2002 Recipient
A One-Year Stay at the University of Florida in Gainesville, College of Medicine, Department of Surgery
By René Voboril, MD
From the Czech Republic
A Recipient of a 2002 ICS Research and Scholarship Grant
During my one-year stay at the University of Florida, I was involved in research studying Nuclear Factor
kappa B (NF-kB) and its relationship to inducible chemoresistance in colorectal carcinoma. NF-kB is a
protein localized in the cytoplasm of cells in an inactive form. This protein when activated is
translocated into the nucleus where it binds to DNA with subsequent induction of target genes.
According recent knowledge, NF-kB is responsible for inducible resistance. Protein products of NF-kB
activating genes protect cancer cells against apoptosis – the main mechanism of action of most
anticancer drugs.
My goal at the University of Florida was to examine the possibilities of NF-kB activation in colon
cancer cells, further evaluate a possible relationship between anticancer drugs and NF-kB and find
out if inhibition of this activation leads to an improvement of anticancer drug efficiency. All
experiments were conducted in vitro in colon cancer cell lines.
After I reviewed the literature and mapped the situation, I made myself familiar with all of the
new lab methods used in experiments, especially with EMSA (Electromobility Shift Assay) –
a method of enabling assessment of NF-kB measured by its nuclear translocation; Supershift Assay,
which helps to distinguish the NF-kB subunits; Western Blot, that detects activation of NF-kB also
by measurement of its nuclear translocation; measurement of NF-kB induced gene activation by
Luciferase Reporter Assay; Cell Proliferation Assay and Caspase-3 Fluorescent Activation Assay.
Because EMSA is a method using radioactive isotopes (32P), it was necessary to attend a Basic
Radiation Safety Course with a written final exam. I have successfully passed this exam and thus
obtained approval to work with the radioactive material.
The first group of experiments that I have performed should elucidate whether 5-fluorouracil
(5-FU), the first line drug in the treatment of colorectal cancer, causes activation of NF-kB and
whether inhibition of this eventual activation leads to drug efficiency improvement. EMSA showed
that treatment with 5-FU causes a dose dependent increase in NF-kB binding. This induction of
NF-kB activation by 5-FU was confirmed by Western Blot analysis. Luciferase Reporter Assay
demonstrated that treatment with increasing doses of 5-FU induced NF-kB dependent transcription
of the luciferase gene in a dose dependent manner. Thus an answer to the question concerning
NF-kB activation in colon cancer cells after exposition to 5-FU treatment was obtained. The next
step was to find out whether inhibition of the NF-kB activation enhanced 5-FU cytotoxicity
(measured by cell growth inhibition and caspase-3 activation). To inhibit NF-kB inducible
activation caused by 5-FU, I have used infection of cancer cells with adenovirus IkB super
repressor (Ad5IkB). This viral vector contains genes for mutant nondegradable IkB (natural NF-kB
inhibitor). The optimal number of viral particles for infection of one cell by causing minimal
cell death was determined by flow cytometry. I have proven that inhibition of NF-kB activation
by Ad5IkB leads to enhanced efficiency of 5-FU (measured by increase of cell growth inhibition
and by increase of caspase-3 activation). Since the relationship between 5-FU and colon cancer
cells was not yet published, I have written a manuscript entitled "Enhanced
Chemosensitivity to 5-fluorouracil in Colorectal Cancer by Inhibition of NF-kB" that was
submitted for publication in Carcinogenesis. I am the first author of this paper.
The next group of experiments I conducted during my stay in the USA was based on previous results
with 5-FU. Because 5-FU is in clinical practice used in combination with leucovorin (folinic acid),
it was interesting to find out whether leucovorin has some impact on NF-kB activation caused by
5-FU. I have confirmed conclusions published previously that leucovorin in combination with 5-FU
improves the efficiency of 5-FU compared to treatment by 5-FU alone. EMSA, Western Blot and
Luciferase Reporter Assay proved that treatment with combination 5-FU and leucovorin activates
NF-kB more than treatment with 5-FU alone. This modulation of NF-kB activation by leucovorin is
dependent on a leucovorin dose. Leucovorin alone doesn’t cause NF-kB activation.
Inhibition of NF-kB activation by AD5IkB delivery improves effectiveness of 5-FU+leucovorin
treatment, i.e. treatment with combination of 5-FU+leucovorin+Ad5IkB is more effective than
treatment with 5-fluorouracil+leucovorin (measured again by increase of cell growth inhibition and
by increase of caspase-3 activation). Because these results have not been published yet, I have
written a manuscript entitled "Modulatory Effect of Folinic Acid on NF-kB Activation Caused
by 5-fluorouracil in Colon Cancer Cells" which will be submitted for publication in
Biochemical and Biophysical Research Communications. I am the first author of this paper too.
A portion of the results was presented as a poster at the Joint Cancer Conference in Orlando, FL,
USA on February 20-22, 2003 (authors: Voboril R., Hochwald S. N., Brank A., Li J., Moldawer L. L.,
Lind D. S., MacKay S. L. D.: Enhancement of Anti-Tumor Activity of 5-fluorouracil in Colorectal
Cancer by Inhibition of NF-kB).
During my stay in the United States, I educated myself in the field of apoptosis, I attended a
course "Application in Apoptosis, Cell Viability and Signaling: Making Sense of Your Options
Workshop" organized by the Interdisciplinary Center for Biotechnology Research of the
University of Florida.
Because I am a surgeon, I have observed work at emergency rooms, standard departments, intensive
care units and operating rooms. I was also interested in a system of general organization of health
care in USA.
Daily contact with native speakers, allowing me to improve my English knowledge, was also very
important. I also attended an English language course where I was included (based upon an entry
screening) in "Advanced Conversational English Class." This all together improved not
only my ability to understand and express myself in the field of medicine but also the ability to
speak and understand the conversational "slang" English.
My contacts established during my stay at the University of Florida may serve as a base for a
future scientific cooperation. My research experience received at the University of Florida will
contribute to the continuing of my research work at the Charles University in the Czech Republic.
René Voboril, MD
Hradec Králové, April 4, 2003
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