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.
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.