Abstract

   Local anesthetics are frequently used in fine-needle aspiration of
   thyroid lesions and locoregional control of persistent or recurrent
   thyroid cancer. Recent evidence suggests that local anesthetics have a
   broad spectrum of effects including inhibition of cell proliferation
   and induction of apoptosis in neuronal and other types of cells. In
   this study, we demonstrated that treatment with lidocaine and
   bupivacaine resulted in decreased cell viability and colony formation
   of both 8505C and K1 cells in a dose-dependent manner. Lidocaine and
   bupivacaine induced apoptosis, and necrosis in high concentrations, as
   determined by flow cytometry. Lidocaine and bupivacaine caused
   disruption of mitochondrial membrane potential and release of
   cytochrome c, accompanied by activation of caspase 3 and 7, PARP
   cleavage, and induction of a higher ratio of Bax/Bcl-2. Based on
   microarray and pathway analysis, apoptosis is the prominent
   transcriptional change common to lidocaine and bupivacaine treatment.
   Furthermore, lidocaine and bupivacaine attenuated extracellular
   signal-regulated kinase 1/2 (ERK1/2) activity and induced activation of
   p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal
   kinase. Pharmacological inhibitors of MAPK/ERK kinase and p38 MAPK
   suppressed caspase 3 activation and PARP cleavage. Taken together, our
   results for the first time demonstrate the cytotoxic effects of local
   anesthetics on thyroid cancer cells and implicate the MAPK pathways as
   an important mechanism. Our findings have potential clinical relevance
   in that the use of local anesthetics may confer previously unrecognized
   benefits in the management of patients with thyroid cancer.

Introduction

   Thyroid cancer is the most common of all endocrine cancers, and the
   incidence of thyroid cancer is increasing worldwide [33][1], [34][2].
   The majority of thyroid cancers are well differentiated with papillary
   and follicular thyroid carcinoma being the most common types. Although
   well-differentiated thyroid cancer has a generally favorable prognosis,
   the overall recurrence rates could be as high as 35% [35][3].
   Persistent or recurrent disease occurs largely in the neck. Moreover,
   development of recurrence is associated with a higher mortality.
   Thirty-year cancer mortality rates were reported to be about 12% in
   patients with local recurrence and 43% in those with distant recurrence
   [36][3].

   The treatment options for persistent or recurrent disease include
   additional surgery and radioactive iodine therapy. Compartmental neck
   dissection for locoregional recurrence is recommended by the American
   Thyroid Association guidelines [37][4]. Another alternative treatment
   with promising results is ultrasound-guided percutaneous ethanol
   injection and radiofrequency ablation [38][5]–[39][7]. During the
   procedure, lidocaine infiltration to the puncture site and soft tissue
   around the recurrent tumor is a simple and effective method of pain
   control [40][8]. No adverse effects were reported except for that
   ultrasound image quality may be compromised with a large amount of
   lidocaine [41][7].

   In addition to the well established action of analgesia and
   antiarrhythmia, studies have demonstrated that local anesthetics have a
   broad spectrum of effects including anti-inflammatory and antimicrobial
   properties [42][9]. Recent observations of their chondrotoxicity call
   for caution in the clinical use of intra-articular injections [43][10].
   Furthermore, there is growing evidence that local anesthetics might
   exert beneficial actions in the treatment of cancer by inhibition of
   cell proliferation, invasion, and migration [44][11]–[45][13]. These
   effects appear unrelated to their modulation of sodium channels
   [46][14]. In this regard, we have shown that apoptosis plays an
   important role in the local anesthetic-induced cell toxicity of breast
   cancer cells [47][15]. Previous studies suggest that neuronal apoptosis
   induced by local anesthetics is mediated, at least in part, by
   mitogen-activated protein kinase (MAPK) pathway [48][16], [49][17].
   However, little is known whether similar mechanisms apply to local
   anesthetic-induced cytotoxicity in cancer cells.

   Given that local anesthetics are frequently used in fine-needle
   aspiration of thyroid lesions and locoregional control of persistent or
   recurrent thyroid cancer, it would be interesting to find out whether
   local anesthetics have an antiproliferative effect on thyroid cancer
   cells. We reported here that commonly used local anesthetics, lidocaine
   and bupivacaine, inhibited cell growth and induced apoptosis of human
   thyroid cancer cells in clinically relevant concentrations.
   Furthermore, in agreement with previous studies and our microarray and
   pathway analysis, we observed that MAPK signaling pathways were
   involved.

