Local anesthetics inhibit kinesin motility and microtentacle protrusions in human epithelial and breast tumor cells
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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.
KeywordsMicrotentacles Glu-tubulin Metastasis Kinesin Tetracaine Breast cancer
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.
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)
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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)
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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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