biomedicines Article 8-Hydroxydaidzein, an Isoflavone from Fermented Soybean, Induces Autophagy, Apoptosis, Differentiation, and Degradation of Oncoprotein BCR-ABL in K562 Cells 1, 2,3, 4,5 2,6 7 Pei-Shan Wu y, Jui-Hung Yen y, Chih-Yang Wang , Pei-Yi Chen , Jui-Hsiang Hung and Ming-Jiuan Wu 1,* 1 Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan; [email protected] 2 Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien 970, Taiwan; [email protected] (J.-H.Y.); [email protected] (P.-Y.C.) 3 Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan 4 Ph.D. Program for Cancer Molecular Biology and Drug Discovery, Taipei Medical University, Taipei 11031, Taiwan; [email protected] 5 Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei 11031, Taiwan 6 Center of Medical Genetics, Buddhist Tzu Chi General Hospital, Hualien 970, Taiwan 7 Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +886-6-2664911 (ext. 2520) Both authors contributed equally to this work. y Received: 29 October 2020; Accepted: 13 November 2020; Published: 16 November 2020 Abstract: 8-Hydroxydaidzein (8-OHD, 7,8,40-trihydoxyisoflavone) is a hydroxylated derivative of daidzein isolated from fermented soybean products. The aim of this study is to investigate the anti-proliferative effects and the underlying mechanisms of 8-OHD in K562 human chronic myeloid leukemia (CML) cells. We found that 8-OHD induced reactive oxygen species (ROS) overproduction and cell cycle arrest at the S phase by upregulating p21Cip1 and downregulating cyclin D2 (CCND2) and cyclin-dependent kinase 6 (CDK6) expression. 8-OHD also induced autophagy, caspase-7-dependent apoptosis, and the degradation of BCR-ABL oncoprotein. 8-OHD promoted Early Growth Response 1 (EGR1)-mediated megakaryocytic differentiation as an increased expression of marker genes, CD61 and CD42b, and the formation of multi-lobulated nuclei in enlarged K562 cells. A microarray-based transcriptome analysis revealed a total of 3174 differentially expressed genes (DEGs) after 8-OHD (100 µM) treatment for 48 h. Bioinformatics analysis of DEGs showed that hemopoiesis, cell cycle regulation, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducers and activators of transcription (JAK-STAT)-mediated apoptosis/anti-apoptosis networks were significantly regulated by 8-OHD. Western blot analysis confirmed that 8-OHD significantly induced the activation of MAPK and NF-κB signaling pathways, both of which may be responsible, at least in part, for the stimulation of apoptosis, autophagy, and differentiation in K562 cells. This is the first report on the anti-CML effects of 8-OHD and the combination of experimental and in silico analyses could provide a better understanding for the development of 8-OHD on CML therapy. Keywords: K562; 8-hydroxydaidzein; apoptosis; autophagy; BCR-ABL; MAPK; NF-κB Biomedicines 2020, 8, 506; doi:10.3390/biomedicines8110506 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 506 2 of 23 1. Introduction Chronic myeloid leukemia (CML) is a hematopoietic disease characterized by the expression of the BCR-ABL fusion oncoprotein, which is caused by a reciprocal translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)] [1]. BCR-ABL fusion protein exerts a deregulated tyrosine kinase activity that activates proliferative and anti-apoptotic signaling pathways and leads to the malignant expansion of pluripotent stem cells in bone marrow [2]. Historically, CML was treated with hematopoietic stem cell transplantation, chemotherapy, and interferon-α [3]. Small molecule tyrosine kinase inhibitors (TKIs), such as imatinib, bosutinib, dasatinib, nilotinib, radotinib, and ponatinib, have been developed to treat CML by blocking the kinase domain of the BCR-ABL oncoprotein. However, a variety of adverse effects, such as off-target and metabolic toxicities, resistance to TKI therapy due to BCR-ABL mutations, and progression to advanced disease, render the need to pursue novel therapeutic strategies [3,4]. Since the BCR-ABL protein plays a crucial role in the pathogenesis of CML, its degradation is a new strategy for overcoming the problem of TKI resistance [5,6]. Targeting approaches include ubiquitin–proteasome, ubiquitin–lysosome, autophagy–lysosome, and caspase-mediated degradation pathways [5]. For example, arsenic sulfide As4S4 and diterpenoid oridonin promote ubiquitin–proteasome degradation of BCR-ABL [7,8]. Andrographolide, geldanamycin (GA), and 17-allylamino-17-demethoxygeldanamycin (17-AAG) downregulate the BCR-ABL oncoprotein by inducing HSP90 cleavage [9,10]. Arsenic trioxide (As2O3) promotes p62/SQSTM1-mediated autophagic degradation of the BCR-ABL [11]. Platinum pyrithione and xanthohumol downregulate BCR-ABL level through caspase-mediated degradation [12,13]. Isoflavones are dietary phytoestrogens mainly produced by Fabaceae family [14]. They are able to compete with estrogen hormone, 17β-estradiol, for the ligand-binding domain of the estrogen receptors and are used in the prevention and/or treatment of cancers, cardiovascular disease, osteoporosis, and postmenopausal symptoms [15–17]. Furthermore, genistein can inhibit tyrosine kinase activity and displays cancer chemopreventive activity [18]. 