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 min at room temperature in the dark. Cells were then fixed with 2% formaldehyde in binding buffer for 5 min. 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.
Analysis of cell apoptosis
Cell apoptosis was analyzed using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences Pharmingen, San Diego, CA) according to the instructions of the manufacturer. Thyroid cancer cells were treated with the indicated concentrations of lidocaine and bupivacaine for 24 to 72 h. Both floating and attached cells were harvested and washed, followed by incubation with 5 μl of annexin V-FITC and 5 μl of PI for 15 min at room temperature in the dark. After the incubation, 400 μl of binding buffer solution was added, and flow cytometry analysis was performed.
Colony formation assay
For colony formation assay, 400 cells/well were seeded into six-well plates, allowed to adhere for 24 h, and treated with the indicated concentrations of lidocaine and bupivacaine from day 2. After 8 to 15 days, colonies were stained with 3% crystal violet and colonies containing >50 cells were counted.
Cell cycle analysis
The effect of lidocaine and bupivacaine on the cell cycle was analyzed by PI staining and flow cytometry [77]. After treatment with lidocaine and bupivacaine for 6, 24, and 48 h, thyroid cancer cells were harvested, gently washed, and fixed in 70% cold ethanol at 4 °C overnight. The fixed cells (1x106) were incubated with RNase A for 30
min at room temperature, and stained with PI solution using the BD Cycletest Plus DNA reagent kit (BD Biosciences, San Jose, CA) in the dark. Subsequently, the cells were analyzed on a FAS Calibur flow cytometer (BD Biosciences) equipped with Cell Quest Pro software. The distribution of cells in G0/G1, S and G2/M phases of cell cycle was estimated using the ModFit LT software (Verity Software House, Topsham, ME). As an estimate of the proportion of apoptotic cells, the percentage of hypodiploid cells was calculated in the DNA histogram.
Determination of mitochondrial membrane potential (ΔΨm)
Mitochondrial membrane potential was determined by flow cytometry using the ΔΨm-dependent fluorescent dye JC-1 (CS0390; Sigma). JC-1 is a lipophilic, cationic dye that that can selectively enter into mitochondria, and undergoes a reversible change in fluorescence emission according to the ΔΨm. Healthy cells with high ΔΨm will form JC-1 aggregates and fluoresce red, whereas those apoptotic cells with low ΔΨm will contain monomeric JC-1 and fluoresce green. After treatment with lidocaine and bupivacaine for the indicated time, thyroid cancer cells were harvested and incubated with JC-1 for 20 min at 37 °C according to the manufacturer's instructions. The samples were then subject to flow cytometry.
Western blot assay
Total breast cellular 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 10-15% SDS-PAGE and then transferred onto a nitrocellulase membrane (Amersham Biosciences/GE Healthcare, Piscataway, NJ). Membranes were blocked in 5% (w/v) non-fat milk and immunblotted 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).
Total thyroid cellular proteins were extracted, quantified, and subjected to gel electrophoresis according to standard procedures as we described previously [78]. The antibodies used in our study, including anti-caspase 3 (#9662), anti-cleaved caspase 3 (#9661), anti-caspase 7 (#9494), anti-poly(ADP-ribose) polymerase (PARP) (P248;
Sigma), anti-Bax (#2774), anti-Bcl-2 (#2872), anti-phospho-ERK (#9101), anti-ERK (#9102), phospho-p38 (#4511), p38 (#9212), phospho-JNK (#9255), anti-JNK (#9252), and anti-actin (A5441; Sigma). Antibodies were obtained from Cell Signaling, Danvers, MA unless specified otherwise. The antigen-antibody complexes were visualized with by chemiluminescence with the Amersham ECL detection system (GE Healthcare, Piscataway, NJ).
