We want to screen pant extracts for anticancer activity by using cell culture or cell line.
We are looking for the best and advance methods?
Please add protocol?
Evaluating Medicinal Plants for Anticancer Activity
November 2014
DOI: 10.1155/2014/721402
SourcePubMed
LicenseCC BY 3.0
Elisha SoloweyMichal LichtensteinSarah SallonSarah SallonShow all 6 authorsHaya Lorberboum-GalskiHaya Lorberboum-Galski
Hi,
Antiproliferative Assay: MTT assay is good to know the cytotoxicity property .
Cell Viability and Cell Morphology
Apoptosis Quantification by ELISA.
RT-PCR
And many more look at the mathodology in published papers.
good luck
ELISA is more accurate but MTT assay is so easy & economical.
I'm not well in RT-PCR, so I have no idea about it.
MTT assay is cheap and basic start. But you should bear in mind that, MTT assay is NOT a cytotoxicity assay. It will indirectly give you the comparison of viable cells between treated (i.e. plant extract added cells) and non-treated cells. However, it will not tell you the difference caused by:
* cytotoxicity : plant extracts kill the cells, thus you saw less viable cells
* cytostatic effect : plant extracts caused cell cycle arrest, thus you saw less viable cells
* anti-adhesive effect : plant extracts affected the adhesive properties of cells so you lost cells in assay, thus you saw less viable cells
* mitochondrial effect : MTT is reduced in mitochondria. If your extracts have effect on mitochondrial enzyme activity, you may see false-negative less viable cells.
Still, I would start with MTT. You can observe cells by microscopy during the assay and eliminate some of the points above. Combine it with a cytotoxicity assay (such as, membrane disruption based LDH assays or propidium iodide uptake).
If you have flow cytometry option, Annexin V/PI staining would also identify cytotoxic mechanisms.
Good luck
You could try testing your compound on a xenograft model (http://altogenlabs.com/pre-clinical-research-services/xenograft-animal-service-immunocompromised-nodscid-mice/). These work by having cell lines turn into tumors, with the drug dosage (namely your plant extract) affecting the tumor size. This shows anticancer activity if it is successful, and helps pinpoint possible side effects (like general cytotoxicity) and if the drug is effective at small dosages. Starting in vitro by testing your compound on cell lines is a good idea, perhaps even using a selection cell lines to test the compound out (http://altogenlabs.com/pre-clinical-research-services/pharmacology-and-toxicology-testing-ic50-for-tumor-cell-lines/) and using the most "curable" cancer cell line as a basis for the xenograft.
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