in paraffin, and stained with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay.
The fluorescein-FragEL DNA fragmentation detection kit (QIA39; Calbiochem, Merck Millipore) was used to detect apoptosis in paraffin-embedded mouse tumor sections according to the manufacturer’s protocol. After deparaffinization and rehydration, the sections were incubated with 20 μg/mL proteinase K at room temperature for 20 minutes for permeabilization, followed by rinsing with substituting tris-buffered saline, and then incubated with equilibration buffer at room temperature for 30 minutes. After equilibration, the sections were incubated with TdT labeling reaction mixture at 37°C for 1.5 hours. During this labeling reaction, TdT labels the exposed 3′-OH ends of DNA fragments with fluorescein-labeled deoxynucleotides and catalyzes the addition of fluorescein-labeled and unlabeled deoxynucleotides. Finally, the sections were embedded in fluorescein-FragEL mounting medium and examined by fluorescent microscope (Olympus, IX71, Japan).
All experiments were performed at least in triplicate. When 3 separate experiments yielded consistently reproducible results, no more duplicate experiments were performed. Otherwise, more experiments were repeated until convincingly consistent results were obtained. The number of independently performed experiments was not determined based on statistical power analysis. Results are expressed as mean ± SEM. For statistical comparisons, Student t test and 1-way analysis of variance (ANOVA) followed by post hoc Dunnett tests were used. All analyses were performed using the SPSS program version 17.0 (SPSS Inc., Chicago, IL). A P value <0.01 was considered statistically significant.
Effects of Lidocaine and Bupivacaine on Cell Viability
The viability of MCF-7 and MCF-10A cells was determined after incubation with lidocaine or bupivacaine at serially diluted concentrations for 6, 24, and 48 hours. Lidocaine and bupivacaine inhibited the growth of both breast tumor cell lines in a dose- and time-dependent manner (Fig. 1A, all P < 0.001). The ED50 values of lidocaine and bupivacaine were significantly lower in MCF-7 cells than in MCF-10A cells (P < 0.001). For MCF-7 cells, the ED50 of lidocaine was 4.5 ± 0.26 mM and that of bupivacaine was 1.3 ± 0.11 mM at 24 hours.
Drug synergy was determined using the combination index (CI) and isobologram analyses according to the median effect methods. In the isobologram graph, combination data points that are on, above, and beneath the oblique line represent additive, antagonistic, and synergistic effects, respectively. As shown in Figure 1B, cotreatment with lidocaine and bupivacaine for 48 hours had a slight antagonistic effect (CI, 1.26 ± 0.12) in MCF-7 cells and a nearly additive effect (CI, 1.05 ± 0.03) in MCF-10A cells.
Induction of Apoptosis
Next, we investigated the mechanism underlying the reduced viability of cells treated with lidocaine and bupivacaine. Apoptosis is characterized by the degradation of nuclear DNA in response to various apoptotic stimuli in a wide variety of cell types. DNA gel electrophoresis revealed that treatment of MCF-7 and MCF-10A cells with lidocaine and bupivacaine for 24 hours caused oligonucleosomal DNA fragmentation in a dose-dependent fashion (Fig. 2A, all P < 0.001 except for P = 0.002 for MCF-10A cells treated with lidocaine).
During apoptosis, translocation of phosphatidylserine from the inner side of the plasma membrane to the outer leaflet is common. Based on its affinity for phosphatidylserine, annexin V can be used as a sensitive probe for cell surface changes. MCF-7 and MCF-10A cells were treated with lidocaine or bupivacaine for 4 hours, fixed, and detected using fluorescence microscopy. As shown in Figure 2B, the fraction of cells in an early state of apoptosis was determined by staining cells with annexin V. The mean percentage of MCF-7 cells that were apoptotic after treatment with lidocaine (7.4 mM) or bupivacaine (2.6 mM) was 74% and 81%, respectively. In the nontumorigenic cell line MCF-10A, treatment with lidocaine (7.4 mM) or bupivacaine (2.6 mM) resulted in apoptosis of 8% and 19% of the cells, respectively (P < 0.001). These results are in concordance with our cell viability data, indicating that lidocaine and bupivacaine exhibit higher cytotoxicities in MCF-7 than in MCF-10A cells.
