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Local anesthetics inhibit kinesin motility and microtentacle protrusions in human epithelial and breast tumor cells

Abstract

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.

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Acknowledgments

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.

Author information

Correspondence to Stuart S. Martin.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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 material 3 (MOV 2696 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)

Supplementary material 8 (AVI 551 kb)

Supplementary material 9 (AVI 1378 kb)

Supplementary Data 6 Additional live-cell movies of GFP-kif5C transfected MCF10A cells treated with vehicle control and 0.25mM Tetracaine. Images of particle tracks generated from movies are also shown. (AVI 2218 kb)

Supplementary Data 12 (AVI 8 mb)

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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 material 3 (MOV 2696 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 material 5 (AVI 3513 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)

Supplementary material 8 (AVI 551 kb)

Supplementary material 9 (AVI 1378 kb)

Supplementary Data 6 Additional live-cell movies of GFP-kif5C transfected MCF10A cells treated with vehicle control and 0.25mM Tetracaine. Images of particle tracks generated from movies are also shown. (AVI 2218 kb)

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Supplementary material 14 (AVI 1947 kb)

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Supplementary material 16 (AVI 3962 kb)

Supplementary Data 17 (JPG 40 kb)

Supplementary material 18 (AVI 7730 kb)

Supplementary Data 19 (JPG 60 kb)

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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

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Keywords

  • Microtentacles
  • Glu-tubulin
  • Metastasis
  • Kinesin
  • Tetracaine
  • Breast cancer