Measurement of cytochrome c release
To determine the release of cytochrome c from mitochondria to cytoplasm, preparations of cytosolic extracts were carried out with the Mitochondria Isolation Kit (#89874; Thermo Scientific/Pierce, Rockford, IL) according to the manufacturer's instructions. In brief, thyroid cancer cells (2x107) were harvested after treatment with lidocaine and bupivacaine for the indicated time periods, washed, and incubated with cytosol extraction buffer in ice for 10 min. The supernatant was collected through
centrifugation at 700 x g for 10 min at 4 °C. The cytosolic fraction was obtained through centrifugation again at 12,000 x g for 15 min at 4 °C, and was analyzed by Western blotting using anti-cytochrome c antibody (#4272; Cell Signaling) as described above.
Assay for caspase 3 activity
The caspase 3 activity was assayed using the ApoTarget Caspase-3 Colorimetric Protease Assay Kit (KHZ0022; Invitrogen) according to the manufacturer's protocol.
Briefly, 5 x 106 thyroid cancer cells were treated with lidocaine and bupivacaine with or without specific inhibitors for the indicated time periods. The cells were harvested using trypsinization and cell lysates prepared as described above. Samples of the cell lysates (100 μg protein per sample) were mixed with reaction buffer and 200 μM substrate (DEVD-pNA) and incubated for 2 hours at 37 °C in the dark. The absorbance was then measured at 405 nm and the sample readings calculated by subtracting the absorbance of blank samples.
Global gene expression analysis
8505C cells were treated with lidocaine (12 mM), bupivacaine (4 mM), or left untreated for 24 hours. Cells were harvested and total RNA was isolated using the TRI Reagent (Sigma). After RNA integrity was verified, cDNA was synthesized using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen), subsequently labeled using the One-Color DNA Labeling Kit (Roche NimbleGen, Madison, WI), and hybridized to a Human HG18 expression array (12x135K) using the NimbleGen Hybridization System. Arrays were scanned and chip images were collected on a NimbleGen MS200 Microarray Scanner. Following the acquisition and initial quantification of array images, raw array data were normalized per chip and per gene and filtered based on raw signal intensity and detection call. Genes with an expression
fold change of ≥ 2 between a treatment and a control were considered to be significant.
To determine the potential mechanistic network, transcripts with differential expression were studied using the MetaCore pathway analysis suite (GeneGo-Thomson Reuters, New York, NY) and Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA).
In vivo xenograft model
The xenograft model was established and modified as previously described.9 In brief, 17-estradiol was implanted into thirty female BALB/c nude mice. Twenty-four hours later, 1×107 MCF-7 cells in 100 μL mixture of PBS 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, sacrificed at two 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 x 5). After 24 and 48 hours, five mice from each group were sacrificed, 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.
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 min for permeabilization, followed by rinsing with substituting tris-buffered saline, and then incubated with equilibration buffer at room temperature for 30 min. After equilibration, the sections were incubated with TdT labeling reaction mixture at 37 ˚C for 1.5 h. 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).
Statistical analysis
All experiments for breast tumor cells were performed at least in triplicate.
When three separate experiments yielded consistently reproducible results, no more duplicate experiments were carried out. 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 ± standard error of the mean. For statistical comparisons, Student's t-test and one-way analysis of variance (ANOVA) followed by post hoc Dunnett's tests were used. All analyses were performed using the SPSS program version 17.0 (SPSS Inc., Chicago, IL). A P value < .01 was considered statistically significant.
Results from thyroid cancer cells were expressed as means ± SEM. Comparisons between groups were performed using a two-tailed Student's t test. Values of P < 0.05 were considered significant.
R
ESULTSEffects of lidocaine and bupivacaine on breast tumor 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. 1, all P < .001). The ED50 values of lidocaine and bupivacaine were significantly lower in MCF-7 cells than in MCF-10A cells (P
< .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 fall on, above, and beneath the oblique line represent additive, antagonistic, and synergistic effects, respectively. As shown in Fig. 2, co-treatment with lidocaine and bupivacaine for 48 hours had a slight antagonistic effect (CI, 1.26 ± 0.12) in 7 cells and a nearly additive effect (CI, 1.05 ± 0.03) in MCF-10A cell
Induction of breast cancer cell apoptosis
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. 3, all P < .001 except for P = .002 for MCF-10A cells
treated with lidocaine).
