Can Yeast Cells Simulate HUMAN TUMOR CELLS FOR CHEMOTHERAPY RESEARCH?

Can Yeast Cells Simulate HUMAN TUMOR CELLS FOR CHEMOTHERAPY RESEARCH?LAUREN PEASEACADEMY OF NOTRE DAME GRADE 1011QUESTIONCan different species of yeast simulate human tumor cells for chemotherapy research?

This experiment will test if yeast can metabolize the chemotherapy drugs Xeloda and 5-Fluorouracil.2RationaleAt least 31% of the 6,000 genes found in yeast cells are also found in human cellsPrevious experimentation has shown that Saccharomyces cerevisiae, a species of yeast, can imitate human cells because the same genes that control its cell cycle and that malfunction in tumor cells, occur in around the same amount in human cellsAn experiment conducted by David Botstein, a researcher at Stanford, led him to conclude that What is true for yeast is also true for human (Pines, 2008) .This experiment tested that theory to determine if yeast cells can metabolize chemotherapy drugs as human tumor cells can3Background ResearchXeloda (capecitabine) is an orally administered chemotherapy drug, most commonly used to treat metastatic breast and colorectal cancers It is a prodrug which means it must be enzymatically converted to its active formThe active form of Xeloda is 5-Fluorouracil (5-FU) which is also a prodrugFor Xeloda to convert to its active form, three enzymes must be present: enzymes carboxylesterase, cytidine deaminase, and thymidine phosphorylase The conversion from Xeloda to 5-FU is a three step processThe final enzymatic reaction in which 5'-deoxy-5'-fluorouridine is converted to 5-FU by thymidine phosphorylase, is highly active in tumor tissue which is one reason why tumors can be treated with Xeloda. Lastly, the active drug, 5-FU is converted in a final catabolic step to fluorodeoxyuridine monophosphate, fluorodeoxyuridine triphosphate, and fluorouridine triphosphate for incorporation into RNA and DNAAll steps must take place for the yeast to metabolize the drug which would result in inhibition of growth

4hypothesisIf minimum inhibitory concentration assays are taken of Xeloda and 5-Fluorouracil, then both will be able to metabolize the drugs to their active form and incorporate them into their DNA and RNA. If Xeloda and 5-Fluorouracil, are compared, then the 5-Fluorouracil will inhibit more yeast growth than the Xeloda.

5MaterialsColony of Saccharomyces cerevisiaeColony of Candida albicansColony of Candida glabrataColony of Candida kruseiColony of Candida guilliermondii Colony of Candida lusitaniaeColony of Tricherosporon asahii.Petri dishesMicrodilution assay plateGlovesGogglesSharpieLab coat1 Xeloda (capecitabine) capsule 500 mg5-Fluouracil 1.68 microlitersSpectrophotometerIncubator at 35.PipettesBunsen BurnerVortexRPMI-1640 28 mL34 microliters of Dimenthol Sulfoxide34 microliters of sterilized waterCamera (to take pictures)Paper (to print assay results)Weighing Paper

6ProcedurePut on goggles, gloves, and lab coat.Prepare the Xeloda stock by transferring one capsule into a test tube.Light the Bunsen Burner and sterilize a metal spatula.. Then crush the pill into small pieces.Weigh one small piece of the broken pill using weighing paper.Measure 34 Lof sterilized water and 34 L of dimenthol sulfoxide (a universal solvent) into a new test tube, and allow the weighed piece to dissolve.Seven species of yeast were used in this experiment: Candida albicans, Candida glabrata, Saccharomyces cerevisiae, Candida krusei, Candida guilliermondii, Candida lusitaniae, and Trichosporon asahii.7Prepare the liquid medium of each yeast by adding 1 mL of RPMI-1640 to 7 different glass test tubes and labeling them accordingly. Take each Petri dish, flame the inoculating loop in the Bunsen burner, take a small piece of a yeast colony, and carefully place it into the RPMI at the bottom of the correctly labeled test tube. Vortex the tube to ensure it is mixed and opaque-looking.Dilute each liquid medium further by adding 2.5 mL of RPMI to new, plastic, labeled tubes. Transfer .60 L of the yeast medium into the new tubes for further dilution.Using an electronic pipette, transfer 100 mL of each yeast to each well in columns one, two, and three, but wells A get 200 mL.Repeat for each yeast giving three columns for each species.8Using a pipette, add 8 L of Xeloda to the first well in the first column. In every second column for each yeast species, add .5 L of Xeloda to the first well, and for every third column, add .21 microliters of 5-FU to the first well.Use a multichannel pipette to create a serial dilution by transferring dilutions from one row to the next. Leave the last row without any drug for a control and dispose of the last 100 L. Put the assay plate into a bag and put it in the incubator which is set at 35.Lastly, clean everything up by placing materials into appropriate biohazard bins.After 24 hours, a spectrophotometer will be used to read each assay.