Results

Inhibition of 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
   ([50]Fig. 1A). 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).

Figure 1. Effects of local anesthetics on cell growth and colony formation of
human thyroid cancer cells.

   [51]Figure 1
   [52]Open in a new tab

   (A) 8505C and K1 cells were treated with serial dilutions of lidocaine
   and bupivacaine, individually or in combinations, for 24 and 48 h.
   Error bars represent standard error of the mean. (B) A conservative
   isobologram demonstrates that lidocaine and bupivacaine acts
   antagonistically to inhibit the cell growth of thyroid cancer cells. ED
   indicates effective dose. The ED50 (red X), ED75 (green crosses), and
   ED90 (blue circles) are graphed. (C) Treatment with lidocaine and
   bupivacaine resulted in reduction of colony formation of the 8505C and
   K1 cell lines. Error bars represent standard error of the mean. *,
   P<0.01 versus control.

   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 ([53]Fig. S1), indicating that the observed effects were
   pharmacological in nature.

   Drug synergy was determined by the combination index (CI) and
   isobologram analyses according to the median effect methods ([54]Fig.
   1B). 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 [55]Fig. 1C. 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.

Induction of 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 ([56]Fig. 2A). 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 [57]Fig. 2B, 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.

Figure 2. Effects of local anesthetics on cell cycle and apoptosis in human
thyroid cancer cells.

   [58]Figure 2
   [59]Open in a new tab

   (A) 8505C and K1 cells were treated with the indicated concentrations
   of lidocaine and bupivacaine for 24 h. Thereafter, the cells were
   washed, fixed, and stained with propidium iodide (PI) and were analyzed
   for DNA content in different phases of the cell cycle by flow
   cytometry. (B) Thyroid cancer cells were treated with lidocaine and
   bupivacaine for 48 h, and cells were subsequently stained with
   fluorescein-conjugated annexin V and PI and analyzed by flow cytometry.
   Error bars represent standard error of the mean. *, P<0.05 versus
   control.

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 ΔΨ[m]. 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 [60]Fig.
   3A, 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 ΔΨ[m] was slightly lower than the
   percentage of apoptotic cells treated with local anesthetics at the
   same concentrations.

Figure 3. Change in mitochondrial membrane potential (ΔΨ[m]) and cytochrome c
release induced by local anesthetics in human thyroid cancer cells.

   [61]Figure 3
   [62]Open in a new tab

   (A) 8505C and K1 cells were treated with the indicated concentrations
   of lidocaine and bupivacaine for 16 h. ΔΨ[m] change was monitored by
   loading with JC-1 and was analyzed by flow cytometry. Error bars
   represent standard error of the mean. *, P<0.05 versus control. (B)
   Thyroid cancer cells were treated with lidocaine (6 mM) and bupivacaine
   (2 mM) for the indicated periods of time. Cytochrome c release from
   mitochondria to cytosol was determined by Western blotting. The blots
   were stripped and reprobed with an antibody against actin for equal
   loading.

   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 ([63]Fig. 3B). 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 ([64]Fig. 4A).
   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 ([65]Fig. 4B). These results
   clearly indicate that caspase activation plays an important role in
   thyroid cancer cell apoptosis induced by local anesthetics.

Figure 4. Expression of apoptotic proteins and caspase 3 activities in human
thyroid cancer cells treated with local anesthetics.

   [66]Figure 4
   [67]Open in a new tab

   (A) 8505C and K1 cells were treated with the indicated concentrations
   of lidocaine (L6, 6 mM; L12, 12 mM) and bupivacaine (B2, 2 mM; B4, 4
   mM) for 16 h. Cells were harvested and samples were prepared for
   Western blot analysis. C, control. PARP, poly(ADP-ribose) polymerase.
   (B) Activity of caspase 3 in cell lysates from 8505C cells treated with
   lidocaine (12 mM) and bupivacaine (4 mM) for different time periods was
   determined using a colorimetric protease assay. Error bars represent
   standard error of the mean.

   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 ([68]Fig. 4A). 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
   [69]Tables 1 and [70]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 pathways reportedly involving molecular mechanism of cancer.
   Integrating microarray data from cells treated with lidocaine and
   bupivacaine, pathway analysis suggested that extracellular
   signal-regulated kinase (ERK) 1/2 and p38 MAPK are significantly up- or
   down-regulated in response to treatment with local anesthetics
   ([71]Fig. S2).

