(280f) Genetic Engineering for the Production of Curcuminoids in Mammalian Cells

Curcumin, (diferuloylmethane), a naturally occurring compound extracted from the rhizome of several curcuma plant species, has been studied extensively for its potent antioxidant, anticancer, and radioprotective properties. Specifically, curcuminoids have shown promise in reducing the oxidative stress of multiple cellular environments and inducing apoptosis in breast cancer cells such as MDA-MB-231. However, the ability for curcumin to act as an effective therapeutic agent is largely limited by poor bioavailability. We have engineered human cells to synthesize three curcuminoids—curcumin, bisdemethoxycurcumin, and desmethoxycurcumin—from either tyrosine or intermediate cinnamic acids, in an effort to circumvent the need for exogenous delivery of curcumin. Genes encoding for enzymes involved in the biosynthesis of curcuminoids in plants—TAL, 4CL1, C3H, CCoAMT, DCS, CUS, CURS1—were individually inserted into mammalian expression vectors and transfected into HEK293 cells using specially modified polyethylenimine derivatives developed within our lab. Curcuminoids were extracted and concentrated from conditioned media 48 h post transfection and quantified via HPLC. Concentration of the starting amino and cinnamic acids, as well as the relative enzyme expression levels, were systematically varied in order to optimize the production of curcuminoids, resulting in media concentrations of 0.5, 0.1, and 0.16 µM of bisdemethoxycurcumin, desmethoxycurcumin, and curcumin, respectively. Upon exposure to 1 mM hydrogen peroxide, curcumin-producing HEK293 (HEK293-ccm) cells exhibited 20-37% decrease in reactive oxygen species compared to unmodified HEK293 cells, as measured by fluorescence of CellRox Deep Red, a dye that fluoresces upon oxidation. To investigate the potential anticancer properties of intracellularly produced curcumin, unmodified or curcuminoid-producing HEK293 cells were co-cultured with MDA-MB-231 cells that were previously modified to stably produce luciferin. The luciferase activity of MDA-MB-231 cells co-cultured with HEK293-ccm steadily decreased over 8 days, reaching as low as 7-8% of the MDA-MB-231 cells co-cultured with unmodified HEK293 cells, suggesting the biosynthesized curcuminoids were secreted and selectively killed the cancer cells. To our knowledge, this is the first synthesis of a plant-derived therapeutic molecule in mammalian cells. After seeing promise during our studies using transient plasmid expression, we plan to pursue our work further by engineering cell lines that will stably produce the enzymes responsible for the biosynthesis of curcuminoids.