I tested some synthesized molecules for antiproliferative activity on MDA MB231, HCT 116, MCF-7 and HT 29 cell lines using MTT assay. They all showed good activity in nano molar range. Can the MTT assay give an insight to the probable mechanism of cell death?
To understand the probable mechanism of cell death, the related assay of cell apoptosis should be done, such as apoptosis rate and caspase3 activity.
Dear Prashanth,
MTT or a more advanced form of in-vitro cytotoxicity assay, i.e. SRB assay can give information only about overall cytotoxicity produced by test compounds which could be attributed to multiple mechanisms.
A single cytotoxic agent can act most of the time, in multiple pathways leading to cellular death.
Mechanisms of cytotoxicity could be of many ways, DNA damage, activating the pro-apoptotic genes or inhibition of anti-apoptotic genes, necrosis, blockade of cell cycle at various phases, activation of tumor suppressor genes, telomere inhibition etc.
However, if you want to check in detailed mechanism of cytotoxicity, you have to further test your test compounds in the respective assays.
For eg. in case of exploring mechanism of apoptosis, you need to test the crucial enzymes involved in apoptosis path ways, i.e. caspase-3, caspase-8, 10 etc. and also the proapototic factors (BAX, BAD etc.) or anti-apoptotic factors like Bcl-xl etc. This can be done using Westernblot analysis or kit methods using specific marker apparatus, like flow-cytometry.
Cell cycle analysis is also of immense help using flow-cytometry to know arrest of cell cycle at different phases (G0/G1, G2, S Phases) by test compounds.
DNA ladder assay will give you an idea about the DNA damage related mechanisms. Various staining assays, like Hoechst, AO/EB assays will also give information on apoptosis/ necrosis mediated cell death.
Thanking you,
Best Regards,
Dr. Grandhi V Ramalingayya
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts