Polyunsaturated fatty acids (PUFAs) as Potential Anticarcinogenic Agents Against U937 Non-Hodgkin's Lymphoma Cancer Cells

Student: Katie Qin
Table: MED4
Experimentation location: Reseach Institution
Regulated Research (Form 1c): No
Project continuation (Form 7): No

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Abstract:

Bibliography/Citations:

Bibliography:

[1] Schmid A., Collomb M., Sieber R., Bee G. Conjugated linoleic acid in meat and meat products: A review. Meat Sci. 2006;73:29–41. doi: 10.1016/j.meatsci.2005.10.010.

[2] Racine N.M., et al.. Effect of conjugated linoleic acid on body fat accretion in overweight or obese children. Am. J. Clin.Nutr.2010;91:1157–1164.doi: 10.3945/ajcn.2009.28404

[3] Gerhard Agatha, Astrid Voigt, Eberhard Kauf, Felix Zintl Conjugated linoleic acid modulation of cell membrane in leukemia cells Cancer Letters, Volume 209, Issue 1, June 2004, Pages 87-103

[4] Ruth E. Patterson, et al., Marine Fatty Acid Intake Is Associated with Breast Cancer Prognosis. J Nutr. 2011 Feb; 141(2): 201–206.

[5] Zorica Cvetkovic, Vesna Vucic, Bora Cvetković, Milan Petrović. Abnormal fatty acid distribution of the serum phospholipids of patients with non-Hodgkin lymphoma August 2010 Annals of Hematology 89(8):775-82 DOI:10.1007/s00277-010-0904-6


Additional Project Information

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Project files
 

Research Plan:

Problem being addressed: 

Non-Hodgkin’s Lymphoma (NHL) is one of the most common cancers in the United States. Current treatment involves chemotherapy and radiotherapy, which have severe side effects. More effective treatments with less side effects are clearly the unmet needs for NHL cancer.

Materials and Methods

  • Culture U937 Lymphoma cell line 
  • Polyunsaturated fatty acids: CLA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), as well as conjugated linoleic acid (CLA) were prepared in various concentrations ranging from 0.1 M to 1 M.
  • Chemokine (C-X-C motif) ligand 10 (CXCL10) assay kit
  • Caspase assay kit
  • The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
  • Phorbol-12-Myristate-13-Acetate (TPA)

Experimental Assays to find PUFA’s effect on NHL cancer cells.

Cell Adhesion Assay:

  1. Place U937 lymphoma cells in a 24-well plate with these PUFAs and Phorbol-12-Myristate-13-Acetate (TPA). 
  2. After a 24-hour incubation period, 300 μL of a fixative solution is pipetted into each well, and the wells are rinsed thoroughly to remove excess waste material.
  3. An OMAX 40X-2000X Digital Binocular Biological Compound Microscope connected to a computer with ImageJ software is used to take pictures of the wells. 
  4. The quantity of cells is measured using the ITCN cell counter software to examine the effects of PUFAs on the proliferation of the cancer cells.

Caspase Assay: 

  1. Prepare a six-well plate containing U937 cancer cells with control, and 10 µL solutions of CLA, EPA, and DHA. 
  2. After 24-hours incubation, well contents are centrifuged and stored at -20°C for 24-hours.
  3. The cells are lysed and transferred to a 96-well plate, where a 5-µL caspase substrate is added. 
  4. Read the resulting solution using a microplate reader at 415-nm in 30-, 60-, and 80-minute intervals.

MTT Assay

  1. Treat U937 cell cultures with CLA, EPA, and DHA at concentrations of 1M, 0.1M, 0.01M, and 0.001M. 
  2. Add 10μL of MTT after 24-hours of incubation
  3. Incubate the assay further for 3-hours. 
  4. Perform Student's t-test on the average of 16-well samples and controls.

