Abstract
Portimine A is a marine cyclic imine toxin that was isolated by the Cawthron Institute in Nelson, New Zealand, and is produced by the dinoflagellate Vulcanodinium rugosum. This compound has been assessed against several cultured cancer cell lines and observed to have potent apoptotic activity. However, despite the high cytotoxicity of portimine A in vitro, portimine A has low toxicity in vivo - far lower than related cyclic imine compounds. The liver is widely regarded as the primary site of drug metabolism and detoxification. Liver S9 fractions and microsomes are fractions of liver lysates that contain key phase I and phase II enzymes involved in xenobiotic metabolism and are widely used for in vitro studies of drug metabolism. We hypothesised that liver metabolism is implicated in the decrease in cytotoxicity of portimine A seen in mice.
The cytotoxicity of portimine A was assessed using the MTT cell viability assay across four murine cancer cell lines: MOPC315 myeloma, Lewis Lung carcinoma (LL/2), EO771 breast cancer, and B16/F10 melanoma. In vitro liver metabolism of portimine A was performed in S9 fractions and microsomes from human and mouse, and cytotoxicity of the product/s was assessed against LL/2 cells. The MTT cell viability assay was used to determine change in metabolic activity, and automated microscopy to determine change in cell number. Mass spectrometry at the Cawthron Institute was used to measure loss of parent compound following liver metabolism.
The concentration of portimine that reduced cell viability by 50% (IC50) across murine cancer cell lines ranged from 2.01 nM to 2.55 nM. We saw no evidence that portimine A was metabolised through exposure to subcellular liver fractions, and there was no observed reduction in cytotoxicity against LL/2 cells compared to parent compound despite a measurable loss in cytotoxicity of the positive control (paclitaxel). This agreed with mass spectrometry data showing minimal loss of parent compound following metabolism. There was no significant difference between human and mouse liver metabolism, or between S9 and microsome metabolism.
The data shows that exposure to hepatic phase I and phase II drug metabolising enzymes does not lead to significant loss of portimine A, suggesting that another mechanism of detoxification may be responsible for the discrepancy observed between in vitro and in vivo toxicity of this compound.