In vitro activity: Brefeldin A is a fungal metabolite and blocks the forward transport between the endoplasmic reticulum and Golgi apparatus, Brefeldin A causes an impaired distribution of the membrane proteins. When HCT 116 human colon cancer cell is treated with Brefeldin A, morphological changes indicating cell differentiation are observed. Brefeldin A exerts its cytotoxic effects mainly by inducing differentiation and apoptosis in tumor cells. The treatment of the strips with 20 μg/mL Brefeldin A for 6 hours completely abolishes the relaxation induced by bradykinin in the presence of 10mM indomethacin and 30 μM L-NOARG. The treatment with 20 μg/mL Brefeldin A substantially abolishes the bradykinin-induced decreases in [Ca2+]i and tension in the range of concentrations between 1 nM and 1 mM. Brefeldin A has no effect on the [Ca2+]i elevation in endothelial cells induced by bradykinin or substance P. Addition of the fungal metabolite Brefeldin A does not affect the spontaneous phospholipid-dependent GTPS binding to myr-rARF1 but totally abolishs the retinal isotonic extract (RIE)-catalyzed exchange, with half-maximal inhibition at 2 μM Brefeldin A. Brefeldin A prevents a wide variety of membrane traffic pathways. Brefeldin A inhibits an ADP-ribosylation factor-specific guanine nucleotide exchange activity present in Golgi membranes or in brain cytosol. The complete prevention by Brefeldin A strongly suggests that the retinal extract contains an ARF-specific guanine nucleotide exchange factor. Retinal isotonic extract (RIE)-catalyzed GTPS release from both ADP-ribosylation factors (ARFs) is only partly inhibited by Brefeldin A, even at 300 μM. Brefeldin A induces fusion of the Golgi apparatus with the ER. Brefeldin A abolishes the inhibitory effect of the CERT inhibitor HPA-12. Brefeldin A treatment, which induces fusion of the Golgi apparatus and the ER, rescues the limonoid-induced prevention of sphingomyelin biosynthesis. BFA treatment of CHO cells causes a 2 to 3 fold increase in sphingomyelin synthesis. Apart from B-CLL cells, Brefeldin A reportedly causes apoptosis in multiple myeloma (U266, NCI-H929), Jurkat, HeLa, leukaemia (HL60, K562, BJAB), colon (HT-29) and prostate, as well as adenoid cystic sarcoma cells. The administration of 25 ng/mL of Brefeldin A completely blocks growth of HF4.9 and HF28RA cells, whereas higher Brefeldin A doses (75 ng/mL) are required to achieve the same effect in HF1A3 cells. Cell proliferation is inhibited within 24 hours in a dose-dependent manner and, depending on the cell line, almost complete cessation of 3H-thymdine incorporation is observed at 50-75 ng/mL of Brefeldin A (26%, 76%, 87% inhibition at 50 ng/ml and 75%, 87%, 92% inhibition at 75 ng/mL for HF1A3, HF4.9 and HF28RA cells respectively. Brefeldin A-induced cell killing is in a dose-dependent manner using YO-PRO 1/PI assay.

Cell Assay: HF1A3, HF4.9 cell viability upon the treatments is tested using double staining of cells with YO-PRO 1/PI and SYTO16/PI probes. To access cell proliferation, cells are treated with 0–100 ng/mL Brefeldin A in complete medium for 20 hours before adding 1 μCi/mL [methyl-3H]-thymidine for additional 4 hours at 37 °C. The incorporated radioactive thymidine is quantified by scintillation counting with Microbeta counter. To examine long-term effects of Brefeldin A treatment, cells are seeded at initial concentration 105 cells/mL and treated with 0-75 ng/mL Brefeldin A for up to 5 days. At the time indicated, a sample of cells is removed and viable cell number is assessed by standard Trypan Blue exclusion assay.