STEM 1

STEM 1 is a five month long class which involves an independent research project. The class, taught by Dr. Crowthers has helped me improve my time management and organization skills while providing experience in a research environment. Scroll to the bottom of this page to see the poster that I presented at our February Fair!

My STEM 1 project looked at apoptosis in C. elegans. I treated groups of these small worms with α-tocopherol acetate and observed their growth. Then, I measured mobility and fertility to determine the effect of this vitamin E analogue on apoptosis induction. My results showed that vitamin E has promise in inducing apoptosis in a controlled manner.

Graphical Abstract

Research Proposal

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Literature Review

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Researchable Question

How does vitamin E affect mobility and fertility in C. elegans?

Hypothesis

It is expected that as the concentration of α-tocopherol acetate increases, numbers of progeny will decrease as well as numbers of thrashes, as a result of apoptosis.

Background

How does variation in vitamin E dosage affect the incidence of apoptosis in c. elegans? If the dosage of vitamin E increases, it is suspected that the frequency of apoptosis will also increase, till a certain point at which the vitamin E dosage becomes lethal. This relationship is due to the ability of certain vitamin E analogues to inhibit protein kinases which normally prevent activation of apoptosis pathways. The pro-apoptotic properties of vitamin E could be used in cancerous cells, which have defective cell cycle checkpoints and in turn, too little apoptosis. Cancers grow rapidly because of their ability to forgo cell cycle checkpoints. These checkpoints, most commonly found before the S and G2/M phases, work by detecting damages to DNA and other cell components. When cancers develop in the body, p53 mutations occur. In a healthy cell, the p53 protein acts as a tumor suppressor. The mutated p53 proteins found in cancerous cells are not effective in the p53 pathway, and in turn result in defective cell checkpoints (Grieco et al., 2016). These checkpoints cannot identify the tumor and the cell continues to receive nutrients, allowing it to grow. Apoptosis can be induced via two pathways, extrinsic and intrinsic. The latter relies on internal cell signals and utilizes the mitochondria. The mitochondria is the most central part of the pathway controlling apoptosis, it trafficks pro and anti- apoptosis proteins. One of these proteins, the p53 tumor suppressor protein, controls cell cycle checkpoints. In a healthy cell, when DNA damage is present, p53 identifies it and signals to Bcl-2 proteins, the main regulators of the intrinsic pathway. Following the activation of Bcl-2 proteins, the mitochondria initiates a series of caspases which eventually produce an apoptotic effect (Mukhtar et al., 2010). Once the mitochondria is activated, a point of no return is reached and apoptosis is inevitable. This activation is accompanied by the movement of cytochrome c from intermembrane space to the cytoplasm. In homeostatic cells, inhibitors bind to a protein produced from the caspase chain, preventing apoptosis from progressing any further (Mukhtar et al., 2006). Vitamins are divided into two categories, water-soluble and lipid-soluble. Vitamin E is lipid-soluble meaning it is found in oils, fats, and dairy products. Once in an organism's body, lipid-soluble vitamins are broken down by acidic substances, called bile. The vitamins are then transported by proteins and carried around the body. This type of vitamins are stored in the liver and stay there until they are needed. This means that lipid-soluble vitamins do not need to be ingested daily and too much of them can be harmful. Vitamin K causes blood clotting, and vitamin E induces apoptosis and acts as an antioxidant. The other type of vitamins, water-soluble vitamins, need to be ingested daily and can be passively transported through the bloodstream. These vitamins are found in fruits and vegetables. Lipid-soluble and water-soluble vitamins differ in the way they are transported, used, and disposed of. Many studies have found that analogues of vitamin E can reliably induce apoptosis. One of these analogues is α-tocopheryl succinate (α-TOS). This analogue has a unique property in that it can selectively promote apoptosis, in a pH-dependent manner, most likely because α-TOS has a negative charge at a neutral pH. This provides an important advantage given that tumors generally have a lower pH than most healthy cells. Based on information gathered through tests with α-TOS, scientists have hypothesized that vitamin E causes apoptosis by membrane destabilization (Neuzil et al., 2003). Later studies have found that α-TOS triggers apoptosis via the mitochondrial-mediated pathway. α-TOS has been found to relocate cytochrome c, and cleave the death substrate, a result of the caspase cascade. These findings were a result of a study done on breast cancer cells which over-expressed the anti-apoptotic receptor protein HER2/erbB2 (Neuzil et al., 2005). In this study α-TOS was successful in promoting apoptosis and reducing tumor size. Doctors could potentially use vitamin E analogues like α-TOS to induce apoptosis in a controlled environment. Some data and findings suggest that analogues of vitamin E could suppress tumors by up to 80% (Jiang 2017). Substances like α-TOS give scientists a promising prospect for selective apoptosis induction and tumor suppression. My goal is to determine the optimal dosage of vitamin E, which induces apoptosis in a model organism.

