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Essay: Investigate cancerous stem cells

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  • Subject area(s): Science essays
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  • Published: September 26, 2021*
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  • Words: 1,868 (approx)
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  • Investigate cancerous stem cells
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The purpose of my research is to investigate cancerous stem cells, which are cells that are able to undergo specialization based on the environmental factors and location of the cell (7). Cancer cells can have stem cell properties in their ability to propagate and reduce the likelihood of normal, programmed cell death (3). This particular article studies the impact of zinc on cancerous lung cells and its role in regulating stem cell properties.

Zinc was thought to suppress cancer stem cells since it plays a vital role in the function of human development the study found that zinc leads to anti-inflammatory response in immune cells, reduced oxidative stress in cancer patients and low zinc concentrations was common in patients with illnesses that went to hospitals across the world. Also, zinc concentrations were lower in patients that had been diagnosed with other cancers such as gallbladder and prostate cancer (6). In reference to lung cancer, it was found to stimulate cells and lead them to anoikis which is a beneficial process (5). The research completed by these scientists was to find whether zinc had a similar response in cancer stem cells in lung cancer, and to find a mechanism that allows zinc to mitigate the effect of cancer cell growth by limiting the stem cell properties (3). Other studies have found that the presence of iron can induce stem cell properties (1). All of these studies uncover the pathways in which ions are being used in the process that lead or inhibit cell migration, cancer cell proliferation, and anoikis. This research illuminates the importance of which protein kinase C (PKC) pathway is involved in cancer stem cell proliferaton. Also, with more research, there can possibly be a cure or prevention method against aggressive cancer phenotypes, in addition bettering modern treatments for cancer (1, 3).
Ninsontia, et al. noticed that zinc was a known ion regulator and is vital to many different functions within the body. It was also known that zinc caused the inhibition of cancer stems cells. In this study, they tested key proteins known to proliferate the aggressive phenotype of stem cells after exposing the cancer cells to zinc to determine the physiological pathway that leads to the inhibition. If was found that the phosphorylation of PKClet to the phosphorylation of -catenin, which lead to the extraneous cell death, ubiquitination, and lower concentrations of -catenin which would stimulate the harmful phenotypes of stem cells. It resulted in lower ALDH1A1 and CD133 proteins, as well as key transcription factors. Also, during testing, it found that the phosphorylation caused by zinc released a superoxide anion early in the mechanism. They hope that this pathway can be used in the future to help reduce metastasis, relapse, and deaths from lung cancer (3).
The cells used for this study were called H460 non-human, small cell lung cancer from the American Type Culture Collection. In a tumor sphere assay, there were two cultures done. The first one was generated from about 5000 cells per well placed in a serum-free well plate. These cells cultivated were reduced down to the initial cells and re-cultured in the same way. Each culture lasted 14 days, and the culture which was observed was the second culture (28 days total). The cells were analyzed for their phenotypic properties under a light-microscope. Then the cells from the culture underwent a Western Blot Analysis to measure the levels of proteins in the cells. The cells were pH and osmotically buffered, as well as treated with a protease inhibitor. Once the cells breakdown, the lysate was collected and underwent a protein assay to measure the concentrations of proteins in each sample.

From the lysate collection, 60g was used per sample, heated, and denatured. Once the proteins separated, they were placed onto nitrocellulose membranes and were blocked in milk and the initial buffering solution. The blocked samples were incubated with primary antibodies then washed then incubated with secondary antibodies. The protein samples were analyzed by chemoluminescence. The level of proteins was calculated using computer software (3).

The western blot analysis is relevant to this research because it compared the relative levels of concentration of the proteins CD133, ALDH1A1, Oct4, Nanog, and Sox4 in the cells after being treated with various concentrations of zinc. These are proteins and transcription factors that are commonly used to measure the upregulation of stem cell properties in cancerous cells. If after the exposure to zinc the cell protein levels for these proteins decrease, it can show that zinc has had an effect on the protein synthesis that leads to the negative phenotypes of stem cells (3).

In Figure 1I, the results from the western blot analysis are shown as the mean relative protein levels when exposed to 0, 5, 10, 25 and 50 M zinc for 72 h. The results were statistically tested using a one-way ANOVA in comparison to the control group (0 M). The western blot analysis tested for the proteins CD133 and ALDH1A1, and transcription factors Oct4, Nanog, and Sox4. The error bars illustrate the standard deviation for each trial and the asterisks illustrate significant differences in comparison to the untreated control group (Fig. 1). This study found that the CSC protein markers and transcription factors were significantly less than the untreated trial. Also, it shows that the also decrease as the concentration of zinc increases. The effect of zinc overall shows inhibition in the molecular expression of the CSC markers (Fig.1).

