Selective Activation of ZAK Β Expression by 3-Hydroxy-2-Phenylchromone Inhibits Human Osteosarcoma Cells and Triggers Apoptosis Via JNK Activation

International Journal of Molecular Sciences

Article Selective Activation of ZAK β Expression by 3-Hydroxy-2-Phenylchromone Inhibits Human Osteosarcoma Cells and Triggers Apoptosis via JNK Activation

1,2, 3,4, 5 6 Chien-Yao Fu † , Ing-Shiow Lay †, Marthandam Asokan Shibu , Yan-Shen Tseng , Wei-Wen Kuo 7 , Jaw-Ji Yang 8, Tso-Fu Wang 9,10, B. Mahalakshmi 11, Yu-Lan Yeh 12,13,* and Chih-Yang Huang 6,14,15,16,*

1 Orthopaedic Department, Armed Forces General Hospital, Taichung 411, Taiwan; [email protected] 2 Department of Orthopaedic, National Defense Medical Center, Taipei 114, Taiwan 3 School of Post-Baccalaureate Chinese Medicine, China Medical University, Taichung 404, Taiwan; [email protected] 4 Chinese Medicine Department, China Medical University Beigang Hospital, Yunlin 651, Taiwan 5 Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan; [email protected] 6 Graduate Institute of Biomedical Science, China Medical University, Taichung 404, Taiwan; [email protected] 7 Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan; [email protected] 8 School of Dentistry, Chung-Shan Medical University, Taichung 402, Taiwan; [email protected] 9 Department of Hematology and Oncology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan; [email protected] 10 School of Medicine Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 97004, Taiwan 11 Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam; [email protected] 12 Department of Pathology, Changhua Christian Hospital, Changhua 500, Taiwan 13 Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli 356, Taiwan 14 Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan 15 Department of Biotechnology, Asia University, Taichung 41354, Taiwan 16 Center of General Education, Buddhist Tzu Chi Medical Foundation, Tzu Chi University of Science and Technology, Hualien 97004, Taiwan * Correspondence: [email protected] (Y.-L.Y.); [email protected] (C.-Y.H.) These authors have equally contributed to this work. † Received: 11 March 2020; Accepted: 21 April 2020; Published: 9 May 2020

Abstract: Although various advancements in radical surgery and neoadjuvant chemotherapy have been developed in treating osteosarcoma (OS), their clinical prognosis remains poor. A synthetic chemical compound, 3-hydroxylﬂavone, that is reported to regulate ROS production is known to inhibit human bone osteosarcoma cells. However, its role and mechanism in human OS cells remains unclear. In this study, we have determined the potential of 3-Hydroxy-2-phenylchromone (3-HF) against OS using human osteosarcoma (HOS) cells. Our previous studies showed that Zipper sterile-alpha-motif kinase (ZAK), a kinase member of the MAP3K family, was involved in various cellular events such as cell proliferation and cell apoptosis, and encoded two transcriptional variants, ZAKα and β. In this study, we show that 3-HF induces the expression of ZAK and thereby enhances cellular apoptosis. Using gain of function and loss of function studies, we have demonstrated that ZAK activation by 3-HF in OS cells is conﬁned to a ZAKβ form that presumably plays a leading role

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in triggering ZAKα expression, resulting in an aggravated cancer apoptosis. Our results also validate ZAKβ as the predominant form of ZAK to drive the anticancer mechanism in HOS cells.

