Category: Keyi Cheng

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.

 

From the experiments we did, we found that the combined drug might affect ferroptosis and lead to cell death. However, until now we haven’t been able to repeat the experiment and acquire more significant data. This makes us believe that the cancer cell number decrease in the combined treatment is not caused by ferroptosis. And then we came up with a third hypothesis, that the combined treatment would interrupt cell invasion and prevent the cell number from growing.

Epithelial-to-mesenchymal transition (EMT) plays important roles in physiologic and pathologic processes such as organ development in embryos, wound healing and cancer metastasis (Wu et Al., 2020). It is the first step of cell invasion. In this process, epithelial cells lose the cell-to-cell adhesion and cell polarity. The cells change morphology and acquire motility, migratory potential, and invasive properties (Wu et Al., 2020). In terms of cancer cells, the EMT process will eventually lead to metastasis, which is the spread of cancer cells from the place where they originally formed to another part of the body, through blood or lymph system (National Cancer Institute, n.d.). They will a new tumor in other organs or tissues, but maintain the same cell type as the primary tumor.

To test the effect of the P+B treatment on cancer cell invasiveness, we first did a wound healing assay in the 231 breast cancer cells. We took four petri dishes with 231 cells in them and treated them with DMSO, Palbociclib, Bempedoic Acid, and P+B respectively (D, P, B, and P+B). Then, we scratched a line in all four dishes to create wounds in the same size. After a certain amount of time, we took pictures of them found that the wounds healed in D and P. Meanwhile, the wound was not completed healed in B. In the P+B petri dish, the healing process was even slower, leaving a wider wound in the cell culture. Our data indicated that the P+B treatment inhibit the invasiveness of cancer cells.

Fibrosarcoma is a malignant mesenchymal tumor derived from fibrous connective tissue. It is a highly invasive cell line. So, we want to use HT 1080 fibrosarcoma cells (HT 1080) to test the effects of P + B on cell invasions, whether the treatment will inhibit it or not. First we want to find what EMT markers are expressed in HT 1080 cells. We did a western blot and found that N-cad, Snail, Zeb are good markers for detecting invasion. Our next step is to treat HT1080 cells with P, B and P + B and see if the treatments would reduce N-cad, Snail, and Zeb expression in them and affect cell invasion.

 

References

National Cancer Institute. (n.d.). Metastatis. In National Cancer Institute Dictionary of Cancer Terms. National Cancer Institute.

Wu, Y., Zhao, Y., He, X., He, Z., Wang, T., Wan, L., …Yan, N. (2020). Hydroxypropyl-[beta]-cyclodextrin attenuates the epithelial-to-mesenchymal transition via endoplasmic reticulum stress in MDA-MB-231 breast cancer cells. Molecular Medicine Reports, 21(1), 249-257.

From previous experiments, we found that the combined treatment of Palbociclib and Bempedoic Acid would lead to the decreases of cancer cell numbers. In order to figure out the pathway that causes the cell death, we have conducted more experiments on breast cancer cells 231 and pancreatic cancer cell Panc1 since the beginning of this semester.

We first assumed that these cancer cells underwent apoptosis after the combined treatment. Caspase-Glo^® 3/7 Assay was used to determine the apoptotic process of 231 cells in a standard 96-well assay plate. On the first day of the experiment, 6,000 cells were counted and placed in each well from well B2 to E7 in the plate. On the second day, we added Media, DMSO, Palbociclib (P), Bempedoic Acid (B), P+B, Staurosporin (ST) to columns 2 to 6 respectively, four trials for each drug (B:E). Staurosposin is a drug that triggers apoptosis in cells. Four days later, we observed that there were fewer cells in the P+B group and almost no cells in the ST group. After we made Caspase Glo reagent, added them to the place and read the plate, the fluorescence level of the cells indicates that most cell death in the ST group was caused by Apoptosis, whereas in the P+B group, not many cells underwent apoptosis. We repeated this experiment once and obtained the same results. Because the plate were read 3 days after we added the drug, the apoptotic process might had occurred between Day 2 and Day 5 so that we failed to catch it. Therefore, we decided to use the RealTime-Glo® Assay in our next experiment. It allowed us to measure apoptosis in the plates multiple times for days without killing the cells.  Meanwhile, we wanted to test if the cell number decrease under the combined treatment is caused by other pathways like ferroptosis.

