Antioxidants and Lung cancer

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Re: Antioxidants and Lung cancer

Postby donnahardy2 » Sat Nov 10, 2012 6:12 pm

and the last 4 pages:

antioxidants part IV.pdf
(166.72 KiB) Downloaded 142 times


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


Donna
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Re: Antioxidants and Lung cancer

Postby 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!
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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.


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Re: Antioxidants and Lung cancer

Postby 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.
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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
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Re: Antioxidants and Lung cancer

Postby 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
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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
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Re: Antioxidants and Lung cancer

Postby 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
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Re: Antioxidants and Lung cancer

Postby LuciaHuo » Sun Dec 02, 2012 3:49 pm

Thank you!
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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!
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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
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Re: Antioxidants and Lung cancer

Postby 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!
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

Re: Antioxidants and Lung cancer

Postby 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
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Re: Antioxidants and Lung cancer

Postby 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.
LuciaHuo
 
Posts: 16
Joined: Sat Nov 03, 2012 10:35 pm
Occupation: Student: 9th grade
Project Question: 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.
Questions: 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?
Project Due Date: The proposal and a mentor must be found before Nov.16 of 2012 and the experiment must be done before Feb.15 of 2013.
Project Status: I am conducting my research

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