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10VariablesIndependent- the concentration of drug tested on each species of yeastDependent- how much yeast grows in each wellControl- the last row of wells that has only grown yeast and no drugConstants- The temperature of the incubator (35C)Pipettes Same stock of Xeloda and 5-Fluorouracil11Data

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16ConclusionHypothesis was partially supportedIt was originally hypothesized that if minimum inhibitory concentration assays were taken of Xeloda and 5-Fluorouracil, then both would be able to metabolize the drugs to their active form. This was rejected because no concentration of Xeloda was able to inhibit yeast growth by 50% or more It was also hypothesized that if Xeloda and 5-Fluorouracil, were compared, then the 5-Fluorouracil would inhibit more yeast growth than Xeloda.This was supported because the 5-fluorouracil was able to inhibit yeast growth by over 50% compared to the control whereas the Xeloda had no impact upon the growth of yeast. The results show that overall, yeast cells cannot effectively represent human tumor cells because they were unable to metabolize the Xeloda, a trait which cancer specific tumor cells have demonstrated.

17Final ConclusionA source of error in this experiment was that although 7 concentrations of drug were tested on each species, one one serial dilution was tested per drugAlso, the assay plates were read within 18-24 hours after they were completed, however it is possible that the 6 hour range could have affected how much growth was read by the spectrophotometerIf this experiment was repeated more trials and more precise reading times would be necessaryThese results are valuable to the field of oncologyThis data shows that yeast contain some of the same enzymes as tumor cells which is why they were able to convert 5-FU to its active form Further research could potentially find a species of yeast that contains all 3 enzymes required to convert Xeloda to fluorodeoxyuridine monophosphate, fluorodeoxyuridine triphosphate, and fluorouridine triphosphate 18References5-FU [Fact Sheet]. (n.d.). Retrieved October 20, 2011, from Scott Hamilton website: http://www.chemocare.com/BIO/fu.aspA.D.A.M. 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Killing Kinetics of Caspofungin, Micafungin, and Amphotericin B against Candida guilliermondii. Abstract retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1538680/Capecitabine [Fact Sheet]. (2011, April 19). Retrieved October 8, 2011, from http://www.drugbank.ca/drugs/DB01101Capecitabine (Xeloda) [Fact Sheet]. (2010, December 1). Retrieved October 8, 2011, from Macmillan Cancer Support website: http://www.macmillan.org.uk/Cancerinformation/Cancertreatment/Treatmenttypes/Chemotherapy/Individualdrugs/Capecitabine.aspxClark, R. A., Levine, R., & Snedeker, S. (2005, March 18). The Biology of Breast Cancer [Fact Sheet]. Retrieved October 8, 2011, from Cornell University website: http://envirocancer.cornell.edu/factsheet/general/fs5.biology.cfm19Diasio, R. B., & Harris, B. E. (1989, April 16). Clinical pharmacology of 5-fluorouracil. Public Medicine, 16(4). Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2656050Doctors Responses. (1999, October 14). Retrieved October 20, 2011, from Medicine Net website: http://www.medicinenet.com/script/main/art.asp?articlekey=10736Ebright, J. R., Fairfax, M. R., & Vazquez, J. A. (2001, September 1). Abstract. Trichosporon asahii, a non-Candida yeast that caused fatal septic shock in a patient without cancer or neutropenia. Abstract retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11477533Fidel, P. L., Jr., Vazquez, J. A., & Sobel, J. D. (1999). Candida glabrata: Review of Epidemiology, Pathogenesis, and Clinical Disease with Comparison to C. albicans. American Society for Microbiology. Retrieved from http://cmr.asm.org/content/12/1/80.fullGilard, V., Desmoulin, F., Malet-Martino, M., & Martino, R. (2002). METABOLISM OF CAPECITABINE, AN ORAL FLUOROURACIL PRODRUG: 19F NMR STUDIES IN ANIMAL MODELS AND HUMAN URINE. Drug Metabolism and Disposition, 30(11), 1222-1229. Retrieved from http://dmd.aspetjournals.org/content/30/11/1221.full.pdfHernday, A. D., Noble, S. M., Mitrovich, Q. M., & Johnson, A. D. (Eds.). (2010). Genetics and Molecular Biology in Candida Albicans. Retrieved from http://johnsonlab.ucsf.edu/sj/wp-content/uploads/2011/01/sdarticle-149.pdfKurtz, J. E., Dufour, P., Bergeat, J. P., & Exinger, F. (2004, September 14). Abstract. Saccharomyces Cerevisiae as a Genetic Model in Anticancer Therapy. Retrieved from http://www.benthamscience.com/cppm/Sample/cpg3-1/001AF.pdfMetastatic Cancer [Fact Sheet]. (2011, May 23). Retrieved October 20, 2011, from National Cancer Institute website: http://www.cancer.gov/cancertopics/factsheet/Sites-Types/metastaticMidgley, R., & Kerr, D. J. (2008, October 21). Capecitabine: have we got the dose right? Nature Clinical Practice Oncology. Retrieved from http://www.nature.com/nrclinonc/journal/v6/n1/full/ncponc1240.html20Nobles, S. M., & Johnson, A. D. (2007). Abstract. Genetics of Candida albicans, a diploid human fungal pathogen. Abstract retrieved from http://www.ncbi.nlm.nih.gov/pubmed/17614788Pfaller, M. A., Diekema, D. J., Gibbs, D. L., Newell, V. A., Nagy, E., Dobiasova, S., . . . Barton, R. (2008, February). Abstract. Candida krusei, a multidrug-resistant opportunistic fungal pathogen: geographic and temporal trends from the ARTEMIS DISK Antifungal Surveillance Program, 2001 to 2005. Abstract retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18077633Pines, M. (2008). Yeast Researchers Get a Head Start. Retrieved October 20, 2011, from Howard Hughes Medical Institute website: http://www.hhmi.org/genesweshare/a110.htmlPray, L. (2008). L. H. Hartwells Yeast: A Model Organism for Studying Somatic Mutations and Cancer. Retrieved October 20, 2011, from Scitable website: http://www.nature.com/scitable/topicpage/l-h-hartwell-s-yeast-a-model-808Raz-Pasteur, A., Ullmann, Y., & Berdicevsky, I. (2011, June 13). Abstract. The Pathogenesis of Candida Infections in a Human Skin Model: Scanning Electron Microscope Observations. Abstract retrieved from http://www.isrn.com/journals/dermatology/2011/150642/Roshan, A. S., Janaki, C., & Parveen, B. (2009). Abstract. White Piedra in a Mother and Daughter, 140-141. Abstract retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938578/Sood, S., Pathak, D., & Rishi, S. (2006). Abstract. Urinary tract infection by Trichosporon asahii, 24(4), 294-296. Abstract retrieved from http://www.ijmm.org/article.asp?issn=0255-0857;year=2006;volume=24;issue=4;spage=294;epage=296;aulast=Sood21Xeloda 1000g/mL Concentration Percent Change From ControlConcentrationCandida albicansCandida glabrataCandida guilliermondiiCandida kruseiCandida lusitaniaeSaccharomyces cerevisaeTrichosporon asahii

