Isobavachalcone Induces ROS-Mediated Apoptosis via Targeting Thioredoxin Reductase 1 in Human Prostate Cancer PC-3 Cells

Abstract

Prostate carcinoma causes a great number of deaths every year; therefore, there is an urgent need to find new drug candidates to treat advanced prostate cancer. Isobavachalcone (IBC) is the chalcone composition of Psoralea corylifolia Linn used in traditional Chinese medicine. Although IBC demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of IBC have not been fully defined. In our study, we found that IBC may induce reactive oxygen species- (ROS-) mediated apoptosis via interaction with a selenocysteine (Sec) containing the antioxidant enzyme thioredoxin reductase 1 (TrxR1), and induce lethal endoplasmic reticulum (ER) stress by inhibiting TrxR1 activity and increasing ROS levels in human prostate cancer PC-3 cells. Furthermore, we also observed that knocking down TrxR1 would sensitized cancer cells to IBC treatment. Our study provides evidence for the anticancer mechanism of IBC with TrxR1 as a potential target.

Figure 1

Effect of IBC on the proliferation of PC-3 cells. After treatment with IBC at a series of concentrations for 24, 48, or 72 h, the viability of PC-3 cells was assessed with an MTT assay. All the data are expressed as percent change compared to the control group, which was arbitrarily designated as having 100% viability. All data are mean ± S.D. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the control group.

Figure 2

Effect of IBC treatment on PC-3 cell morphology and apoptosis. Cells were exposed to 15, 30, or 45 μM IBC for 24 h. (a) After treating cells for 24 h, the morphology of PC-3 cells was visualized and imaged by inverted phase contrast microscopy. (b) The morphological changes in PC-3 cells were examined following staining with Hoechst dye 33258. (c) Rate of apoptotic cells of IBC-treated PC-3 cells as detected by flow cytometry. (d) Quantitative analysis of the rate of apoptotic cells after IBC treatment. All data are presented as mean ± S.D. ^∗p < 0.05 and ^∗∗p < 0.01 compared with the control group.

Figure 3

IBC activates ER stress, which contributes to the IBC-induced lethality of PC-3 cells. Cells were exposed to 15, 30, or 45 μM IBC for 24 h. (a) Quantitation of levels of GRP78, XBP-1, CHOP, and ATF4 mRNA by RT-qPCR. (b) Protein levels of GRP78, p-eIF2α, ATF4, Chop, eIF2α, pro-caspase-3, and cleaved caspase-3 were determined by Western blot. (c) Quantitation of GRP78, p-eIF2α, eIF2α, ATF4, Chop, pro-caspase-3, and cleaved caspase-3 protein levels. All data are presented as mean ± S.D. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the control group.

Figure 4

IBC directly binds and inactivates TrxR1 in PC-3 cells. (a) The activity of the TrxR1 enzyme. (b) The TrxR1 expression was determined by Western blotting after knockdown with siRNAs for 12 h. (c) Knockdown of TrxR1 in PC-3 cells significantly increased the ROS levels (d) and apoptotic cells. All data are presented as mean ± S.D. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the control group.

Figure 5

IBC-induced PC-3 cell apoptosis is dependent on intracellular ROS generation. (a) Time course of IBC-induced ROS production. As indicated, cells were treated for different lengths of time with 45 μM IBC and then stained with 10 μM DCFH-DA. The fluorescence intensity was measured by flow cytometry. (b) Intracellular IBC-induced ROS generation was measured by flow cytometry. (c-d) Representative images of ROS production in cells treated with IBC in the presence or absence of NAC or BOS. PC-3 cells were preincubated with NAC or BSO for 2 h before incubating with 45 μM IBC for 6 h. Intracellular ROS production was measured by flow cytometry. (e) Representative images of apoptotic cells identified by staining with annexin V-FITC/PI. Cells were treated with 45 μM IBC in the presence or absence of NAC or BSO for 24 h. Percentage of apoptotic cells was determined by annexin V/PI staining and flow cytometry. (f) Quantitative analysis of the rates of apoptotic PC-3 cells after IBC treatment. Data from three independent experiments are presented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 6

IBC-induced ER stress was mediated by ROS generation. PC-3 cells were treated with 45 μM IBC with or without 5 mM NAC or 1 mM BSO for 24 h, and then ER stress marker expression levels were measured by Western blot. (a) Protein levels of GRP78, ATF4, Chop, and p-eIF2α were examined by Western blot. (b) Quantitative analysis of GRP78, ATF4, Chop, and p-eIF2α protein levels. All data are presented as mean ± S.D. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the control group.