CXCL10 Assay:

  1. Incubate U937 NHL cells with control and 10 µL solutions of CLA, EPA, and DHA at 0.1 concentration for 24 hours. 
  2. Centrifuge the resulting samples
  3. Measure the CXCL10 protein in the supernatant by ELISA assay following the protocol supplied by manufacturer (R&D Systems, Minneapolis, MN). 
  4. Perform the Student's t-test on the results of the 6 timepoints of the sample and controls.       

Risk and Safety

  1.       Potential risk factors: U937 cell culture, PUFAs solutions, MTT solution, TPA 
  2.       Individual protection measures: 
    1.       Hygiene: Wash hands, forearms, and face thoroughly after handling chemical products, before eating, using the lavatory and at the end of the working period. Ensure that eyewash stations and safety showers are close to the workstation location.
    2.       Use the following for personal protection: Safety eyewear, Chemical-resistant, impervious gloves complying with an approved standard should be worn at all times.
    3.       Disposal methods: Disposal of these products, solutions should at all times comply with the requirements of environmental protection and waste disposal legislation and any regional local authority requirements. Dispose of surplus and non-recyclable products via a licensed waste disposal contractor. Waste should not be disposed of untreated to the sewer.

Data analysis

 

  1.       For cell attachment assay, effects of PUFAs on NHL cells will be compared with controls by looking at amount of cancer cells attached to surfaces of incubation plate wells. The images of the cells on the walls of micro-plate wells are counted using ITCN cell counter for EPA, DHA and CLA treated NHL cell samples and control. The well images of the cells will be in figures to visually compare difference between PUFA treated sample and control. The cell attachment reduction will be calculated and displayed in a bar chart with standard errors.
  2.       For MTT assay, the cellular metabolic activity will be measured after exposure to PUFAs. Based on a cell’s ability to metabolize compounds, MTT is converted from a yellow tetrazole into a purple formazan. The amount of formazan is recorded using a microplate reader, from which NHL cancer cell survival rates are calculated for PUFA samples and control. These calculations will be shown in a graph with standard errors. Student T-test will be conducted to compare the PUFA treated samples to control to demonstrate the significance of the effect of PUFA on NHL cells survival rates.
  3.       For Caspase-3 assay, the percent increase of caspase-3 concentrations will be compared with control and the results will demonstrate the PUFA’s effects on activation of caspase-3 enzyme and therefore increase cancer cell death through enhanced cell apoptosis. The difference in the caspase-3 activities will be displayed in a bar chart and standard errors will be added.
  4.       For CXCL10 assay, the percent increase of amount of CXCL10 will be calculated and compared with control and student t-test will be used to demonstrate the significance of the PUFAs’ effect on the cancer cells stimulation of CXCL10 enzyme expression and therefore increase the cancer cell death through enhanced NHL cell suppression caused by T-cells.

Questions and Answers

1. What was the major objective of your project and what was your plan to achieve it? 

       a. Was that goal the result of any specific situation, experience, or problem you encountered?  

       b. Were you trying to solve a problem, answer a question, or test a hypothesis?

It all started with my 9th grade Biology final project. I chose to research microalgae and its effect on its surrounding environment. The project showed that algae could have a devastating effect on its environment. They can outcompete other organisms for nutrients, congest the water, and ultimately destroy ecosystems. So my question was, could I somehow turn this problem into an opportunity? And I later found out that there are compounds in algae that potentially could help fight against cancer. 

There are three long chain polyunsaturated fatty acids or PUFAs, linoleic acid, LA, docosahexaenoic acid, DHA, and eicosapentaenoic acid, EPA, that can be found in algae. For this study, I used conjugated linoleic acid, CLA, which is basically a mixture of the different forms, or isomers, of linoleic acid. Algae is rich in these PUFAs due to multiple reasons. One reason is its adaptation to aquatic environments, where fatty acids are important in maintaining the fluidity and function of cell membranes, another is that they are also used for energy storage. These compounds are essential for the human body as well. For example, linoleic acid is very important for many different functions, one being brain function, and they cannot be synthesized in the human body, but they can, however, be found in vegetable and nut oils. Both docosahexaenoic acid and eicosapentaenoic acid can be found in fish or fish oil supplements, which are known to be good for our health. At the same time, I was learning more about the lymphatic system, which is responsible for maintaining our body’s fluid levels, defending it from infection, but also managing fats (or lipids). I wondered if these PUFAs could be helpful with fighting Non-Hodgkin’s lymphoma, one of the most deadly cancers in the US. Current treatment only consists of radiotherapy and chemotherapy, which have severe side effects, so hopefully this study would help develop treatments that are less invasive. 

