Upstream eventBinding of MSAs to microtubules
Disturbance in microtubule dynamic instability
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Microtubule interacting drugs lead to peripheral neuropathy||adjacent||Not Specified||Not Specified|
Life Stage Applicability
Key Event Relationship Description
Evidence Supporting this KER
It is well known that the binding of taxol and MSAs like epothilones and discodermolide to microtubules stabilizes microtubules thereby promoting polymerization and concomitantly suppressing depolymerisation. Therefore, they directly disturb microtubule dynamic instability. [1-9]
It is assumed that the M-Loop, which is part of the taxane pocket, undergoes conformational changes and gets more structured as a short helix is formed upon MSA binding. This structuring promotes the assembly and stabilization of microtubules as it is needed for lateral tubulin interactions. [10, 11]
Mutations in the β-tubulin gene were identified in patients with taxol-resistant non-small-cell lung cancer. Patients with β-tubulin mutations did not respond to taxol-treatment, whereas patients without β-tubulin mutations had complete or partial responses and survived longer. β-tubulin mutations were therefore identified as predictor of taxol-response thereby confirming β-tubulin as the binding and interaction site of taxol. 
In two taxol-resistant ovarian cancer cell lines, two point mutations were identified in the β-tubulin gene. Taxol-driven polymerization was shown to be impaired in these cells. Taxol-resistant cells did not exhibit microtubule polymerization upon taxoll treatment whereas the parental cells show increasing tubulin-polymerization with increasing doses of taxol. 
In two epothilone-resistant ovarian carcinoma cell lines two point mutations were identified in the β-tubulin gene. Epothilone- as well as taxol-driven polymerization was shown to be impaired in these cells while parental cells exhibit dose-dependent increase in tubulin polymerization upon epothilone A- and taxol-treatment. 
- Taxol known to bind along the lumen of microtubules was proven to block microtubule dynamics. Various parameters of microtubule dynamic instability were proven to be changed upon taxol treatment in a dose-dependent manner. The growth or shortening rates were shown to be decreased and the attenuation time was increased with increasing taxol concentrations. [6, 15, 16]
- Taxol was shown to decrease the lag-time for microtubule assembly and also the critical concentration of tubulin needed for microtubule assembly. The latter was proven by centrifugation and turbidity measurements in absence and presence of taxol using different tubulin concentrations. [1, 4, 17] The critical tubulin concentration was also proven to be decreased by discodermolide and epothilone A and B. [4, 17]
- Microtubules that were polymerized in the presence of taxol were shown to be resistant to cold- or CaCl2-depolymerization. [1, 2, 5] Cold- and CaCl2-depolymerization resistance could also be proven for epothilone A and B treated  and also for discodermolide treated [3, 5] microtubules.
- Tubulin polymerization assay proved that taxol-resistant cells exhibit less/impaired taxol-driven tubulin polymerization compared to parental cells.  The same was true for epothilone-resistant cells. 
- Taxol, discodermolide and epothilone A and B were shown to enhance in vitro tubulin-polymerization. [2, 3, 5] Furthermore, MSAs arrest cells in mitosis shown by quantification of cells in G1 phase or at G2-M transition, quantification of mitotic figures, overall assessment of the cell number or analysis of cyclin B1 expression. [2, 3, 5, 18] It was also shown that cells, which were treated with taxol and therefore arrested in mitosis, re-enter cell cycle after washout of taxol. 
Uncertainties and Inconsistencies
The binding of MSAs to microtubules is extensively studies and well established. Its impact of this interaction on microtubule dynamic instability is addressed in numerous studies and the findings are largely consistent in the point of stabilization of microtubules accompanied by the disturbance of microtubule dynamic instability.
It has to be noted that microtubule destabilizing agents like the vinca alkaloids are known to bind to microtubules and disturb microtubule dynamic instability as well. However, vinca alkaloids differ in their mode of action as they bind to the end of microtubules and, in case of stoichiometric binding, promote depolymerization. 
