Drugs that Bind DNA to Treat Diseases - Case Study Example

Topic: Drugs that bind DNA to treat diseases (selected disease: cancer) Part A- (word count excluding the three citations = 459 The following keywords and keyword combination were used to search for articles on drugs that bind DNA to treat diseases: “Drug-DNA interactions AND cancer;” “DNA binding AND anti-cancer drugs” 2. To find articles on the given topic, PubMed Central was selected as the database for the search. Pubmed Central (PMC) is a digital archive or electronic storage of biomedical and life science journals of the U.S. National Library of Medicine at the National Institutes of Health. It is a feature of the homepage of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Pubmed Central has several advantages over other databases: it can be accessed by anybody who has internet connection, registration is free and does not require any fee; and articles are linked to free full text. Moreover, the collection in the database is very extensive. In fact, there are more than 1,500,000 articles from over 450 international journals. Pubmed Central is linked to Pubmed, which is a bigger database. The only difference between the two databases is that the texts of some articles in Pubmed are not free. 3. Pubmed Central was accessed at http://www.pubmedcentral.nih.gov/. On this webpage, the “advanced search” option was clicked. Then the tab for “Limits” was chosen to apply only to journal articles and reviews that were published in the last five years (2005-2009). A list of articles came out after typing the keywords. The links to the free full text of the selected articles were then checked to access the html file and pdf of the articles. 4. Citations for three articles 1. Galea, A., & Murray, V. (2008). The anti-tumour agent, cisplatin, and its clinically ineffective Isomer, transplatin, produce unique gene expression profiles in human cells. Cancer Informatics , 6, 315-355. 2. Hajihassan, Z., & Rabbani-Chadegani, A. (2009). Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins. Journal of Biomedical Sciences , 16 (1), 31 (online). 3. Lemke, K., Wojciechowski, M., Laine, W., Bailly, C., Colson, P., Baginski, M., et al. (2005). Induction of unique structural changes in guanine-rich DNA regions by the triazoloacridone C-1305, a topoisomerase II inhibitor with antitumor activities. Nucleic Acids Research , 33 (18), 6034-6047. The three articles were selected because they show a wide range of techniques and results of experiments that explained how different anti-cancer drugs interact with DNA and DNA structure. Their findings show different ways in how cancer can be attacked, and how future drugs can be designed to be more effective. The study by Hajihassan & Rabbani-Chadegani (2009) compared the binding affinity of anticancer drug mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), and histone proteins using UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, gel electrophoresis, and equilibrium dialysis. In the second paper, the antitumor triazoloacridone, compound C-1305, which is a topoisomerase II inhibitor, was characterized with respect to its DNA interactions in vitro with the use of surface plasmon resonance, chemical probing with DEPC, and molecular modelling. (Lemke, et al., 2005). Gene expression profiling techniques were employed to explain the molecular events in the DNA-damaging activity of the anti-cancer agent cisplatin (Galea & Murray, 2008), which is used to a wide range of tumours but has toxic side effects associated with dosing quantity. Gene microarrays identified genes that were affected by the action of cisplatin and its inactive isomer, transplatin. 5. All articles are available in their electronic form from the Pubmed Central Database. Part B- Essay (1026words) Biochemical Techniques Help to Understand Drug-DNA Interactions in Developing Treatment for Cancer Cancer is undoubtedly the number one killer disease in the world. Cancerous cells proliferate uncontrolled, and this could be seen in the formation of tumours. Cancers develop because the information carried in one’s DNA is altered in a manner that leads to uncontrollable cell multiplication. One of the objectives in developing drugs for cancer therapy is to find agents that can bind DNA to stop the uncontrolled replication and production of cancerous cells. During and after an anti-cancer drug is developed, biochemical and molecular studies verify, validate and explain how the drug interacts with DNA and its associated proteins, its effects on gene expression and regulation. Biochemical studies are also employed to check for improvements that are brought about by drug modification. Hajihassan and Rabbani-Chadegani (2009) used SDS gel electrophoresis and agarose gel electrophoresis to detect the interaction of the drug mitoxantrone with histones (these are proteins that bind nucleosomes) and DNA. Electrophoresis is the process that exploits the property of a molecule with a net charge to move in an electric field (Figure 1). It is a means to separate proteins, nucleic acids (DNA and RNA) and other molecules based on charge and size. The speed of migration of the molecules will depend on the strength of the electric field, the net charge of the molecules, and friction. The molecules will travel towards the electrode that carries an opposite charge (Berg, Tymoczko, & Stryer, 2002). The electrophoretic separations are usually carried out in a gel or other solid support like paper or nitrocellulose, which is immersed in a weak buffer. The gel also acts like a sieve that improves the separation. This is the reason why molecules that are smaller than the gel pores move easily through the gel. Larger molecules move at a smaller rate