Apoptosis is largely controlled by a family of intracellular cysteine proteases known as caspases. Caspases can be grouped into initiators (caspase 2, 8, 9, and 10) and effectors (caspase 3, 6, and 7). PARP, an enzyme involved in DNA damage and repair, is cleaved by caspase 3 and caspase 7 during apoptosis. This cleavage inactivates PARP and contributes to a cell’s commitment to undergo apoptosis. Previous studies have shown that MCF-7 cells do not express caspase 3 or caspase 10.10,11 As such, caspase 7 might compensate for the lack of caspase 3, while caspase-3-deficient MCF-7 cells are still sensitive to apoptotic cell death. As shown in Figure 3A, lidocaine and bupivacaine induced proteolytic activation of caspase 7 in a dose- and time-dependent manner (P < 0.001). Similarly, increased PARP cleavage was observed in MCF-7 cells treated with lidocaine and bupivacaine (Fig. 3B, P < 0.001).Approximately 2 to 6 hours after lidocaine treatment of 4.5 mM and bupivacaine treatment of 1.3 mM showed significantly increased cleavage of caspase 7 and PARP compared with the control group.
Caspases are activated by 2 major signaling routes, the extrinsic death receptor and the intrinsic mitochondrial pathway. The binding of death receptor ligands to their respective receptors activate the initiator caspase 8, while the intrinsic (mitochondrial) pathway is mediated by the release of apoptogenic proteins which result in the activation of caspase 9. In MCF-7 cells treated with lidocaine and bupivacaine, both caspase 8 and caspase 9 were cleaved and activated about 6 hours after treatment (Fig. 3C, P = 0.006 and 0.009 for caspase 8, P < 0.001 for caspase 9). These results suggest that apoptosis induced by local anesthetics involves both the extrinsic and intrinsic pathways.
Effects of Caspase Inhibitors
To further confirm the involvement of the extrinsic and intrinsic pathways in this process, MCF-7 cells were pretreated with a caspase-8 inhibitor, caspase-9 inhibitor, or a pan-caspase inhibitor before incubation with lidocaine or bupivacaine for 24 hours. All caspase inhibitors reduced the proteolytic activation of caspase 7 (Fig. 4A, P < 0.001) and the downstream cleavage of PARP (Fig. 4B, P < 0.001) induced by lidocaine and bupivacaine. Collectively, these data indicate that local anesthetics induce apoptosis in MCF-7 cells through activation of the caspase-dependent extrinsic and intrinsic apoptosis pathways.
Effects in Xenograft Tumors
The effects of lidocaine and bupivacaine were evaluated in a xenograft model to determine whether local anesthetics could induce apoptosis in vivo. Clinical concentrations of lidocaine (21.3 mM) and bupivacaine (4.3 mM) were infiltrated around xenograft tumors. The tumors that were treated with local anesthetics had higher expression of cleaved caspase 7 than did those treated with saline (Fig. 5A, P = 0.003 at 24 hours and P = 0.008 at 48 hours). Identification of apoptotic cells by DNA fragmentation assays revealed the presence of a multitude of DNA strand breaks in treated tumor cells (Fig. 5B). These findings indicate that lidocaine and bupivacaine also induce apoptosis of breast tumor cells in vivo.
Our data demonstrate that lidocaine and bupivacaine induce apoptosis of breast tumor cells at clinically relevant concentrations. It is noteworthy that the apoptotic action of local anesthetics is more pronounced in malignant breast cancer cells than in mammary epithelial cells. Furthermore, our results suggest that the in vitro apoptotic effects of local anesthetics are reproducible in vivo.
Local anesthetics may directly or indirectly influence the oncologic outcomes of breast cancer. The direct effects of local anesthetics on tumor cells include induction of apoptosis,12 inhibition of invasion,3 and suppression of metastatic efficiency.13,14 Indirect actions of local anesthesia include attenuation of the neuroendocrine response to surgery, followed by improved preservation of immunocompetence.15 Moreover, local anesthetics can render tumor cells more sensitive to the effects of chemotherapy16 and systemic hyperthermia.17,18 In breast tumor cells, we found that local anesthetics induce apoptosis more effectively in breast cancer MCF-7 cells than in nontumorigenic mammary epithelial MCF-10A cells. These results are encouraging from a clinical perspective, because a lower level of cytotoxicity toward normal breast epithelial cells may minimize collateral damage during treatment.
