Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFα-induced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase

Abstract

Background: Matrix-metalloproteinases (MMP) and cancer cell invasion are crucial for solid tumour metastasis. Important signalling events triggered by inflammatory cytokines, such as tumour necrosis factor α (TNFα), include Src-kinase-dependent activation of Akt and focal adhesion kinase (FAK) and phosphorylation of caveolin-1. Based on previous studies where we demonstrated amide-type local anaesthetics block TNFα-induced Src activation in malignant cells, we hypothesized that local anaesthetics might also inhibit the activation and/or phosphorylation of Akt, FAK and caveolin-1, thus attenuating MMP release and invasion of malignant cells.

Methods: NCI-H838 lung adenocarcinoma cells were incubated with ropivacaine or lidocaine (1 nM-100 µM) in absence/presence of TNFα (20 ng ml(-1)) for 20 min or 4 h, respectively. Activation/phosphorylation of Akt, FAK and caveolin-1 were evaluated by Western blot, and MMP-9 secretion was determined by enzyme-linked immunosorbent assay. Tumour cell migration (electrical wound-healing assay) and invasion were also assessed.

Results: Ropivacaine (1 nM-100 μM) and lidocaine (1-100 µM) significantly reduced TNFα-induced activation/phosphorylation of Akt, FAK and caveolin-1 in NCI-H838 cells. MMP-9 secretion triggered by TNFα was significantly attenuated by both lidocaine and ropivacaine (half-maximal inhibitory concentration [IC50]=3.29×10(-6) M for lidocaine; IC50=1.52×10(-10) M for ropivacaine). The TNFα-induced increase in invasion was completely blocked by both lidocaine (10 µM) and ropivacaine (1 µM).

Conclusions: At clinically relevant concentrations both ropivacaine and lidocaine blocked tumour cell invasion and MMP-9 secretion by attenuating Src-dependent inflammatory signalling events. Although determined entirely in vitro, these findings provide significant insight into the potential mechanism by which local anaesthetics might diminish metastasis.

Keywords: anesthetics, local; inflammation; neoplasm metastasis.

© The Author 2015. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figures

Fig 1

Summary of proposed mechanism by which amide-linked local anaesthetics inhibit cancer cell invasion and metastasis. As demonstrated previously, local anaesthetics (LA) block tumour necrosis factor α (TNFα)-induced activation of Src tyrosine protein kinase (Src) by inhibiting signal propogation downstream of TNF receptor 1 (TNF-R1). Through this mechanism, amide-linked LAs indirectly prevent activation of Akt kinase (Akt), phosphorylation of caveolin-1, and activation of focal adhesion kinase (FAK). These signalling steps are crucial during metastasis, as they promote cancer cell migration by regulating the cytoskeleton and are necessary for the secretion of matrix-metalloproteinases (MMP), thus enabling the cells to break up the basal lamina and the extracellular matrix (ECM) and further invade into the surrounding tissue.

Fig 2

(a) Representative Western blots of Akt phosphorylated at threonine 308 (pT308 Akt, row 1), total Akt (row 2), and GAPDH, (row 3) after treatment with either (i) lidocaine or (ii) ropivacaine (1 nM–100 µM) for 20 min in absence (a) or presence (b) of tumour necrosis factor α (TNFα) (c) Quantitative analysis of densitometry of Western blots showing the ratio of pT308 Akt over total Akt in NCI-H838 cells treated with (i) lidocaine or (ii) ropivacaine compared with untreated cells (set as 1.0, dashed line) in absence (green bars) or presence (blue bars) of TNFα (20 ng ml−1). Data shown are mean (sd) (n=10 for lidocaine; n=7 for ropivacaine; *P<0.05 compared with TNFα alone. (d) Representative Western blots of FAK phosphorylated at tyrosine 397 (pY397 FAK, row 1), total FAK (row 2), and GAPDH (row 3) after treatment with either (i) lidocaine or (ii) ropivacaine (1 nM–100 µM) alone for 20 min, or (e) after treatment with TNFα in absence or presence of (i) ropivacaine or (ii.) lidocaine (1 nM–100 µM) for 20 min. (f) Quantitative analysis of densitometry of Western blots showing the ratio of pY397 FAK over total FAK in NCI-H838 cells treated with (i.) lidocaine or (ii.) ropivacaine compared with untreated cells (set as 1.0, dashed line) in absence (blue bars) or presence (green bars) of TNFα (20 ng ml−1). Data shown are mean (sd), n=8 for lidocaine, n=7 for ropivacaine; *P<0.05 compared with TNFα alone, #P<0.05 vs. control.

Fig 3

Representative Western blots of caveolin-1 (Cav-1) phosphorylated at tyrosine 14 (pY14 Cav-1, row 1), total Cav-1 (row 2), and either (i.) β-actin or (ii.) (GAPDH, row 3 respectively) after treatment with either (i.) lidocaine or (ii.) ropivacaine (1 nM–100 µM) alone for 20 min (a), or following (b) treatment with TNFα in absence or presence of (i.) ropivacaine or (ii.) lidocaine (1 nM–100 µM) for 20 min. (c) Quantitative analysis of densitometry of Western blots showing the ratio of pY14 Cav-1 over total Cav-1 in NCI-H838 cells treated with (i.) lidocaine or (ii.) ropivacaine compared with untreated cells (set as 1.0, dashed line) in absence (green bars) or presence (blue bars) of TNFα (20 ng ml−1). Data shown are mean (sd), n=7 for lidocaine; n=8 for ropivacaine; *P<0.05 compared with TNFα alone.

Fig 4

(a) Amount of MMP-9 (in pg normalized to mg of cell protein content) in cell culture supernatants of NCI-H838 cells treated with 100 nM Wortmannin (an inhibitor of phosphoinositide-3 kinase) or 5 µM FI 14 in absence (green bars) or presence (blue bars) of TNFα (20 ng ml−1) for 4 h. Data shown are mean (sd), n=6; *P<0.05 compared with TNFα alone. (b) Normalized resistance of NCI-H838 lung cancer cell monolayers over time after electrical injury with 2.5 V at 40 000 Hz for 10s (indicated by arrow). Treatment with either TNFα (20 ng ml−1, green), TNFα+lidocaine (10 µM, pink), TNFα+ropivacaine (1 µM, gold), TNFα+wortmannin (100 nM, turquoise, pre-treatment for 30 min before addition of TNFα), TNFα+FI 14 (5 µM, orange, pre-treatment for 30 min before addition of TNFα), or vehicle (control, blue). Data shown are mean (sd) of normalized resistance. (c) Quantification of normalized resistance at 3 h. Data shown are mean (sd); untreated cells (blue bar), TNFα alone (green bar), lidocaine at 10 µM (pink bar), TNFα+ropivacaine at 1 µM (gold bar), TNFα+wortmannin (turquoise bar) and TNFα+FI 14 (orange bar); n=10 for control, TNFα, TNFα+ropivacaine, TNFα+lidocaine; n=8 for TNFα+wortmannin, TNFα+FI 14; #P<0.05 vs untreated cells, *P<0.05 compared with TNFα alone.

Fig 5

Quantitative analysis of the number of invading cells found per field of view in DAPI stained images of Matrigel coated membranes. Cells were treated with and without TNF plus lidocaine, ropivacaine, wortmannin or FI 14. Data presented as boxplots showing the median, with interquartile and full range. N=5, whereas the values for each slide were calculated as the mean of 8 fields of view. #P<0.05 vs untreated cells, *P<0.05 compared with TNFα alone.