During apoptosis, translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer leaflet is common. Based on its affinity for PS, annexin V can be used as a sensitive probe for cell surface changes. 7 and MCF-10A cells were treated with lidocaine or bupivacaine for 4 hours, fixed, and detected using fluorescence microscopy. As shown in Fig. 4, 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 non-tumorigenic 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 < .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.
Caspase activation induced by lidocaine and bupivacaine
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 [79, 80]. 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 Fig. 5, lidocaine and bupivacaine induced proteolytic activation of caspase 7 in a dose- and time-dependent manner (P < .001). Similarly, increased PARP cleavage was observed in MCF-7 cells
treated with lidocaine and bupivacaine (Fig. 6, P < .001). Compared with the control group, lidocaine treatment of 4.5 mM and bupivacaine treatment of 1.3 mM showed significantly increased cleavage of caspase 7 and PARP approximately 2 to 6 hours after treatment.
Caspases are activated by two 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. 7, P = .006 and .009 for caspase 8, P < .001 for caspase 9). These results suggest that apoptosis induced by local anesthetics involves both the extrinsic and intrinsic pathways.
Effects of caspase inhibitors on breast tumor cells
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. 8, P < .001) and the downstream cleavage of PARP (Fig. 9, P < .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 breast 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. 10, P = .003 at 24 h and P = .008 at 48 h). Identification of apoptotic cells by DNA fragmentation assays revealed the presence of a multitude of DNA strand breaks in treated tumor cells (Fig. 11). These findings indicate that lidocaine and bupivacaine also induce apoptosis of breast tumor cells in vivo.
Inhibition of thyroid tumor cell growth and colony formation by local anesthetics Cell viability of 8505C and K1 thyroid cancer cells was determined by incubating with lidocaine and bupivacaine at serially diluted concentrations for 24 and 48 hours. Lidocaine and bupivacaine inhibited the growth of both thyroid cancer cells in a dose-dependent manner (Fig. 12). At 24 hours, the median effective dose (ED50) of lidocaine was 7.3 mM for 8505C cells and 6.8 mM for K1 cells, respectively. These were lower than the commonly used concentration of 1% (w/v) lidocaine (42.67 mM).
Similarly, the ED50 at 24 hours of bupivacaine was 3.1 mM for 8505C cells and 1.3 mM for K1 cells. Both were much lower than the clinical concentration of 0.5% (w/v) bupivacaine (17.34 mM).
Drug synergy was determined by the combination index (CI) and isobologram analyses according to the median effect methods (Fig. 13). The CI was 1.17 ± 0.03 for 8505C cells and 1.14 ± 0.06 for K1 cells. These results suggest that combination of lidocaine and bupivacaine yielded slight antagonism.
Furthermore, the effects of local anesthetics on tumor cell growth were determined by clonogenic assay. The results are shown in Fig. 14. A significant dose-dependent
reduction in colonies was observed in 8505C and K1 cells. Thyroid cancer cells did not form colonies following treatment with lidocaine > 4 mM or bupivacaine > 0.8 mM.
To examine whether the cytotoxic effect is restricted to amide-type local anesthetics, thyroid cancer cells were also treated with an ester-type local anesthetics, procaine. The ED50 was 39.6 mM and 30.9 mM, respectively, suggesting that the effect correlated with the potency, but not the class, of local anesthetics. Furthermore, to exclude the possibility that changes in pH or osmolality would account for the cytotoxic effects, pH and osmolality of the culture media for experiments were determined. There were no significant variations in pH or osmolality (Fig. 15), indicating that the observed effects were pharmacological in nature.