The mechanisms of cytotoxicity appear to be unrelated to the primary action of local anesthetics.19 Lidocaine has been shown to directly inhibit the tyrosine kinase activity of epidermal growth factor receptor (EGFR) in corneal epithelial cells20 and in human tongue cancer cells.21 A previous study reported that MCF-7 cells have higher levels of cell surface and cytoplasmic EGFR expression than do MCF-10A cells.22 Therefore, 1 plausible explanation for our results is that apoptosis induced by local anesthetics is associated with inhibition of EGFR tyrosine kinase activity. Another potential mechanism whereby local anesthetics may influence tumor growth is by interaction with the tumor epigenome.23 Genome stability and normal gene expression are maintained by a fixed and predetermined pattern of DNA methylation. Increased methylation frequently leads to downregulation of tumor suppressor genes, favoring tumor progression.23 Procaine, the prototypic ester-type local anesthetic, has been shown to demethylate DNA and inhibit tumor growth in breast cancer cells.24 Similar results have been obtained for lidocaine, the prototypic amide-type local anesthetic.25
Caspase 8, an initiator capsase, is the key mediator of the extrinsic pathway.26 This pathway involves the activation of caspase 8 through dimerization and autoproteolytic cleavage; the activated protein then processes the downstream effector caspase 7, which subsequently cleaves specific substrates, resulting in cell death. Activated caspase 9 also initiates a caspase cascade involving the downstream effector caspase 7, ultimately resulting in cell death. In the present study, we found that caspases 8 and 9 both mediate apoptotic signal pathways, leading to apoptosis of breast tumor cells treated with local anesthetics. In addition, a slight antagonism was observed with a combination of lidocaine and bupivacaine. It is therefore likely that lidocaine and bupivacaine share a similar mechanism of apoptosis-inducing activity. These findings are reminders that our understanding of the mechanisms underlying local anesthetic function is far from complete.
Despite concerns that surgery may inhibit host defenses and facilitate the development of metastases,15 surgical tumor removal remains a highly relevant treatment option for cancer patients. Surgical trauma, stress, anesthetics, and other drugs can interact with the cellular immune system and affect long-term outcomes. Regional anesthetic procedures and IV administration of local anesthetics have both been shown to reduce perioperative surgical stress.27,28 Experimental studies in murine models of breast cancer have shown that regional anesthesia may also reduce the metastatic burden.29,30 Apart from the use of lidocaine as a regional anesthetic, its administration via IV infusion during the perioperative period is increasing. Perioperative lidocaine infusion reduces postoperative pain, decreases the need for opioids, and reduces nausea/vomiting.31 Our results suggest that the apoptotic effects of local anesthetics deserve further clinical evaluation for use in breast cancer surgery. The peripheral continuous infiltration of local anesthetics or IV infusion of lidocaine is an effective analgesic technique6,32 that, because of its simplicity, may prove to be an important instrument in our armamentarium for breast cancer surgery.
In summary, this study is the first to find that clinically relevant concentrations of lidocaine and bupivacaine effectively induce apoptosis of human breast tumor cells. Local anesthetics are more cytotoxic for malignant than nonmalignant cells, offering a compelling rationale for exploiting these drugs in breast cancer surgery. Emerging evidence indicating a relationship between local failure and outcome in breast cancer emphasizes the importance of the surgeon’s role in reducing local recurrence and systemic failure after surgery. Administration of local anesthetics at the wound site is a rational approach to reducing the afferent nociceptive barrage. Our results demonstrate previously unrecognized benefits of local anesthetics and suggest that lidocaine and bupivacaine might be ideal infiltration anesthetics for breast cancer surgery.
Name: Yuan-Ching Chang, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Yuan-Ching Chang has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Chien-Liang Liu, MD.
Contribution: This author helped design the study and analyze the data.
Attestation: Chien-Liang Liu, has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Ming-Jen Chen, MD, PhD.
Contribution: This author helped design the study.
Attestation: Ming-Jen Chen has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Yung-Wei Hsu, MD.
Contribution: This author helped design the study and conduct the study.
Attestation: Yung-Wei Hsu has seen the original study data and approved the final manuscript.
Name: Shan-Na Chen, BSc.
Contribution: This author helped conduct the study.
Attestation: Shan-Na Chen has seen the original study data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Chi-Hsin Lin, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Chi-Hsin Lin has seen the original study data and approved the final manuscript.
Name: Chin-Man Chen, MS.
Contribution: This author helped conduct the study.
Attestation: Chin-Man Chen has seen the original study data and approved the final manuscript.
Name: Feng-Ming Yang, PhD.
Contribution: This author helped conduct the study.
Attestation: Feng-Ming Yang has seen the original study data and approved the final manuscript.
Name: Meng-Chun Hu, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Meng-Chun Hu has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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