Quantitative Understanding of the Linkage
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
1. Schiff, P.B., J. Fant, and S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol. Nature, 1979. 277(5698): p. 665-667.
2. Bollag, D.M., et al., Epothilones, a New Class of Microtubule-stabilizing Agents with a Taxol-like Mechanism of Action. Cancer Research, 1995. 55(11): p. 2325-2333.
3. Hung, D.T., J. Chen, and S.L. Schreiber, (+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest. Chemistry & Biology, 1996. 3(4): p. 287-293.
4. Kowalski, R.J., et al., The Microtubule-Stabilizing Agent Discodermolide Competitively Inhibits the Binding of Paclitaxel (Taxol) to Tubulin Polymers, Enhances Tubulin Nucleation Reactions More Potently than Paclitaxel, and Inhibits the Growth of Paclitaxel-Resistant Cells. Molecular Pharmacology, 1997. 52(4): p. 613-622.
5. ter Haar, E., et al., Discodermolide, A Cytotoxic Marine Agent That Stabilizes Microtubules More Potently Than Taxol. Biochemistry, 1996. 35(1): p. 243-250.
6. Derry, W.B., L. Wilson, and M.A. Jordan, Substoichiometric Binding of Taxol Suppresses Microtubule Dynamics. Biochemistry, 1995. 34(7): p. 2203-2211.
7. Dumontet, C. and M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer therapeutics. Nature Reviews. Drug Discovery, 2010. 9(10): p. 790-803.
8. Jordan, M.A. and L. Wilson, Microtubules as a target for anticancer drugs. Nature Reviews Cancer, 2004. 4: p. 253.
9. Carozzi, V.A., A. Canta, and A. Chiorazzi, Chemotherapy-induced peripheral neuropathy: What do we know about mechanisms? Neurosci Lett, 2015. 596: p. 90-107.
10. Prota, A.E., et al., Molecular mechanism of action of microtubule-stabilizing anticancer agents. Science, 2013. 339(6119): p. 587-590.
11. Snyder, J.P., et al., The binding conformation of Taxol in β-tubulin: A model based on electron crystallographic density. Proceedings of the National Academy of Sciences, 2001. 98(9): p. 5312-5316.
12. Monzó, M., et al., Paclitaxel Resistance in Non–Small-Cell Lung Cancer Associated With Beta-Tubulin Gene Mutations. Journal of Clinical Oncology, 1999. 17(6): p. 1786-1786.
13. Giannakakou, P., et al., Paclitaxel-resistant Human Ovarian Cancer Cells Have Mutant β-Tubulins That Exhibit Impaired Paclitaxel-driven Polymerization. Journal of Biological Chemistry, 1997. 272(27): p. 17118-17125.
14. Giannakakou, P., et al., A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proceedings of the National Academy of Sciences, 2000. 97(6): p. 2904-2909.
15. Witte, H., D. Neukirchen, and F. Bradke, Microtubule stabilization specifies initial neuronal polarization. The Journal of Cell Biology, 2008. 180(3): p. 619-632.
16. Jordan, M.A., et al., Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proceedings of the
National Academy of Sciences of the United States of America, 1993. 90(20): p. 9552-9556.
17. Kowalski, R.J., P. Giannakakou, and E. Hamel, Activities of the Microtubule-stabilizing Agents Epothilones A and B with Purified Tubulin and in Cells Resistant to Paclitaxel (Taxol®). Journal of Biological Chemistry, 1997. 272(4): p. 2534-2541.
18. Risinger, A.L. and S.L. Mooberry, Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel. Cell Cycle, 2011. 10(13): p. 2162-2171.
19. Dumontet, C. and B.I. Sikic, Mechanisms of Action of and Resistance to Antitubulin Agents: Microtubule Dynamics, Drug Transport, and Cell Death. Journal of Clinical Oncology, 1999. 17(3): p. 1061-1061.