5-ATAN, A TAN Derivative, Screened As A Lead In Pro-apoptosis Effects And Its Preliminary Mechnism On MCF-7Cells

Aged citrus peel as a Traditional Chinese herb was well documented by theCompendium of Material Medical at least as early as the sixteenth century. Likewise, it hasgained great popularity to be a natural alternative agent against both inflammation andtumor in western medicine. From medicinal chemistry, the chemical diversity ofstructures may lead flavonoids to be different in their pharmacological activities.Flavonoids and their synthetic analogs have attracted much attention in drug discovery. Forexample, although TAN is one of the most archived PMFs found exclusively in citrus peels,its inhibitory effect on cancer cell growth is quite weak compared with itsmono-demethylated derivative at C5-position in the original TAN skeleton. Increasingreports have concentrated on the antitumor effect of5-desmethyltangeretin. However, littlehas been known about whether the acetyl modified derivative of TAN has more potentanti-cancer activity than its original skeleton of TAN. In the current study, we providedclear data that5-ATAN possess more powerful ability against MCF-7breast cancer cellsthan that of TAN.The pro-apoptotic toxicity is critical for PMFs against tumors. Herein, by using themost appropriate approach in the study of the apoptosis, namely, the combination ofmorphological and biochemical methods,we presented sufficient evidence that5-ATANinduced apoptosis on MCF-7cells, such as nuclear morphological changes, DNAfragmentation.The process of apoptosis is complicated and controlled by manifold of cell signals.During this process, caspase is crucial because it plays the central role in the transduction ofapoptotic signals and proteolytically degrades loads of intracellular proteins to run the celldeath program. We first investigated the caspase-dependent and caspase-independentapoptosis process in the prevention against MCF-7breast cancer cell by5-ATAN.The translocation of AIF and phosphorylation of H2AX are required incaspase-independent apoptosis pathway. In previous studies, AIF and H2AX are used forevaluating the impact of phytochemicals-induced caspase-independent apoptosis pathway,especially in flavonoid research. With the pan-caspase inhibitors, the flavonoid apigenin isdemonstrated to induce phosphorylated H2AX preceding the activation of apoptotic process.Our results clearly supported the notion that pro-apoptosis of5-ATAN acted throughcaspase-independent mechanisms because we also found that translocation of AIF andphosphorylation of H2AX took place in MCF-7cells when treated with5-ATAN.Active caspase-3is a critical mediator of caspase-dependent apoptosis and leads tocleavage of PARP and fragmentation of DNA. Caspase-dependent apoptosis can beinitiated by two alternative pathways: either through extrinsic pathway by death receptors or through the intrinsic pathway by mitochondria. We first tested whether5-ATANinduction of apoptosis in MCF-7cells involves extrinsic apoptosis. However, we found noclear evidence about activation of caspase-8, cleavage of BID and regulation of FADD. Ourresults indicated that caspase-dependent extrinsic pathway was not required under thiscircumstance. In caspase-dependent intrinsic pathway, lethal signals result in Bcl-2familymembers interacting with the mitochondria to induce apoptosis. Then, mitochondrial outermembrane permeabilization (Δψm) was dissipated, leading to the release of cytochrome cfrom the mitochondrial inter-membrane space into the cytosol. The cyt C was assembled toa multi-protein apoptosome, which in turn triggers the pro-enzyme of caspase-9into theactive form. In our study,Δψm dissipation, release of cytochrome C and cleavage ofcaspase-9were observed in MCF-7cells after exposure to5-ATAN in a time-dependentmanner, clearly indicating that pro-apoptotic role of5-ATAN is also related tocaspase-dependent intrinsic pathway.In Summary, our current study demonstrates that5-ATAN plays pro-apoptoticcytotoxic roles in MCF-7cells through both caspase-dependent intrinsic apoptosis andcaspase-independent apoptosis. Development of acetylated derivatives from originaltangeretin represents a promising direction in providing a potential agent in anti-breastcancer research.