Table 1. Top ten pathways identified by pathway enrichment analysis of
differentially expressed genes of 8505C human thyroid cancer cells following
treatment with lidocaine (12 mM) for 24 hours.

   Pathway P value FDR
   Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling 8.467E-10
   5.92707E-07
   Apoptosis and survival_FAS signaling cascades 5.344E-08 1.87052E-05
   Regulation of lipid metabolism_RXR-dependent regulation of lipid
   metabolism via PPAR, RAR and VDR 1.674E-07 3.90509E-05
   Blood coagulation_Blood coagulation 1.958E-07 0.000129219
   Development_Hedgehog signaling 7.555E-07 0.000132204
   Development_Role of HDAC and calcium/calmodulin-dependent kinase (CaMK)
   in control of skeletal myogenesis 1.032E-06 0.000144549
   Development_G-CSF-induced myeloid differentiation 1.316E-06 0.00043431
   Neurophysiological process_Receptor-mediated axon growth repulsion
   2.335E-06 0.00024149
   Signal transduction_cAMP signaling 2.415E-06 0.00024149
   Immune response_HMGB1/RAGE signaling pathway 2.982E-06 0.000252541
   [72]Open in a new tab

   FDR, false discovery rate.

Table 2. Top ten pathways identified by pathway enrichment analysis of
differentially expressed genes of 8505C human thyroid cancer cells following
treatment with bupivacaine (4 mM) for 24 hours.

   Pathway P value FDR
   Protein folding and maturation_POMC processing 1.843E-10 1.2825E-07
   Immune response_IL-17 signaling pathways 2.347E-07 0.000101697
   Development_G-CSF-induced myeloid differentiation 3.077E-07 0.000101697
   Protein folding and maturation_Bradykinin/Kallidin maturation 7.610E-07
   0.000167678
   DNA damage_Role of Brca1 and Brca2 in DNA repair 1.150E-06 0.000400323
   Immune response_IFN alpha/beta signaling pathway 1.874E-06 0.000434714
   Apoptosis and survival_FAS signaling cascades 3.884E-06 0.000675776
   Immune response_MIF-mediated glucocorticoid regulation 4.375E-06
   0.000722955
   Protein folding and maturation_Angiotensin system maturation\Human
   version 6.366E-06 0.000841555
   Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling 8.117E-06
   0.000953231
   [73]Open in a new tab

   FDR, false discovery rate.

Modulation of mitogen-activated protein kinase signaling

   Our microarray and pathway enrichment analysis indicates that MAPK
   pathways are probably involved in the mechanisms mediating action of
   local anesthetics on thyroid cancer cells. To examine this possible
   relationship, total cellular proteins were extracted from 8505C and K1
   cells treated with lidocaine (12 mM) and bupivacaine (4 mM) for various
   time periods, and lysates were immunoblotted with specific antibodies
   against phosphorylated and total ERK1/2, p38, and c-Jun N-terminal
   kinase (JNK), respectively. As shown in [74]Fig. 5, a decreased
   phosphorylation of ERK1/2 was observed. Lidocaine and bupivacaine
   significantly increased phosphorylation of p38 and JNK. Lidocaine and
   bupivacaine had no remarkable effect on total p38 and JNK protein
   level.

Figure 5. Mitogen-activated protein kinase signaling in human thyroid cancer
cells treated with local anesthetics.

   [75]Figure 5
   [76]Open in a new tab

   8505C (A) and K1 (B) cells were treated with lidocaine (12 mM) and
   bupivacaine (4 mM) for various periods of time, and activities of ERK,
   p38, and JNK were examined by Western blot analysis using
   phospho-specific antibodies. The total protein levels of ERK, p38, and
   JNK were also measured.

