Objective: In this study, 25 synthetic cyclic lipopeptides (CLPs) were investigated for their anticancer potential against mouse melanoma (B16F10) cells, human prostate cancer (PC-3), human colorectal adenocarcinoma (HT-29) and mouse embryonic fibroblast (NIH3T3) cells.

Methods: The cytotoxic activity of investigated compounds was evaluated using MTT and CV assays. In order to examine the mechanism of action of the most potent compound cell cycle analysis, apoptosis assay, caspase activity, CFSE and DHR staining, DAF-FM, autophagy and immunocytochemistry caspase-3 assays were performed.

Results: During the fast screening, compound 9, was identified as prospective active CLP against B16F10 cell line at 10 μM concentration. MTT and CV assays exhibited at least four times higher cytotoxic potential of 9 (IC₅₀ = 8.4±1.3 μM, MTT; 10.6±1.1 μM, CV) in comparison to control drug natural occurring CLP surfactin (IC₅₀ = 50.3±0.6 μM, MTT; 40.4±0.3 μM, CV). The use of flow cytometry analysis confirmed that apoptosis was involved in the death of B16F10 cells after treatment with 9, as demonstrated also by DAPI staining. Caspase activity could be detected during cell death (ApoStat assay, immunocytochemistry caspase-3 assay). Compound 9 provokes enhancement of nitric oxide (NO) production in B16F10 cells but does not trigger ROS/RNS generation or autophagy.

Conclusion: The study highlights that synthetic macrocycle 9 has superior tumor-specificity and potential as an anticancer agent compared to surfactin and cisplatin. These findings could guide the development of more selective and less harmful macrocyclic lipopeptides for cancer therapy.