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Cancer is a vast disease category that requires constant research for a cure. Over the decades, different treatment plans have arisen to try and combat the range of cancer cells. Some treatment has resulted in success, some end in death, and others are still an ongoing battle for the patients. Anti-cancer drugs have many side effects that come with them such as fatigue, inflammation, infection, and necrosis of cancerous cells. Cancer medicine has come a long way and now many drug treatments can mimic the natural process of apoptosis. Apoptosis is known as programmed cell death. As advance as the drugs are, they still need the fine tuning to accurately attack cancer cells while protecting the healthy cells. This is a challenge for many researchers in moving forward with cancer medicine. NoGro is a company that is looking to develop an anti-cancer drug that works with the best drug uptake mechanism of mammalian cells. The goal of the experiment was to understand how environmental factors such as temperature and time could influence the way the drug is taking up to the cell. An analysis of these factors would allow the company to make an informed decision based on the effectiveness and success of an anti-cancer drug with the use of the drug possibly accompanied by a cytoskeleton inhibitor. This experiment concluded that the use of transferrin in uptake is the most effective and gets stronger as time goes on. Also, the in the presence of a cytoskeleton inhibitor with the drug is not effective as without.
Cancer is a diverse collection of diseases that are characterized by multiple etiologies with constantly changing combinations of genetic and epigenetic alterations (1). Key features of cancer cells include resisting cell death, replicative immortality, evading growth suppressors, and sustaining proliferative signaling. The ability of cancer to resist cell death or apoptosis is a major factor in cancer promotion and survival. Apoptosis is a gene-directed process of programmed cell death in multicellular organisms (2). There are many different ways to treat cancer. Some more effective than others and some that don’t work with the particular patient at all. A few treatment procedures can include surgery, radiation therapy, chemotherapy, immunotherapy, and stem cell transplant (3). The new go-to treatment for cancer has been anti-proliferative drugs that slow or halt tumor progression while being significantly less toxic to normal cells (4). Another common anti-cancer drug that has been introduced are cytoskeleton inhibitors. The cytoskeleton interacts with actin and microtubules playing a crucial role in cell division. Because of this connection, cytoskeleton inhibitors can be used against cancer cells.
How it works
To be able to use these drugs, there must be an understanding of the mammalian cell uptake mechanisms and effectiveness of drug delivery to specific cells. Anti-cancer drugs must have a high level of specificity and direct targeting, which is a major challenge in designing drugs for cancer treatment (5). In this experiment, multiple uptake mechanisms were looked at to assist in designing the best delivery of drug to the cell. Cells are able to take up material by phagocytosis and endocytosis. Endocytosis can be broken down even further into pinocytosis and receptor mediated endocytosis (RME). In phagocytosis, cells can envelope and internalize bacteria or other larger particles. Relatively few cells carry out the process of phagocytosis, but the ones that do are generally lower eukaryotes. With endocytosis, the plasma membrane invaginates to form a vesicle. Most cells carry out this kind of process. Endocytosis can then be divided into pinocytosis, which happens by non-specifically taking up small droplet of extracellular fluid. On the other hand, RME has cell surface receptors that specifically binds to ligands, the ligands are released into the intracellular endosome, and the receptor can be recycled back to the plasma membrane (6).
In this study, the environmental factors such as temperature and incubation time were examined to see what kind of effect it would have on the drug uptake process. Two different ligands, low-density lipoprotein (LDL) and transferrin (Tf) will be used in the RME process and as variables of this study. LDLs bind to their receptor, form a vesicle to early endosome, undergo a conformational change, and releases into the cell as free cholesterol. Whereas Tf binds to iron as a protein and uses this connection to release iron ion into the cell. Both of these processes allow for specificity of uptake. Cytoskeleton inhibitors will be used in this experiment as well to see if it assists in the uptake mechanism. Examining the uptake mechanism of cells is of utmost importance because it will provide a better understanding to target specific cancer cells and how to make the most effective anti-cancer drug. We hypothesize that the drug will be most effective at 37°C with transferrin and its effectiveness will increase will time, but if it is treated with the cytoskeleton inhibitor, it will lose its effectiveness.