Keywords: osteosarcoma; 3-Hydroxy-2-phenylchromone; ZAKβ; apoptosis

1. Introduction Osteosarcoma (OS) is a rare cancerous tumor that commonly affects children and young adults [1]. Boys are twice as likely to have osteosarcoma as girls, and most cases of osteosarcoma involve the bones around the knee [2]. Osteosarcoma (OS) is also the most common histological form of primary bone cancer and sometimes spreads elsewhere [3]. In spite of advancements in the therapeutic strategies such as radical surgery and neoadjuvant chemotherapy, the clinical outcome of OS remains poor [4]; therefore, therapies that are more efficient must be explored. Our previous reports show that the chemotherapeutic agent doxorubicin induces cell apoptosis and attenuates cell viability of human bone OS cells by triggering Zipper sterile-alpha-motif kinase (ZAK) expression [5]. However, doxorubicin, being a chemotherapeutic agent, causes serious side effects, and therefore finding an alternative that induces ZAK expression is desirable in treating human OS. A synthetic flavonol 3-Hydroxy-2-phenylchromone (3-HF) [6] has been shown to inhibit endogenous Aurora B and hinder cancer cell growth [7]. In human bone osteosarcoma cells, U2OS and 143B cells, 3-HF can inhibit cell metastasis and reduce tumor growth in vivo [8,9]. However, its mechanism of action remains unclear. ZAK is a novel mixed lineage kinase-like protein containing a leucine-zipper (LZ) and a sterile-alpha motif (SAM) [10]. ZAK acts as a signal transduction kinase of the MAP3K family and encodes a protein with an N-terminal kinase catalytic domain, also termed MLTK (for MLK-like mitogen-activated protein triple kinase) [11,12]. Previous studies showed that there are two transcriptional splice variants encoding the different isoforms of ZAKα and ZAKβ, respectively; these isoforms have been characterized [11]. All of them have common structural characteristics that are unique among the protein kinase family: a catalytic domain bearing the amino acid motifs found in serine/threonine and tyrosine kinases, and one or two leucine-zipper motifs [13]. ZAKα contains a kinase domain followed by a short LZ motif and an SAM domain, and ZAKβ is composed of 455 amino acids and is identical to ZAKα from the N-terminus to the LZ motif; however, the ZAKβ sequence then diverges and lacks an SAM domain [11]. Numerous studies have indicated that ZAKα can play different roles in normal cells and in cancer cells. In our previous reports, we found that ZAKβ plays different roles in human OS cells and in H9c2 cardiomyoblast cells. In H9c2 cells, we found that ZAKβ plays an antagonistic role to antagonize and ameliorate the cardiac hypertrophic and apoptotic effects induced by ZAKα [13]. In addition, both the overexpression of ZAKβ in cardiac tissue and the beta-adrenergic stimulation of ZAKβ can activate the p38 and JNK pathways and lead to cardiac myocyte hypertrophy in transgenic mice [14]. ZAKβ can also activate JNK in response to saturated free fatty acids (FFA) [15]. In human OS cells, we found that ZAKβ could enhance ZAKα expression, resulting in a synergistic apoptotic effect [5,16]. In this study, we demonstrate that 3-HF can induce ZAK overexpression to trigger human OS cells apoptosis, and ZAKβ can play a leading role in activating ZAKα and a synergistic apoptotic effect.

2. Results In the present study, we elucidated the eﬀect of 3-HF against human OS cells through the ZAKβ signaling axis. Int. J. Mol. Sci. 2020, 21, 3366 3 of 12

2.1. 3-HF Reduces the Viability of Human Osteosarcoma Cells, which Correlates with Simultaneous Upregulation in ZAKα, β Levels and Cleaved Caspase Levels To investigate whether 3-HF inhibits cell viability of human OS cells, we performed an MTT assay, andInt. J. the Mol. results Sci. 2020 show, 21, x thatFOR PEER the cell REVIEW viability decreased as the concentration of 3-HF increased (Figure3 of1a). 13 Subsequently, the Western blot analysis showed that ZAKα, β expression levels increased upon 3-HF (Figure 1a). Subsequently, the Western blot analysis showed that ZAKα, β expression levels increased treatment in a dose-dependent manner. In addition, the levels of cleaved-Caspase 3, a prominent upon 3-HF treatment in a dose-dependent manner. In addition, the levels of cleaved-Caspase 3, a apoptosis marker, increased with a simultaneous decrease in survival protein Bcl-xL. The senescence prominent apoptosis marker, increased with a simultaneous decrease in survival protein Bcl-xL. The marker β-gal also increased with high concentrations of 3-HF (Figure1b). According to the results, senescence marker β-gal also increased with high concentrations of 3-HF (Figure 1b). According to we confirmed the enhancing effect of 3-HF on ZAK expression and its influence in suppressing the the results, we confirmed the enhancing effect of 3-HF on ZAK expression and its influence in viability of human OS cells. suppressing the viability of human OS cells.

FigureFigure 1. 1. Dose-dependentDose-dependent e effectffect of of 3-Hydroxy-2-phenylchromone3-Hydroxy-2-phenylchromone (3-HF)(3-HF) onon humanhuman osteosarcoma osteosarcoma (HOS)(HOS) cells.cells. (a(a)E) Effectffect of of 3-HF 3-HF treatment, treatment which, which in in a dose-dependenta dose-dependent manner manner (0.5–20 (0.5–µ20M) μM) decreased decreased the viability of human OS cells, measured with MTT assays. Data are shown as the mean SEM of three the viability of human OS cells, measured with MTT assays. Data are shown as the mean± ± SEM of independentthree independent experiments. experiments. (b) Western (b) Western blot analysis blot analysis of the of expression the expression of Zipper of Zipper sterile-alpha-motif sterile-alpha- β kinasemotif kinase (ZAK), ( apoptoticZAK), apoptotic protein proteinC-Caspase C-Caspase 3, survival 3, proteinsurvival Bcl-xL protein and Bcl--galxL and with β 3-HF-gal with treatment 3-HF µ intreatment a dose-dependent in a dose-dependent manner (0.5–20 mannerM). (0.5 ***-20μM).p < 0.001 *** p represents< 0.001 represents significance significance with respect with respect to the controlto the control group. group.

