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CAN YEAST CELLS SIMULATE HUMAN TUMOR CELLS FOR CHEMOTHERAPY RESEARCH? LAUREN PEASE ACADEMY OF NOTRE DAME GRADE 10 1 QUESTION Can 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. 2 RATIONALE At least 31% of the 6,000 genes found in yeast cells are also found in human cells Previous 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 cells An 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 can 3 BACKGROUND RESEARCH Xeloda (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 form The active form of Xeloda is 5-Fluorouracil (5-FU) which is also a prodrug For 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 process The final enzymatic reaction in which 5'-deoxy-5'-fluorouridine is converted to 5FU 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 DNA All steps must take place for the yeast to metabolize the drug which would result in inhibition of growth 4 HYPOTHESIS If 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 5Fluorouracil will inhibit more yeast growth than the Xeloda. 5 MATERIALS Colony of Saccharomyces cerevisiae Colony of Candida albicans Colony of Candida glabrata Colony of Candida krusei Colony of Candida guilliermondii Colony of Candida lusitaniae Colony of Tricherosporon asahii. Petri dishes Microdilution assay plate Gloves Goggles Sharpie Lab coat mg 5-Fluouracil 1.68 microliters Spectrophotometer Incubator at 35℃. Pipettes Bunsen Burner Vortex RPMI-1640 28 mL 34 microliters of Dimenthol Sulfoxide 34 microliters of sterilized water Camera (to take pictures) Paper (to print assay results) Weighing Paper 1 Xeloda (capecitabine) capsule 500 6 PROCEDURE Put 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 µLof sterilized water and 34 µL 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. 7 Prepare 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 µL 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. 8 Using a pipette, add 8 µL of Xeloda to the first well in the first column. In every second column for each yeast species, add .5 µL 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 µL. 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. 9 PHOTOS 10 VARIABLES Independent- the concentration of drug tested on each species of yeast Dependent- how much yeast grows in each well Control- the last row of wells that has only grown yeast and no drug Constants1. The temperature of the incubator (35˚C) 2. Pipettes 3. Same stock of Xeloda and 5-Fluorouracil 11 DATA 12 13 14 GRAPH 15 16 CONCLUSION Hypothesis was partially supported It 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. 17 FINAL CONCLUSION A 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 drug Also, 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 spectrophotometer If this experiment was repeated more trials and more precise reading times would be necessary These results are valuable to the field of oncology This 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 18 REFERENCES 5-FU [Fact Sheet]. 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