Effects of inhibition of mitogen-activated protein kinases

   Subsequently, we would like to know whether the modulations of ERK1/2,
   p38 MAPK, and JNK signaling pathways account for apoptosis induced by
   local anesthetics. To this end, we examined the effects of specific
   inhibitors on caspase activation and PARP cleavage. PD98059 is an
   inhibitor of MAPK/ERK kinase (MEK), thereby inhibiting the
   phosphorylation and the activation of MAPK. SB203580 is a selective,
   reversible, and ATP-competitive inhibitor of p38 MAPK, whereas SP600125
   is an anthrapyrazolone JNK inhibitor that competes with ATP to inhibit
   the phosphorylation of c-Jun. As shown in [77]Figs. 6A and 6B, PD98059
   and SB203580, but not SP600125, reduced the activation of caspase 3 and
   cleavage of PARP. These results were confirmed by caspase 3 activity
   assay ([78]Fig. 6C). Cotreatment with PD98059 or SB203580 suppressed
   caspase 3 activity, whereas SP600125 paradoxically increased caspase 3
   activity following treatment with local anesthetics. These data are
   accordant with the results of pathway enrichment analysis ([79]Fig. S2)
   that ERK1/2 and p38 MAPK signaling pathways are involved in apoptosis
   induced by local anesthetics.

Figure 6. Effects of inhibition of mitogen-activated protein kinase signaling
on apoptosis in human thyroid cancer cells treated with local anesthetics.

   [80]Figure 6
   [81]Open in a new tab

   8505C (A) and K1 (B) cells were cotreated with 30 μM PD98059 (PD), 30
   μM SB203580 (SB), or 30 μM SP600125 (SP) in addition to 12 mM lidocaine
   (L) or 4 mM bupivacaine (B) for 16 h. Cells were harvested and samples
   were prepared for Western blot analysis. C, control. C3, caspase 3.
   PARP, poly(ADP-ribose) polymerase. (C) Activity of caspase 3 in cell
   lysates from 8505C cells treated with lidocaine (12 mM) and bupivacaine
   (4 mM) with or without specific inhibitors for 24 h was determined
   using a colorimetric protease assay. Error bars represent standard
   error of the mean. *, P<0.01 versus control.

Discussion

   It has been more than a century since the first synthetic local
   anesthetic was introduced in the early 1900s. Local anesthetics bind
   reversibly to a specific receptor site within the sodium channels and
   block ion movement. It was believed that the effects are reversible
   with recovery of nerve function and without damage to neuronal cells.
   However, reports of permanent neurologic injury have generated concern
   about the neurotoxicity of local anesthetics [82][18]. Accumulating
   evidence suggests that local anesthetics can cause rapid neuronal death
   through triggering apoptosis and necrosis [83][19], [84][20]. Moreover,
   a number of studies have reported local anesthetic toxicity on other
   cell types [85][21]–[86][23]. In the present study, we firstly
   demonstrated the direct effect of lidocaine and bupivacaine inhibiting
   cell growth and colony formation of thyroid cancer cells.

   The mechanisms of cell toxicity from local anesthetics have not been
   fully elucidated. In U937 histiocytic lymphoma cells, lidocaine induced
   apoptosis at concentrations below 12 mM and induced necrosis at
   concentrations above 15 mM [87][24]. Similar observations have been
   reported by others [88][19], [89][20], [90][25]. In this study, both
   apoptosis and necrosis participate in cell death induced by local
   anesthetics. Intriguingly, a slight antagonism was identified with
   combination of lidocaine and bupivacaine. It seems likely that
   lidocaine and bupivacaine induce cell death by a similar mechanism.
   When a cell is unable to die by apoptosis, it may undergo necrotic cell
   death. It is well known that ATP levels are a determinant of
   manifestation of cell death [91][26], [92][27]. Lidocaine and
   bupivacaine were shown to decrease ATP levels and viability of melanoma
   cells [93][28]. The mode of cell death likely depends on the duration
   of exposure and concentrations of local anesthetics.

   Mitochondrial energetics affected by local anesthetics may account for
   the decrease in the ATP levels. Local anesthetics can reach
   mitochondria and this effect is highly dependent on the
   lipid-solubility of the local anesthetic [94][29]. Clinical and
   experimental myopathy induced by bupivacaine has been reported to
   result from mitochondrial dysfunction and reduced oxidative energy
   metabolism [95][30], [96][31]. Specifically, bupivacaine suppressed
   cell respiration through inhibition of complexes I and III, accompanied
   with production of reactive oxygen species [97][32]. Irwin et al. have
   shown that bupivacaine myotoxicity relied on active oxidative
   metabolism because highly glycolytic extensor digitorum longus were
   resistant to bupivacaine-induced toxicity [98][30]. Nonetheless, tumor
   cells predominantly produce energy through glycolysis (Warburg effect)
   [99][33]. Our findings are in agreement with those of a few reports
   demonstrating that mitochondrial dysfunction could also be elicited by
   local anesthetics in malignant tumor cells [100][24], [101][25],
   [102][34].

