Antioxidants and Lung cancer

LuciaHuo · Post by **LuciaHuo** » Sat Nov 03, 2012 10:44 pm

I hope to find the affect of one type of antioxidant on one type of lung cancer tumor cell. Groups of the cells will be each injected with a different substance to increase the amount of it in the cell. Dose-response to antioxidants will be tested and comparisons will be made.

How can I increase the amount of a substance in a cell? Does the amount of a substance in one's body reflect the amount of it in one's cell? What are better procedures for this experiment?

donnahardy2 · Post by **donnahardy2** » Sun Nov 04, 2012 7:37 pm

Hi Lucia,

Welcome to Science Buddies! This is an excellent topic for a science fair project. I don't think there is any way to increase the uptake of antioxidants inside the cell. I assume you will be growing the cancer cells in tissue culture medium, so you can just add different concentrations of antioxidant to the growth medium. It's very good to include the dose response as part of your experimental design.

What type of lung cancer cells will you be testing? What type of antioxidant will you be using? The best way to ensure an optimum experimental design is to read about the research that others have done on the topic, and then design an experiment that will answer a unique question.

What information about the effect of antioxidants on lung cancer cells have you found so far? Antioxidants are generally considered to be protective against the oxidative processes that lead to the development of cancer, but I'm not sure what effect they have once the cancer cells have developed.

If you have not found a mentor yet, please check out the guidelines for this process on the Science Buddies website. This article contains lots of helpful advice:

https://www.sciencebuddies.org/science- ... tors.shtml

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Sun Nov 04, 2012 10:11 pm

I have been having trouble finding a mentor who has a lab with lung cancer cells lines that I can use. Thus, I have decided to take a step back and take other types of cancer into consideration as well, such as skin and lymphoid cancer. I haven't decided on what antioxidant to use, but I do know that I want to use one that has not been frequently tested before.

I know that free radicals- unstable molecules- and ROS (reactive oxygen species) result from oxidative stress. Free radicals are unstable so they tend to steal electrons from neighbouring cells to stabilize themselves. This sets off a chain reaction and this entire process is very damaging to DNA. ROS are basically free radicals with oxygen and they cause damage to DNA, oxidations of polyunsaturated fatty acids in lipids, oxidations of amino acids in proteins and inactivation of specific enzymes by oxidations of co-factor. Antioxidants an terminate chain reactions by removing free radical intermediates, and inhibit other oxidation reactions.

Thank you very much for your reply and your help

. This is a very challenging topic for me. Please offer any suggestions you have.

donnahardy2 · Post by **donnahardy2** » Mon Nov 05, 2012 9:23 am

Hi Lucia,

It sounds like you have done your background reading and have a definite objective in mind. I think it would be worthwhile to do this project with any type of cancer cell that you might have available to work with, if you are not able to find lung cancer cell lines to work with.

Donna

LuciaHuo · Post by **LuciaHuo** » Mon Nov 05, 2012 11:22 pm

I have contacted many researchers and have not received a positive response. I understand their reasons for not saying yes though; my project asks for a lot from the mentor and it's hard for them to trust a grade 9 student with a professional lab because everything is very precise in terms of using the equipments and things like that. Thus, I'm planning to take a step back and try to simplify my project to its core, so much that I will not need a lab and cells to complete my project, even though that's the ideal situation. I'll continue to try to find a mentor with a lab, but I will start researching more about the chemistry involved in this topic, which I think is the most simple way to achieve an accurate answer to this kind of topic. I'm thinking of testing the reactions of different oxidations to antioxidants. Any suggestions on how I can make my experiment simple but still have a high level of accuracy?

deleted-108366 · Post by **deleted-108366** » Wed Nov 07, 2012 2:21 pm

Dear LuciaHuo,
Testing Antioxidants can become quiet complicated or simple depending on what kind of equipment you have available. From what I have seen to be relatively accurate the Folin-Ciocalteau Total Phenolic Assay, seems to be relatively simple but has a degree of accuracy and all you need is a spectrophotometer with some solutions. Check out this web page, it contains the majority of antioxidants testing that food companies perform and their relative inaccuracy/irrelevancy to this parent company’s claim. I hope this helps.

http://foodscience.cornell.edu/research ... -assay.pdf

As far as, Cancer cells lines go, I would be very cautious when obtaining these. The purchase and shipping of potentially pathogenic samples is highly regulated and typically requires the facility to have a certain level of Biosafety rating, especially if the facility plans on growing them.

