Synthesis of BODIPY FL-tethered ridaifen-B, RID-B-BODIPY, and its localization in cancer cells.

We synthesized ridaifen-B boron dipyrromethene (RID-B-BODIPY) using 2-methyl-6-nitro benzoic anhydride (MNBA)-mediated dehydration condensation reaction between amino alkyl-tethered RID and BODIPY FL. Comparative experiments between dicyclohexylcarbodiimide (DCC) and MNBA for their coupling reactions demonstrated that MNBA is an effective condensation reagent for amines and BODIPY FL. A cell staining study with RID-B-BODIPY showed intracellular localization of BODIPY FL fluorescence, attributed to the RID-B structure, indicating the successful development of a tool for analyzing intracellular molecular behavior efficiently.

Anti-proliferative effect of ridaifen-B on hepatoma cells.

Ridaifens (RIDs), a novel series of tamoxifen derivatives, exhibit a potent growth-inhibitory effect against numerous tumor cells regardless of the expression of estrogen receptors, and are thus promising candidates as novel anti-tumor drugs. RID-B is a first generation RIDs, and inhibits the proliferation of several tumor cell lines. However, the potentially growth inhibitory effect of RID-B against hepatoma cells, and the detailed mechanism underlying RID-B-mediated tumor cell death remain to be elucidated. The purpose of the current study was to evaluate the anti-proliferative effect of RID-B against hepatoma cells. The anti-proliferative effect of RID-B against human hepatoma Huh-7 cells was investigated by cell proliferation assay using WST-1 reagent, and caspase-3 activity was evaluated by using specific fluorescent substrate. In addition, DNA fragmentation in Huh-7 cells induced by RID-B was estimated by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay, and binding of RID-B to double-stranded DNA was confirmed by mass spectrometry. RID-B (0.5, 1 and 2 µM) inhibited the growth of Huh-7 cells, seemingly dose-dependently, but did not inhibit the growth of normal primary rat hepatocytes in the same concentration range. Furthermore, the caspase-3 activity of Huh-7 cells was increased by RID-B (0.5 and 5 µM), and the anti-proliferative effect of RID-B (1 µM) on Huh-7 cells was partially suppressed by the addition of the caspase inhibitor, Z-VAD-FMK. Additionally, RID-B (10 µM) directly bound to double-stranded DNA, and the addition of DNA suppressed RID-B-mediated cell growth inhibition and DNA fragmentation in Huh-7 cells. From these data, it may be concluded that RID-B inhibited cell growth and induced apoptosis via activating caspase-3 and binding to DNA directly, leading to DNA fragmentation in hepatoma cells.