Differential Phosphorylations of NFkB and Cell Growth of MDA-MB 231 Human Breast Cancer Cell Line by Limonins

The present research focused on investigating cell growth inhibition and total and differential NFkB serine residue phosphorylations on estrogen insensitive MDA-MB 231 breast cancer cells by various citrus limonoids and camptothecin. A cell growth assay was carried out after a 24 hr incubation with various concentrations of limonin glucoside, limonin, obacunone and obacunone glucoside (1, 5 and 10 mu M) and camptothecin (10 mu M), as a standard chemotherapy drug, along with DMSO solvent control. NFkB phosphorylations were assayed using a commercially available NFkB Elisa profiler kit (Active motif, Carlsbad CA) on these cells. The specific serine residue phosphorylation was studied using respective anti-phospho serine antibodies (anti-phospho ser-468 and ser-536, respectively) supplied in the kit. The WST-1 cell growth assay showed that limonoids, at all tested concentrations, did not show a significant decrease in MDA-MB 231 cell growth when compared to camptothecin (37-40% growth inhibition). The NFkB p65 profiler Elisa assay with 10 uM concentration of three types of limonoids (limonin glucoside, obacunone and obacunone glucoside) exhibited a significant increase (50%) and limonin showed only a 10% increase in ser-468 residue phosphorylations than DMSO treated cells. However, camptothecin treated hormone insensitive cells exhibited a 50% increase in the phosphorylations of both ser-536 and ser-468 residues than DMSO treated cells and inhibiting the cell growth. The total constitutive NFkB phosphorylation activity of limonoids treated, show a decrease than DMSO treated control cells. On the other hand, camptothecin treated cells showed a significant increase in total constitutive NFkB phosphorylation activity. These results may suggest that hormone insensitive MDA-MB 231 cell growth were not inhibited by limonin glucoside, limonin, obacunone and obacunone glucoside, but perhaps was due to differential phosphorylations of serine residues of NFkB.