The amount of substance in ones body does not directly effect the amount in one's cells. (A good example is a diabetic patient- their blood glucose is high but their cells are unable to internalize the sugar for different reasons)

A good way to increase the amount of a substance in a cell is to exfoliate the properties of the lipid bilayer or to use a intermembrane transporter, or you could directly inject the cells but this requires highly specialized equipment. Your best bet is to plate your cells, grow them, and simply coat them with your desired chemical (low concentrations) and rely on diffusion to carry the chemical across the membrane.

Tyber Cheever

donnahardy2 · Post by **donnahardy2** » Thu Nov 08, 2012 8:20 am

Hi LuciaHao,

Tyber has provided excellent information for measuring antioxidant levels in various foods and a good explanation of why it is difficult to increase the concentration of substances inside cells.

If you are not able to find a mentor and access to a tissue culture lab, I think you could do an excellent project by using one of the analytical methods to determine the levels of antioxidants in various foods. If you had access to a spectrophotometer, for example, you could do the project in your high school lab.

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Sat Nov 10, 2012 12:24 am

I can't put into words how much I appreciate the suggestions!
I have done more research and my revised plan is to test antioxidants and their effects on oxidations in biological systems. I hope to test a type of antioxidant (undetermined) on different reactive oxygen species produced in cells which include hydrogen peroxide (H2O2), hypochlorous acid (HClO), and free radicals such as the hydroxyl radical (·OH) and the superoxide anion (O2−) and then compare the results. This idea originated from the existing idea of that oxidative stress induces cancer; antioxidants are molecules that inhibit the oxidations of other molecules, which helps terminate chain reactions that cause negative effects on DNAs, lipids and proteins. My plan is to test the different doses of the type of antioxidant on the different unstable molecules to look at the effectiveness of the antioxidant in balancing unstable molecules.
Tyber, I really appreciate you sending me the link. I'm currently researching and looking over the different methods. My current issue right now remains to be how I'm going to test the number of molecules that have been balanced.
Donnahardy2, simplifying my experiment plan is my goal right now. I'm trying to simplify it as much as possible while still achieving my goal and high accuracy. I think the key is to eliminate materials that are difficult for me to access.
My plan is to do more research and look at some past researches and think about my question more.

donnahardy2 · Post by **donnahardy2** » Sat Nov 10, 2012 9:57 am

Hi LuciaHuo,

Thanks for the additional information about your research plan.

You are smart to carefully consider your experimental options at this point. Here is a paper that you can access that includes the details of 4 different antioxidant assays. There are some details included as well as references to previous publications that you will need to look for.

http://www.google.com/#hl=en&sugexp=les ... 36&bih=475

Why don’t you decide if you can adapt any of these assays for your experiment with the resources that you have available? You only need one method for your project, and any of these techniques would work very well for a science project as they will give you measurable results.

Let us know if you have questions.

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Sat Nov 10, 2012 10:24 am

Thank you for the link! Every time I click on it, it just directs me to google though. Can you maybe tell me what to search up on google?

donnahardy2 · Post by **donnahardy2** » Sat Nov 10, 2012 11:00 am

Hi,

I apologize, I sent you the wrong link. For your article, I did a search for “how to measure the antioxidant effect on hydrogen peroxide,” and this is the link I meant for you to have:

http://www.google.com/#hl=en&sugexp=les ... 36&bih=475

It’s a citation from Int. J. Mol. Sci, 2007, 8, 797-809.

Measurement of Antioxidant Activity of Wine Catechins,
Procyanidins, Anthocyanins and Pyranoanthocyanins
Jan Muselík 1, María García-Alonso 2, María P. Martín-López 2, Milan Žemlička 3 and
Julián C. Rivas-Gonzalo 2,*

Please let me know if you can’t access it, and I will try to upload a copy for you.

