When we want to screen or test the effect of a natural product in cancer cells, we have to know how safe is it. Why do some articles mention antiproliferative and others cytotoxicity assays for this screening or testing?
Proliferation literally means rapid growth of any stuff and in case of cells it is cell proliferation which is nothing but cancer. In my view antiproliferative activity is the ability of a compound to stop the growth of cells. This means that not allowing the cells to multiply rapidly. While cytotoxicity refers to causing harm to cells thereby killing them. Let me explain with an example.
Assume 100 cells are incubated in a plate and after a certain time the cells become 200. After the addition of a drug, if the number of cells in the plate are between 100 and 200 then this refers to growth inhibition which is a measure of antiproliferative activity. On the other hand if the number of cells in the plate are less than 100 then this is referred as cytotoxicity.
Cells incubated = 100
Cells after incubation without drug = 200
Cells after incubation with drug = 100-199 (antiproliferative activity)
Cells after incubation with drug < 100 (cytotoxicity)
Several reports use the terms, antiproliferation and cytotoxicity, interchangeably. The term, “cytotoxic” means causing toxicity to cells; many anticancer agents aim to kill cancer cells. Chemotherapy is cytotoxic therapy. The objective of other treatments is to arrest the multiplication or proliferation of cells; they stop cancer growth, which is cytostatic, meaning “cell stopping; at rest or stationary”. An example is hormone therapies. DNA damage and the consequent induction of apoptosis are principal cytotoxic mechanism of several anticancer agents; therefore, certain cytotoxicity assays measure DNA damage and apoptosis.
Assays for cell proliferation determine the number of cells over a period, metabolic activity, the number of cell divisions, or DNA synthesis. Cell counting using viability based dyes are able to estimate both the rate of proliferation and the percentage of viable cells. Some assays perform cell counts to determine the number of living cells; a dye reduction procedure for estimating viable cells is also performed. Assays designed to measure metabolic activity may be applied for determining cell proliferation, viability and cytotoxicity. Actively proliferating cells increase their metabolic activity while cells exposed to toxic molecules will have diminished activity. The MTT assay system, based on the reduction of the tetrazolium derivatives in living cells by mitochondrial dehydrogenases, allows the estimation of the activity of metabolically active cells. This assay accomplishes the assessment of cell viability and survival (cell counting) and of the proliferation of cells (cell culture assays), an indicator of mammalian cell survival and growth.
Important points to consider regarding approaches to assess cytotoxicity and proliferation:
1) Viability assays report the number of remaining viable cells while offering little insight into the mechanisms concerning antiproliferative versus cytotoxic effects. A viability assay alone could have misreported the compound as cytotoxic owing to lower number of cells in the treated sample as compared to that of the untreated control, which has continued to proliferate; however inclusion of a concurrent cytotoxicity assay in the wells could show no impaired membrane integrity, indicating that the combined data elicit strong likelihood of cell growth inhibition.
2) Dye reduction assay to assess cytotoxicity followed by the addition of another reagent to determine cell viability in the same sample well is also performed.
3) In certain cytotoxicity determinations, addition of a specific dye with affinity to DNA binds it, and causes cell killing.
Several reports use the terms, antiproliferation and cytotoxicity, interchangeably. The term, “cytotoxic” means causing toxicity to cells; many anticancer agents aim to kill cancer cells. Chemotherapy is cytotoxic therapy. The objective of other treatments is to arrest the multiplication or proliferation of cells; they stop cancer growth, which is cytostatic, meaning “cell stopping; at rest or stationary”. An example is hormone therapies. DNA damage and the consequent induction of apoptosis are principal cytotoxic mechanism of several anticancer agents; therefore, certain cytotoxicity assays measure DNA damage and apoptosis.
