Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway

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.

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

(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.

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

(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.

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

(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.

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

(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.

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

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.

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

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.