I'm going to study the cytotoxic potential of some natural compounds. Can anyone please suggest what compounds should be kept as positive controls? Besides this, is there any further information about controls to be kept?
Dear Sonaly,
I fully agree with your comments: the MTT assay does not measure cytotoxicity, but "cell viability".
Cytotoxic means direct "cell killing effects" induced by the drug of interest.
Cytostatic means that the compound of interested lowers the growth rate of a given cell population without direct cell killing effects. Cell death will occur as a consequence of a too long cytostatic effect.
A colorimetric assay can only bring "relative global growth inhibition information" because it is a relative test in which you compare the ODs of a treated cell population to the ODs of a control condition (untreated cells) arbitrarily scaled at 100%.
Thus, the IC50 / GI50-related values obtained by means of a colorimetric assay do actually not translate “cytotoxic” effects.
When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days (usually 2 or 3)), i.e. the GI50 concentration (or the IC50 as commonly used in the literature) you do not know whether your compound of interest killed 50% of the cells (cytotoxic effects), whether it inhibited 50% of the cell proliferation (cytostatic effects), whether it detached 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...
Once you have determined the GI50 / IC50 concentration for a given compound on a given cell line, you must use complementary biochemical and/or morphological techniques to determine whether your compound is cytotoxic, cytostatic, anti-adhesive, etc..., etc...
The two attached articles by Galluzzi and colleagues (2012, 2015; Appendix-1 and Appendix-2) are of great help in this domain.
The attached article by Kornienko et al. (2013; Appendix-3) reviewed various chemicals that are able to induce non-apoptotic cell deaths in cancer cells.
Coming back to the IC50 / GI50 values obtained by means of a colorimetric assay (as for example the MTT one):
in the Mathieu et al. (2009 (Appendix-4) and 2015 (Appendix-5)) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.
In the Lefranc – Nunzo et al. (2013; Appendix-6) article, the MTT test-related GI50 concentrations relate to cytotoxic effects that in turn do not relate to apoptosis … This means that each cytotoxic effect does not “universally” translate into pro-apoptotic ones.
In the Van Goietsenoven et al. (2010; Appendix-7) article, the MTT test-related GI50 concentrations relate to cytostatic effects, neither to cytotoxic nor to pro-apoptotic ones.
Be aware that you cannot always translate the MTT test-related growth inhibition of a given compound into a precise GI50 value. Some compounds reach a “plateau” of inhibition (see Lefranc – Nunzo et al., 2013; Appendix-6).
Lastly, you can also have "false" data generated with colorimetric assays (see the attached article by Chan et al. (2013; Appendix-8) and the first NCI-60-cell line-related article (Shoemaker, 2006 (Appendix-9)).
The US NCI set up a fantastic tool to characterize the effects of a given drug in terms of growth inhibition in a panel of 60 cancer cell lines belonging to >10 histopathological types (Shoemaker, 2006; Appendix-9).
The US NCI clearly defined by means of the combination of the GI50 (growth inhibition), the LD50 (lethal dose by 50%) and the TGI (total growth inhibition) how to make the difference between a cytotoxic and a cytostatic compound:
The US NCI-related GI50 value corresponds to a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to an untreated control condition (= 100%) grown during the same time;
The US NCI-related LD50 value corresponds to the a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to the initial number of cells in the untreated control condition;
The TGI is the US NCI-related parameter to determine the concentration needed to kill 100% of the treated cells.
It is by comparing the GI50 to the LD50 value that one can determine whether a compound is cytotoxic or cytostatic, and not at all with the sole GI50 value.
We are using morphological approaches in the research unit to which I belong for determining whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013 (Appendix-6); Mathieu et al. 2009 (Appendix-4), 2015 (Appendix-5); Van Goeitsenoven et al., 2010 (Appendix-7)).
The US NCI is not so far for having tested about 800,000 anticancer drugs, whose data are publicly available on the NCI website https://dtp.cancer.gov/databases_tools/data_search.htm
I actually benefited several times from the amazing help of the NCI in identifying the mechanism of action of an innovative anticancer compound (see for example Frederick et al. JMC 2011 (Appendix-10)).
Hoping that this long explanation would not be too boring,
Best regards
Robert
As per natural compound you are using before going for MTT assay, you should check that the natural compound is not reducing the MTT.
Further,
Positive control for cytotoxicity : cells treated with a cytotoxic drug/chemical + MTT + solubilizing buffer
Negative control for cytotoxicty : cells left untreated + MTT + solubilizing buffer.
Blank : medium without cells + MTT + Solubilizing buffer
The term of viability should be used with a general definition comprising cytotoxic effect, cytostatic effect, antiproliferative effect represented by decrease in cell viability.
Moreover for detail, you follow conversation between Ubenako Njende and Engin Ulukaya for question : can someone advise on MTT assay for blank and controls in topic - cell ceulture.
Whether you are using normal cell lines or diseased like cancer cell lines?
Also to add to Sonali's comment,
When analysing the results you should represent data as relative viability
RV= O.D of sample (drug treated)- O.D of medium only/ O.D of untreated cells (without drugs)-O.D of medium.
For control positive you have to use the drug that working on the disease that you are looking for. For e.g. I used doxorubicin, ATRA and 5-flurouracil for leukaemia, suramine for liver cancer, tamoxifen for breast cancer, plaxitaxil for cervical cancer. While in the case of hypercholesterolaemia I used simvastatin as control positive, and nystatin for fungus infection and so on. Please kindly at first target your disease then search for control positive and perform MTT. Regarding control negative, also depends on your work or research you want to run. For e.g. I used 3T3, RAW 127, vero cells in case of murine cancer to check the antiproliferative activity of some natural products in vivo. While I used MCF-10, chang cell, HUVEC, PBMNCs as control negative when I tried to check antiproliferative activities of some natural products in case of human cancer. TQVM
I want to check the cytotoxic effects of one type of Antifreeze Protein on the normal cell line.Can anyone give some Idea about the controls, that what should be a negative and positive control ?