The major objective of my research project was to study these PUFAs anti-cancer effects on the Non-Hodgkin Lymphoma cells. The hypothesis is that when Long-chain polyunsaturated fatty acids (PUFAs) are incubated with Non-Hodgkin’s Lymphoma (NHL) cancer cells, they will facilitate the NHL cancer cell apoptosis and therefore decrease the survival rate of NHL cells. Due to the involvement of cells and compounds derived from natural sources, I plan to use the experimental methods that most suitable for such research, namely Enzyme-Linked Immunosorbent Assay (ELISA) assays and other common assays for cells as well as immunology related studies.

2. What were the major tasks you had to perform in order to complete your project?

       a. For teams, describe what each member worked on.

After the literature search and determining the best methods to test the proposed hypothesis, the major tasks of the project included preparing the research materials, for example the PUFAs compounds, the ELISA assay kits, adhesion assay and MTT assay materials. Then the important steps were running the different assays according to the research plan and the assay protocols, and accurately recording the data to ensure data integrity. The next major tasks were Performing data analysis, generating graphs or charts, and interpreting results.

3. What is new or novel about your project?

       a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?

       b. Is your project's objective, or the way you implemented it, different from anything you have seen?

       c. If you believe your work to be unique in some way, what research have you done to confirm that it is?

The novel part of this project is that polyunsaturated fatty acids do have an effect on the cancer cells, in this case non-Hodgkin’s lymphoma. They can decrease the amount of cell adhesion to slow down cancer cell growth, increase caspase-3 activity to encourage apoptosis, decrease the survival rate of cancer cells, and increase the expression of CXCL10 for tumor suppression and apoptosis. If these results can eventually be utilized in the form of a less invasive cancer therapy with no or mild side effects, it can help make non-Hodgkin’s lymphoma a lot less dangerous. What is unique about this study is that these PUFAs can be derived from algae, which is naturally abundant and there are a good source of them. In fact, they might be too abundant at times, so I hope that by exploring algae’s potential in human health benefits, it would also help use algal blooms sustainably to mitigate their negative effects.

 

4. What was the most challenging part of completing your project?

      a. What problems did you encounter, and how did you overcome them?

      b. What did you learn from overcoming these problems?

The most challenging part of the project is that carrying out the experiment required the utmost precision and accuracy, and I learned that even one mistake could result in weeks of setbacks. I have encountered problems with handling the pipettes when transferring samples between samples in test tubes and incubation wells, as the speed of drawing liquid samples and how to deposit the samples into incubation wells could affect the experiment results. I practiced this using tap water and eventually learned to control my thumb movements to smoothly transfer the samples.

5. If you were going to do this project again, are there any things you would you do differently the next time?

Due to the time constraints, the project did not have a large sample size. If I were to do this project again, I would evaluate increasing the number of replicates because larger sample sizes can enhance the statistical power of this project. And this can also give more options for exploring different data analysis methods so I can more accurately interpretate results. 

6. Did working on this project give you any ideas for other projects? 

This project involved compounds derived from algae and their anti-cancer effects on the cell level. This gave me two different directions. On a macro point of view, I would love to work on projects concerning environmental protection and sustainability while on the micro level, I am very interested in cancer treatment and prevention, especially immunology related studies as cancer vaccines and targeted therapies are two biggest aspects of immunotherapies. 

7. How did COVID-19 affect the completion of your project?

No effects since the COVID-19 pandemic was not as severe as before.