Looks like that your compound has no effect at lower concentrations and is highly effective at higher ones, killing almost all of your cells.

Try to use your compound in 0.5 μmol/l concentration increments, i.e. 4.0, 4.5, 5.0, 5.5., 6.0 or even lower increments to determine the threshold of the concentration and finding the IC 50 value.

Sabine Strehl Thank you very much. We have discussed this and plan to try smaller increments in the next round.

The sharp increase in the percentage of apoptotic cells and the sudden decrease in the OD value observed in your experiments can be indicative of a concentration-dependent cytotoxic effect of the semisynthetic compound of betulinic acid (BA) on the lung cancer cell lines you tested. There are several possible explanations for this phenomenon:

1. Dose-dependent response: The compound may have a dose-dependent cytotoxic effect, where higher concentrations of BA lead to increased apoptosis and decreased cell viability. This response suggests that BA at higher concentrations is more effective in inducing cell death.

2. Threshold effect: The compound might have a specific threshold concentration beyond which its cytotoxic effects are amplified. In your case, this threshold could be around 6μmol/l, as indicated by the sudden increase in apoptotic cells and the decrease in live cells.

3. Saturation of cellular machinery: At higher concentrations, BA may overwhelm the cellular machinery responsible for cell survival, leading to increased apoptosis and reduced cell viability. This saturation effect can explain the sharp change observed at the 6μmol/l concentration.

4. Off-target effects: BA or its metabolites could have off-target effects at higher concentrations, triggering cellular responses that induce apoptosis. It is possible that BA interacts with specific cellular components or pathways that are crucial for cell survival, leading to the observed cytotoxic effects.

5. Time-dependent effects: The time of exposure to BA might also influence the cytotoxic response. It is possible that longer exposure times at higher concentrations are necessary for the compound to exert its cytotoxic effects fully.

To further investigate and understand this phenomenon, it would be beneficial to conduct additional experiments. These experiments could include:

1. Time-course experiments: Assess the cytotoxic effects of BA at different time points to determine whether the observed effects are time-dependent.

2. Lower concentration range: Perform experiments with a wider range of lower concentrations to establish a dose-response relationship and determine if there is a concentration threshold.

3. Mechanistic studies: Investigate the underlying mechanisms by which BA induces apoptosis. This could involve examining specific cellular pathways related to apoptosis or conducting gene expression profiling to identify potential targets.

4. Additional cell lines: Test BA on different lung cancer cell lines or other cancer cell lines to determine if the observed effects are cell line-specific or generalize across different cancer types.

By conducting further experiments and exploring the mechanisms underlying the cytotoxic effects of BA, you can gain a better understanding of its potential as an anti-cancer compound and identify the optimal concentration for future studies or potential therapeutic applications.

Saif Wahid Thank you very much for your kindly reply. Indeed we have suspected these reasons. We will make further discussions and conduct additional experiments accordingly.