The purpose of this experiment was to describe the mechanism that allows for zinc to inhibit the CSC phenotypes. It was hypothesized that zinc is involved in the protein kinase C-PKC) regulation of the -catenin pathway. There is a direct connection between the purpose and hypothesis. The hypothesis can answer the purpose by testing to see if the mechanism proposed is real, feasible, and likely. The researchers tested this hypothesis by exposing lung cancer stem cells to varying conditions of zinc exposure. They evaluated the three-dimensional growth of tumor growth, cell viability, the relative levels and density of key proteins linked to the proliferative phenotype as well as -catenin. If zinc could impact any the tested characteristics associated with cancer stem cell growth, it could allow them to piece together the missing links in the pathway, and how zinc impacts the mechanism of PKC regulation of -catenin, which had not been well understood initially. The results showed that zinc was effective in reducing CSC phenotypes. It did so by showing that the key CSC markers were less abundant and less dense. It also showed inhibition by increases in PKCand decreases in -catenin as zinc concentrations increased within the cells. It showed less cell proliferation and less spheroid formation, which is a clear sign that the phenotype is inhibited, after exposure of zinc. These results show the expected result that zinc inhibits the phenotype of CSC, but the mechanism is established in the conclusion of the paper. They were able to establish that the inhibition from zinc was in fact related to PKCIt was found that zinc ions help phosphorylate PKCs which release a superoxide anion. The phosphorylated PKC, in turn, phosphorylates catenin. This signals for the ubiquitination of the cell as the phosphorylated -catenin increases and the non-phosphorylated -catenin decreases. The generation of the superoxide ion also assists in reducing tumor size and affects the concentration of the hydroxide radicals. The conclusions about the mechanism come from the results of zinc exposure for the mechanisms along the PKC and -catenin pathway (3).

Compared to other review articles on cancer stem cells, these results were the only ones that I thought were optimistic in a fight against cancer. I reviewed articles about the effect of iron (II), caveolin-1, nitric oxide (NO), and the E-cadherin expression; all of which, positively impacted the cancer stem cell proliferation through one or more characteristics. I also noticed a difference in the articles’ methods, results, and discussion, and the way each researcher present the information. I think that the graphs were very cluttered, and not very well explained for the zinc inhibition (3). In comparison to the zinc inhibition article, the graphs for iron and NO inducing effects were presented in a manner in which was easier to visualize (1, 8). I also noticed in the paper about caveolin-1 that the description of the methods about the results were more descript about what tests were used to collect the results. Both papers had done as such, but for the paper about caveolin-1, it was clearer what each method was testing (proteins tested were listed for the western blot analysis), and what results that specific test generated (2). Since all the papers I reviewed revolved around cancer stem cells in lung cancer, I liked that a migration, an invasion, or both assays were done in the caveolin-1, NO, and iron (1, 2, 8). I think that it is an important piece of information, since CSCs often lead to metastasis, to mention the effect in which these compounds have on the spread of cancer to other tissues. The study about zinc lacked this. I think it could be improved if their proposed mechanism could have been linked to not only tumor growth but the ability for lung cancer CSCs to spread within a non-cancerous sample of lung tissues, but to other organ tissues and propagate there, the research could have offered more to readers and fellow researchers.

I think the research from this paper was better than the other reviews because it went into detail about the CSC phenotypes. Since a mechanism was proposed and tested, the biochemical analysis and physiology of CSCs were very descriptive, as well as how each part of the mechanism could be linked to a result. This was much easier to read and link to other studies compared to the NO article. The NO article had similar tests and tested similar mechanisms with similar proteins, but I found it difficult linking NO to the mechanism, and how each test connected to that pathway (8). It also seems as if there is more background information on the role of zinc in oncology compared to other studies. For example, iron is prevalent in the human body and has a multitude of obdurate functions from blood circulation to human metabolism, and can even be toxic. On the topic of CSCs, there are many proposed mechanisms, and it is difficult to explain the results or determine a mechanism because there are many uncertainties (1). For the article about zinc, every result could be describing a part of a mechanism along a pathway, and for iron and NO research, it was more difficult to generate a “big picture” (1, 8). Lastly, the articles about zinc and the E-cadherin expression mention how their data can be used in modern treatments of cancer (4). Each article talks about how their data can affect a patient undergoing chemotherapy or radiation. The article about zinc inhibiting effects mentioned that in the future could be used in supplementation with modern treatments (1). The article about E-cadherin expression is strongly linked to the resistance of modern treatments and how it can be strongly linked to metastasis (4).


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