Can Yeast Cells Simulate HUMAN TUMOR CELLS FOR CHEMOTHERAPY RESEARCH? LAUREN PEASE ACADEMY OF NOTRE DAME GRADE 10
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.
Rationale • 31% of the 6,000 genes found in yeast cells are also found in human cells • Saccharomycescerevisiaehas shown an ability toimitate human cells • “What is true for yeast is also true for human (Pines, 2008) .”
Background Research • Xeloda (capecitabine) -orally administered chemotherapy drug • Used to treat metastatic breast and colorectal cancers • It is a prodrug • 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: • carboxylesterase, • cytidinedeaminase • thymidinephosphorylase
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 5-FU by thymidinephosphorylase, is highly active in tumor tissue 5-FU is then converted to fluorodeoxyuridinemonophosphate, fluorodeoxyuridinetriphosphate, and fluorouridinetriphosphate 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
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 5-Fluorouracil will inhibit more yeast growth than the Xeloda.
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 • 1 Xeloda (capecitabine) capsule 500 mg • 5-Fluouracil 1.68 microliters • Spectrophotometer • Incubator at 35℃. • Pipettes • Bunsen Burner • Vortex • RPMI-1640 28 mL • 34 microliters of DimentholSulfoxide • 34 microliters of sterilized water • Camera (to take pictures) • Paper (to print assay results) • Weighing Paper
Procedure • 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 µLofsterilized water and 34 µL of dimentholsulfoxide (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.
Prepare the liquid medium of each yeast by adding 1 mL of RPMI-1640 to 7 different glass test tubes and labeling them accordingly. • Transfer 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. • 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. • Transfer 100 µL of each yeast to each well in columns one, two, and three (wells “A” get 200 µL). • Repeat for each yeast giving three columns for each species.
Add 8 µL of Xelodato well A1. Add .5 µL of Xelodato well A2. Add .21 microliters of 5-FU to A3. Repeat for each species of yeast. • Create serial dilutions 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℃. • Place waste materials into appropriate biohazard bins. • After 24 hours, a spectrophotometer was used to read each assay.
Variables • Independent • The drug and its concentration • Species of yeast • Dependent • Yeast growth • Control • Wells with no drug • Constants • The temperature of the incubator (35˚C) • Pipettes • Same stock of Xeloda and 5-Fluorouracil
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.
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
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