I am not an expert on this phenomenon, but I have seen it myself with my own data. Let's discuss in more detail using some figures. These figures are obtained from my own paper that can be accessed from links below.Article Chelerythrine Chloride Downregulates β-Catenin and Inhibits ...

Article Identification of Phytochemical-Based β-Catenin Nuclear Loca...

Let's look at the figure with the name 1.jpg. When looking at panel A, B, and C that represent continuous monitoring of cell viability throughout 72 hr of observations, you will notice that the compound applied at the highest concentration (red line) does not always have the lowest normalized cell index as a measure of cell viability. This is in part probably contributed by the rapid killing action of the said compound that did not allow enough time to be completely devoid of cell attachment. For your information, the machine I used here relies on cell attachment to estimate the overall health of adherent cells. In this case, however, you might say that the difference is minor, so we can safely assume that the cell viability values are probably at the same level or range.

Now, let's see the figure with the name 2.jpg. When looking at panel B with the label "Monolayer", you will notice substantial concentration-independent effect, e.g., with compound PI at 3.13 ug/mL, the growth inhibition is > several other higher concentrations. In this assay, I used an ATP-measuring luciferase assay for 3D culture. I did not think that the reagent was that incompatible for monolayer because for 3D culture, it is modified to have an enhanced penetration and lysis, which should not differentiate much between monolayer and 3D culture. However, as you can tell, it is more concentration-dependent when looking at the Spheroid (3D) model.

So, from these observations, you can probably conclude that the difference response you saw might be contributed by:

• type of cell viability assay, i.e., mitochondrial activity, cell attachment, protein content, ATP content, etc.
• growing mode of cells, i.e., monolayer, spheroid
• mechanism of actions of compound that occurs at a very specific range of concentrations
• differential range of concentrations inducing cytostatic (stopping growth without killing) or cytotoxic (killing)

The combination of these factors is likely the reasons for this phenomenon. I would say it is not uncommon; this type of data is just less interesting and people mostly could not explain exactly its occurrence.

Hello Tenzin Adon ,

This phenomenon can definitely happen, and the different factors provided by Win Sen Heng could play a substantial role in this.

If still interested in digging more about this phenomenon, these thread links below provide additional factors to consider (aggregation of your test compound, interference of test compound with assay, drug-resistance mechanisms, negative feedback, concentration dependent-crosstalk with other pathways with a net result of lower drug sensitivity at high concentration).

https://www.researchgate.net/post/What_are_some_reasons_why_some_drugs_lose_efficacy_at_higher_concentrations_instead_of_behaving_in_a_dose-dependent_manner

https://www.researchgate.net/post/How-can-a-low-concentration-of-a-drug-be-toxic-to-cells-and-a-higher-concentration-enhance-the-growth-of-the-cells/1

If you haven't performed this already, I would suggest to repeat the experiments, this time including 100 uM, essentially a range you can plot in log scale on the X-axis, if possible (something similar to the graphs provided by Win Sen Heng ).

This would not only allow you to confirm your initial observations, but also to have an overview of the growth inhibition kinetics of your test compound.

Best of luck!

U-shaped dose effect curves seemed to be quite common in pharmacology with various examples (take a look here: https://endocrinesciencematters.org/non-monotonic-dose-responses-2/non-monotonic-dose-responses-technical-overview/ ) and the effect could also explained by Hormesis.

Best wishes Soenke

Win Sen Heng I am grateful for your willingness to share your knowledge. Thank you so much.

Sönke Weinert I went through the link shared and found it to be helpful. Thank you