Procedure

Chunking was performed the most in the beginning stages of experimentation. Chunking involves removing a piece of agar, containing E. coli and C. elegans. This chunk is placed onto a new, empty plate, most often on the edge of E. coli growth to encourage the worms to leave their chunk. Chunking is a very quick process that is important to maintaining worm growth. It is not the most precise and is hard to track which organisms are being moved, but in areas where large groups are required to be moved, chunking is highly effective.

Picking is another technique for transporting C. elegans. Picking involves using a tiny pick (about the size of an artificial eyelash) to remove an individual worm from one plate and transport it to a new plate. This is very useful in areas when precision is greatly needed, for example fertility assays. Picking allows for a specific worm of a desired sex, age, or size to be selected. However, worms can sometimes be killed in the process of picking. In this experimentation, picking was used to guarantee that a gravid, large adult was successfully placed onto a smaller plate. This meant that the number of embryos and L1/L2s could be expressed as number of offspring per worm. If a chunking technique has been used, this assumption would have been lost. Picking was also used to select individual worms for the thrashing assay.

As earlier mentioned, bleaching synchronizes the life cycles of a population. This is important to guarantee that the worms on the vitamin E treated plates, lived their whole lives on the treated plates, eliminating the risk of original worms being selected. Because of the casing surround the embryos prior to hatching, they are not affected by the bleach. Therefore, a solution of only embryos can be put onto a certain plate. To start, a chunk was taken out of a plate to keep the population alive, should the treatment kill all worms, and create an opening in the agar. The plate was then covered in [amount] water and a clean, gloved hand wiped the C. elegans and E.coli material, along with excess water into the trough (created from the removed chunk). This solution along with [] of bleach and [] of NaCl were mixed in a test tube. This tube was put into a centrifuge to force all material to the bottom. The excess material on top pipetted off and filled up with water once again, this rinses the embryos of the remaining bleach. The tube was spun in the centrifuge and rinsed again. This process was repeated 3 times until there was only the embryos remaining. This material was divided into three groups and pipetted onto the three treatment plates. This process is important to remove the age factor from growth and fertility.

The thrashing assay was performed by selecting an individual worm about L4 life stage. This worm was dropped into 25 µL of water. Under a microscope, the number of thrashes in 30 seconds were counted. A thrash occurs whenever the worm, suspended in water, curls to make a ‘C’ shape. This is an indicator of mobility. Thrashing was performed in order to collect additional data about C. elegans health and mobility.

The fertility assay counted the number of progeny per worm. After the worms were picked onto individual plates, their progeny was counted. This was done by creating stripes on the bottom of the plate, to aid in visualization during counting. Two [brand, model?] counters were used to count the numbers of embryos and young larvae. Counting was done under a microscope, for 3 worms of each concentration. This was important because it provided data on the number of eggs in the oocytes of the worms, which is an indicator of apoptosis occurring.

Figure 1
C. elegans under microscope
Performing fertility assay
Figure 2

Analysis

As seen in Figure 1, the results of the thrashing assay showed that C. elegans grown on plates containing 400 µg/mL concentration of vitamin E had significantly less thrashes than the worms treated with only ethanol (**p = 0.0019). Worms grown on plates containing the 200 µg/mL concentration did not display a significant difference from the ethanol only thrashing data (p = 0.2874). As shown in Figure 2, a fertility assay shows a significantly less progeny in 400 µg/mL treated worms than ethanol treated worms (**p = 0.0028). C. elegans treated with 200 µg/mL did not have a significant difference from the ethanol treated plates (p = 0.0805).

