Tag Archives: cancer

Using CRISPR to Explain the ART of Artemisinin Analogs

My Research Project

Diagram showing how the CRISPR-Cas9 editing tool works

Artemisinin is a drug derived from the Artemisia annua plant. It is known for its anti-malarial properties, but has also been found to have anti-cancer properties. The active portion of artemisinin is an oxygen-oxygen bond called an endoperoxide. When in contact with free iron in a cell, this endoperoxide breaks and creates oxygen radicals which are extremely reactive. These oxygen radicals then proceed to react with cellular components such as membranes and proteins which eventually leads to cell death. Previously, it had been found that DMR1 and HSM2—two analogs of artemisinin— are particularly effective in inducing cell death in cancer cell lines but not in normal cell lines. This summer, my lab and I worked on figuring out why this is so.

 

It has been found that cancer cells contain a higher iron concentration than normal cells. This higher iron concentration is due to higher concentrations of transferrin receptors—the receptor that transports iron into the cell— in cancer cells when compared to normal cells. We believe that the specificity of our artemisinin analogs to cancer cells is due to the higher concentration of iron in cancer cells. To test this, we planned to use CRISPR, a gene editing technique that can remove the transferrin receptor gene in lung cancer cells. Then, we would test our analogs on these transfected cells to determine if a lower iron concentration would show the analogs as ineffective. However, we were unable to test our analogs on the transfected cells because the transfected cells died three days after successful transfection. This proved to us that transferrin receptor is required for cell growth and the proliferation of cancer cells, and that cancer cells cannot survive with low iron concentrations. In the future, we plan on using CRISPR to overexpress the transferrin receptor gene in normal lung cells and testing our analogs on these cells to see if the specificity of our artemisinin analogs is indeed due to iron concentration within the cell.

Realities of Research

Cell culture flasks and media in a laminar airflow hood

Doing research this summer has been very enlightening. In all honesty, before starting research, I imagined it to be a bit boring. I couldn’t see myself really enjoying sitting at a bench and waiting for experiments to run and cells to grow. Surprisingly, when doing research on a subject that you enjoy, it all becomes very exciting. I have learned so much about cell culture techniques and how to maintain a lab this summer. I found myself waiting in anticipation for an experiment to finish because I was so curious to know the results.  I couldn’t wait for cells to grow to large, usable percentages because I wanted to get the next experiment running. Admittedly, it was always disappointing when certain experiments didn’t go as planned or when a lengthy experiment needed to be done multiple times due to errors in previous runs. However, I have learned that even when experiments yield unexpected results, those results still contribute to the research we are conducting. It is not uncommon for an experiment to produce strange results that only make sense after hours of thinking “How could this have happened?” Fortunately, all data that we obtained this summer—expected and unexpected—contributed to my original hypothesis

Life as a Scientist

My day-to-day life as a scientist consisted of waking up early, getting to lab and checking on the cells. Every Monday, Wednesday and Friday the cells have to be fed. If they have grown exponentially, they needed to be split into a new flask. The cell media must be warm, so I had to turn the water bath on and place tubes of media in the bath well before I needed them. I checked the cells under a microscope and estimated the amount of cell growth of each individual flask. If a flask had less than 80% cell growth, the media needed to be discarded and replaced. If a flask had cell growth of 80% or above, then the cells needed to be removed from the current flask and placed into a new one to give them more room to grow. After feeding and splitting was completed, I met with my research mentor and discussed what needed to be done for the rest of the day. The biggest surprise about being a scientist was realizing how little I know about my field of research. Going into research, I believed that I had decent knowledge of physiology and biochemistry. Despite this, I spent every day learning something new and interesting about these fields. My favorite part about research is that there always seems to be more to do. Because of this, there was never a moment where I was bored with nothing to do. That being said, my least favorite part was that there were certain days where an experiment was particularly long and I found myself either overwhelmed with the amount of work to be done or exhausted by the amount of work I completed. Fortunately, working as part of a lab team took a huge amount of stress and burden off of my shoulders. It was very nice to have people to talk to and help me out whenever I need help with a task. Overall, life as a scientist is very rewarding and I have learned so much since I started research this summer.

