The work I’ve been doing with Dr. Nancy Krucher involves the very invasive disease, Pancreatic cancer. We have worked on a possible solution to the invasive behavior of cancer cells that comprises of knocking down a specific phosphatase subunit and its effect on the Retinoblastoma tumor suppressor protein. We also studied the effects of the drug Erlotinib on cell death. At the start of the year, I worked with 96-well plates for various experiments. Doing this allowed my pipetting skills to improve greatly and I gained more knowledge of knockdowns and cell growth. Some experiments included creating and testing several concentrations of Erlotinib and finding it’s optimal concentration for cell death. Then after finding this concentration, we treated the MiaPaCa-2 pancreatic cancer cells with PNUTS knockdown to inhibit invasion for 48 hours and proceeded with Erlotinib for 24 hours. Afterwards, to count cells we used Cell Titer-Glo spectrophotometer assays to see how effective the combined treatment was in killing off cells. This process took several experiments for me to get the hang of the techniques but the results were great. There were plenty of experiments that were flops, and some that were good but had very big error bars for our data (which isn’t so great). However, multiple experiments of mine showed a decrease in cell count when the cells were treated with both PNUTS knockdown and Erlotinib.
To further look into how PNUTS knockdown would affect the cells, we studied its effects on invasion. I had to perform immunoblotting experiments to see the differences in expression for several EMT (Epithelial-Mesenchymal Transition) markers. Before this experiment, I didn’t know a thing about EMT and it honestly helped me so much in one of my classes afterwards when reviewing other scientific papers. Our main focuses were the markers Zeb, N-cadherin, E-cadherin, and Vimentin. This took me so long to get used to. I had to learn how to lyse cells and extract protein and create samples to run a gel with. The hardest part was finding the concentrations of protein and creating samples properly. If this wasn’t done correctly then my gels were pointless and the bands on my nitrocellulose paper wasn’t reliable. I was able to finally work through my mistakes with some practice and obtained some good results. I found a decrease in both Zeb and N-cadherin expression; which signifies a reduction in invasiveness due to the knockdown. I’m still currently working on experiments like what was just mentioned but with other markers and even touching on different pathways affecting invasion as well.
Throughout this whole experience I’ve realized how great it is to work in the lab and conduct such interesting research. My goal in life is to become a doctor but with my experiences this past year I actually am looking into joining a program to obtain an MD-PHD degree. I want to continue researching in the future. Yes, it gets frustrating when experiments don’t go as planned or when results get messed up, but as Dr. Krucher would always remind me, everything takes time and practice. It is always a learning experience. Even when I thought I knew how to do everything properly there was always something else to be learned and practiced. I am so proud and happy with what I’ve done in this past year and so thankful I’ve been able to do it. I’m very appreciative of the UGR program and Dr. Krucher (for putting up with all my mistakes!). Her guidance has helped me greatly and I hope the project continues to bring positive results!
These past few months have definitely been a great experience for me. In the beginning, and even a little bit now, I would find myself getting frustrated and discouraged if my experiments weren’t working or I couldn’t get a technique right. There has been a big improvement and I’m thankful to have the experience I am having.
Pipetting seems like it would be such an easy task. However, when pipetting <10 uL substances, there is such a big chance for error. A lot of my experiments have turned for the worst because I left just a drop of substance on the pipette tip by accident. I have greatly improved on my pipetting technique and a few of my experiments have shown great results too! I am currently working on gels showing the effects of PNUTS knockdown on certain EMT markers. My recent experiment with the MiaPaCa2 cell line showed a decrease in the EMT regulator Zeb when PNUTS was knocked down, which is what we were hoping to see!
My project with Dr. Krucher has taught me so many useful techniques that I wouldn’t have access to learning in just a regular classroom. I have even learned so much more about different markers of invasion in cancers and pathways of cancer.
Not much has gone on since the last time I have posted a blog. We were unfortunately set back by a loss of MiaPaCa2 cells. It seemed as though they were growing too slow after a long time of splitting. This affected our results greatly and started to make them unreliable. New cells were ordered, a new line called PanC1 cells, but the major setback was that the shipment was delayed. We were unable to work on any cells for some time. However, we have received those cells (which we were very excited about!) and started conducting the same experiments to see if we would get the same results as before.
My skills at splitting, transfection, and pipetting have greatly improved. Before, I was unable to pipette evenly (something which seems so easy to do yet it takes so much practice). As far as this new experiment I have worked on, the results (and cell counts within each well) have been fantastic! We achieved the same result as with the MiaPaCa2 cells. These results lead us to believe that the combined effect of both drug and knockdown yield a much lower cell count. I am to conduct another experiment like this to confirm our results, which I have no doubt will go as smoothly as this one has.
The title of my research project is “The Combined Effect of Erlotinib and PNUTS Knockdown in Pancreatic Cancer Cells.” The purpose for this project is to compare the effectiveness of the drug Erlotinib and PNUTS Knockdown alone versus the combination of the two. Cells that are cancerous are constantly dividing and resist apoptosis (cell death). Pancreatic cancer is hardly diagnosed, and even when diagnosed in its early stages, it progresses very quickly. Erlotinib is a drug treatment used in pancreatic cancer patients. However, we also want to see if the effect of that drug would be better if used with a method called PNUTS Knockdown. This is a technique used to manipulate a tumor suppressor protein, Rb, which is constantly active in cancer cells.
Working under Dr. Krucher, I know I’d learn a lot in the field of cancer research. Already, she has taught me the basis of our experiment along with the methods she has been using for years. I have learned only a percentage of what I know I will experience in the next year. In order to research how to treat cancer cells, it’s important to know how they work first. That was the first thing I learned so far, specifically with the tumor suppressor protein we’re focusing on. Until now, I have already learned how to split cells and count them microscopically, and to plate them in 96-well plates. Then I also learned how to dilute the drug concentration multiple times and add them to some wells while also being able to do a PNUTS knockdown for others. Using a spectrophotometer, we can see if my experiment has supported our hypothesis or not by finding the cell count for each specifically treated well. So far the experiment is going well and Dr. Krucher has already told me within the next week or so I’ll also be learning to do Western Blotting and by the end of the semester we will start to form the cancer cells in a three-dimensional models. I have already learned so much from this experience. My first week, I was making mistakes everywhere since I’ve never had the opportunity to work in a lab before. However, I can say I’ve greatly improved in the techniques I’ve learned and will only continue to improve for the next year.