Ferroptosis is a non-apoptotic, iron-dependent, oxidative cell death primarily implicated in inflammation (Skouta et al., 2014) Accumulation of reactive oxygen species (ROS) in a cell can lead to degradation of cell membrane and other damages that are lethal to the cell. The cell can detect such damages and trigger ferroptotis. It has been found to be involved in diseases like cancer, myocardial infarction and many neurological diseases (Li et al., 2019). Researches done by Skouta et al. indicate that ferrostatin is a potent inhibitor of Ferroptosis (2014). Ferrostatin (F) inhibits cell death by processing through a reductive mechanism to prevent damage to membrane lipids (Skouta et al., 2014). Our second assumption is that the cell number decrease is caused by Ferroptosis. If our assumption is right, we should observe cell death rescue when we add ferrostain to the combined treatment.

In our fifth experiment, Panc1 cells were placed in the standard 96-well assay plate (3000 cells/well, B2:E9). On the second day, we added Media, DMSO, P, B into Columns 2 to 5 respectively, and P+B into Columns 6 to 9. In Column’s 7 to 9, ferrostatin of different concentrations (10µM, 1µM, 100nm respectively) were added. From Day 3 to Day 6, the cell number change was observed under the microscope and any apoptosis was observed using the RealTime-Glo® assay and reagent. We did not detect significant apoptosis in the P+B group. Thus, it was drawn that the cell number decrease was not caused by apoptosis, and we focused on detecting ferroptosis rather than apoptosis in the following experiments. In the meantime, ferrostatin did not have a significant effect on rescuing cell death. We believed that the Panc1 cells we used were old and did not actively react to the drugs. Also, the ferrostatin concentration might be so high that it caused cell death.

In Experiment 6, new Panc1 cells (3000 cells/well) were placed in a new plate (B2:E11). On the second day, we added Media, DMSO, P, B into Columns 2 to 5 respectively, and P+B into Columns 6 to 8. In Columns 7 and 8, ferrostatin of different concentrations (10µM, 1µM respectively) were added. In Columns 10 and 11, only ferrostatin was added to the cells (10µM, 1µM respectively). On Day 6, the Cell-Titer Fluor Assay was used to read the plate, which measures the relative number of viable cells in culture. As a result, we found that ferrostatin (Column 10 & 11) did not lead to cell death, meaning it is safe to use ferrostatin as the ferroptosis inhibitor. In the meantime, ferrostatin still did not show signs of rescuing cell death.

In Experiment 7, we counted and plated Panc 1 cells using the same data from Experiment 6. On the second day, we placed the same drugs into Columns 2-5 and added P+B into Columns 6-11. In Columns 10 and 11, ferrostatin of different concentrations (10µM, 1µM respectively) were added. In Columns 7 and 8, we added N-acetyl-L-cysteine (NAC, 1µL & 2µL respectively) to the P+B treatment. NAC is a ROS inhibitor. If the Panc1 cells did undergo ferroptosis, NAC would prevent ROS accumulation from signaling the cell death, which means the number of viable cells in these two columns will be higher than that in the P+B group. Cell-Titer Fluor was used to detect the numbers of viable cells in each column. Our results from Experiment 7 suggested that there wasn’t enough cell death in the P+B group. We decided to decrease the cell confluence in each well by placing 1000 cells/well in Experiment 8 while keeping other data the same. Results from Experiment 8 indicate that the cell number in the P+B group significantly decreased, and the cell death was rescued in the P+B+1µM F group. There was no significant cell number increase in the P+B+NAC group. We will repeat more experiments to compare cell numbers in the P+B and P+B+1µM F group in order to draw our conclusion that the cells underdo ferroptosis. We found that the cell numbers in the wells at the corner of the plate are relatively lower than those in other wells. We believed that it resulted from evaporation. Therefore, in the following experiments, we will add 200µL phosphate buffered saline (PBS), a salt solution used to wash and transport cells, around the wells we plated cells in, to minimize the effect of evaporation.

These are the data and results we have obtained so far. We have found that the cell number decrease under P+B combined treatment did not result from apoptosis but was very likely caused by ferroptosis. In future experiments, we need to manipulate the concentration of P & B in the treatment in order to get a larger decrease in cell number. Also, we plan to do the same experiment on breast cancer cell 231 to see if ferrostatin is able to rescue cell death in different cell types.

