INTRODUCTION Even as a toddler, I would identify the most amusing object in our house to be the unshapely skull on our bookshelf. I always had an interest in the human body and its wonders. All the questions I would ask my father, a doctor, and the researches I would make to find out even more, I justify to have led me to this research today. Whenever my father had an upcoming surgery my mother would ask ‘What is it?’ and if ‘glioblastoma’ the answer, my mother’s shoulders would drop with a glimpse of pity in her eyes; an encouraging pat on my father’s back would follow. The reason for that scenery I learned years later when I could pronounce the word glioblastoma: the most malignant brain tumor leaving its victims approximately 12 months of survival time. Curious, I began to research this fatal disease. My research showed me that glioblastoma multiforme (GBM) in its full name is a grade IV brain tumor deriving from the cancer of astrocyte cells in the brain1. (see Appendix 1) According to the grading of the World Health Organization (WHO) the most distinctive characteristics of the grade IV tumor would be its rapid growth so much as to include necrosis regions, blocking the way of other cells forcing them to break down and thus forming a dead area inside of the tumor. Next, I decided to talk to experts on the issue and ended up speaking to a biologist2. He named me some drugs that help eradicating glioblastoma and advised me to observe the antioxidant, oxidant, and cytotoxic activity of these different drugs on the cell line. However, I was not looking for drugs. I was looking for something more natural so I began my own research again.

INTRODUCTION Even as a toddler, I would identify the most amusing object in our house to be the unshapely skull on our bookshelf. I always had an interest in the human body and its wonders. All the questions I would ask my father, a doctor, and the researches I would make to find out even more, I justify to have led me to this research today. Whenever my father had an upcoming surgery my mother would ask ‘What is it?’ and if ‘glioblastoma’ the answer, my mother’s shoulders would drop with a glimpse of pity in her eyes; an encouraging pat on my father’s back would follow. The reason for that scenery I learned years later when I could pronounce the word glioblastoma: the most malignant brain tumor leaving its victims approximately 12 months of survival time. Curious, I began to research this fatal disease. My research showed me that glioblastoma multiforme (GBM) in its full name is a grade IV brain tumor deriving from the cancer of astrocyte cells in the brain1. (see Appendix 1) According to the grading of the World Health Organization (WHO) the most distinctive characteristics of the grade IV tumor would be its rapid growth so much as to include necrosis regions, blocking the way of other cells forcing them to break down and thus forming a dead area inside of the tumor. Next, I decided to talk to experts on the issue and ended up speaking to a biologist2. He named me some drugs that help eradicating glioblastoma and advised me to observe the antioxidant, oxidant, and cytotoxic activity of these different drugs on the cell line. However, I was not looking for drugs. I was looking for something more natural so I began my own research again.

 I found that wheat germ extract (WGE) is recently being used to cure cancer. All studies I found were on the impacts of WGE on different cancer types, though I never came across a study which focused only on glioblastoma multiforme in this sense. I decided to use WGE and to choose one of the three activities of antioxidant (inhibiting oxidation), oxidant (promoting oxidation) or cytotoxic (the quality of being toxic to cells) as to make the core of my research question clear and straightforward. I wanted the experiment to be genuinely mine and desired the least amount of help from professionals. Therefore I resolved to choose cytotoxic activity as it is directly correlated with the amount of cells that die from the exposition to WGE. For the other two; I would have to work with biochemists. In light of these decisions, I have settled my research question to be: How does the cytotoxic activity measured by the percentage of remaining living cells found in MTT test (Methylthiazol Tetrazolium Assay) of glioblastoma multiforme cells differ according to their exposition to different concentrations of wheat germ extract? This paper will display how the experiment was planned and carried out as well as evaluate the results provided by the analyzing of the data obtained.HYPOTHESIS Since most studies focus on the cytotoxic activity of Wheat Germ Extract (WGE) on cell lines like ovarian and colon cancer, there is limited information about WGE’s effects on glioblastoma cell line. 