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Sat Nov 10, 2012 1:30 pm

Hi Donnahardy2,
I still cannot access it. Please try to upload it for me. Once again, thank you!

donnahardy2 · Post by **donnahardy2** » Sat Nov 10, 2012 6:04 pm

Hi,

I will post the article a few pages at a time. Here are the first 3 pages:

antioxidants part I.pdf: (152.63 KiB) Downloaded 399 times

Donna

donnahardy2 · Post by **donnahardy2** » Sat Nov 10, 2012 6:07 pm

Here's the second section.

antioxidants part II.pdf: (131.88 KiB) Downloaded 336 times

donnahardy2 · Post by **donnahardy2** » Sat Nov 10, 2012 6:10 pm

pages 7-9

antioxidants part III.pdf: (98.23 KiB) Downloaded 370 times

donnahardy2 · Post by **donnahardy2** » Sat Nov 10, 2012 6:12 pm

and the last 4 pages:

antioxidants part IV.pdf: (166.72 KiB) Downloaded 364 times

Read and try to understand as much of the articles as you can, especially the methods sections.

Donna

LuciaHuo · Post by **LuciaHuo** » Sun Nov 11, 2012 5:24 pm

Hi Donnahardy2,

Thank you for the information you gave me! I still have to read it over a few more times to understand the procedures for testing concentration of unbalanced molecules but the information in it is very very useful. I narrowed down and thought carefully about my question. I want to choose the two most promising antioxidants (one water soluble and one fat soluble) and use the two in different doses on hydrogen peroxide (a type of ROS that are generated by cells in the body). Then, I can compare the reactions to each other and analyze my data. However, I currently face the problem of producing a hypothesis for this experiment. Do you know of any ways for me to produce a logical guess on this type of chemistry experiment? If someone asks me why my hypothesis is what it is, I want to be able to give the person an explanation.

By the way, I have to submit my proposal (meaning find mentor and come up with hypothesis) by Nov.16 and finish my experiment before the beginning of February. Thank you!

donnahardy2 · Post by **donnahardy2** » Mon Nov 12, 2012 1:30 pm

Hi Lucia,

Thanks for the additional explanation about your research plan. It seems like a very good plan to me and very worthwhile doing.

If you have access to a spectrophotometer you could do the FRAP assay at 593 nm or the TEAC assay at 734 nm and these are experiments that could be done in a high school lab. The experiments would involve setting up standard curves and measuring the results of your samples with the spectrophotometer. So you might not need access to a research laboratory.

However, if a spectrophotometer is not available, and you do need a mentor who is doing research on your topic, then you need to focus on writing e-mails, and perhaps making phone calls.

Please post again if you have any other questions.

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Tue Nov 13, 2012 12:14 am

Hi Donnahardy2,
I'm going to be measuring the amount of ROS left in the result, not the amount of antioxidants. The FRAP and TEAC methods both measure the antioxidant capacity of a given substance. Are they effective in measuring ROS capacity as well? What I'm looking for are methods for measuring free radical amounts.

donnahardy2 · Post by **donnahardy2** » Tue Nov 13, 2012 8:12 am

Hi Lucia,

I can tell that you have done a good job in your background reading. The FRAP and TEAC methods do not measure reactive oxygen directly, but instead measure the potential of the the sample to prevent oxidation. Both methods use a spectrophotometer and would give you a measurable results.

Here is a good review article that includes methods for measuring reactive oxygen species. Do you have access to the equipment and reagents needed for any of these methods?

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1574951/

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Wed Nov 14, 2012 12:20 am

Hi Donnahardy2,

Thank you for the article! It's very helpful! I have to choose my antioxidants for the experiment and I don't know how to make the selection. I know for sure that I want to choose antioxidants of different categories or that differ from each other. I read articles on antioxidant strength, but I could not find a comparison of the antioxidant strengths to each other. I also read an article that talked about the best antioxidants used for preserving meats, also meaning terminating lipid oxidations, which I thought was quite interesting and that is something I can think about while determining my antioxidants. I also read articles that categorized antioxidants into categories such as: synthetic and natural, and water soluble and liquid soluble. I'm not sure how to choose the antioxidant in a logical manner! Are there any suggestions that you have for me?

LuciaHuo · Post by **LuciaHuo** » Thu Nov 29, 2012 10:18 pm

Hi! This is an update on my project. I have access to cancerous and benign prostate cells, cancerous and benign breast cells and some others that have not been specified to me yet. I'm planning to test the effects of antioxidants on cell growth. So, I'm assuming that the cancerous cells have a higher level of superoxides than normal cells. I apply the antioxidants to both types of cells and my results will be measured with a cell counter, which measures the growth.

However, I'm still having trouble determining the type of antioxidant I want to use. I have read many articles that states specific antioxidants work, but I find it hard to compare the results of the papers to each other. Does anyone know of a good research paper that compares the antioxidant groups to each other or has a theory regarding the effectiveness of antioxidants?

