Hello Parth!

In a technical way, MTT viability assay consists of a quantification of how many cells are able to reduce MTT to formazan crystals (which dissolved in DMSO gives the typical purple color).

However, this reduction is mediated by mitochondrial enzymes, so, indeed, what you are actually assessing is how many cells are metabolically active.

So maybe the reviewers think that:

A) You should use a more precise technique such as Crystal Violet staining (CVS), like in this article that compare CVS with MTT:

Article The Comparison of MTT and CVS Assays for the Assessment of A...

B) MTT is more a raw estimation based of the mitochondrial state, so you may over-stimate cell death of a compound (because a cell can be metabolically more inactive, but does not mean that it's dead). And that's why you can't say a drug is anti-cancer only based only in MTT (which may be a more preliminary experiment).

In case they demand you any additional assays, I would personally consider to perform Mass Spect, Western Blot or qPCR to see if the drug can alterate/inhibit a mitogenic pathway such as MAPK (Ras, Raf, Mek, ERK...), Notch, JAK/STAT... (this pathways are involved in cancer).

This depends entirely of the mechanism of the anti-drug compounds or agents you are using or the pathway you are interested in. You can write me in private message if you want to go deeper exploring this idea.

I hope that helps

Best regard :)

Francesc

Francesc Estrany Thank you, sir, for clarifying my question; the objective is to determine whether or not the plant extract has anticancer activity; prior to this, I performed LCMS to determine how many chemicals have such activity. So, at the preliminary stage of the test, the MTT assay (cytotoxic assay) and anticancer activity are the same.

Okay!

So then it's very important to clarify this: MTT is a viability test, can estimate cell mortality but may not be considered an anti-cancer assay.

Maybe is good to try an alternative such as crystal violet staining (CVS)

best regards!

Francesc

Hello Parth Trivedi

The MTT assay is one of the most widely used assays for evaluating preliminary anti-cancer activity of the plant extract. Cytotoxicity is the primary testing process to predict the presence of new cytotoxic drugs from natural ingredients. The cytotoxicity assay will provide information on drug concentrations that still allow cells to survive. This assay will give you an indication of cytotoxicity. As Francesc Estrany rightly mentioned, you cannot consider MTT as an anti-cancer assay.

You have performed LCMS to determine how many chemicals have such activity So, to further prove the anti-cancer activity of your plant extract, you will need to perform other assays to determine the exact molecular target. One of these assays that I would recommend is the kinase inhibition assay, which is the most widespread enzyme inhibition screening assays performed.

Kinases are enzymes that play a key role in several physiological processes and their inhibitors exhibit anti-cancer activity against various human cancer cell lines. A kinase assay works by simply measuring the activity of kinase. A kinase is an enzyme that aids in the transfer of a phosphate from ATP to another specific molecule. They regulate many activities in the human body. The measurement is used to test potential drugs to see how they affect kinase activity.

If you succeed in showing that one of the chemicals from your extract inhibits kinase activity in vitro, then it can be further studied to see if it has pharmacological characteristics that can make it suitable as a drug in the future.

Quantifying kinase activity requires detecting either the formation of the phosphorylated product or measuring the amount of ATP used or ADP formed. Almost any reaction that produces ADP is appropriate for detection with a universal kinase assay kit. These universal kinase assay kits are activity assays that are highly flexible. For instance, immunoassays that directly detect the formation of ADP using anti-ADP antibodies.

I hope your doubt is cleared.

Best.

Thank you Malcolm Nobre for providing useful information on the MTT assay and cytotoxicity testing for determining anti-cancer efficacy in plant extracts. The idea of using the kinase inhibition experiment to discover molecular targets and confirm anticancer activity is very appreciated. The information on checking kinase activity and the variety of universal kinase test kits was extremely useful. Your advice has been great, and I am excited to pursue this further.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is commonly conducted to assess the potential of active substances, compounds, or drug formulations as agents capable of killing cancer cells. Its principle is somewhat similar to staining. Although the MTT assay can also be utilized for antibacterial testing.

Thank you Adi Hermawansyah