Suhayl Khan is a senior majoring in health science at Benedictine University in Lisle, Ill. He is a 2019 Short-Term Research Education Program to Increase Diversity in Health-Related Research (STRIDE) Fellow working in Dr. Jayashree Sarathy’s lab at Benedictine University. Suhayl’s fellowship is funded by the American Physiological Society and a grant from the National Heart, Lung and Blood Institute (Grant #1 R25 HL115473-01). After graduation, Suhayl plans to pursue a Master of Healthcare Administration or Master of Public Health.

Apoptosis! How Endoperoxides Could Be a Difference

Artemisinin – also known as Qinghoasu – is produced by the sweet wormwood tree Artemisia annua. For hundreds of years, unaware of its potential in treating cancer and malaria, the sweet wormwood tree was used in ancient Chinese medicine to treat fevers, which we now know were caused by the Malarial parasite. It wasn’t until 1972 that the Chinese scientist Youyou Too and her collaborators isolated the active anti-cancer and anti-malarial ingredient from Artemisia annua, Artemisinin. The active portion of Artemisinin is an oxygen-oxygen bond that forms free radicals when exposed to iron. These free radicals then disrupt cellular function, thereby inducing cell death. In the case of cancer cells, research has shown that most types of cancer cells have increased intake of iron compared to non-cancerous cells. As a result, iron reacts with Artemisinin, producing free radicals, inducing apoptosis, and causing cell death. Therefore, Artemisinin may also be effective when treating cancer. However, despite Artemisinin’s effect on cancer and malaria, there are disadvantages to its usage. Since Artemisinin constitutes less than only about 1% dry weight of the sweet wormwood plant it has limited availability in developing countries and it is very costly to extract. Additionally, the original Artemisinin molecule has trouble reaching its target due to its limited bioavailability. Therefore, we have synthesized analogues of Artemisinin that have the same oxygen-oxygen bond as the original Artemisinin molecule but are smaller and inexpensive to make. This Summer, my lab and I have been testing the novel analogues on A549 lung cancer, MCF7 breast cancer, BEAS-2B normal lung, and MCF10A normal mammary cell lines to see the effect of the analogues on inducing cell death. We have witnessed an increase in cell apoptosis in cancerous cells and not in normal cells and will continue testing the various analogues to find the one with the greatest efficacy at the lowest dose. 

Realities of Research

In my journey as a researcher, I have learned a lot about the advantages and downfalls of researching. Before entering Benedictine University, there was a stigma in my mind towards researching. I couldn’t imagine myself sitting in a lab because the idea of this sounded monotonous and unpleasing. Once I began researching, I realized the importance of it, making me love what I do now. Witnessing the novel drugs killing cancer cells was fascinating and exciting because I was able to make useful discoveries. Furthermore, I have gained knowledge on how to maintain various cancer and normal cell lines using proper cell culture protocol. I have seen just how easily cells can become contaminated and the headache involved with sterilizing everything and starting over. I have learned to follow safety protocols better to prevent future contamination. Additionally, I have become fluent in the usage of various lab equipment and techniques including the flow cytometer, absorbance reader, fluorescence microscope, Western Blotting, and protein assays. Having to perform some of these experiments multiple times due to errors I’ve made has helped me better my technique. Although not all the experiments I completed turned out how I wanted due to human error, the experiments that went correctly supported my original hypothesis.