References

Li, X., Duan, L., Yuan, S., Zhuang, X., Qiao, T., & He, J. (2019). Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via down-regulation of TGF-β1. Journal of Inflammation, 16:11.

Skouta, R., Dixon, S. J., Wang, J., Dunn, D. E., Orman, M., Shimada, K., … Stockwell, B. R. (2014). Ferrostatins Inhibit Oxidative Lipid Damage and Cell Death in Diverse Disease Models. Journal of the American Chemical Society, 136(12), 4551-4556.

The title of our project is The Effect of the Combined Treatments of the CDK4/6 inhibitor Palbociclib and the ACL inhibitor Bempedoic Acid on Apoptosis in Breast Cancer Cells.

The Retinoblastoma (Rb) family is a group of instrumental tumor suppressors in human cancer. Constant phosphorylation of Rb due to the overexpression of cyclin dependent kinases (CDKs) activates further oncogenic signaling pathways and drives the cell cycle, resulting in human cancers like pancreatic, breast or colon cancer (Wang et Al., 2017). In order to keep Rb active, scientists have tried to developed CDK inhibitors as cancer treatments, for example, Palbociclib – one of the drugs we use in our experiments. ATP citrate lyase (ACL) is an enzyme that serves to synthesize cytosolic acetyl-CoA from citrate during fatty acid and cholesterol biosynthesis (Burke & Huff, 2017), which are responsible for the formation of phospholipid cell membrane bilayer and other necessary cholesterols in cells. During cancer cell replication, the cell membrane has to be generated all the time, meaning ACL is constantly working to convert citrate to acetyl-CoA in preparation for phospholipid production. An ACL inhibitor, Bempedoic Acid that is used in our experiments, has been discovered to inhibit this procedure.

Our aim is to prove that the apoptotic process of breast cancer cells has occurred in the single drug treatments where Palbociclib (P group) or Bempedoic Acid (B group) is applied to the cells. And in the combined treatment (P+B group) where both Palbociclib and Bempedoic Acid are added to the cells, the percentage of apoptosis would be higher. So far, we have found that the cell numbers significantly decreased in the P+B group, which is the evidence of cell death. Furthermore, we want to determine what kind of cell death has been triggered.

Caspase is a family of protease enzymes that plays essential roles in apoptosis. If we are able to locate caspase in a cell, it tells us that the cell undergoes apoptosis. Using a Caspase-Glo^® 3/7 Assay, we are able to measure caspase-3 and -7 activities. On the first day of an experiment, we count cancer cells and place them into wells of the assay, usually around 6000~10000 cells per well depending on the cell type. On the second day, we add drugs into the wells in P, B and P+B groups. Three or four days later, we will measure the caspase level in these wells. The Caspase-Glo^® 3/7 reagent will cause cell lysis. Caspase enzyme inside the cell will cleave the substrate from the reagent so that it generates a luminescent signal, indicating the cells have processed apoptosis (Promega Corporation). For now, I just get used to this technique and have done 2 of these experiments. If the results come out well, we will use different techniques to measure the apoptosis level at different times after they are treated with drugs. In this case, we are able to find out when the apoptosis actually happens between Day 2 and Day 5 or 6.

Not many researches have been done on Bempedoic Acid and the combination treatment of Bempedoic Acid and Palbociclib. Since we have discovered that cancer cell numbers would decrease after the treatment, we expect to find the pathway that leads to the cell death. For me, I think it is meaningful as I will learn different cell division pathways and how to inhibit cancer cell division through drugs that can block one of the pathways and trigger cell death.

 

 

References

Burke, A. C., & Huff, Murray. (2017) ATP-citrate lyase: genetics, molecular biology and therapeutic target for dyslipidemia [Abstract]. Current Opinion in Lipidology. 28(2),193-200.

Promega Corporation. (n.d.) Caspase-Glo® 3/7 Assay Technical Bulletin: instructions for use of products. Wisconsin. Promega Corporation. Retrieved from https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/101/caspase-glo-3-7-assay-protocol.pdf?la=en.

Wang, D., Sun, Y., Li, W., Ye, F., Zhang, Y., Guo, Y., …Suo, J. (2018). Antiproliferative effects of the CDK6 inhibitor PD0332991 and its effect on signaling networks in gastric cancer cells. International Journal of Molecular Medicine, 41(5), 2473-2484.