   Mitochondria are essential modulators of apoptosis, necrosis and
   autophagy [103][27]. The intrinsic (mitochondrial) pathway of apoptosis
   is mediated by the release of pro-, and anti-apoptotic proteins,
   including cytochrome c. In our experiments, local anesthetics increased
   pro-apoptotic Bax expression and downregulated Bcl-2 expression,
   leading to a higher ratio between pro-apoptotic and anti-apoptotic
   Bcl-2 family proteins. That increased Bax/Bcl-2 ratio facilitates the
   release of mitochondrial cytochrome c, which subsequently induces
   activations of caspase cascade that cleave PARP into inactive
   fragments. However, lidocaine and bupivacaine promoted mitochondrial
   injury to an extent less than that of apoptosis induced by the same
   concentrations of the local anesthetic. This suggests that a
   mitochondria-independent process may also play a role. This hypothesis
   is supported by the results of pathway enrichment analysis ([104]Fig.
   S2), in which transcriptional alterations in the death receptor pathway
   components were involved.

   Cellular microarrays are powerful experimental tools for identification
   of novel drug targets and pharmacogenomics. Unami and colleagues
   firstly reported that apoptosis-related genes were transcriptionally
   regulated by bupivacaine according to microarray analysis [105][34].
   Recently, Lucchinetti et al. demonstrated that multiple transcriptional
   programs related to cell differentiation, tumorigenesis, and metastasis
   were negatively affected by ropivacaine [106][35]. The authors
   postulate that this means novel rationales for the perioperative use of
   local anesthetics in patients with cancer. Our data are in concordance
   with these results and showed a strong association between local
   anesthetics-induced alterations in gene expression and apoptosis.
   Furthermore, we identified that MAPK pathway is involved in the
   molecular mechanisms of apoptosis induced by local anesthetics.

   In primary neuron cultures, bupivacaine and ropivacaine were found to
   activate p38 MAPK and JNK but not ERK1/2 [107][16]. Furthermore,
   pharmacological inhibition of these kinases attenuates neurotoxicity in
   vitro. The authors noted that axonal degeneration induced by lidocaine
   is prevented by p38 MAPK inhibitor but not by caspase inhibition
   [108][36]. Harato et al. also demonstrated that p38 MAPK inhibitor
   treatment attenuated the caspase 3 activity and cell death induced by
   bupivacaine [109][37]. In another study, p38 MAPK pathway is implicated
   in bupivacaine-induced apoptosis in human neuroblastoma SH-SY5Y cells
   [110][17]. In line with the findings in neuronal apoptosis, we found
   that p38 MAPK activation plays a crucial role in local
   anesthetics-induced apoptosis in thyroid cancer cells. Negative
   regulation of proliferation is a highly conserved and important
   function of p38α in various cell types [111][38]. Interestingly,
   upregulated p38α activity has been reported in papillary and follicular
   thyroid cancer [112][39]. The notion is supported by the basal
   expression of phospho-p38 proteins in this study ([113]Fig. 5). The
   dual role for p38 MAPK in thyroid cancer biology remains a virtually
   untapped area in spite of Ras-Raf-ERK pathway having been extensively
   studied in thyroid cancer.

   In the present study, ERK1/2 activity was suppressed by lidocaine and
   bupivacaine. Joo et al. demonstrated that lidocaine significantly
   suppressed the increased ERK1/2 response in a rat model of neuropathic
   pain [114][40]. More recently, lithium was shown to protect against
   bupivacaine-induced neuronal injury through reversing the suppression
   of ERK1/2 signaling [115][41]. There is increasing evidence for a
   crosstalk between MAPK pathways. For example, p38 and JNK pathway
   activation in the induction of apoptosis may negatively regulate the
   ERK pathway [116][42]. It is also worth noting that there is a
   paradoxical decrease in caspase activation following PD98059 treatment.
   Many genetic alterations have been implicated in thyroid cancer, and
   the aberrant activation of the Ras-Raf-ERK pathway is most frequent
   [117][43]. Inhibition of the MAPK pathway arrests thyroid cancer cells
   in G1 phase [118][44]. Therefore, a plausible explanation for our
   results is that ERK1/2 inhibition increased susceptibility of thyroid
   cancer cells to other mechanisms of growth inhibition like cell cycle
   arrest and necrosis, which do not involve caspase activation.

