Download Anti-microtubule drugs kill cancer cells by inhibiting mitosis

Anti-microtubule drugs kill cancer cells by inhibiting mitosis
Written by:
Tutored by:
Verdi, Luana, Kollegium Spiritus Sanctus Brig
Anna-Maria Olziersky, M.sc
Nüssel, Jessica, Kantonsschule Olten
Laboratory:
Dr. Prof. Patrick Meraldi
Department of cellular Physiology and
Metabolism, Faculty of Medicine
University of Geneva
1211 Geneva
Switzerland
Abstract
As cancer cells are characterised by their very fast replication rate, common treatments against cancer
target mitosis. The aim is to eliminate all the cancer cells by perturbing the process of duplication.
Cancer cells treated with the anti-microtubule drug Eribulin, mostly prescribed to metastatic breast
cancer patients, show an abnormal mitosis. The aim of our project was to define how eribulin acts on
microtubules. To answer this question, we used two different cancer cell lines: MCF7 and HCC1954.
We treated the cells with a multidrug resistance pump inhibitor, valspodar and Eribulin and we
performed an assay to check the stability of the microtubules. Both cancer cell lines had less stable
microtubules upon eribulin treatment compared to non-treated cells.
Introduction
Cells undergo cell division to guarantee preservation and in order to do so successfully, all the
different steps of the cell cycle need to be faithfully completed1. The process of asexual reproduction
of a cell is called Mitosis and is divided into 5 phases. In Prophase, the chromatin condenses and at
the same time, the centrosomes start nucleating microtubules that help them to migrate on the two
opposite sides of the cell. Microtubules are polymers consisting of α-Tubulin and β-Tubulin. In
Prometaphase the nuclear envelope breaks down and the microtubules shrink and grow in search of
chromosomes in the cytoplasm. This dynamic motion of microtubules happens thanks to their constant
polymerisation and depolymerization. Microtubules coming from the two opposite poles have to
attach on both sides of the chromosomes in order to be able to later pull the sister chromatids apart. In
Metaphase, the chromosomes are aligned along the spindle equator. In Anaphase, the chromosomes
consisting of two sister chromatids are pulled apart to the two opposite poles. In a normal cell division,
the genetic material is now parted equally, thus both cells are identical and survivable. During
Telophase, the nuclear envelope is rebuilt and the chromosomes start to de-condense.
Successful cell division depends on the faithful segregation of chromosomes. If the DNA is not
properly segregated, the cells recognise the mistake and undergo apoptosis, a process that leads to cell
death. In general, cancer cells are duplicating very fast and a common therapy to cure cancer is
chemotherapy. The aim is to eliminate all the cancer cells by perturbing proliferation. Not only the
cancer cells are affected by chemotherapy but also every other fast growing cells in the body like hair,
blood and intestine. That explains the side effects of chemotherapy.
One of the drugs used in breast cancer chemotherapy is called Eribulin. It is used in the late stages of
metastatic breast cancer. Eribulin is an anti-microtubule drug and targets mainly mitosis. The drug
binds to the polymerizing microtubules and blocks polymerization2,3. Therefore, during mitosis it
perturbs the dynamicity of microtubules, the chromosomes cannot properly attach to microtubules and
the cell remains in prometaphase. As the cell cannot properly proceed into anaphase, it goes into
Apoptosis4,5. Here, we show that overall eribulin destabilizes microtubules at a cellular level.
Material and Methods
Two different breast cancer cell lines (MCF 7, HCC1954) were used to investigate the effect of
Eribulin in breast cancer cells. Four 3cm plates with coverslips inside were prepared for each cell line.
1. We seeded the cells 3cm dishes containing coverslips and the next day we added 1nM eribulin
and the pump inhibitor Valspodar (2uM final concentration).
2. 15-20h after, we changed the medium of the cells to a cold one and left the cells on ice for 10
minutes.
3. The coverslips were rinsed one time with Cytoskeleton Buffer (CB, 10mM MES, 150mM
NaCl, 5mM EGTA, 5mM MgCl2, 5mM glucose in water).
