Intracellular Processing of Riboflavin in Human Breast Cancer Cells

A variety of polarized epithelial cells, such as human breast cancer (MCF-7), have mechanistically evolved the ability to adapt to the dynamic cellular environment and maintain homeostasis of an array of micronutrients which display conditional requirements. Active absorption mechanisms, including endocytosis, are able to control cell surface recognition and protein expression which are associated with a substance's intracellular processing and kinetics. Riboflavin (RF), or vitamin B2, has been recognized as an important factor in a multitude of terminal disease states, most notably in breast cancer, where its cellular absorption is significantly enhanced. In order to delineate the regulatory mechanisms and kinetics associated with RF control in human breast cancer tissue, this study aimed to model its absorption profile and identify its intracellular regulatory components. Using both the Michaelis-Menten equation and a modified version of it, incorporating both active internalization and passive diffusion, RF absorption displayed better correlation ( r (2) > 0.998) with the mixed, active and passive, model exhibiting kinetic parameters characteristic of a receptor-mediated uptake mechanism ( J max = 2.58 pmol/5 min, K m = 106 nM) at extracellular RF concentrations under 5 muM and a passive component existing at RF concentrations greater than 5 muM. Following internalization, RF was able to recycle back to the membrane with a half-life of 13.7 min at 37 degrees C, which occurred more rapidly with increasing extracellular RF concentrations ( t 1/2 = 5.4 min at 1 muM) and decreasing temperatures ( t 1/2 = 6.4 min at 4 degrees C). Furthermore, modification to endosomal pH using the lysomotropic agents monensin (25 muM) and primaquine (300 muM) significantly inhibited the exocytosis of RF (61 and 30% of control), whereas biochemical modification of endocytic trafficking with okadaic acid (1 muM) led to a significant increase in RF exocytosis (208%). In conclusion, RF homeostasis in MCF-7 cells is a well regulated process which is dependent upon RF concentration, temperature, and endosomal acidification.