Detached breast tumor cells produce dynamic microtubule protrusions that promote reattachment of cells and are termed tubulin microtentacles (McTNs) due to their mechanistic distinctions from actin-based filopodia/invadopodia and tubulin-based cilia. McTNs are enriched with vimentin and detyrosinated α-tubulin, (Glu-tubulin). Evidence suggests that vimentin and Glu-tubulin are cross-linked by kinesin motor proteins. Using known kinesin inhibitors, Lidocaine and Tetracaine, the roles of kinesins in McTN formation and function were tested. Live-cell McTN counts, adhesion assays, immunofluorescence, and video microscopy were performed to visualize inhibitor effects on McTNs. Viability and apoptosis assays were used to confirm the non-toxicity of the inhibitors. Treatments of human non-tumorigenic mammary epithelial and breast tumor cells with Lidocaine or Tetracaine caused rapid collapse of vimentin filaments. Live-cell video microscopy demonstrated that Tetracaine reduces motility of intracellular GFP-kinesin and causes centripetal collapse of McTNs. Treatment with Tetracaine inhibited the extension of McTNs and their ability to promote tumor cell aggregation and reattachment. Lidocaine showed similar effects but to a lesser degree. Our current data support a model in which the inhibition of kinesin motor proteins by Tetracaine leads to the reductions in McTNs, and provides a novel mechanism for the ability of this anesthetic to decrease metastatic progression.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Nicolson GL, Fidler IJ, Poste G (1986) Effects of tertiary amine local anesthetics on the blood-borne implantation and cell surface properties of metastatic mouse melanoma cells. J Natl Cancer Inst 76:511–519
Whipple RA, Cheung AM, Martin SS (2007) Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment. Exp Cell Res 313:1326–1336
Whipple RA, Balzer EM, Cho EH, Matrone MA, Yoon JR, Martin SS (2008) Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cells. Cancer Res 68:5678–5688
Korb T, Schluter K, Enns A, Spiegel HU, Senninger N, Nicolson GL, Haier J (2004) Integrity of actin fibers and microtubules influences metastatic tumor cell adhesion. Exp Cell Res 299:236–247
Machesky LM (2008) Lamellipodia and filopodia in metastasis and invasion. FEBS Lett 582(14):2102–2111
Donaldson DJ, Dunlap MK (1981) Epidermal cell migration during attempted closure of skin wounds in the adult newt: observations based on cytochalasin treatment and scanning electron microscopy. J Exp Zool 217:33–43
Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:619–626
Reed JC (1998) Dysregulation of apoptosis in cancer. Cancer J Sci Am 4(Suppl 1):S8–S14
Mialhe A, Lafanechere L, Treilleux I, Peloux N, Dumontet C, Bremond A, Panh MH, Payan R, Wehland J, Margolis RL, Job D (2001) Tubulin detyrosination is a frequent occurrence in breast cancers of poor prognosis. Cancer Res 61:5024–5027
Webster DR, Gundersen GG, Bulinski JC, Borisy GG (1987) Assembly and turnover of detyrosinated tubulin in vivo. J Cell Biol 105:265–276
Janmey PA, Euteneuer U, Traub P, Schliwa M (1991) Viscoelastic properties of vimentin compared with other filamentous biopolymer networks. J Cell Biol 113:155–160
Gurland G, Gundersen GG (1995) Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts. J Cell Biol 131:1275–1290
Sharp DJ, Rogers GC, Scholey JM (2000) Microtubule motors in mitosis. Nature 407:41–47
Vale RD, Reese TS, Sheetz MP (1985) Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42:39–50
Coy DL, Hancock WO, Wagenbach M, Howard J (1999) Kinesin’s tail domain is an inhibitory regulator of the motor domain. Nat Cell Biol 1:288–292
Endow SA, Waligora KW (1998) Determinants of kinesin motor polarity. Science 281:1200–1202
