β-Glucosidase activity in fetal bovine serum renders the plant glucoside, hypoxoside, cytotoxic toward B16-F10-BL-6 mouse melanoma cells

Abstract

By using p-nitrophenyl-beta-D-glucopyranoside as substrate, beta-glucosidase activity was observed in fetal bovine serum (FBS). This activity could be inhibited by heat inactivation of the serum. Gel chromatography of FBS indicated the presence of beta-glucosidase activity with an apparent molecular mass of 29 kDa. In McCoy's 5A medium supplemented with non-heat inactivated FBS, the diglucoside hypoxoside ([E]-1,5-bis[4'beta-D-glucopyranosyloxy-3'-hydroxyphenyl]pent-4-en - 1-yne) showed cytotoxicity toward B16-F10-BL-6 mouse melanoma cells. In incubations where the media were supplemented with FBS previously heat inactivated at 56 degrees C for 1 h or more, no cytotoxicity was observed in the presence of hypoxoside. The aglucone of hypoxoside, rooperol ([E]-1,5-bis[3',4'-dihydroxyphenyl]pent-4-en-1-yne), showed cytotoxicity regardless of whether the serum was heat inactivated or not. The kinetics of the heat inactivation of the beta-glucosidase activity in FBS coincided with the loss of apparent cytotoxicity of hypoxoside. High performance liquid chromatography analysis showed that rooperol could be generated by incubation of hypoxoside in non-heat inactivated FBS, but that this ability was lost in serum that was heat inactivated for 1 h or longer. Newborn bovine serum did not contain any beta-glucosidase activity whereas it was found in three different commercial sources of FBS. This observation is of practical importance because conventional heat inactivation of FBS at 56 degrees C for 30 min was not sufficient to inactivate the beta-glucosidase activity completely.