8-Hydroxydaidzein (8-OHD, 7,8,40-trihydroxyisoflavone, NSC-678112) is a hydroxylated derivative of daidzein commonly isolated from fermented soybean products. It has recently attracted research attention due to its strong anti-inflammatory, antioxidant, antitumor, anti-melanogenesis, and hepatoprotective activities [19–24]. It was found that 8-OHD had strong anti-proliferative activity toward HL-60 human promyelocytic leukemia cells [25]. In this study, we evaluated the anti-CML effects of 8-OHD with a focus on cell cycle arrest, apoptosis, autophagy, differentiation, and BCR-ABL levels in K562 cells, which is a model cell line derived from a female CML patient in blast crisis [26]. High-throughput microarray analysis is a valuable tool for early-stage drug discovery decision-making due to its potential to detect adverse effects at the transcriptomic level [27]. Thus, microarray analysis was employed to explore the differentially expressed genes (DEGs) in 8-OHD-treated K562 cells. Then, Gene Ontology (GO), (Kyoto Encyclopedia of Genes and Genomes) KEGG, and BioCarta pathways were analyzed [28–30]. We further established a protein–protein interaction (PPI) network of DEGs to screen out the hub genes with a high degree of connectivity in the biological process. Our data showed that 8-OHD induces reactive oxygen species (ROS) overproduction and cell cycle arrest at the S phase by upregulating p21Cip1 and downregulating cyclin-dependent kinase 6 (CDK6) and cyclin D2 (CCND2) expression. Caspase-7-dependent apoptosis and p62/UVRAV-dependent autophagy may be responsible for the decreased level of BCR-ABL. Furthermore, 8-OHD promoted Early Growth Response 1 (EGR1)-mediated megakaryocytic differentiation. 8-OHD also activated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, and both may be responsible for the stimulation of apoptosis, autophagy, and differentiation in K562 cells. This is the first transcriptomics report on the anti-leukemic effects of 8-OHD, and the obtained results can provide a more thorough understanding about potential development of 8-OHD in CML therapy. Biomedicines 2020, 8, 506 3 of 23 Biomedicines 2020, 8, x FOR PEER REVIEW 3 of 23 2. Experimental Section 2. Experimental Section 2.1. Preparation of 8-Hydroxydaidzein (8-OHD, 7,8,40-trihydoxyisoflavone, NSC-678112) 2.1. Preparation of 8-Hydroxydaidzein (8-OHD, 7,8,4′-trihydoxyisoflavone, NSC-678112) 8-OHD was isolated from soybean fermented by Aspergillus oryzae, and the NMR spectral data and8-OHD purification was isolated of 8-OHD from was soybean reported fermented previously by [20 Aspergillus,21,24]. The oryzae chemical, and the structure NMR ofspectral 8-OHD data was andshown purification in Figure of1a. 8-OHD was reported previously [20,21,24]. The chemical structure of 8-OHD was shown in Figure 1a. 120 24 h 48 h 100 * * ** ** 80 ** ** 60 ** 40 Cell proliferation (%) proliferation Cell 20 0 (a) veh 12.5 25 50 100 8-OHD (μM) (b) 120 24 h 48 h 100 * ** ** 80 ** 60 ** ** 40 ** Cell viability (%) viability Cell 20 0 veh 12.5 25 50 100 8-OHD (μM) (c) FigureFigure 1. 1. EffectsEffects ofof 8-hydroxydaidzein8-hydroxydaidzein (8-OHD) (8-OHD) on on the the cell viabilitycell viability of K562 of cells.K562 (acells.) Chemical (a) Chemical structure structureof 8-OHD. of K5628-OHD. cells K562 were cells treated were with treated vehicle with (0.1% vehicle DMSO) (0.1% or DMSO) 8-OHD or (6.25–100 8-OHD µ(6.25–100M) for 24 μ andM) 48for h. 24(b and) Cell 48 viability h. (b) Cell was viability measured was by measured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) bromideassay. (c )(MTT) Cell viability assay. was(c) examinedCell viability using was trypan examined blue exclusion using test.trypan The blue experiments exclusion were test. repeated The experimentsthree times. were These repeated data represent three times. the mean These dataSD of represent three independent the mean ± experiments. SD of three *independentp < 0.05 and ± experiments.** p < 0.01 represent