Induction of thyroid cancer cell apoptosis by local anesthetics
To determine the basis of reduced cell viability, cell cycle analysis was performed. There was no cell cycle arrest in thyroid cancer cells treated with lidocaine and bupivacaine for 6, 24, and 48 hours (data not shown). However, we observed a dose-dependent increase in the sub-G1 (hypodiploid) fraction following treatment with lidocaine and bupivacaine (Fig. 16). We also used annexin V/propidium iodide (PI) dual staining for further confirmation of local anesthetics-induced apoptosis in thyroid cancer cells. As shown in Fig. 17, treatment with lidocaine and bupivacaine resulted in apoptosis in a dose-dependent manner. Necrosis was also observed in high concentrations of lidocaine and bupivacaine. Taken together, these results suggest that growth suppression by local anesthetics in thyroid cancer cells involves induction of apoptosis.
Assessment of mitochondrial dysfunction
A decrease in mitochondrial membrane potential is one of the earliest events in
apoptosis. Mitochondrial membrane integrity was evaluated using the cationic dye JC-1, a highly specific probe for detecting changes in mitochondrial ΔΨm. JC-1 forms red aggregates in intact mitochondria, while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential. As shown in Fig. 18, lidocaine and bupivacaine significantly increased the formation of JC-1 monomers in 8505C and K1 cells in a dose-dependent way. The percentage of cells with low ΔΨm was slightly lower than the percentage of apoptotic cells treated with local anesthetics at the same concentrations.
We next sought to determine whether cytochrome c was released from mitochondria to the cytosol. As expected, a higher level of cytochrome c was measured in cytosol in both cell lines after treatment with lidocaine and bupivacaine (Fig. 19).
Collectively, these data suggest that treatment of thyroid cancer cells with local anesthetics leads to activation of the mitochondrial apoptotic pathway.
Activation of caspases by local anesthetics
Release of cytochrome c has been shown to activate the downstream caspases that are ultimately required to induce apoptosis. We therefore examined whether caspase activation was involved in the induction of apoptosis by local anesthetics.
Treatment with lidocaine and bupivacaine resulted in the activation of caspase 3 and caspase 7, and cleavage of PARP in 8505C and K1 cells dose-dependently (Fig. 20).
Furthermore, caspase colorimetric substrate assay showed that caspase 3 activity increased in a time-dependent manner in 8505C cells after treatment with lidocaine and bupivacaine (Fig. 21). These results clearly indicate that caspase activation plays an important role in thyroid cancer cell apoptosis induced by local anesthetics.
The proteins of the Bcl-2 family participate in the apoptotic process by
functioning as promoters (e.g., Bax) or inhibitors (e.g., Bcl-2). To activate the mitochondrial apoptotic pathway, activated Bax forms an oligomeric pore and results in the permeabilization of the mitochondrial outer membrane. We found that treatment with lidocaine and bupivacaine lead to a reduction in the Bcl-2 levels with a concomitant increase in the Bax levels (Fig. 20). Therefore, local anesthetics alter the protein levels of key members of the Bcl-2 family in a manner that favors an increase in the ratio of Bax/Bcl-2, which may contribute to the susceptibility of thyroid cancer cells to apoptosis.
Analysis of gene expression signatures affected by local anesthetics
To identify gene expression signatures that are associated with biological functions of local anesthetics, microarray and pathway enrichment analysis was carried out to compare expression patterns in 8505C cells treated with lidocaine and bupivacaine. The top ten pathways identified by the pathway enrichment analysis are listed in Tables 1 and 2. It is noteworthy that the most prominent transcriptional change in thyroid cancer cells treated with local anesthetics is apoptosis. Other pathways common to lidocaine and bupivacaine treatment include cytoskeletal remodeling and myeloid differentiation. Furthermore, we used in silico tools from Ingenuity to identify
To identify gene expression signatures that are associated with biological functions of local anesthetics, microarray and pathway enrichment analysis was carried out to compare expression patterns in 8505C cells treated with lidocaine and bupivacaine. The top ten pathways identified by the pathway enrichment analysis are listed in Tables 1 and 2. It is noteworthy that the most prominent transcriptional change in thyroid cancer cells treated with local anesthetics is apoptosis. Other pathways common to lidocaine and bupivacaine treatment include cytoskeletal remodeling and myeloid differentiation. Furthermore, we used in silico tools from Ingenuity to identify