Similarly to well-known thapsigargin, trilobolide is a naturally occurring sesquiterpene lactone of guaianolide type. Both of them are potent SERCA (Ca2+-ATPase) inhibitors. Thapsigargin is currently in phase II of clinical trials for a cancer treatment. Recently, we have isolated trilobolide from Laser trilobum (L.) Borkh.

The aim of the project was to better understand intracellular kinetic and biological effects of natural trilobolide. New semi-synthetic trilobolide analogues were synthesized and evaluated for cytotoxicity and immunomodulatory properties. Targeted delivery and quantification of trilobolide in samples were also investigated.

Trilobolide molecules were modified with acyl chain linkers of various length at 0-8 position. These derivatives were used for conjugation with fluorescent dye Bodipy. Intracellular localization (live-cell imaging) and cytotoxicity (WST-1) were performed in a panel of human cancer cell lines (PC-3, LNCaP, MCF-7, MiaPaCa-2, HT-29, U-2 OS, HeLa) and mouse C2C12 and RAW 264.7 cells. Effects on production of nitric oxide (NO) and cytokines were evaluated. Supernatants of rat peritoneal cells were analysed for NO production by Griess reagent. Concentrations of cytokines were determined by ELISA kits. SERCA inhibition was analysed by Fura2/AM. Microarray analysis (Illumina USA) of 1 µM trilobolide was performed in LNCaP (prostate cancer cells). Liposomes with trilobolide were prepared. Immunoassay ELISA for detection of trilobolide was developed and optimized.

Trilobolide and its conjugates exhibited sufficient intracellular accumulation and specific localization. Cytotoxic effects were observed in submicromolar concentrations in some drug-resistant cancer cell lines. Microarray data confirmed stress of endoplasmic reticulum, but revealed also down-regulation of cell cycle, involvement of metabolic pathways and disruption of cytoskeleton. Trilobolide is less toxic for fibroblasts and macrophages. Specific immunostimulatory effect (NO production, IFN-gamma, IL-2 secretion) of trilobolides is dose- and structure-dependent (0.01- 100.0 microM). Trilobolide-porphyrin conjugates with low toxicity, represent an interesting tool for photodynamic therapy. Finally, antimycobacterial tests revealed that trilobolide-testosterone and -estradiol derivatives potentiated the anti-mycobacterial activity up to IC50 of 10.6 microM.

In summary, trilobolide and its conjugates could be a useful tool in anti-cancer, anti-infectious and immunomodulatory therapies.

Supported by the Czech Science Foundation 14-04329S, MSMT No. 20/2016, No. 20 /SVV/2017, IGA LF2016/019 Olomouc.