Kreitzer G, Liao G, Gundersen GG (1999) Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol Biol Cell 10:1105–1118
Liao G, Gundersen GG (1998) Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. Selective binding of kinesin to detyrosinated tubulin and vimentin. J Biol Chem 273:9797–9803
Fink BR, Kennedy RD, Hendrickson AE, Middaugh ME (1972) Lidocaine inhibition of rapid axonal transport. Anesthesiology 36:422–432
Richards CD (1978) The action of anaesthetics on synaptic transmission. Gen Pharmacol 9:287–293
Miyamoto Y, Muto E, Mashimo T, Iwane AH, Yoshiya I, Yanagida T (2000) Direct inhibition of microtubule-based kinesin motility by local anesthetics. Biophys J 78:940–949
Spector I, Shochet NR, Kashman Y, Groweiss A (1983) Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219:493–495
Casella JF, Flanagan MD, Lin S (1981) Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature 293:302–305
Martin SS, Leder P (2001) Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2. Mol Cell Biol 21:6529–6536
Gyoeva FK, Gelfand VI (1991) Coalignment of vimentin intermediate filaments with microtubules depends on kinesin. Nature 353:445–448
Prahlad V, Yoon M, Moir RD, Vale RD, Goldman RD (1998) Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks. J Cell Biol 143:159–170
Dunn S, Morrison EE, Liverpool TB, Molina-Paris C, Cross RA, Alonso MC, Peckham M (2008) Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells. J Cell Sci 121:1085–1095
Ingber DE (2002) Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ Res 91:877–887
Ingber DE (2003) Tensegrity II. How structural networks influence cellular information processing networks. J Cell Sci 116:1397–1408
Blick T, Widodo E, Hugo H, Waltham M, Lenburg ME, Neve RM, Thompson EW (2008) Epithelial mesenchymal transition traits in human breast cancer cell lines. Clin Exp Metastasis 25:629–642
Matrone MA, Whipple RA, Thompson K, Cho EH, Vitolo MI, Balzer EM, Yoon JR, Ioffe OB, Tuttle KC, Tan M, Martin SS (2010) Metastatic breast tumors express increased tau, which promotes microtentacle formation and the reattachment of detached breast tumor cells. Oncogene 1–11
Hammond JW, Huang CF, Kaech S, Jacobson C, Banker G, Verhey KJ (2010) Posttranslational modifications of tubulin and the polarized transport of kinesin-1 in neurons. Mol Biol Cell 21:572–583
Krylyshkina O, Kaverina I, Kranewitter W, Steffen W, Alonso MC, Cross RA, Small JV (2002) Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1. J Cell Biol 156:349–359
Tsuda Y, Mashimo T, Yoshiya I, Kaseda K, Harada Y, Yanagida T (1996) Direct inhibition of the actomyosin motility by local anesthetics in vitro. Biophys J 71:2733–2741
Crevel IM, Lockhart A, Cross RA (1996) Weak and strong states of kinesin and ncd. J Mol Biol 257:66–76
Hirose K, Lockhart A, Cross RA, Amos LA (1995) Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. Nature 376:277–279
Morris VL, MacDonald IC, Koop S, Schmidt EE, Chambers AF, Groom AC (1993) Early interactions of cancer cells with the microvasculature in mouse liver and muscle during hematogenous metastasis: videomicroscopic analysis. Clin Exp Metastasis 11:377–390
Tsuji K, Yamauchi K, Yang M, Jiang P, Bouvet M, Endo H, Kanai Y, Yamashita K, Moossa AR, Hoffman RM (2006) Dual-color imaging of nuclear-cytoplasmic dynamics, viability, and proliferation of cancer cells in the portal vein area. Cancer Res 66:303–306
This work was supported by 1R01CA124704-01 from the National Cancer Institute (to S.S.M.), a Breast Cancer Idea Award (BC061047) from the USA Medical Research and Materiel Command (to S.S.M.) and a Clinical Innovator award from the Flight Attendant Medical Research Institute (FAMRI, CIA-062497).
