Dear Varun,

Here below you have my "own point of view about how assessing "antiproliferative versus cytotoxic effects".

With respect to medium color changes, in no way the medium color can bring you any reliable info about "cytotoxicity versus anti-proliferative effects". A "purple or pink" medium becoming yellow just indicates you that your cells are "suffering", as for example when they reach confluence and need medium replacement.

Coming back to cytotoxicity and anti-proliferative effects ...

"Cytotoxicity" cannot be determined by means of the colorimetric MTT assay. You must use other tests than the MTT one if you want to measure cytotoxicity (such as for example the LDH assay). The MTT test enables you to evaluate "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 have been too boring,

Best regards

Robert

Dear Varun,

Here below you have my "own point of view about how assessing "antiproliferative versus cytotoxic effects".

With respect to medium color changes, in no way the medium color can bring you any reliable info about "cytotoxicity versus anti-proliferative effects". A "purple or pink" medium becoming yellow just indicates you that your cells are "suffering", as for example when they reach confluence and need medium replacement.

Coming back to cytotoxicity and anti-proliferative effects ...

"Cytotoxicity" cannot be determined by means of the colorimetric MTT assay. You must use other tests than the MTT one if you want to measure cytotoxicity (such as for example the LDH assay). The MTT test enables you to evaluate "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 have been too boring,

Best regards

Robert

Dear Varun,

MTT assay actually tells you about the metabolic activity of the cells .MTT IS reduced by metabolically active cells mainly by  the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. Conversely it can be used for cytotoxicity and is a widely accepted method. LDH based method relies on the release of intracellular LDH into the supernatant after the loss of cell membrane integrity. Therefore In my opinion MTT will predict the cytotoxic stress much earlier and might be even at lower conc of compound when compared to LDH. But  you cannot tell the antiproliferative effect of the test compound by these assay. The easiest way would be to do cell cycle analysis using PI. But I would recommend to do Annexin V/PI staining. this will give you detailed information about cell cycle phases, early and late apoptosis and necrosis.

Thanks

Dr Kiss, thank you very much for so complete answer!!  And also for papers attached.

Best regards,

Marian

hello dr. robert, thanks for writing. no, your long explanation didn't bore me. just made me think, so much for me to learn in this field. i will read the references one by one. thanks again for writing.

Dear Prafull,

I share at "0% " your explanations ..., which seem to me more than personal because you forgot to attach valuable references validating you "personal point of view".

And the MTT test cannot assess toxicity ...

As it was widely accepted by the scientific community at a certain period that Earth was flat ...

May be you should have a more careful reading of my previous response to Varun.

Dear Marian,

Dear Varun,

Thank you very much for your kind comments.

Best regards

Robert

Dear Dr Kiss,

Thank you very much for providing valuable answers and helping us in understanding the complex questions of  biology. I really appreciate your efforts. 

Coming to the question of MTT; I do agree that MTT is not a direct measurement of cytotoxicity. I clearly mentioned this in the very first line that MTT tells you about the metabolic activity (viability) of the cells and if cells are under stress then the MTT reduction will decrease. Thus it can be a indirect measurement of cytotoxic stress.  I completely agree that LDH is a better measurement of cytotoxicity because it suggest irreversible damage to plasma membrane. LDH is routinely used in clinics and drug testing. My point was that LDH is more specific while MTT is more sensitive and as with every test with high sensitivity chances of false positives are also high.

Thanks

http://www.ncbi.nlm.nih.gov/pubmed/16111842

Dear colleagues,

Thank you very much for the valuable discussion. It is very interesting and helpful (not only) for me. I have been trying to find the answer of this question for years, but, I have to be honest – it is not so easy.  Cytotoxic or cytostatic effect – how to distinguish them?

First of all, toxicity has many faces – we can say that a compound or treatment is toxic for  cells when it influences some (one or more) morphological/functional cell characteristics – for example, when prevents cell adhesion, induces morphological changes, inhibits proliferation or kills the cell. So, the antiproliferative effect is also a kind of toxicity. Let us ask again – how to distinguish both effects – the ability of the compound to kill the cell and its ability to inhibit proliferation?

And is it possible to do this? We must consider many things including:

-          “Dose makes toxicity”! The concentration modulates both – the “quantity” and the “quality” of the toxicity. Applied at one concentration the compound can be “more killing” when the other concentration can be “more cytostatic”;

-          The period of treatment is also important – decreasing the treatment interval we increase the chance to measure mainly (but not only!) the killing effect of the compound. Colony-forming methods are suitable for long-treatment and proliferation studies; 

-          Cell specific response – each cell culture is a unique system and can express different sensitivity to one and the same treatment;

-          And what about the methods? There are different groups – we can see and count (live and dead) cells using light microscope or electronic counter (using trypan blue for example), we can determine the percent of viable cells as compared to the control (MTT, neutral red uptake cytotoxicity assay etc), we can measure DNA and/or protein, we can study cell cycle, we can identify the type of cell death (apoptosis/necrosis), etc.  No one of them is perfect. In the case of trypan blue dye exclusion method – what about treatments that can cause temporary increased permeability of the cell membrane? The decreased percent of viable cells found by MTT and Neutral red uptake cytotoxicity assay can be the result of cell death, inhibited proliferation or both…

In my opinion may be it is better to use a combination of different methods, including: 1) two-three cytotoxicity assays (MTT as the gold standard and 1-2 more) with different cellular/molecular targets and mechanisms of action (the compounds that we test also can have different cellular/molecular targets). The results obtained by different methods can be also “different”. Sometimes it is not easy to discuss such data, but it is always helpful and more objective); 2) “morphological” methods –  it is very helpful to combine “mathematical” data (percent of viability as compared to the control obtained by MTT for example) with “picture” demonstrating the ability of the compound to induce morphological changes and even cell death (the results can be “surprising” from time to time – low viability measured by MTT for example but at the same time the cells look very well – does it mean that the treatment used affect mainly the proliferation?). You can use double staining with propidium iodide and acridine orange (cheap, easy and informative method according to my opinion) and you can continue with different variants of TUNEL test or Annexin V; 3) It would be perfect if we have information about the cell number. Unfortunately, trypan blue dye exclusion technique is not suitable for experiments with many samples even with electronic counter, you will need a lot of time…

Briefly, we have to (at least try to) find the best experimental design for each system “compound/cell culture” and try to obtain the information about cell viability using different methods and conditions. And, of course we need some professional luck! So, Good Luck!!!

Dear Prafull,

We are now singing in the same tempo!!

And thank you so much Radostina for your very helpful comments!

Best regards

Robert