Conclusions

   In summary, our study deciphered the molecular mechanisms responsible
   for the cytotoxicity induced by local anesthetics in thyroid cancer
   cells, presenting the involvement of MPAK signaling pathways and
   suggesting potential benefits of the use of local anesthetics in
   clinical practice.

Materials and Methods

Cell culture and reagents

   The human thyroid carcinoma cell lines 8505C and K1 were purchased from
   the German Collection of Microorganisms and Cell Cultures (DSMZ,
   Braunschweig, Germany) and the European Collection of Cell Cultures
   (ECACC, Salisbury, United Kingdom), respectively. 8505C cells were
   cultured at 37°C in a humidified atmosphere of 5% CO[2] in RPMI 1640
   medium (Invitrogen/Gibco, Carlsbad, CA) supplemented with 10% fetal
   bovine serum (FBS). K1 cells were cultured in Dulbecco's modified
   Eagle's medium (Gibco) mixed with Ham's F12 (Gibco) and MCDB 105
   (Sigma, St. Louis, MO) medium in 2∶1∶1 proportions, supplemented with
   10% FBS and 2 mM L-glutamine. Both 8505C and K1 have been authenticated
   to be unique thyroid cancer cell lines [119][45]. Lidocaine,
   bupivacaine, procaine, PD98059, SB203580, and SP600125 were obtained
   from Sigma. Osmolality of the culture media for experiments was
   analyzed by the freezing point depression method using an Advanced 3320
   Micro Osmometer (Advanced Instruments, Norwood, MA).

Cell viability assay and drug combination analysis

   Cell viability assay was performed in triplicate for each experiment as
   previously described [120][46]. Briefly, 8505C and K1 cells were seeded
   into 96-well plates one day before lidocaine and bupivacaine
   (individually or in combinations) was added at the indicated
   concentrations. For the thiazolyl blue tetrazolium bromide (MTT)
   colorimetric assay, 100 µl of MTT reagent (5 mg/ml; Sigma) was added to
   the cell culture and cells were incubated at 37°C for 4 h. The formazan
   crystals converted from tetrazolium salts by viable cells were
   solubilized with acidified isopropanol. The absorbance was measured
   with Varioskan Flash (Thermo Fisher Scientific, Waltham, MA) at
   wavelength of 570 nm. The absorbance of control cells (incubated
   without drugs) was defined as 100%.

   Drug synergy was determined by the CI and isobologram analyses
   according to the median effect methods described by Chou and Talalay
   [121][47]. On the basis of the dose-response curves from MTT assays,
   the CI values were calculated. Synergism, additivity and antagonism are
   defined as CI <1, CI  = 1 and CI >1, respectively. In the isobologram
   graph, combination data points that fall on, above, and beneath the
   oblique line represent an additive, antagonistic, and synergistic
   effect, respectively.

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 [122][48]. 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 (1×10^6) 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 FASCalibur flow
   cytometer (BD Biosciences) equipped with CellQuest 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.

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.

Determination of mitochondrial membrane potential (ΔΨ[m])

   Mitochondrial membrane potential was determined by flow cytometry using
   the ΔΨ[m]-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 ΔΨ[m]. Healthy cells with high ΔΨ[m] will
   form JC-1 aggregates and fluoresce red, whereas those apoptotic cells
   with low ΔΨ[m] 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 analysis

   Total cellular proteins were extracted, quantified, and subjected to
   gel electrophoresis according to standard procedures as we described
   previously [123][49]. 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), anti-phospho-p38 (#4511), anti-p38 (#9212),
   anti-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 (2×10^7) 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×g for 10 min at 4°C. The
   cytosolic fraction was obtained through centrifugation again at
   12,000×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, 5×10^6 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 (12×135K) 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).

Statistical analysis

   Results 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.

Supporting Information

   Figure S1

   Final pH and osmolality of the culture media for experiments.

   (TIF)
   [124]Click here for additional data file.^ (513.3KB, tif)
   Figure S2

   Molecular mechanism of cancer from Ingenuity Pathways Analysis.

   (TIF)
   [125]Click here for additional data file.^ (4.9MB, tif)

Funding Statement

   This study was supported by grants from National Science Council of
   Taiwan (NSC 100-2314-B-195-001-MY3), and from Mackay Memorial Hospital
   (MMH-10206 and MMH-E-101-10). The funders had no role in study design,
   data collection and analysis, decision to publish, or preparation of
   the manuscript.

References