Life of a Scientist

The day in the life of a scientist begins early in the morning. I wake up, get ready, and am in the lab by 9:00 am daily. Every Monday, Wednesday, and Friday I begin the day by placing media to feed the cells in the water bath. While the media is warming up, I check confluency of the cells to determine whether I need to split them or just feed them. From there, I feed or split cells, clean the hood, and continue with the rest of the day. I then go to my research mentor’s office to determine which experiments need to be completed first, conduct those experiments, and end the day discussing the results. The best part of being a student researcher is the flexibility. I can do so many unique experiments with the cells I am growing, allowing me to test various things simultaneously. Additionally, I have a phenomenal research team and we enjoy conversing with one another. The worst part of researching is the long hours spent in the lab. It does get exhausting to be in the lab all day, however, with my great research group I find ways to help the time pass by. Researching has shown me the importance of interdisciplinary work with the collaboration between the organic chemistry lab and my lab, as well as the importance of effective communication.

 

Mohammed U. Haq is a senior majoring in Health Science at Benedictine University in Lisle, IL. He is a 2018 Undergraduate Student Research Fellow (UGSRF) working in Dr. Jayashree Sarathy’s physiology lab at Benedictine University in Lisle, IL. Mohammed’s fellowship is funded by APS. After graduation, Mohammed plans to pursue a career in medicine with an interest in conducting research in medical school.
Diving Cells and Humans

This summer, I was fortunate enough to continue my research through the American Physiological Society Undergraduate Research Excellence Fellowship (APS UGREF). Their support, along with that of my mentors, has allowed for a unique and interesting project to progress- the investigation of high-dose vitamin C and hyperbaric oxygen therapy (HBOT) for cancer treatment. While the mention of vitamin C for cancer may invite skepticism, the literature teems with evidence that supports additional research exploring vitamin C as a supportive piece of an integrative cancer treatment plan. Fascinatingly, vitamin C can affect the body differently when taken as a supplement (orally) versus administered clinically (intravenously). When given intravenously, vitamin C can actually act as a “pro-oxidant” in cancerous tissue, meaning that it can increase levels of highly reactive oxygen-containing molecules that can stress and sometimes kill cancer cells. Interestingly, with vitamin C, this pro-oxidative effect does not appear to take place in normal cells, making it likely safe for patients that are suitable candidates. HBOT, a medical treatment for severe wounds and other health ailments, delivers 100% oxygen at elevated pressure, suggesting that it may increase the pro-oxidative, anti-cancer effects of vitamin C. So far, we have seen compelling results in isolated mouse brain cancer cells, particularly that high concentrations of vitamin C (> 0.5 millimolar) kill ~80% of cells after 24 hours of treatment and decrease their growth, and that HBOT can enhance these anti-cancer effects. We are also in the process of running additional studies to better understand how these therapies work in combination (i.e. quantifying oxidative stress, studying expression of proteins relevant to cancer), with the ultimate goal to potentially improve patient care.

Aquanauts move across the ocean floor similar to how they would across an asteroid. Photo Credit: NASA.

While concurrently working on my honors thesis, I also had the opportunity to assist with data collection for NASA Extreme Environment Mission Operations (NEEMO) 22 on which my mentor, Dr. D’Agostino, was a crewmember. On this mission, crewmembers live ~60 feet underwater as “aquanauts” at the world’s only undersea laboratory, Aquarius. The goal of NEEMO is to simulate a space flight mission, simultaneously allowing researchers to study the effects of saturation on human physiology. Saturation refers to the aquanauts’ tissues being saturated with nitrogen at a pressure 2.5 times greater than the atmospheric pressure of air at sea level. Before they can return to the surface after the mission, the aquanauts must “decompress” for about 17 hours, where the habitat is gradually depressurized and the crew breathes 100% oxygen for about an hour in total; the latter process is similar to what my cancer cells go through when I put them in a hyperbaric chamber! Our research group looked at the effects of chronic saturation on body composition, autonomic function/dysfunction (heart rate variability and sleep), the gut microbiome (genetic makeup of bacteria in our gastrointestinal tract), and cognition/sensory motor function. It was a great opportunity to learn more about the future of space exploration, research the effects of extreme environments on human health, interact with astronauts, and to work with such a brilliant team of individuals.