Assays for cell proliferation determine the number of cells over a period, metabolic activity, the number of cell divisions, or DNA synthesis. Cell counting using viability based dyes are able to estimate both the rate of proliferation and the percentage of viable cells. Some assays perform cell counts to determine the number of living cells; a dye reduction procedure for estimating viable cells is also performed. Assays designed to measure metabolic activity may be applied for determining cell proliferation, viability and cytotoxicity. Actively proliferating cells increase their metabolic activity while cells exposed to toxic molecules will have diminished activity. The MTT assay system, based on the reduction of the tetrazolium derivatives in living cells by mitochondrial dehydrogenases, allows the estimation of the activity of metabolically active cells. This assay accomplishes the assessment of cell viability and survival (cell counting) and of the proliferation of cells (cell culture assays), an indicator of mammalian cell survival and growth.
Important points to consider regarding approaches to assess cytotoxicity and proliferation:
1) Viability assays report the number of remaining viable cells while offering little insight into the mechanisms concerning antiproliferative versus cytotoxic effects. A viability assay alone could have misreported the compound as cytotoxic owing to lower number of cells in the treated sample as compared to that of the untreated control, which has continued to proliferate; however inclusion of a concurrent cytotoxicity assay in the wells could show no impaired membrane integrity, indicating that the combined data elicit strong likelihood of cell growth inhibition.
2) Dye reduction assay to assess cytotoxicity followed by the addition of another reagent to determine cell viability in the same sample well is also performed.
3) In certain cytotoxicity determinations, addition of a specific dye with affinity to DNA binds it, and causes cell killing.
Dear fellows, many thanks for your scientific contribution and sharing. Regards
Thank you for all your wonderful sharing. its helped me also
Yes, these two assays are very confusing whether we have to do antiproliferative assays again after cytotoxicity MTT assay. Some said NO because the same meaning and other said here it make sense to do further study for anti-proliferative assays.
So, here I would like to raise one question. If I decided to do anti-proliferative assay after MTT test, whether do I need to treat the cells with different concentration as in MTT or just with the IC50 concentration of compound but check different time point 24, 48, 72 hr if there are proliferations. If I am not wrong, using IC50 concentration only to study proliferation makes more sense. Any opinions for this point?
If we do RTCA after MTT assay, do anyone suggests to do antiproliferative assay too or not necessary?
Dear Heshu,
Your question is very interesting!
And here it is my own opinion among the various ones you already received.
Excellent question from Jamail also: I really think that "just the IC50 concentration from MTT" is sufficient to proceed with anti-proliferative versus pro-death analyses post MTT determination of cell viability.
"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. 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 be too boring,
Best regards
Robert
Dear All,
Following on my previous "rather theoretical" message, I am adding here some "practical" examples.
In order to have a more than rough idea about compound-of-interest-induced in vitro anticancer effects, we routinely perform computer-assisted phase-contrast microscope analyses of living cells during 48 to 72h of observation. The cells (whatever normal or cancerous) are cultured in plastic flasks containing buffered media, but in an incubator without CO2 regulation (while thermoregulated at 37°C). The media we use are described in the attached articles (see below).
You can see in the movies here attached the “morphological effects” observed with two very distinct compounds. Each film results from 1,080 digitized images taken every 4 min during 72h and compressed into a 40-60- sec-film. Each film can be seen with softwares freely available from the net. The counter with yellow numbers that you can see in some of the attached films gives you an estimation of the “dynamics” (in terms of number of hours of observation) of the cell behavior.
The attached films illustrate two markedly distinct situations.
The first set of experimental condition refers to human HS683 malignant oligodendroglioma cells (see QVM-1 Le Mercier et al., Neoplasia 2009) left untreated (control; QVM-2 Film Hs683 Control) or treated (QVM-3 Film Hs683-Ferrocifene) with a ferrocifene derivative. These are these morphological observations relating mainly to the enlargement of the treated Hs683 cells as compared to the control ones (without “frank” death processes (see below what we mean as “frank death processes for sphaeropsidine A)) that led us to formulate the hypothesis about potential induction of senescence. The rest of the story is detailed in QVM-4 Bruyere-Mathieu et al. J Inorg Biochem 2014). We estimate that we are facing here CYTOSTATIC effects.
