Summary of the results of the cell fate analysis of the A549 cell line when treated with nocodazole at different doses. Summary of the results of the cell fate analysis of the A549 cell line when treated with taxol at different doses. Summary of the results of the cell fate analysis of the A549 cell line upon treatment with vinorelbine at different doses.
Summary of the results of cell fate analysis of the U-2OS cell line under treatment with Nocodazole at different doses. Summary of the results of cell fate analysis of the U-118 cell line under treatment with Nocodazole at different doses. In the active growth phase of a microtubule, GTP from beta-tubulin is not hydrolyzed, and this GTP cap ensures rapid growth.
Mitotic spindle
When it reaches a certain length, all GTPs suddenly begin to hydrolyze, leading to a change in the conformation of the beta-tubulin molecule. In all cellular processes, microtubules operate in a state of dynamic instability (Alberts, Johnson, & Lewis, 2020). Therefore, in cells, the minus-ends of microtubules are located in the pericentriolar region and grow towards the cell periphery with their plus-ends (Alberts, Johnson, & Lewis, 2020).
However, when microtubules are attached to both kinetochores of sister chromatids, Aurora B kinase is inactivated and Ndc80 complex firmly binds to microtubules in the end-on attachment (Alberts, Johnson, & Lewis, 2020).
Spindle Assembly Checkpoint and Anaphase Promoting Complex
However, the main step in this phase of mitosis is to ensure the correct attachment of microtubules to both kinetochores of sister chromatids, i.e. when microtubules attach to the kinetochore of only one chromatid or from only one side, the kinetochore is positioned laterally on the microtubule. . In this lateral attachment, Aurora B kinase is active and phosphorylates several kinetochore proteins, including the Ndc80 complex.
Aurora B kinase as described above plays a role as a voltage-sensitive inhibitor (Mussachio and Salmon, 2007).
Drugs inhibiting microtubule dynamics
Subsequently, MAD2 binds to CDC20 and recruits the BUBR1-BUB3 complex to form MCC, which inhibits CDC20. Due to unsuccessful clinical trials, Nocodazole is currently used as an experimental tool (Risinger, Giles, & Mooberry, 2009). Vinorelbine is a microtubule destabilizing agent that interacts with tubulin through a Vinca binding site located on beta-tubulin near the GTP binding site.
Vinorelbine is used as a chemotherapeutic agent in lymphoma, non-small cell lung cancer, advanced ovarian carcinoma and metastatic breast cancer (Jordan, 2002).
Aim of the thesis
Taxol and its derivatives are widely used in the treatment of Kaposi's sarcoma, prostate, lung, ovarian, breast, cervical, gastroesophageal, endometrial cancer, and various types of lymphomas and leukemia (Jordan, 2002; Weaver, 2014). The focus of the other studies was on the effect of these drugs on cells. They have shown that Nocodazole, Vinca alkaloids and Taxol cause accumulation of cells in metaphase-like stage starting from the low (nanomolar) concentrations of these drugs.
Also the concentration, which leads to a 50% decrease in the anaphase to metaphase ratio (Kana/met ) was introduced (Jordan, 1991; Jordan, 1992). The proportional increase in the percentage of the blocked-in-mitosis cells occurred with an increase in the concentration of anti-microtubule agents. This blockade of mitosis was accompanied by a proportional inhibition of cell proliferation in the population assay.
Further investigation was conducted into the effect of selected concentrations of antimicrotubule drugs on the behavior of mitotic cells. They defined the effect when at a certain dose of a drug the outcome is not uniform, i.e. in addition, studies at the chosen high concentrations have shown that the cells arrested in mitosis for a longer period of time can either die during mitosis or pass over progress to mitotic slippage. , when the cell exited mitosis without division, usually as a multinucleated cell (Brito and Rieder, 2009).
The aim of this thesis is to study the drug- and dose-dependent effect of the microtubule drug inhibitors on the cancer cells by treating them with the wide range of concentrations of anti-microtubule drugs and the fate of mitotic cells directly under the microscope to observe.
Materials
Methods
The flask with the cells was placed in the incubator and after 24 hours the medium was changed. The cells were grown in two T25 flasks to have the other as an emergency alternative. After the third passage, the cells were seeded at 60% confluence on 48-well plate for the experiment.
After the cells were seeded, the cells were incubated for 24 hours in 37oC 5% CO2 to spread them fully. This is done because the heated chamber of the microscope stage has no CO2 supply. The cell fates observed in the wide range of all three anti-microtubule agents were normal division (Figure 4, A), division with significant delay (Figure 4, B), abnormal/uneven division (Figure 4, C), mitotic slippage (Figure 4, D), and death in mitosis (Figure 4, E).