It’s really incredible to think of how science has positively impacted my life; growing up, I never imagined myself working in a research lab, let alone becoming a scientist. After having the opportunity to immerse myself into the scientific research culture, however, I do not know if any other path would have been as gratifying and intellectually stimulating. It has been enlightening to see the level of dedication and knowledge required of scientists to run a lab, design experiments, analyze data, and translate scientific discoveries to improve the lives of others. Performing research in a lab requires a great deal of patience and perseverance; as a scientist, one must accept the fact that failures are inevitable, but that each setback may illuminate new pathways and discoveries that would have otherwise remained hidden. I am constantly challenged in the lab, always learning new techniques and understanding that methods, theories, and questions are constantly evolving. I continue to find literature that influences my perspective and approach to research and have great appreciation for the guidance I’ve received on my journey, as well as for the techniques available to decipher our most deep-rooted inquiries. Whether counting cells under a microscope or “diving” cells in a hyperbaric chamber, I am grateful for all the amazing experiences, mentorship, support, and insight research has given me, and hope that other students have similar opportunities to unveil their passions and learn more about the world.

References

  1. NEEMO 16: Traversing with Coral [Online]. https://www.nasa.gov/sites/default/files/660151main_coral-traverse_full_0.j
Janine DeBlasi is a senior cell and molecular biology major at the University of South Florida (USF) in Tampa, FL, where she works as an undergraduate research assistant in Dr. Dominic D’Agostino’s laboratory. She is a recipient of the Undergraduate Research Excellence Fellowship supported by the American Physiological Society and has plans to pursue a career in translational medicine and cancer research.
My Summer Researching and Learning About Worms and Alternative Medicine

My research project explores the possible future uses of the alternative medicine Astragalus, which is a Chinese herb known for its immune stimulating effects, such as decreasing melanoma tumor growth. I am specifically looking for immune system responses in transparent roundworms called C. elegans. I am treating the worms with different concentrations of the Astragalus medicine, then observing how their immune system pathways have reacted. My research expands the information available on alternative medicines and their validity. This project could lead to further, more specific studies as well as studies on other animals and potentially humans. Astragalus could be proven to be a viable medication for disease or replace current cancer treatments and medications.

Working in a research lab has proved to be very interesting and exciting. I started out the summer unsure of myself and the techniques involved with my project. There were many standard procedures and instruments I needed to familiarize myself with to successfully and effectively perform tests. Over time I became more comfortable and could do most tests and procedures on my own, only after being instructed by my research host, of course. My research was unsuccessful at first as I couldn’t get the desired result from polymerase chain reaction (PCR) with the house-keeping gene actin. We reran the end-point PCR and ran another gel but still had no results. After taking another step back and rerunning the cDNA synthesis with my RNA sample, we finally had a successful PCR result. I know there are many techniques I have yet to master in my research journey.

I was surprised by how long certain experiments took and how much everyday maintenance was required to continue working with the worms. Much of my time was spent counting the worms for survival assays or synchronizing the worms to prepare for a test. Also, there was constant upkeep of Nematode Growth Media plates and E. coli stock. I also had to adjust to working independently on my research as opposed to working in a class. I had to prepare all the materials I needed and go through the steps on my own instead of having everything laid out for me. The best part of research was getting successful results and making progress, the worst part was becoming frustrated when results were undesirable or no results were produced at all.

Alyssa Knudson is a junior at Coe College majoring in Biology and Molecular Biology and minoring in Music and Chemistry. She is a part of the Coe College Cross Country and Track and Field teams. She is also involved in Pre-Health Club, Biology Club, and Intergenerational Connections at Coe and a member of the co-ed service fraternity Alpha Phi Omega. She has participated in a 10 week research program working under Dr. Cassy Cozine the summer after her sophomore year. After college she plans to go on to graduate school and get her degree in Genetic Counseling.