The second set of experimental conditions refers to human SKMEL-28 melanoma cells left untreated (QVM-5 SKMEL-28 Control) or treated with sphaeropsidin A (QVM-6 SKMEL-28 Sphaeropsidine A). These experiments actually helped us to appreciate the CYTOTOXIC effects of sphaeropsidin A (a more “in-depth” analysis is provided in QVM-7 Mathieu et al. CMLS 2015). The attached document labeled QVM-8 Van Goietsenoven et al. FASEB J 2010 provides a view about various sensitivity levels of melanoma cell lines to pro-apoptotic cytotoxic insults.
The “morphological approach” that we routinely employ can also be used to see how develop primary cultures from a human cancer biopsy (here a GBM biopsy, in the left-handed bottom part of the film "biopsy" – QVM-9 GBM Biopsy).
All the best
Robert
In vitro, cancer cells represent an actual component of a cancer as it behaves in vivo, i.e. cancer cells developing into a tumor microenvironment, which is amazingly complex from the cellular point of view (see the attached articles).
In vitro, "normal" cells correspond to no "clinical" reality, e.g. to no in vivo biological reality.
Agaibst all odds, a cancer is not at all a cancer cell population destroying / attacking / developping among normal cells (ince more see the attached articles).
Major compounds routinely and daily used to efficiently treat cancer patients can be more toxic in vitro against normal than against cancer cells (see the attached article relating to etoposide).
The use of "normal" cells in vitro is of no value ...
Best regards
Robert
The anticancer and anti-proliferative effect of medicinal plants involves the use of natural products in the treatment of cancer. Cancerous cell lines such as Hela, MCF7, CaCo2, Hep G2 and A549 are generally used. In these kinds of assays, you should be interested on the plant species with low IC50 OR LC50 as they are likely to inhibit the growth of cancer cells in vivo. Howver, you may also be interested in testing the safety of these plant materials as well against normal human and animal cell lines. Vero monkey kidney and Bovine dermis are mostly used. The plants of greater interest should be those that have higher IC50 and are likely to be safe for human use either internally or externally. I hope this clarifies the mist.
Thank you very much for all your informative answers and efforts, Best
Thanks a lot, your answers have made it clearer to me, now i can go on with the concluding part of my research..
Regards.
https://www.researchgate.net/profile/Robert_Kiss2/post/What_is_the_difference_between_an_antiproliferative_assay_and_a_cytotoxicity_assay/attachment/59d638a479197b8077995d54/AS%3A398486802583552%401472018065283/download/Normal+versus+cancer+cells+-+in+vitro+-+2013.pdf
I'm collecting toxicology data and receive datapoints with various metrics (CC50, EC50, GI50, IC50, IC90, LC50, MCC, MIC, TGI). Are there some described values of said metrics (in ug/ml) that could delimit the molecules as toxic/nontoxic? Are there some standards in this matter?
@ Krzysztof Rataj Any toxicological measurement depends on exposure/measurement time.
In in vitro cultures, many cytological processes can take 12-24 hours or more to fully manifest, and so what may seem non or mildly toxic at short times may be very toxic at later times. Advice would be to measure at 48 hours at least, and then a "standard" would be to quote the EC50.
The best was is to report your data in micro grams per ml. It is more of a comparable standard to the research in the literature. Toxicity may be the lower LC 50 when dealing with normal cell lines. However, that may be the opposite when dealing with cancerous cell lines. It is important and much easier to publish when you have data with lower LC 50 against cancerous cell lines and higher against normal cell lines. Its selective inhibition of cancerous cell lines then. Some important information may also be retrieved from American Cancer organization which recommends that LC 50 les than 30 micrograms per ml is of toxic level. Hope I gave the clue
I may have misspoke earlier. I'm not collecting biological data, but extracting literature data. Different groups and papers used different measures and I'm trying to compile some basic database of toxic and non-toxic compounds. I have a lot of data, but need to standardize it in some way.
@Nkoana Ishmael Thanks, at least there is some starting point for me with the 30ug/ml number!
Cytotoxicity of an extract or drug is the ability of such material to reduce the population of cells below the control limit or baseline (in the absence of any growth promoter). The antiproliferation implies the ability of the material to reduce or inhibit the growth of the cells below the control (in the presence of growth promoter, in a favorable environment). The antiproliferation may also be defined cytotoxic, particularly if the cell growth is grossly below the control baseline.
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