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The effects of doxorubicin and pxd101 on diffuse large b-cell lymphoma cell lines db and su-dhl-4
The effects of doxorubicin and PXD101 on diff
use large B-cell lymphoma cell lines DB and SU
First of all, I would like to thank my research methods and biotechnology teacher at Tucson High Magnet School, Margaret Wilch for all of her support, mentoring, and guidance. She taught me basic laboratory techniques that were vital to my research, edited, and gave me suggestions on all of my research papers and presentations. She also assisted me in finding the lab that I conducted the majority of my research in, whom I also owe many thanks to. I would like to thank Catharine Smith Ph.D at the University of Arizona’s College of Pharmacy, for allowing me the use of her lab and equipment and for her guidance in beginning my research. I would also like to aknowledge Ana Alejandra Tula Sanchez, a graduate student in Dr. Catharine Smith’s laboratory, for all of her assistance in helping me to learn how to use the equipment needed for my research and for helping me learn various laboratory techniques necessary for my project. Many thanks are also due to my research methods class at Tucson High Magnet School for their support throughout my research. And above all, I would like to acknowledge my parents, Barbara and Robert Loomis, for their never ending support and guidance.
One aim of this research was to determine the concentration at which 50% of cells die after 24 ho
urs of treatment (IC50) with doxorubicin in the cell lines tested (DB and SU-DHL-4). Knowing the IC50 of doxorubicin on each cell line made it possible to compare the treatment to PXD101 in checking for DNA damage, as both drugs needed to be used at the IC50 concentration. Comparing pharmaceuticals must be done by using the IC50 concentration so the drugs will be working in similar conditions, and therefore will be comparable.
Comparing the effects of doxorubicin and PXD101 was also another aim of this research. By com
paring the two pharmaceuticals and their effects on the four DLBCL cell lines being tested, the effects of PXD101 will be better understood.
Epigentics is the study of what takes place within the process of gene expression. The roo
t ‘epi’ means ‘above’, so epigentics means “above genetics”. Epigenetics helps to define the changes in gene expression not caused by change in the actual gene, but by non-genetic factors causing the expression of genes to be changed. DNA, made up of deoxyribose, phosphate, and nitrogen bases, wraps around histones, which are small proteins. Histones then coil after DNA has been wrapped around them, forming chromosomes. Histone coiling and uncoiling can effect gene expression. Acetylation of the histones can also have a major effect on transcription upregulation and/or supression. Acetylation or deacetylation is the addition of removal, respectively, of an acetyl group. (Fig.1)
Acetylation of histone proteins plays a large role in the overall scheme of gene expression.
Acetylation has been associated with chromatin assembly, DNA repair, and recombination. Much more research needs to be done to understand the implications of inhibiting acetylation of histone proteins and the effect of chromatin modifications.
Histones can be acetylated by histone acetyltransferases (HATs), or deacetylated by histo
ne deacetylases (HDACs). Acetylation of a protein occurs when an acetyl group is added into a chemical compound; Deacetylation is the removal of an acetyl group. Within the subject matter of this research, the chemical compound of focus for acetylation are histones. Previous studies have shown that deacetylation of histones is correlated with cancer. (Catharine L. Smith; 2007). This is because if a protein is acetylated or deacetylated, transcription can be upregulated or repressed. Inhibition of HDAC has been shown to cause apoptosis associated with tumor repression, therefore, HDAC inhibitors (HDACi), such as PXD101, PXD77, and trichostatin A (TSA), are of interest for use in the treatment of cancer.
Certain genes have also been found to cause cancer or keep cancer cells alive, these are k
nown as oncogenes. Oncogenes are genes that, when mutated, possess the ability to morph a normal cell into a cancerous cell. Oncogenes include c-myc, c-jun, and c-src. Any of these oncogenes could possibly play a role in the development of diffuse large B-cell lymphomas. Currently, all diffuse large B-cell lymphomas are being treated the same way. It has been proven that while some genetic subtypes respond to the current treatment, others may appear to be responding but come back when treatment is discontinued.
The current standard of treatment for diffuse large B-cell lymphomas is called R-CHOP.