Studies focusing specifically on ovarian cancer3 , colon cancer4 , and leukemia5 indicate that WGE has anti‐proliferative and anti‐metastatic effects on cancer cell lines. It is suggested that the high redox potential of 2,6‐dimethoxy‐p‐benzoquinone and 2‐ methoxy benzoquinone found in wheat germ causes cell cycles to be disturbed, thereby acting as an inhibitor.6 Apparently, WGE keeps malignant cells from growing by changing their metabolism and induces apoptosis4 (the triggering of a pathway in a cell which is naturally programmed to kill it). Both apoptosis and the limited growth of a cell are considered as cytotoxic activity for a cell. Therefore it can be hypothesized that, as the concentration of applied WGE increases, the cytotoxic activity in glioblastoma cells will also increase. It is expected that the rapidly proliferating cells will be slowed down and largely killed as the WGE concentration increases.METHOD DEVELOPMENT AND PLANNING Glioblastoma cell line was chosen to work with because it is one of the most malignant cancer cells. Wheat Germ Extract (WGE) was chosen to expose the cells to because it is a natural substance newly being tested on cancer cells. 

A laboratory was needed as such an experiment could not be conducted at home. Talking to a few doctors, I was lead to the molecular biology department of Ankara University. There, I explained the aim of my experiment and it was shown great interest. I was told that I could receive assistance with using the lab equipment. The glioblastoma cell line was provided from the Neurosurgery Department Neuro‐oncology Laboratory of Ankara University. The materials needed to inoculate cells and feed them throughout the experiment such as RPMI‐1640, Trypsin Hormone, Fetal Bovine, and L‐glutamine were obtained from the medical firm Sigma. (See Appendix 2) I was also lead to a research which explains the method of WGE production.7 (see Appendix 7) Next came designing the experiment. The cells were too valuable to waste. To find an interval of WGE concentration which would probably contain the cytotoxic activity threshold of glioblastoma cells was needed to determine the amount of WGE to give the cells. 

With this aim, the threshold levels of other malignant cancer cells were researched as there were not many articles on glioblastoma cell lines. It was found that there is a general IC50 (half maximal inhibitory concentration is a measure of the effectiveness of a compound in inhibiting biological or biochemical function8) interval from 10% to 35% in various cancer cell 7 Oya Sena AYDOS, Aslıhan AVCI, Tülin ÖZKAN, Aynur KARADAĞ, Ebru GÜRLEYİK, Buket ALTINOK, Asuman SUNGUROĞLU. Antiproliferative, apoptotic and antioxidant activities of wheatgrass (Triticum aestivum L.) extract on CML (K562) cell line. Turk J Med Sci 2011; 41 (4): 657‐663 © TÜBİTAK doi:10.3906/sag‐0912‐425 http://journals.tubitak.gov.tr/medical/issues/sag‐11‐41‐4/sag‐41‐4‐13‐0912‐425.pdf 8 “IC50.” Wikipedia, The Free Encyclopedia http://en.wikipedia.org/wiki/IC50 Figure 1: Molecular Biology Laboratory of Ankara University YAĞMUR UĞUR 001129‐093 MAY 2014 7 lines9, IC50 being the value that causes 50% of cells to stop growing or go through apoptosis which is interpreted as cytotoxic activity. So, the WGE concentrations given to cell lines were determined as 10%, 15%, 20%, 25%, 30%, 35%. Now, knowing how long the cells would be exposed to WGE was needed. It was found that 48 hours is usually the best fit for cells to react to WGE as 24 hours can sometimes be insufficient for significant change in cell activity.1

0 As glioblastoma cells are considered malignant cells, 48 hours was chosen for the duration of WGE exposure to cells before conducting a viability test. At the end of the experiment, there was the need to carry out a viability test to measure what percentage of the cells had died to see cytotoxic activity. As earlier mentioned, cell apoptosis is cytotoxic activity. Research for possible viability assays showed that tetrazolium salts (reflect a purple color when the enzymes in a living cell reduce them to their insoluble formazan dye) are used in experiments in which cells go through apoptosis. During apoptosis, deformations in the mitochondria occur and the mitochondria die.11 These tetrazolium salts can be reduced by an enzyme present in only metabolically active cells, thus, cells with healthy mitochondria.12 Among these tests with tetrazolium salts, were the commonly used MTS, MTT, and XTT tests. As the laboratory only had MTT assay available, MTT assay13 was determined as the test to be conducted to measure cell viability in this experiment. MTT was provided the firm Roche in Germany. To reach viability statistics after the viability assay, a control group to which the cytotoxic activity of cells exposed to WGE can be compared was needed. Since distilled water was used as a solvent in the production of WGE, it was appropriate to give the same percentages of distilled water and medium of all wells to a new set of wells which would form the control group. So 10%, 15%, 20%, 25%, 30%, and 35% distilled water would be given to cells in 90%, 85%, 80%, 75%, 70%, and 65% medium respectively. At this state, determining the controlled variables was the issue.

