Enhancement Of Auranofin-induced Apoptosis In Human Breast Cancer Cells By Organic Selenocompounds, A Synergistic Inhibitor Of Thioredoxin Reductase

Thioredoxin system plays an important role in the regulation of intracellularredox balance and various signaling pathways. Thioredoxin reductase (TrxR) isoverexpressed in many cancer cells and has been identified as a potential target ofanticancer drugs. Auranofin (AF) is a potent TrxR inhibitor with novel in vitro and invivo anticancer activities. Selenocystine (SeC) is a nutritionally available selenoaminoacid with selective anticancer effects through induction of apoptosis. The thioredoxinsystem plays an important role in regulation of intracellular redox balance and varioussignaling pathways. The anticancer activities of selenium (Se) and its mechanismshave been well clarified. However, the target of Se is still unclear. In the present study,we demonstrate the synergistic effects and the underlying molecular mechanisms ofSe in combination with AF on cancer cells and suggest that TrxR is a potential targetto achieve the synergistic chemotherapy. Also, our results enlightened a novelstrategy to use selenocompounds-based combination chemotherapy. Flow cytometry,fluorescence microscope and Western blotting in the present study were employedand the results found that:1. The expression level of TrxR in three breast cancer cell lines, MCF-7，MDA-MB-231and RMCF-7DOX, was detected by Western blot assay. The resultsshowed that the max level of TrxR was detected in MCF-7, followed by that inMDA-MB-231, whereas the level of TrxR in MCF-7RDOXis almost nondetectable.The results of MTT assay found that the effect of Se and AF was positively relate tothe TrxR expression level. For details, the synergistic effect of Se and AF on TrxRhigh-expressed MCF-7and MDA-MB-231breast cancer cells was significantlystronger than that of TrxR low-expressed MCF-7RDOXcells. These results highlightedthe combination of anticancer effect of Se and AF in TrxR high-expressed cancercells.2. TrxR high-expressed MCF-7is selected for further investigation about themolecular mechanism of synergetic apoptosis induced by SeC and AF. In this part,MTT assay was employed to detect the cell viability, the cell cycle distribution was analyzed by PI-flow cytometry, the cell apoptosis was further confirmed byTUNEL-DAPI co-staining assay, JC-1fluorescent probe was employed to measurethe mitochondrial membrane potential, the intracellular ROS generation was probedby DCF fluorescence and the important signaling molecules involved in the apoptosiswere detected by Western blotting. The results reflected the enhanced inhibition ofTrxR activity by SeC and AF resulted in the ROS accumulation, which activated thep53signaling pathway through the regulation of AKT and ERK phosphorylation andfinally induced the mitochondria-mediated apoptosis in MCF-7human breast cancercells. These results highlighted the strategy to use Se and AF combination therapy toachieve synergism by targeting TrxR activity. These works have been published inPLoS ONE (2013,8(1): e53945).3. Moreover, the enzyme kinetics of the synergetic inhibition of TrxR by SeCand AF is further investigated. Michaelis-Menten plots, Lineweaver-Burk plots andEisenthal plots derived from DTNB reduction assay were employed to measureenzyme kinetics. The results showed that SeC may act as a potential substrate of TrxRcompeting with Trx, whereas AF inhibited the TrxR in a non-competitive manner.The combination of SeC and AF inhibited the TrxR in a mix type, and SeC and AFshow a stronger inhibition to the free TrxR than the enzyme-substrate complex.Taken together, a potential target of Se cooperated with AF induced apoptosis isset up and these suggest the strategy to use Se and AF in combination could be ahighly efficient way to achieve anticancer synergism by targeting TrxR.