4. The cells were fixed with Glutaraldehyde fixation buffer (3% Formaldehyde, 0.05%
Glutaraldehyde and 0.1% Triton X in CB buffer) for 15 minutes and were washed twice with
CB for 10 minutes each and once with Phosphate Buffered Saline (PBS).
5. We blocked with blocking buffer (BB) for 30 minutes at room temperature.
6. The cells were incubated with primary antibodies (a-tubulin and CREST) for 1 hour at RT and
were washed three times with PBS.
7. The cells were incubated with secondary antibodies (anti-rabbit alexa 488, anti-human alexa
647) for 30 minutes at RT and were washed 3 times with PBS.
8. The coverslips were mounted on slides with mounting medium containing DAPI.
9. We looked at the cells under the microscope and we took pictures.
Results and Discussion
In the figures below, we show control and eribulin treated cells after cold treatment. The entire spindle
is built out correctly in the CTR cells but not in the Eribulin treated cells. The chromosomes are
correctly aligned in the middle of the cell in the control cells whereas there are a lot of unaligned
chromosomes in the eribulin treated cells.
Figure1: Control and eribulin treated cells after 10min cold treatment. Cells were stained for a-tubulin
(microtubules), CREST (kinetochores, complex of proteins on the centromere) and DAPI (nucleus).
In both cancer cell lines, the drug Eribulin destabilized the microtubules, leading to a collapse of the
entire spindle after cold treatment. We also noticed that the eribulin treated cells were stuck in
prometaphase, as the number of the mitotic cells was increased upon eribulin treatment. This happens
because the microtubules are destabilized upon eribulin treatment and thus they are not able to attach
to chromosomes. As a result, the chromosomes cannot properly align along the spindle equator. The
reason we used cold treatment is because it contributes also to the destabilization of the microtubules
and helps us to see the difference between control and treated cells clearer.
Conclusion and significance
From the above experiment, we can conclude that overall eribulin destabilizes microtubules and thus
leads to a mitotic arrest which leads to cell death.
Eribulin causes cell death and is used for the treatment of breast cancer. However, only one out of
three breast cancer patients responds to this drug and the cause of this differential response is not
known. If we understand why only some of the patients respond to this drug, the treatment will be
more personalized. Studying and understanding the mode of action of eribulin is important to also
understand why some patients respond very well to Eribulin while others do not respond at all.
References
1. Walczak, C. E., Cai, S. & Khodjakov, A. L. Mechanisms of chromosome behaviour during
mitosis. Nat Rev Mol Cell Biol 11, 91–102 (2010).
2. Eribulin Mesylate: Mechanism of Action of a Unique Microtubule-Targeting Agent, Nicholas F.
Dybdal-Hargreaves, April L. Risinger, and Susan L. Mooberry, April 2, 2015
3. Mary Ann Jordan, Kathryn Kamath, Tapas Manna, Tatiana Okouneva, Herbert P. Miller, Celia
Davis, Bruce A. Littlefield, and Leslie Wilson, The primary antimitotic mechanism of action of
the synthetic halichondrin E7389 is suppression of microtubule growth. Mol Cancer Ther, 4(7):
1086– 95 (2005)
4. James D. Orth, Alexander Loewer, Galit Lahav, and Timothy J. Mitchison, Prolonged mitotic
arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction.
Molecular biology of the cell, 23, 567–576 (2012)
5. Karen E. Gascoigne and Stephen S. Taylor, How do anti-mitotic drugs kill cancer cells? Journal
of Cell Science 122, 2579-2585 (2009)
Acknowledgements
We would like to thank the team of Dr. Prof. Patrick Meraldi Laboratory, especially Anna-Maria
Olziersky for showing us around the whole week. We learnt many new things but what was most
valuable was the experience of working in a research laboratory and to get an impression of how it
could be after our studies at university. Because of this insight, we will surely be able to tell whether
this is something we can imagine for the future or not. We also like to thank Swiss Youth in Science
for the great opportunity we had to take part in this research week. To meet people with the same
interests gave us the opportunity to exchange our thoughts and imaginations. Especially the closing
ceremony at EPFL gave us the chance to exchange with other people who had different projects at
different universities. It was most interesting to collaborate our results. Thank you very much once
again for this opportunity.