Dear Sonaly,
I fully agree with your comments: the MTT assay does not measure cytotoxicity, but "cell viability".
Cytotoxic means direct "cell killing effects" induced by the drug of interest.
Cytostatic means that the compound of interested lowers the growth rate of a given cell population without direct cell killing effects. Cell death will occur as a consequence of a too long cytostatic effect.
A colorimetric assay can only bring "relative global growth inhibition information" because it is a relative test in which you compare the ODs of a treated cell population to the ODs of a control condition (untreated cells) arbitrarily scaled at 100%.
Thus, the IC50 / GI50-related values obtained by means of a colorimetric assay do actually not translate “cytotoxic” effects.
When you obtain a concentration (for a given compound) decreasing by 50% the global growth (after x days (usually 2 or 3)), i.e. the GI50 concentration (or the IC50 as commonly used in the literature) you do not know whether your compound of interest killed 50% of the cells (cytotoxic effects), whether it inhibited 50% of the cell proliferation (cytostatic effects), whether it detached 50% of the cells (anti-adhesive, i.e. "in vitro antimetastatic" effects), etc..., etc...
Once you have determined the GI50 / IC50 concentration for a given compound on a given cell line, you must use complementary biochemical and/or morphological techniques to determine whether your compound is cytotoxic, cytostatic, anti-adhesive, etc..., etc...
The two attached articles by Galluzzi and colleagues (2012, 2015; Appendix-1 and Appendix-2) are of great help in this domain.
The attached article by Kornienko et al. (2013; Appendix-3) reviewed various chemicals that are able to induce non-apoptotic cell deaths in cancer cells.
Coming back to the IC50 / GI50 values obtained by means of a colorimetric assay (as for example the MTT one):
in the Mathieu et al. (2009 (Appendix-4) and 2015 (Appendix-5)) articles, the MTT test-related GI50 concentrations relate to actual cytotoxic effects.
In the Lefranc – Nunzo et al. (2013; Appendix-6) article, the MTT test-related GI50 concentrations relate to cytotoxic effects that in turn do not relate to apoptosis … This means that each cytotoxic effect does not “universally” translate into pro-apoptotic ones.
In the Van Goietsenoven et al. (2010; Appendix-7) article, the MTT test-related GI50 concentrations relate to cytostatic effects, neither to cytotoxic nor to pro-apoptotic ones.
Be aware that you cannot always translate the MTT test-related growth inhibition of a given compound into a precise GI50 value. Some compounds reach a “plateau” of inhibition (see Lefranc – Nunzo et al., 2013; Appendix-6).
Lastly, you can also have "false" data generated with colorimetric assays (see the attached article by Chan et al. (2013; Appendix-8) and the first NCI-60-cell line-related article (Shoemaker, 2006 (Appendix-9)).
The US NCI set up a fantastic tool to characterize the effects of a given drug in terms of growth inhibition in a panel of 60 cancer cell lines belonging to >10 histopathological types (Shoemaker, 2006; Appendix-9).
The US NCI clearly defined by means of the combination of the GI50 (growth inhibition), the LD50 (lethal dose by 50%) and the TGI (total growth inhibition) how to make the difference between a cytotoxic and a cytostatic compound:
The US NCI-related GI50 value corresponds to a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to an untreated control condition (= 100%) grown during the same time;
The US NCI-related LD50 value corresponds to the a global growth decrease by 50% induced by a compound on a given cell line “x” days after having cultured the cells with the drug and in comparison to the initial number of cells in the untreated control condition;
The TGI is the US NCI-related parameter to determine the concentration needed to kill 100% of the treated cells.
It is by comparing the GI50 to the LD50 value that one can determine whether a compound is cytotoxic or cytostatic, and not at all with the sole GI50 value.
We are using morphological approaches in the research unit to which I belong for determining whether a compound is cytotoxic or cytostatic (see Lefranc-Nunzo et al., 2013 (Appendix-6); Mathieu et al. 2009 (Appendix-4), 2015 (Appendix-5); Van Goeitsenoven et al., 2010 (Appendix-7)).
The US NCI is not so far for having tested about 800,000 anticancer drugs, whose data are publicly available on the NCI website https://dtp.cancer.gov/databases_tools/data_search.htm
I actually benefited several times from the amazing help of the NCI in identifying the mechanism of action of an innovative anticancer compound (see for example Frederick et al. JMC 2011 (Appendix-10)).
Hoping that this long explanation would not be too boring,
Best regards
Robert
Second major concerns also highlighted by Sonali: be aware of false positive versus false negative data generated by the MTT assay (see four attached articles).
Lastly, it is a loss of time to study "cytotoxicity selectivity" between normal and cancer cells in vitro.
A cancer (i.e. the disease that kills patients) is much more complicated than a single cancer cell population growing as prisoneers in a plastic flask and facing a single normal cell population.
You should have an in-depth reading about the notion of tumor microenvironment (see the articles here attached).
And the cherry on the cake, the 10th article I am attaching here clearly (published in 2013) shows that etoposide (one of the potent anticancer drug routinely used to combat nasty cancers and actually helps patients) kills about 10x more normal than cancer cells ... in vitro .... in plastic flasks ...
Best regards
Robert
Thanks Robert for your extensive in details statements with references on Cytotoxicity assays
Cheers
Dr.Ranjith Kumavath
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