Towards personalized medicine: analysis of the combined effectiveness of antimitotic drugs and inhibitors of the bcl-2 family of proteins in complex chemotherapy

Project title: Towards personalized medicine: analysis of the combined effectiveness of antimitotic drugs and inhibitors of the bcl-2 family of proteins in complex chemotherapy.

IRN AP08857554

 Principle investigator – Professor Ivan Vorobyev.

ORCID No:  0000-0002-1815-7829

Scopus ID:  “Vorobjev, Ivan A."  6506052936

Web of Science ResearcherID:   B-2776-2015

 

Introduction

The project is dedicated to studying combined effectiveness of mitotic inhibitors and inhibitors of anti-apoptotic proteins to determine optimal sequence and duration of their application for effective elimination of cancer cells. This project is a continuation of the research, which was completed within the framework of the previous MES project (project AP05134232). We previously showed that significant part of the cells arrested in mitosis does not die but transitions to interphase without chromosome segregation. Moreover, some cells survive even in the presence of high (millimolar) concentrations of mitotic inhibitors. As a result, mitotic inhibitors demonstrate, as a rule, cytostatic, but not cytotoxic effect. Thus, we asked whether it is possible to increase the frequency of cell death during mitotic arrest and after mitotic slippage with the help of additional treatment.

Based on the data of our own and literature, we assume that mitotically arrested cells and multinucleated cells that were formed as a result of mitotic slippage reside in stress and are more sensitive to induction of apoptosis than normal interphase cells or cells undergoing normal division. Thus, our central hypothesis states that cells after mitotic arrest are more sensitive to inductors of apoptosis in comparison with normal interphase cells and can be selectively eliminated by using inhibitors of anti-apoptotic proteins. To test this hypothesis, it is supposed to study the effect of combined treatment of two groups of inhibitors – mitostatics for accumulation of dividing cells and inhibitors of anti-apoptotic proteins of Bcl-2 family (Bcl-2, Bcl-xL, Mcl-1) for selective elimination of cancer cells.

Results, obtained during the completion of the project:

Minimal and more effective mitostatic concentrations of Nocodazole, Taxol, Vinorelbine, Taxol and Epothilone B for eleven cell lines (normal cells 3T3, primary human fibroblasts, immortalized keratinocytes HaCaT and cancer cells MCF-7, HeLa, A549, HT1080, PC-3, Du-145, U2-OS, U-118) were determined. It was shown that at effective concentrations of inhibitors all cells are arrested in mitosis for prolonged time (mitotic arrest) – from 4-5 to 30-40 hours. As a result of this treatment only two outcomes are possible: death during mitotic arrest and mitotic slippage without chromosome segregation. We showed that cell death during mitotic arrest proceeds via apoptotic pathway. HaCaT and HeLa cells, in majority of cases, die during mitotic arrest. Other cell lines demonstrate increased resistance and transition from mitotic arrest to interphase without chromosome segregation (mitotic slippage). As a result of mitotic slippage, multinucleated polyploid cells are formed. Part of these cells die soon after mitotic slippage into interphase. Another part of multinucleated cells in some cell lines (A549, PC-3, HT1080) remain viable for prolonged time and can produce clones.

   We showed that the number of nuclei in the cells that underwent mitotic slippage depends on the inhibitor used. The number of nuclei is maximal in case of Taxol (average number of nuclei is 5-8 per cell) and is minimal in case of Nocodazole (as a rule, two or three nuclei are formed, sometimes one large nucleus is formed). Multiple nuclei that were formed after treatment with Taxol in the cells undergoing mitotic slippage were often connected with each other by thin constrictions. (Figure 1).

Figure 1. Changes in the morphology of nuclei of A549 cells after mitotic slippage. Stained with Hoechst 33258. Confocal microscopy. Scale – 5 µm. On the left – two control cells, in the middle – two mononucleated cells at the bottom and one multinucleated cell at the top after incubation in Nocodazole (100 nM, 24 hours). On the right – multinucleated cell after incubation in Taxol (300 nM).

 Multinucleated cells after mitotic slippage start to proliferate after washing out of the inhibitors and can produce multinucleated as well as mononucleated cells (Figure 2), whose offspring does not lose the ability to proliferate. Timing of division initiation and clonogenicity of these cells depends on the drug used and is minimal in case of Taxol.

 

Figure 2. A549 cells after washing out of Nocodazole and Taxol demonstrate proliferative capacity.

 

On some cell lines, experiments on combined effect of three microtubule inhibitors (Nocodazole, Taxol, Vinorelbine) and five inhibitors of anti-apoptotic proteins (two inhibitors of Bcl-xL, one inhibitor of Bcl-2 and two inhibitors of Mcl-1) were conducted. None of these inhibitors was cytotoxic against normally cultivated cells below 10-30 uM. It was shown that during combined treatment with inhibitors of mitosis and inhibitors of anti-apoptotic proteins, only Bcl-xL inhibitors have specific effect of inducing cell death during mitotic arrest and preventing mitotic slippage. These inhibitors (Navitoclax, A-1155463) are effective against mitotically arrested cells at low (nanomolar) concentrations and demonstrate dose-dependent effect, by accelerating death during mitotic arrest at micromolar concentrations (Figure 3). Inhibitors of two other anti-apoptotic proteins (Bcl-2 and Mcl-1) do not show any effect against mitotically arrested cells.

 

Figure 3. Cells fates of A549 and PC-3 cell lines (in %) after mitotic arrest under the treatment with minimal effective doses of Nocodazole, Taxol and Vinorelbine and after combined treatment with given inhibitors and Navitoclax. 

 

Methods of research: In this work, inhibitors of microtubule dynamics Vinorelbine, Taxol and Eothilone B are used. To study effect of these drug on various processes (cell division, motility, induction of apoptosis and others), high-throughput fluorescent time-lapse microscopy, confocal microscopy and flow cytometry are used.

 

 Within the framework of the project two articles were published in international peer-reviewed journals (Q1):

  1. Kauanova S., Urazbayev A., Vorobjev I. 2021. The frequent sampling of wound scratch assay reveals the “opportunity” window for quantitative evaluation of cell motility-impeding drugs. Frontiers in cell and developmental biology 9: 640972. Published online 2021 March 11. doi: 10.3389/fcell.2021.640972
  2. Urazbayev A., Serikbayeva A., Tvorogova A., Duisenbayev A., Kauanova S., Vorobjev I. 2021. On the relationship between EB3 profiles and microtubules growth in cultured cells. Frontiers in Molecular Bioscience. 8: 745089. Published online: 08 November, 2021. doi: 10.3389/fmolb.2021.745089.

Poster at ASCB/EMBO Annual meeting (USA) was presented:

Temirgaliyev A., Kauanova S., Ten M., Vorobjev I.A. 2021. Improved initial cell spreading on the substrate depends on the cell size. Molecular Biology of the Cell Vol. 32, No. 22. ASCB Annual Meeting abstracts, ID#P777. The American Society for Cell Biology. Published Online: 29 Nov 2021. https://doi.org/10.1091/mbc.E21-11-0545