New Research Topic

[deleted-336487]

Hello, I'm ALANG.

I would like to start lung cancer research, and I would like to know if this topic I came up with is a viable research topic: A Novel: Genetically Engineering a plasmid (gene extracts from cayenne and green fluorescent protein) to atrophy the A-427 lung cancer cell to treat carcinoma. I'm still in the early stages of researching this experiment.

I'm thinking of genetically engineering a plasmid (having both a cayenne extract gene and green fluorescent protein< the glo used to better view the cell, after transformation {glowing cells may show that this cayenne extract has entered into the cancer cell}>, and additional reporter genes ). After the genetic engineering is done, I'll use viral transduction or transformation and transfection to insert this plasmid into the A-427 cancer cell and seeing it's effect.

After further research, this is what I found: Both turmeric and Cayenne are non-protein molecules and therefore cannot be produced by DNA sequences (and hence cannot be genetically engineered). It may be possible to covalently attach the GFP to either of these molecules for the purposes of quantifying cellular internalization but the permeability characteristics would change, rendering the data close to meaningless in-vivo situations.

I asked someone and they said I could possibly test and have a study of concentration dependent apoptosis of transgenic lung cancer cell lines (mimicking smokers' DNA) using curcumin and/or cayenne. <The person who helped me come up with the study of concentration dependent apoptosis, didn't exactly tell me what I should do research wise. I'm really confused about that topic. Does this directly mean that I will test how how different concentrations of curcumin and cayenne effect the process of cell death on transgenic lung cancer cell lines? I'm a bit confused about that.

Thanks

[SciB]

Hi,

Curcumin is a strong candidate for anticancer treatments and would make a good science project subject. The major problem with curcumin is that it is only slightly soluble in water. There are many researchers and companies trying various strategies to make Cur more soluble or to encapsulate it in nanoparticles that can be delivered to cancer cells.

Apoptosis is a kind of cell death that is triggered by certain proteins and receptors in cells. It is a natural way for the body to eliminate cells that are abnormal, like tumor cells. There are also direct ways to kill cells or at least to stop them from dividing. This causes necrosis which is different from apoptosis but has the same result of killing the cell.

In your post you said that you wanted to target the human lung cancer cell line A427. I have used A549 which is an epithelial carcinoma and I know that it is inhibited by Cur (http://www.atcc.org/products/all/CCL-185.aspx). Your initial idea was to target the cancer cells with a plasmid but you did not say what the plasmid would code for other than the GFP reporter gene. As you said, curcumin and capsaicin are not proteins so cannot be encoded in DNA. But you could deliver these compounds to cancer cells using a polymer such as Chitosan which is derived from chitin, the polymer that makes up crustacean shells: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3090273/
http://www.azonano.com/article.aspx?ArticleID=3232

To do an experiment you would load the chitosan nanoparticles (CNPs) with curcumin or cayenne extract (capsaicin?) and add it to cultures of the A427 cells. The controls would be no addition and CNPs alone. There are many ways to measure the effect of drugs on cancer cells but viability is the simplest. You just need an inverted tissue culture microscope and some trypan blue vital stain. I can give you the procedure for this. If you want to look at more specific changes such as apoptosis then you need to do enzyme assays. There are kits for this but they are quite expensive and require fluorescence plate readers or flow cytometers. If you will be working in a university research lab with a mentor then you may be able to perform these advanced assays.

I hope this information helps. Let us know what you want to do and we can help you design the experiments.

Sybee

[deleted-336487]

Your initial idea was to target the cancer cells with a plasmid but you did not say what the plasmid would code for other than the GFP reporter gene.

At first I had the crazy idea that it would code for the cayenne plant, but now I've learned that it's not very possible to do that.

But you could deliver these compounds to cancer cells using a polymer such as Chitosan which is derived from chitin, the polymer that makes up crustacean shells

I'm very interested in this. How would that actually work. I know that to move a plasmid into a sell, you use transformation and transfection, through heat shock, I believe. How would a polymer be transferred?

I can give you the procedure for this. If you want to look at more specific changes such as apoptosis then you need to do enzyme assays. There are kits for this but they are quite expensive and require fluorescence plate readers or flow cytometers. If you will be working in a university research lab with a mentor then you may be able to perform these advanced assays.

I'll appreciate the procedures! I believe I would use the MTT or annexin based assays right?

I'm now working on finding labs to work in over summer. To cover materials costs, I'm applying to multiple scholarships!

-Thanks

[deleted-332001]

Hi ALANG! I'd like to add that there are more than two types of cell death. The main kinds of cell death include apoptosis, necrosis, and autophagy. Here is a review article of cell death and cross-talk between these processes: http://www.sciencedirect.com/science/ar ... 8913002243 .

Also, from experience, principal investigators are less likely to accept a student that wants to do his/her own project independent from the rest of the lab. You're best trying to find a cancer lab that does this kind of work and has someone who can teach you techniques. I work in a cancer lab, and most students who enter the lab are assigned a project.

Best regards,
Jen

[SciB]

To answer your questions:

Nanoparticles are called 'nano' because they are REALLY tiny. A typical size would be 0.1 micrometers ('microns') while the diameter of a human epithelial cell would be around 20 microns. Cells are able to take in small particles through their membranes by a regulated process called endocytosis. Chitosan nanoparticles (CNPs) have free amino groups that are positively charged and this allows them to bind to negatively charged protein molecules on a cell's membrane. Once the CNP binds, it can be 'carried' through the cell membrane into the cytoplasm where it can release its cargo--a plasmid, a drug or a combination of these.

The simple method of determining whether a cell is alive or not is to take a portion of the cell culture and add a small amount of trypan blue to it. Trypan is what is called a 'vital' stain. Living cells have intact cell membranes that block the influx of dye molecules while dead cells do not. You need a microscope and a hemocytometer to do the cell counts. You put the cells with the trypan blue on the counting chamber slide, focus on the grid pattern and count the clear cells (live) and the blue cells (dead). Then you express the viability as % live cells. Any lab that works with cultured cells knows how to do this type of cell count.

As Jen said there are many ways for a cell to die and determining this requires specific assays. You are right that a common assay for apoptosis is the annexin-V. The MTT assay shows viability but does not indicate the 'cause of death'. Again, a cancer research lab will surely know about these assays and probably have the kits and instruments to use them.

Good luck finding a mentor for your summer project. If you can get a scholarship to pay for the cost, that will really help your chances of finding a good position. You will need to get as familiar as possible with the scientific jargon related to this field and learn how to do the various molecular biology methods and what they are used for. There are lots of good molecular biology tutorials on youtube and biotech company websites have tons of information about their assays.

And, if you have questions just post here and we will be there to help you understand.

Good luck!

Sybee

[deleted-336487]

Thanks for the very interesting and enriching information @Sybee and @Jen.

I'll digest this information, read more, and let you know if I need help.

Best,
ALANG