( ( ( ( The Effects of Etoposide on Acute Leukemia HL-60 and Colon/ Rectum HT-29 Cancer Cells Thomas Bodnar1 Caio Lucena2 , Advisor Prof. Demetrius Araujo2 1Suny Oswego, New York 2 Federal University of Paraíba, Paraíba, Brazil Abstract The major objective of this research is to further the understanding of cytotoxin techniques to practice the most fundamental procedures to cancer research. The overall goal was practicing maintaining and testing how Luekimiea Hl-60 and Colon/Rectum HT-29 responds to the chemical Etoposide at different concentrations. Changing the medium When changing the medium of the Hl-60 cells, the first step is to decontaminate all of the equipment with the pressure chamber and to spray anything going into the hood with alcohol. Once in the hood, I would take all of the cells and medium and put them in 15ml centrifuge tubes and centrifuge them so all of the cells fall to the bottom creating a cell pellet. It is now easy to remove the old medium (the supernatant) to the discard bucket. The next step is to add approximately 9 ml of complete medium to the new bottle and about one ml of SBF. After that add about one or two ml of cells from the cell pellet. It is important to homogenize the cell pellet and anything else when mixing solutions together. To change the colon/rectum Ht-29 cells it is a very similar procedure. The only difference is that these cells attach to surfaces. To remove the adherent cells I use Trypsin. First I took all of the medium from the cell container out and used PBS to wash any other medium out. If this is not done it will affect the Trypsin to not function properly. Next I would add about two ml of Trypsin to the bottle and place in the hood for five minutes. It is vital that it stays in the hood for not any longer because the Trypsin could damage the cell membrane which will lead to its death. After I remove the cells from the bottle’s surface the procedure is the same for the medium change. MTT Test Procedure Prepare different concentrations of toxin of interest. Acute Luekimia Hl-60 cellls 200 100 50 25 12.5 6.25 (µM) Colon/Rectum HT-29 cells Find the cell viability by taking a portion of the stock cells and using TripanBlue to distinguish healthy cells. At least 90% of the sample needs to have viable cells. Count only the cells in the corners of the Neubauer Chamber Background Etoposide is a very popular chemotherapy drug that is derived from the chemical podophyllotoxin. This toxin is found in the plant of Podophyllumpetatum and hexandum, commonly known as Himalayan Mayapples. Etoposide is thought to work by blocking the enzyme topoisomerase II which is used in keeping the DNA’s proper morphology. With this enzyme blocked, the DNA strands break causing cell death. This makes Etoposide a very effective drug against lung and testicular malignancies. Calculate the cell density to distribute into each well and put 100 µl into each well except the control. Making a Complete Medium To make a complete DMEM medium for the cells to live in, I took two 50 ml centrifuge test tubes and added 45 ml of incomplete medium to each. When incomplete, the solution has different concentrations of glucose. Next I would add 5ml of Fetal Bovine Serum to each tube as a usable nutrient for both types of cancer cells. The last solution that is needed to the medium is 500 micro-liters of the anti body Penicillin Streptomycin. This is used to help prevent contamination with bacteria by inhibiting the 16S rRNA of the 30S subunit of the bacterial ribosome, which leads to the death of microbial cells keeping the cells of interest from a contamination. ((number of viable cells)*(1.1*10^5))(1/4)=Concentration one Solve for V1 to find out how much of cell solution to put in each well. C1*V1= (3*10^5)*3ml or Trypsin Protein (5*10^5) MTT test in well plate After mixing the cell solution and the toxins in the well plate wait the desired time and then wash the old medium out and add the MTT for three and a half hours. After the MTT has been metabolized clean the wells with PBS and put on a shaker overnight. In the next day take light absorbency readings of each well to construct a absorbency curve to determine the most effective toxin concentration. Penicillin Streptomycin Incomplete medium containing glucose Fetal Bovine Serum References Medical marvels : the 100 greatest advances in medicine By: Straus, Eugene http://www.nlm.nih.gov/medlineplus/druginfo/meds/a684055.html , Medline Plus http://www.cancer.gov/cancertopics/druginfo/etoposide, National Cancer Institute at the National Institutes of Health.