 To make sure the same amount of cells was inoculated into each well I learned to use the technique used in the laboratory of the university called the Thoma cell counting chamber which allows one to count the number of cells in 1mm3 on the chamber under the microscope and then reach the total number of cells per ml by direct correlation. A 96 well plate as was going to be used as need 60 wells for 5 trials in 6 different concentrations and their control groups were needed. Research showed that 30,000 to 40,000 cells should be inoculated in each well to reach a 90% confluence after incubation.14 So, 40,000 were inoculated in each well. Another important controlled variable was temperature. The CO2 incubator in the laboratory was used and was set to 37oC as it is the body temperature. Naturally, pH levels could not be a controlled variable as the difference in WGE concentrations would also affect the pH levels. The amounts of any liquid put into the wells would be performed by pipettes which are professional tools to prevent errors and get precise amounts of liquids. The wells are also all the same sizes so any other cells in another well will not have more space to grow as sometimes, inadequate place to grow may also cause of cell deaths. 

The kind of serum given to the cells for them to feed on will be the same but the amounts will change due to the concentrations of WGE. This serum would be the one always used by the laboratory for cell cultures. It contains 89% RPMI 1640 Medium, 10% Fetal Bovine serum which is rich of proteins and further supports the growth and survival of cells, and 1% L‐glutamine which is an amino acid which also assists cell growth. It is important to note that a different pipette end must be used for each different substance handled by the pipette. The experiment must be conducted in a sterilized laboratory and the person conducting must wear medical gloves and a face mask. The tools used, the area that the experiment will be conducted in, and the gloves should be sterilized with a 75% ethanol solution before the experiment starts and every time an object like the incubator or a chair that is outside the area of experiment is touched.

The glioblastoma cells are by nature, cells that grow by sticking to surfaces. So when there is not enough surface area, some cells begin to die. That is why, when the flask full of glioblastoma cells is first taken out, 1 ml of the hormone Trypsin is added to the flask and kept in the incubator with 37oC for 3 minutes. The hormone Trypsin helps separate the cells from the surface so that they can be inoculated in wells. After 3 minutes, the flask is put into a centrifuge which spins 2000 times per minute. This makes the healthy cells precipitate to the bottom so that the dead cells can easily be extracted from the top of the liquid with a pipette. When the liquid formed of the Trypsin hormone and dead cells is extracted, 2 ml of medium is added to the healthy cells. The cells and the medium are mixed with pipetting technique. 100 µl of the cell solution is put on the Thoma Cell Counting Chamber (see Appendix 3) using the pipette. This chamber is composed of microscopic square areas on which cells can be counted under the microscope. Numbers of cells in two squares are counted and their average is taken. This number is multiplied by 104 which give the expected number of cells in 100 µl. The two squares counted gave the numbers 12 and 13. Their average, 12.5, multiplied by 104 made 50,000 cells in every 100 µl. In the 96‐well plate, a 6 by 10 rectangular area in the middle of the set is selected to plant the cells in. 

This is to further prevent any temperature changes or air circulation that might happen in the outer wells that are closer to the outside environment. In order to plant 40,000 cells per well, 80 µl is put into each well that is selected. Then 200 µl of medium is added to each well and the well plate is left in the incubator for 24 hours for the cells to stick to the bottom and grow. Figure 4: Flask containing glioblastoma cells Figure 5: Glioblastoma cells under light microscope. The clearly outlined ones are the ones that have died and been dispatched to the top. YAĞMUR UĞUR 001129‐093 MAY 2014 12 After 24 hours, it is time to expose the cells to WGE. For %10, %15, %20, %25, %30, %35 concentrations of WGE, 20, 30, 40, 50, 60, and 70 µl of WGE respectively is needed for every 5 wells as there will be 5 trials of each concentration and the same amount of distilled water for their control group. This makes the total need of WGE 1.35 ml and the total need for distilled water also 1.35 ml. In correspondence to WGE, 180, 170, 160, 150, 140, 130 µl of medium respectively is needed for every 10 wells for both the variable group and the controlled group making the total amount 9.3 ml medium. 6 rows are allocated for 6 different concentrations. The first 5 in a row is for the variable group with WGE and the second 5 is for the controlled group corresponding to that concentration. Taking these into consideration, the used up medium is quickly removed from wells and the new appropriate amount of medium is put into the wells with the order that they have been emptied.