while very large molecules can remain immobile. Polyacrylamide gel electrophoresis (or PAGE) is performed in a vertical slab of gel. Under denaturing conditions, the proteins are separated largely based on mass. This is made possible by dissolving the protein mixture in a solution with sodium dodecyl sulfate (SDS), an anionic detergent that destroys noncovalent interactions in proteins. The addition of mercaptoethanol or dithiothreitol reduces disulfide bonds. With the use of SDS, the protein change is covered by a large negative net charge. Because all proteins in the mixture are negatively charged, the migration in the gel during the electrophoretic process will now be wholly dependent on the protein mass alone. Figure 1. A representation of how SDS gel electrophoresis works. Figure is available from Molecular Station.com http://www.molecularstation.com/no/molecular-biology-techniques/gel-electrophoresis/ After the completion of electrophoresis, the separated proteins are visualized by staining the gel with silver or Coomassie blue dye. The proteins appear as a series of bands. Radioactive labels also are detected using autoradiography, where an X-ray film is used to cover the finished gel (Berg, Tymoczko, & Stryer, 2002). SDS-PAGE is a classical technique that is rapid and sensitive, giving a high degree of resolution. Proteins weighing 0.1 m g (~two pmol) gives a distinct band when stained with Coomassie blue, while less than this can be detected with a silver stain. Differences in mass by as little as 2% or of about 10 residues) can usually be distinguished. If the purification process is very accurate, the number of bands in SDS-PAGE will be very clear with the band of interest highly prominent. (Berg, Tymoczko, & Stryer, 2002). The principles that apply to SDS-PAGE also apply to agarose gel electrophoresis, except that SDS-PAGE separate proteins while agarose gels separate DNA or RNA. These nucleic acids are negatively charged in solution and will therefore move towards the positively charged end of the gel. Movement is due to differences in size, with larger DNA molecules moving slowly compared to shorter DNA fragments. The gels are stained with ethidium bromide, which intercalates between DNA strands and fluoresces under ultraviolet light. SDS gel electrophoresis showed the binding of mitoxantrone to histones and DNA (Hajihassan & Rabbani-Chadegani, 2009). Mitoxantrone was first mixed with sodium EDTA soluble (SE) –chromatin components. Then the supernatants resulting from the mixture were run on SDS-PAGE and gel electrophoresis (Figure 2). It is shown that in the absence of mitoxantrone, the proteins coming from the chromatins are mostly histones. However, with the addition of the mitoxantrone at higher concentrations, chromatin becomes compacted and the histones are no longer present in the supernatant. This is why the bands for histones are no longer observed in the gel (lanes 5-7). When the DNA coming from the mitoxantrone treated samples were run on the agarose gels, it was shown that increasing mitoxantrone concentration lessened the DNA concentration. Figure 2. (A) SDS gel electrophoresis of the supernatants from the mixture of mitoxantrone and histone shows that increased mitoxantrone concentrations lessened the amount of free histones in solution. (B). Agarose gel of the DNA extracted from supernatant of mitoxantrone-DNA mixture. Increased drug concentration reduced DNA in the supernatant. Electrophoresis provided a simple, visual means to understand the mechanism of action of mitoxantrone. The results using SDS-PAGE and agarose gel electrophoresis show that the action of mitoxantrone is to associate with both histones and DNA. This is very clear because the increasing the dosage of mitoxantrone results in the decrease of the amount of DNA and histones in solution. Examining the gels help us to understand that the drug interacts with DNA and secondly, the interaction is dependent on the amount of dose that is given. An avenue for research is to look at how the drug interacts with both histones and DNA, comparing the affinity of the drug for either histones or DNA. The implication of the results is that mitoxantrone preferentially binds to nucleosome structure, which is associated with histone proteins, or partially histone free DNA. Naked DNA without histones appears to be not a preference for the action of mitoxantrone shows strong affinity to histone proteins. Based on the results, it can be safely concluded the action of mitoxantrone is binding and compacting the chromatin. Once the chromatin is compacted, then the process of DNA replication is affected. Results of the experiment also imply that the protein component or histone proteins of the chromatin can be used as targets for anti-cancer drug agents. However, the results are not enough to give information if mitoxantrone would prefer to bind histones over DNA, or if the drug binds them simultaneously. This is another avenue for research. References Berg, J., Tymoczko, J., & Stryer, L. (2002). Biochemistry, Fifth Edition. W.H. Freeman and Co. Galea, A., & Murray, V. (2008). The anti-tumour agent, cisplatin, and its clinically ineffective Isomer, transplatin, produce unique gene expression profiles in human cells. Cancer Informatics , 6, 315-355. Hajihassan, Z., & Rabbani-Chadegani, A. (2009). Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins. Journal of Biomedical Sciences , 16 (1), 31 (online). Lemke, K., Wojciechowski, M., Laine, W., Bailly, C., Colson, P., Baginski, M., et al. (2005). Induction of unique structural changes in guanine-rich DNA regions by the triazoloacridone C-1305, a topoisomerase II inhibitor with antitumor activities. Nucleic Acids Research , 33 (18), 6034-6047. Read More