Conflicts of interest
The authors declare no conflicts of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Data 1Lidocaine and Tetracaine do not disturb membrane integrity in Mammary Epithelial and Breast Tumor Cells. A-C) Propidium Iodide exclusion assay. A) Representation cellular morphology of PI Assay, MDA-MB-436 B,C) MCF10A and MDA-MB-436 were suspended in pre-warmed phenol-free DMEM with propidium iodide stain (1:3000), plus 1mM Lidocaine or 0.25mM Tetracaine +/- Latrunculin A. Addition of Lidocaine and Tetracaine does not increase the disruption of membrane integrity compared to that of the control (DMEM). N=6 Factorial ANOVA and Fischer’s tests performed. No significant differences in PI counts for both MCF10A and MDA-MB-436 cells. (TIFF 11418 kb)
Supplementary Data 2Tetracaine inhibits vimentin trafficking in mammary epithelial cells. Time-lapse movies of MCF10A cells expressing GFP-N-vimentin were taken with and without Tetracaine treatment. In cells treated with vehicle control, GFP-motion persists, When cells are treated with 0.25mM Tetracaine, GFP particle movement ceases after approximately 4 minutes. Each movie is Quicktime format and is looped once to emphasize the difference between the end of the drug treatment and the motion at the beginning of the time course. (MOV 2705 kb)
Supplementary Data 3 Recent studies have shown that McTNs facilitate efficient formation of cell-cell and cell-matrix attachments (2). To determine the effects of Lidocaine and Tetracaine on McTN function, we tested the rate of homotypic aggregation in suspended populations of MCF10A, MCF10A-Bcl2, and MDA-MB-436 cells in the presence of Lidocaine or Tetracaine. A single-cell suspension was prepared using a 0.2% methylcellulose medium, with or without appropriate drug. A 25-gauge syringe was used to separate cells into a single-cell suspension. Aggregation was observed 30-60 min after suspension in all cell lines (Figure 4). Tetracaine impeded the rate of aggregation of suspended cells compared to DMEM control wells. Lidocaine, did not significantly reduce the aggregation rate (Figure 4A-C). At 90 min, the differences between the integrated density of each treatment for each cell line can be seen with the greatest separation between Tetracaine and DMEM treatments in MDA-MB-436 cells (Figure 4D-F). (AVI 3046 kb)
Supplementary Data 4Tetracaine causes the retraction of McTNs in suspended mammary epithelial cells. MDA-MB-436 cells were allowed to settle on glass coverslips precoated for 30 minutes with 2%BSA/PBS to prevent cell adhesion. Time-lapse movies of cells imaged with differential interference microscopy (DIC) were collected and are shown at 100x speed (12 minutes total). (A) McTN protrusion motion persists for an extended period of time in cells treated with vehicle (DMEM) alone. (B) Upon addition of 125µM Tetracaine, a rapid reduction in McTN length and frequency was observed, accompanying a complete retraction of all protrusions by 12 minutes. (TIFF 37749 kb)
Supplementary Data 5Tetracaine causes the inhibition of GFP-kif5c motion in MCF10A mammary epithelial cells. To examine inhibition of kinesin motility with anesthetic treatment, time-lapse movies of MCF10A cells expressing GFP-kif5C (kinesin-1) were taken with and without Tetracaine treatment. GFP particles were recorded at 5 frames/min for vehicle control and Tetracaine treatment. (A) In vehicle control, GFP-kif5c motion persists for an extended period of time. (B,C) In cells treated with 0.25mM Tetracaine, particles could be seen slowing by 2 minutes (B) with almost complete inhibition of movement between 3-5 minutes after treatment (C). (AVI 1135 kb)
About this article
Cite this article
Yoon, J.R., Whipple, R.A., Balzer, E.M. et al. Local anesthetics inhibit kinesin motility and microtentacle protrusions in human epithelial and breast tumor cells. Breast Cancer Res Treat 129, 691–701 (2011). https://doi.org/10.1007/s10549-010-1239-7
- Breast cancer