Thank you very much!

Regards,
Lucia

donnahardy2 · Post by **donnahardy2** » Sun Dec 02, 2012 9:28 am

Hi Lucia,

Congratulations on securing access to the lab and materials that you will need to do your project. You have the resources now to be able to do a really great project. The selection of the antioxidant you will use is important, but this is a decision that you will have to make. However, here are some things that you could consider in making the decision:
If I were doing the project, I would not choose one the antioxidants that has been studied a lot, such as ascorbic acid, vitamin E, or beta-carotene, although you could use one of these as a positive control. Instead, I would look for something unique and not studied as much. Here is a paper with full access to the text that includes a list of foods with potential antioxidants:

http://ajcn.nutrition.org/content/84/1/95.full

Is there a particular food that you like, such as chocolate? Is there a local crop grown, such as broccoli, that you would be interested in studying? Is anyone else in the lab where you will be working investigating a similar project? In your background reading, have you read about any antioxidants with results that you think need to be verified? I'm sure there is a perfect antioxidant for your study.

Good luck!

Donna Hardy

donnahardy2 · Post by **donnahardy2** » Sun Dec 02, 2012 9:35 am

Hi Lucia,

Here's one more suggestion. Here is a summary of a research report on apples. In a survey of diets of 2,500 men, the authors found that the one dietary factor associated with improved lung function was the consumption of 5 of more apples a week. Since you are interested in lung cancer, perhaps you use apples or one of the antioxidants found in apples for your study.

http://www.livestrong.com/article/46489 ... breathing/

Donna

LuciaHuo · Post by **LuciaHuo** » Sun Dec 02, 2012 3:49 pm

Thank you!

LuciaHuo · Post by **LuciaHuo** » Mon Dec 24, 2012 2:09 pm

Hi do you know any information on a cell viability and proliferation assay called crystal violet?

Thanks and Happy Holidays!

donnahardy2 · Post by **donnahardy2** » Fri Dec 28, 2012 5:23 pm

Hi Lucia,

Cell proliferation assays use a visible dye, either crystal violet, or a fluorescent dye to stain cells growing in tissue culture. The dye is visible for a few cell divisions, so the eventual fate of the dye can be tracked. This type of technique could be adapted to your objective, however if you want to evaluate the effect of a substance on cell growth, you could compare the rate of overall cell growth compared to a control.

For example, you could measure the time that it takes the cell culture to cover the surface of a tissue culture well. Or, here is an abstract that reports a method for measuring the mitotic index using an antibody to phosphorylated proteins.

http://www.ncbi.nlm.nih.gov/pubmed/11879713

Do you have access to a tissue culture lab at this time?

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Sun Dec 30, 2012 11:26 pm

Hi Donnahardy2,

I do have access to a tissue culture lab and all the equipments I need to perform crystal violet and WST-1 assays. I can choose one.

Thank you!

donnahardy2 · Post by **donnahardy2** » Mon Dec 31, 2012 8:15 am

Hi Lucia,

It's great that you have access to a tissue culture lab, and a choice of methods to use. From what I can tell, either assay will give you the quantitative results that you need for your experiment. Here is some more information that might help you decide which assay to use?

The WST-2 assay measures overall general metabolic activity of the cells.

http://en.wikipedia.org/wiki/MTT_assay

The crystal violet assay has been shown to measures cytotoxicity in carcinoma cells reproducibly and precisely.

http://cancerres.aacrjournals.org/conte ... art_1/1114

Here is a reference that includes a comparison of the WST-2 assay (MTT) and crystal violet (CV). From this reference, can you see any differences in the results? Can you find any similar references? Look for information that would show a difference in sensitivity or reproducibility between the two assays.

http://www.medicine.nevada.edu/wps/proc ... _10-14.pdf

Have you read through the protocols for each assay to see if there is a difference in the amount of work involved? Have you talked to anyone in your lab who has done both assays? If so, which assay do they prefer?

So, what is your decision?

Donna Hardy

LuciaHuo · Post by **LuciaHuo** » Wed Jan 02, 2013 8:50 pm

Hi Donna,

I have decided to use crystal violet and not WST-1 (also MTT) because of it's relative simplicity. Also, I believe it takes less time to perform. Both are very similar, achieving quantitative results of the same quality so it doesn't matter a lot.

Thank you for the papers.

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