Discussion and Conclusion

It can be concluded from the results that the presence of a-tocopherol acetate does increase apoptosis in C. elegans. A highly significant difference between the number of thrashes in 400 µg/mL treated worms and control worms shows that the vitamin E treatment decreased both reaction time and muscle strength of the organisms. This can be attributed to apoptosis occurring in muscle tissue. However, 200 µg/mL treated worms did not show a statistical difference from the control treatment in number of thrashes. This means that the treatment was not strong enough to affect the apoptotic pathways. The p value of 0.0019 between 400 µg/mL treated worms and the control group supports the hypothesis that an -tocopherol acetate treatment will decrease the number of thrashes. Similarly, fertility assay results show that the presence of a-tocopherol acetate increased germ cell apoptosis. Worms treated with 400 µg/mL had significantly less progeny than the control group. This can be attributed to more apoptosis occurring in the oocytes of the adult worms. This will lead to increased cell death and less progeny. With a p value of 0.0028 the alternate hypothesis can be supported, and it is concluded that increased apoptosis took place because of the vitamin E treatment. In the 200 µg/mL treatment group there was no significant difference from the control group. In both assays, significant results were seen in the organisms treated with the most vitamin E. This is expected, however, at a certain threshold the concentration of vitamin E could become lethal. This work supports claims made by other studies however it provides additional data from two types of assays. It differs in its use of thrashing assays to measure muscle health. This could provide a different avenue for quantifying muscle strength and response time. It could also be used as an indirect measure of nerve and stress response. These results could be improved by using protein assays to measure apoptosis on a molecular scale.

Expanding this research using different organisms or quantifying methods would offer more insight into the activation of apoptotic pathways. Strains of C. elegans with mutations for CED-3, a caspase 3 equivalent are intended to be used to repeat these methods and gain information about where in the apoptotic pathway is most affected by vitamin E.

References

Angulo-Molina, A., Reyes-Leyva, J., López-Malo, A., & Hernández, J. (2013). The Role of Alpha Tocopheryl Succinate (α-TOS) as a Potential Anticancer Agent. Nutrition and Cancer,66(2), 167-176. doi:10.1080/01635581.2014.863367

Corsi, A. K. (2015). A Transparent window into biology: A primer on Caenorhabditis elegans. WormBook,1-31. doi:10.1895/wormbook.1.177.1

Khan, N., Afaq, F., & Mukhtar, H. (2006). Apoptosis by dietary factors: The suicide solution for delaying cancer growth. Carcinogenesis,28(2), 233-239. doi:10.1093/carcin/bgl243

Khan, N., Adhami, V. M., & Mukhtar, H. (2010). Apoptosis by dietary agents for prevention and treatment of prostate cancer. Endocrine-Related Cancer,17(1). doi:10.1677/erc-09-0262

Lee, G., & Han, S. (2018). The Role of Vitamin E in Immunity. Nutrients,10(11), 1614. doi:10.3390/nu10111614

Pfeffer, C., & Singh, A. (2018). Apoptosis: A Target for Anticancer Therapy. International Journal of Molecular Sciences,19(2), 448. doi:10.3390/ijms19020448

Reagan-Shaw, S., Nihal, M., Ahsan, H., Mukhtar, H., & Ahmad, N. (2008). Combination of vitamin E and selenium causes an induction of apoptosis of human prostate cancer cells by enhancing Bax/Bcl-2 ratio. The Prostate,68(15), 1624-1634. doi:10.1002/pros.20824

Roser, M., & Ritchie, H. (2015, July 3). Cancer. Our World in Data. Retrieved January 25, 2022, from https://ourworldindata.org/cancer

You, W., & Henneberg, M. (2017). Cancer incidence increasing globally: The role of relaxed natural selection. Evolutionary Applications,11(2), 140-152. doi:10.1111/eva.12523

Zaman, S., Wang, R., & Gandhi, V. (2014). Targeting the apoptosis pathway in hematologic malignancies. Leukemia & Lymphoma,55(9), 1980-1992. doi:10.3109/10428194.2013.855307

February Fair Poster