The cell entered mitosis and divided into two equal daughter cells in a time period equal to the duration of mitosis under control (approximately 1 hour). As with normal division, this fate resulted in morphologically similar two daughter cells, but the duration of mitosis for this category was at least three times longer compared to the control group. The duration of mitosis for the cells belonging to this category was also at least three times longer compared to the control group.
For further analysis, the categories 'distribution with significant delay' and 'abnormal/unequal distribution' have been combined. The duration of mitosis for this category of cells was extremely long (>5 hours and sometimes up to 30 hours), and as a result of mitotic arrest, only one multinucleated cell was formed.
Analysis of dose-dependent changes in cell fates
At a concentration of 30 nM nocodazole, 61% of A549 cells divided with a significant delay, while the rest divided normally. At a concentration of 10 nM taxol, almost 50% of the cells were arrested in mitosis, and most of these cells had divided, but a few. At a concentration of 3 nM vinorelbine, exactly 50% of the cells divided with a significant delay, while the rest divided normally.
Starting from the concentration of 300 nM Nocodazole, 100 nM Taxol and 100 nM Vinorelbine, no significant change in the ratio of different fates was observed, and most cells underwent mitotic slippage. At the concentration of 10 nM Taxol, 63% of cells divided with a significant delay, and some slipped cells were observed. At the concentration of 1 nM Vinorelbine, all cells were arrested in mitosis for a significant period of time, and as a result, 83% of cells divided and other cells underwent mitotic slippage.
The dose-dependent effect of Nocodazole and Vinorelbine was sharp, but the dose-dependent effect of Taxol resulted in a gradual increase in the proportion of stacked in mitotic cells. Starting from the concentration of 300 nM Nocodazole, 1000 nM Taxol and 30 nM Vinorelbine cells underwent mitotic drift or died in mitosis. At a concentration of 30 nM Nocodazole, the majority of cells were stalled in mitosis for a considerable time, while at 10 nM the majority of cells divided normally.
At a concentration of 30 nM Taxol, the majority of cells divided with significant delay or underwent mitotic slippage, and sometimes death in mitosis was also observed. Starting from the concentration of 100 nM Nocodazole, 300 nM Taxol and 10 nM Vinorelbine cells underwent mitotic slippage or death in mitosis.
Analysis of dose-dependent changes on the duration of mitosis
Dependence of the duration of mitosis of A549 cells on the concentration of a mitosis inhibitor. The red bars show the 95% confidence band of the best-fit line for the selected interval analyzed by linear regression. Dependence of the duration of mitosis of U-2OS cells on the concentration of a mitosis inhibitor.
Dependence of the duration of mitosis of U-118 cells on the concentration of an inhibitor of mitosis. For all cells and drugs at MMC, a gradual increase in the duration of mitotic arrest was observed. Summary of p-values indicating the significance of the deviation of the slope from zero for linear regression analysis of the selected concentration ranges (detailed report of the analysis in Appendix 1).
The aim of this study was to study the drug and dose-dependent effect of the microtubule inhibitor agents on the cancer cells by treating them with the wide range of concentrations of anti-microtubule agents and the fate of mitotic cells directly under the microscope where to take. Further increase in the concentration led to the increase in the duration of mitotic arrest. Vinorelbine resulted in a gradual increase in the proportion of cells arrested in mitosis, which mostly divided successfully after considerable delay.
Based on the earliest studies done on isolated microtubules, which determined two distinct IC50 concentrations - IC50 of shortening rate and IC50 of dynamics (Jordan 2002), it can be concluded that the effect of anti-microtubule drugs observed in MMC occurs due to inhibition of the shortening rate. In the case of the T2 threshold concentration, it may correspond to the situation when the dynamic IC50 was reached inside the cells. This leads to the activation of reaction cascades, which determine the further fate of the cell that is dictated only by its internal features.
Therefore, further studies should be focused on determining the key players that determine these properties of the cell, which dictate the outcome of prolonged mitotic arrest in the concentrations above the T2 threshold.
Detailed report of the linear regression analysis on the chosen concentrations
To quantify this effect, the concept of minimal mitostatic concentration was introduced and defined as a concentration which induces mitotic arrest in at least 50% of cells. This arrest mostly ended with division into two or more cells, but could sometimes lead to mitotic slippage. It was also shown that above T2 the outcome of mitotic arrest is mitotic slippage or death in mitosis, and the proportions of these two fates do not change with further increase in concentration.
In addition, the duration of prolonged mitotic arrest above T2 does not depend on the concentration and remains constant.