Six humid chambers were made and then set into the ice bucket to pre-chill. Six cover slips with cells were taken and labeled. The next step was to remove the media and wash three times with 2 mL of ice cold 1X PBS. Leaving the last rinse in the petri dish, petri dishes was put into the ice bucket to pre-chill as well. Petri dishes quickly transferred into separate humid chambers and treated with one 50 µL drop of labeling solution. All six chambers with the cover slip petri dish were placed into the ice bucket and incubated for 5 minutes. To test the effect of temperature on the uptake mechanism, three of the chambers were taken out and labeled for the incubator, ice, and room temperature. The incubator dish was put into the incubator at 37°C for 15 minutes, the ice dish was left in the ice bucket at 4°C for 15 minutes, and the room temperature dish was placed in a drawer for 15 minutes. To test the effect of time on the uptake mechanism, the other three chambers were labeled 5 minutes, 10 minutes, and 15 minutes. All three chambers were placed into the incubator at 37°C for their respective labeled times. After all chambers completed the amount of time needed under their own conditions, media was removed and cells were washed twice with 2 mL of ice-cold 1X PBS. The cells were fixed by pipetting 2 mL of 4% paraformaldehyde solution in PBS into the petri dish. All chambers were left to incubate for 10 minutes at room temperature. The paraformaldehyde solution was removed and the slip was washed twice with 2 mL of room temperature 1X PBS. Lastly, a drop of vectashield (with DAPI) was put on the slip, the cover slip was placed cell side down on the glass slide, and the corners were sealed with clear nail polish for all slips.
The following experiment tested diluted-LDL and FITC-Tf without inhibitor (control) and diluted-LDL and FITC-Tf and nocodazole inhibition (treatment). The first few steps are the same to set up the control and treatment. A humid chamber was made for control and treatment and labeled in that respective way. The chamber was then put into the ice bucket to pre-chill. A cover slip with cells in a petri dish was obtained for control and treatment. For each, the media was removed and washed three times with 2 mL of ice-cold 1X PBS. Leaving the last wash in the petri dish, the dish was placed into the ice-bucket to pre-chill. For the control, after pre-chilling, the petri dish was transferred to the humid chamber. The PBS in the control was removed and then a 50 µL drop of labeling solution was added. The control was incubated for 5 minutes on ice and then incubated at 37°C for 20 minutes. For the treatment, after pre-chilling, PBS was removed and 1 mL of medium was added to be followed by incubation on ice for 15 minutes. The petri dish was transferred to the humid chamber and one 50 µL drop of nocodazole-treated was added to the cover slip. Treatment dish was incubated on ice for 5 minutes and then put into the incubator for 20 minutes. Lastly, to fix the cells the same procedures for fixation from the first experiment was performed.
The results of this experiment showed that in the variables of temperature and time overall transferrin was the better method of an uptake mechanism for drugs to get into the cell. This can be seen from the results in Table 1 with the higher average percentage of transferrin taken up in the experiments of temperature and time. This result makes sense because the use of LDL takes a longer amount of time to release into the cell and also has more stopped checkpoints to go through before it reaches the cell. It can also be seen that there was more of a take up of transferrin and those percentages only increased as time went on. This can be seen in the time section of Table 1. In the values to the left of transferrin averages, it can be seen that LDL had a much smaller uptake. LDL did increase as well as time increases, but nowhere near the value that of transferrin. With the factor of temperature in Table 1, it shows that the temperatures below 37°C did not have as much uptake. This result makes sense because mammalian cells typically function normally at temperatures near 37°C. The temperatures of this range are considered abnormal and cells have to try to adjust possibly leading them not to function properly (7). In Table 1 also, the control showed to be 60.19% and treatment showed to be 0.23%. This tells that the cytoskeleton inhibitor did not do much to help the drug in its uptake. All of these results collected from this experiment support our hypothesis. The results greatly help in the possible design of a better drug. It would need to take into consideration to be able to release the drug at a specific temperature, have it stay in as long as possible, and not need to use a cytoskeleton inhibitor.
During the experiment, it was said to not pipette directly on the cover slip because cells could be wash into the petri dish. Many times while pipetting, it would be done very close to the cells. This could have caused error in losing a viable number of cells because after washing the rinse is thrown out. A mistake like this could have caused the experiment to not have as many cells and ultimately mess with the cell count. Another pit fall of the experiment could be that the chambers were not kept chilled after the first pre-chilling. This could occur from the constant taking out of the chambers to wash and to add labeling solutions to the cells. It was mentioned several times that keeping the cells cold is important for the experiment. One last error that could have caused results to be skewed is the transfer of the cells from the cover slip to the slide. On several occasions, it was difficult to pick up the slide with the needle hook and in one case the cover slip broke in a corner. Here, there must have been a loss of cells.
For future research into anti-cancer drugs, there needs to be great specificity in the temperature that the drug can be released at and the length of time that it can stay effective at the temperature. There should also be a deeper look into how to get transferrin or LDL to work better with cytoskeleton inhibitors. The results showed that transferrin could not take up as much. Thus, the inhibitor basically stopped the effectiveness of the drug and that is not what should be happening. The use of Tf receptors on the LDL receptor to treat tumor cells should be considered because it is already seen that TF is most effective, but LDL is more structured. The use of the drug with inhibitor should be looked into to make a better combination. This is because using it at the same time does not do anything for the cells (8,9). It might be a better idea to use just the drug or the drug then the inhibitor.
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