In my project, I investigated the effect of the combination of Palbociclib and Bempodoic acid on cell number, cell death, and invasiveness, three important processes in carcinogenesis. In order to examine the treatment’s effect on cancer cell number and cell death, we first assumed that the cell underdo apoptosis when treated with the drugs. Immunoblotting experiments shows that the combination treatment led to an increased express in BAD pro-apoptotic markers and a decreased express in Mcl1 anti-apoptotic markers apoptosis. The result reveals that the combination treatment led to an increase in apoptosis (cell death).
I also wanted to see whether the ferroptosis pathway was triggered under the combination treatment. I learned the techniques about how to split, count, and place cells in the 96-well plates, how to make drugs at certain concentrations and treat the cells. Cell Titer-Fluo® assay from Promega was used to determine cell number decrease, cell viability of the cells in the combination treatments. A ferroptosis inhibitor, Ferrostatin, was used to block the ferroptotic process and rescue cell numbers if the cells do undergo ferroptosis in the combination treatment. As a result, I found in one of my experiments where the cell number decreased in the combination treatment group and was rescued in the presence of ferrostatin. However, the result has not been replicated so far so we believe the cells do not under go ferroptosis when the cell number decreases in the combination treatment.
Another hypothesis of mine is that the combined treatment inhibits the process of cancer cell invasion, thus leading to the cell number decrease. Expression of EMT markers like Snail indicates cell invasion in immunoblotting experiments. I have found suggest that the expression of snail in the immunoblotting decreased with the combined treatment. This proves my hypothesis that the application of the combination of Palbociclib and Bempodoic acid to cancer cells would lead to decrease in invasiveness.
In conclusion, all the data from my study suggest that combining CDK4/6 inhibition with ACL inhibition may be an effective treatment strategy in cancer, as it promotes cell apoptosis and block the cell invasion process.
My research experience with Dr. Krucher helped me understand the cell-division cycle and how any disruptions to the cell cycle could lead to cancer cell and tumor development. I also learned about different pathways that would lead to cell proliferation and cancer, such as the cyclin-CDK and the AKT/mTOR pathways, and the epithelial-to-mesenchymal transition (EMT) that leads to cell invasion. It is not easy to come up with a hypothesis and prove it ourselves. It requires our understanding of the topics we are interested in, access to the techniques and equipment from which we could data, and the ability to integrate, analyze the data and draw the conclusion. However, the entire research process is never as simple as having a hypothesis, completing the experiments, and drawing the conclusion. When we don’t have any protocols to follow, we had to develop one on our own. For example, when we added the ferrostatin to the combination treatment, we didn’t know how much we should add since there were no previous researches and data found on conducting this experiment. As a result, we had to figure the concentration out by ourselves, by adding Ferrostatin at different concentrations to the combination treatment. When the hypothesis was proved wrong, we have to find a new direction to study and keep working on it until we actually found something. With the help of Dr. Krucher, who has been in the cancer research field for more than ten years, we knew where to go when our hypothesis was proved wrong, and I could learn my lessons when I failed to follow the protocols when performing the experiments and obtain bad results. Overall, cancer cells develop resistance against single treatments easily. As we are studying new combination treatments that could actually be applied to human patients, it’s important to know the efficacy of them and. In the future, we may study if the cancer cells would develop resistance to this combination treatment. If so, we would like to know how long does the process take. And hopefully our results could help in finding an effective treatment strategy.