8 L-0.65%27.40%7.50%370%-2.26%6.48%271%

4 L-21.40%24.70%-7.50%452%-7.02%13.00%360%

2 L -15.20%44.10%-15.60%410%-16.50%24.10%312%

1 L-29.10%27.80%-11.80%412%-12.80%15.70%338%

0.5 L-21.40%25.50%-12.30%395%-7.02%17.60%293%

0.25 L-24.90%4.60%-13.20%433%1.50%11.10%361%

0.125 L-23.60%-27.20%-9.00%316%0.75%1.85%265%

Xeloda 62.5g/mL ConcentrationConcentrationCandida albicansCandida glabrataCandida guilliermondiiCandida kruseiCandida lusitaniaeSaccharomyces cerevisaeTrichosporon asahii

.5 L-6.90%15.10%10.20%94.60%45.30%-0.65%158%

.25 L4.83%19.90%-6.34%153%17.10%-18.70%188%

.125 L8.97%21.40%4.39%158%-12.40%-2.58%149%

.0625 L16.50%25.10%4.88%152%-4.95%-14.80%146%

.03125 L12.40%28.40%-1.46%152%-6.31%-6.45%139%

0.015625 L11.40%18.90%-12.70%138%-5.18%-31.60%98.80%

.0078125 L1.72%-10.70%-3.41%63.70%-3.83%-20%35.30%

5-Fluorouracil 16g/mL ConcentrationConcentrationCandida albicansCandida glabrataCandida guilliermondiiCandida kruseiCandida lusitaniaeSaccharomyces cerevisaeTrichosporon asahii

.21 L-9.46%-89.50%-74.10%-92.20%-90.20%-86.40%-85.20%

.105 L-2.41%-89.50%-56.50%-92.30%-81.40%-87.50%-84.60%

.0525 L1.48%-89.70%-32.70%-73.00%-88.90%-85.60%-84.30%

.02625 L2.41%-87.20%-21.10%-77.30%-77.60%-70.30%-84.90%

.013125 L4.67%-15.50%-20.80%-7.83%-90.00%15.60%-73.90%

.0065625 L8.16%15.50%-12.40%-7.22%-90.00%2.30%-84.60%

.00328125 L7.42%15.50%-10.30%-4.92%-89.80%1.79%-84.30%