The “R” portion of R-CHOP treatment includes the use of rituximab, which is an antibody that destroys cells with CD20 protein on them. The majority of CD20 protein is found on the surface of B-cells, therefore allowing this antibody to destroy B-cells (Mohammad A. Tabrizi 2007). The antibody is an IgG1 mAb isotype. Rituximab is an infusion only medication, which can cause some severe infusion reactions. There can be many dangerous adverse reactions to the drug, some serious side effects including (but not limited to): possibly fatal severe infusion reactions, tumor lysis syndrome (leading to a depletion of electrolytes and eventually renal failure), Steven-Johnson syndrome or toxic epidermal necrolysis, pemphigus, an array of dermatological reactions, serious infections secondary to immunocompromisation, and cardiac failure (Epocrates).
CHOP therapy is a chemotherapy that is a combination of mulitple drugs. “C” stands for
cyclophosphamide, “H” stands for Hydrodaunorubicin, “O” stands for Oncovin, and “P” stands for Prednisolone. BCL2 is a gene involved in apoptosis. BCL6 is a gene involved in the development of diffuse large B-cell lymphoma. Rearrangements of the BCL6 gene have been associated with development of diffuse large B-cell lymphoma.
R-CHOP therapy used in patients with diffuse large B-cell lymphoma with MYC rearran
gement have a poor prognosis. (Sharon Barrans et al,;2010). While R-CHOP therapy has increased the number of patients who enter remission, 40% of patients still die from their condition within 3 years. In patients with MYC rearrangement, deregulation of BCL2 or BCL6 may have a synergistic type effect on lowering prognosis when being treated with R-CHOP.
HDACs are much more promising for the treatment of lymphomas than R-CHOP. Howev
er, HDACs can target non-histone proteins, and the effects of inhibiting these other proteins is not well known. Before we can fully understand HDACs and the effects of inhibiting acetylation, we will need to discover other HDAC substrates. Other HDAC substrates may include non-histone proteins as well as non-protein molecules including polymines or metabolic intermediates. (Saveria Minucci et al.; 2006).
Chemotherapy agents may have cytostatic effects, cytotoxic effects, or both. A cytostatic
al effect is where the agent causes cell arrest (inhibited growth), but not cell death. Cytotoxicity causes cell death. It has been shown that HDACi PXD101 is an effective cytostatic and cytotoxic drug against three DLBCL cell lines: SU-DHL-6, OCI-LY19, and DB. It has also been shown that PXD101 produces cytostatic but not cytotoxic effects in a fourth DLBCL cell line: SI-DHL-4. Other research groups have proposed that HDACi’s antiproliferative effects are either due to DNA damage or induction of funtional cell cycle checkpoints. Cell cycle checkpoints occur throughout cell replication, and is basically a way for a cell to check for damage, repair that damage if possible and continue to replicate, or to decide it is unrepairable and begin the process of apoptosis. Normal cells stop growth after substantial DNA damage, but cancer cells may be missing these
checkpoints altogether, or possibly ignoring the damage and continuing with the replication process.
Doxorubicin is an anthracycline used in the treatment of cancers. It works by causing DN
A double strand breaks. Normal cells are able to repair the damage that doxorubicin causes, cancer cells on the hand, cannot. The damage caused by the drug in cancer cells causes the cancer cells to die. Doxorubicin is a known inducer of DNA damage. It works by causing DNA strand damage. By comparing the effects of doxorubicin versus PXD101 on two different DLBCL cell llines (DB and SU-DHL-4), it will be possible to know more about PXD101 and it’s effects on cell cycle arrest and death.
PXD101 is currently in various Phase I and II clinical trials for the treatment of bladder c
ancer, T-cell lymphomas, and cancers of undetermined origins. It had been shown to be effective against these cancers in vitro and in animal studies. If PXD101 produces DNA double strand breaks, it is possible that it would be able to be used in a wide variety of cancers.
In drug development, an IC50 must be determined to be able to research the drug. IC50 is t
he term used to describe the concentration of a pharmaceutical at which half of the treated cells die within 24 hours. EC50 measures a pharmaceutical's potency and is defined as the half maximal effective concentration.
●Treated two cell lines (DB: cytostatic/cytotoxic versus SU-DHL-4: cytostatic) with varying
concentrations of doxorubicin (0, 25, 50, 100, 200, 400, 800, 1200, 1600, 3200, 6400, and 12800 μM).
●Both of these cell lines were seeded in a 96 well plate, treated with varying concentrations o
f doxorubicin, and manually counted after 24 hours of treatment.
●The concentration of doxorubicin that kills nearly 50% of cells is the IC50.
●The experiment was set in duplicate and replicated at least 3 times.
●Cells were stained with trypan blue to discriminate between alive and dead cells
Comparing the effects of doxorubicin (a known DNA damage inducer) versus PXD101
●Treated two cell lines (DB: cytostatic/cytotoxic versus SU-DHL-4: cytostatic) in parallel wi
●Cells were seeded (10ml) in T12.5cm flasks and treated with doxorubicin or PXD101 IC50s.
●Cells were harvested at certain time points (0, 4, 8, 24, 48, and 72 hours). 0 hours is the neg
ative control and required only one sample.
●15ml of cells were used in histone extraction, and 5ml were used in DNA extraction.
●Histone extracts were used in western blotting to check for increasing H2AX phosphorylati
on (an established biomarker for DNA double strand breaks) upon PXD101 or doxorubicin treatment.
●Isolated DNA was run in a 2% agarose gel to check for DNA shearing (an apoptosis marke
ResultsMTS assays (a colorimetric bioassay measuring cell death) results of both cell lines treated with doxorubicin for 24 hours. The concentration at which half of the cells died at 24 hours of treatment is the IC50. The IC5
Fig.2: MTS assay results of 3 trials of L4 cells treated with doxorubicin for 24 hours.
and 1.25 µM in the DB cell line (Fig. 3).
Fig. 3: MTS assay results of 5 trials of DB cells treated with doxorubicin for 24 hours.
Fig. 4: Western blot analysis checking for phosphorylation of H2AX histone extracts from DB cells treated with doxorubicin and PXD101 harvested at 0, 2, 4, 8, and 24 hours of treatment.
The Western blot analysis to check for phosphorylation of the H2AX protein (a biomarker for DNA double strand breaks), showed a positive correlation between treatment with both doxorubicin (as expected) and PXD101 in the DB cell line, with increased phosphorylation as time progressed.
Thus far, this research has accomplished the first objective of finding the IC50 of doxorubicin in both cell lines. Experiments comparing PXD101’s effects to doxorubicin’s effects on the DB and L4 cell lines are still in progress. The DNA gel of the treatments in the DB cell line did not show any DNA fragmenting as was expected. It was later discovered that DNA double strand breaks caused by intercalation of a molecule into DNA does not cause DNA fragmenting, therefore, separate fragments would not be expected to be shown in a gel electrophoresis analysis of the DNA extracts from the cells treated with either drug. Although it was not possible to determine if PXD101 produced DNA double strand breaks similar to doxorubicin from a gel electrophoresis, the Western blot shows evidence of double strand breaks in the PXD101 treatment’s histone extracts from DB cells that increases over time (Fig. 6).
In the protein gel electrophoresis of the histone extracts, it was expected to see three clear bands which illustrate histones. However, the results (not shown on this poster) did not show three clear bands as expected. The histone extraction protocol needs to be further optimized. Comparison of early Western blots (Fig. 7) and more recent blots (Fig. 6) shows that the protocol is becoming further optimized. These Western blots suggest that PXD101, like doxorubicin, is capable of producing DNA double strand breaks. These data are similar to what Lee (2010) discovered in a study of a different HDACi (SAHA).
The IC50 of doxorubicin in the L4 cell line is 1.54 µM. The IC50 of doxorubicin in the DB cell line is 1.25 µM. Initial results suggest that PXD101 is capable of producing genotoxicity similar to doxorubicin, shown through phosphorylation of the H2AX protein.
TimelineSeptember-October 2010: Began reading background literature and learning laboratory techniques.
November-December 2010: Developed project ideas and methods, continued learning laboratory techniques.
January 2011-Present: Began working on specific project and continued up until now.
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LEY ORGANICA DE PROCEDIMIENTOS ADMINISTRATIVOS Gaceta Oficial: N° 2.818 Extraord. de fecha 1-7-81 Desde art. TITULO I Disposiciones Fundamentales 1 1 7 TITULO II De la Actividad Administrativa 30 30 36 41 44 TITULO III Del Procedimiento Administrativo 47 47 48 51 60 67
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