2.2. 3-HF Can Trigger Cell Apoptosis and Decrease Mitochondrial Membrane Potential Further, to understand the effect of 3-HF treatment on human OS cells, we used a TUNEL assay to detect apoptosis following 3-HF in human osteosarcoma cells. The results show that the apoptosis rate increased with 3-HF treatment in a dose-dependent manner compared with the control cells (Figure 2a). On the other hand, we used a JC-1 mitochondrial membrane potential assay to monitor mitochondrial health. These results showed a decrease in mitochondrial membrane potential through a reduction in red fluorescence, indicating an event of apoptosis under 3-HF treatment in a dose- dependent manner in human OS cells (Figure 2b). Simultaneously, we determined the proportion of apoptotic cells using a flow cytometer by the double staining of cultures with propidium iodide (PI)

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2.2. 3-HF Can Trigger Cell Apoptosis and Decrease Mitochondrial Membrane Potential Further, to understand the effect of 3-HF treatment on human OS cells, we used a TUNEL assay to detect apoptosis following 3-HF in human osteosarcoma cells. The results show that the apoptosis rate increased with 3-HF treatment in a dose-dependent manner compared with the control cells (Figure2a). On the other hand, we used a JC-1 mitochondrial membrane potential assay to monitor mitochondrial health. These results showed a decrease in mitochondrial membrane potential through a reduction in red fluorescence, indicating an event of apoptosis under 3-HF treatment in a dose-dependent manner in human OS cells (Figure2b). Simultaneously, we determined the proportion of apoptotic cells using a flow cytometer by the double staining of cultures with propidium iodide (PI) and annexin V-FITC. We found that dose-dependent increments in apoptotic cells among 3-HF-treated human OS cells were Int.clearly J. Mol. demonstratedSci. 2020, 21, x FOR (Figure PEER 2REVIEWc). The proportion of apoptotic cells following 3-HF treatment increased5 of 13 significantly compared with the control group.

Figure 2. Cont.

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Figure 2. Effect of 3-HF on mitochondrial membrane potential and apoptosis in HOS cells. (a,b) Cells Figure 2. Effect of 3-HF on mitochondrial membrane potential and apoptosis in HOS cells. (a), (b) were seeded on 6-well plates and treated with 0, 1.0, 4.0, 8.0, 16.0, or 20.0 µM of 3-HF for 24 h. Cells(a) The were apoptotic seeded e ffonect 6 detected-well plates by TUNEL and treated assay with and DAPI0, 1.0, staining 4.0, 8.0, in 16.0, human or 20.0μM OS cells. of ( b3)-HF Fluorescence for 24 h. (imagea) The of apoptotic human OS effect cells detected stained withby TUNEL JC-1 after assay 24 h and incubation DAPI staining with di infferent human concentrations OS cells. (b of) Fluorescence3-hydroxyflavone. image Photograph of human OS showing cells stained JC-1 red, with JC-1 JC green-1 after and 24 merge h incubation image. The with JC-1 different green concentrationsfluorescence indicates of 3-hydroxyflavo a decrease inne. mitochondrial Photograph showing membrane JC potential,-1 red, JC- an1 green event and in apoptosis. merge image. Increased The JCconcentrations-1 green fluorescence of 3-HF enhanced indicates the a decrease loss of mitochondrial in mitochondrial membrane membrane potential. potential, Scale bars an event indicate in apoptosis.100 µm at 20Increasedmagnification concentrations (c) The percentageof 3-HF enhanced of apoptotic the loss cells of in mitochondrial 3-HF groups increasedmembrane significantly potential. × Scalecompared bars indicate with the 100 control µm at group 20× magnification (parental cells). (c Flow) The charts:percentage Q4, annexinof apoptotic V-positive cells in and 3-HF propidium groups increasediodide (PI)-negative significantly cells compared indicate with early the apoptotic control cells;group Q2, (parental annexin cells). V- and Flow PI-positive charts: Q4, cells annexin represent V- positivelate apoptotic and propidium cells. ** p

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. Figure 3. Role of ZAK in 3-HF-induced apoptosis in HOS cells. Western blot analysis of the expression Figure 3. Role of ZAK in 3-HF-induced apoptosis in HOS cells. Western blot analysis of the expression of ZAK, apoptotic protein C-Caspase 3, survival protein Bcl-xL and β-gal by transient transfection of of ZAK, apoptotic protein C-Caspase 3, survival protein Bcl-xL and β-gal by transient transfection of shZAK in a dose-dependent manner (0.1–4.0 µg/mL) following 3-HF treatment (20 µM). shZAK in a dose-dependent manner (0.1–4.0 μg/mL) following 3-HF treatment (20μM). 2.4. Cell Apoptosis Induced by 3-HF is Due to ZAKβ Overexpression not ZAKα in Human OS Cells 2.4. Cell Apoptosis Induced by 3-HF is Due to ZAKβ Overexpression not ZAKα in Human OS Cells In the previous studies, we demonstrated that ZAKβ might play a novel role in the regulation of ZAKαIn-dependent the previous molecular studies, mechanisms we demonstrated in cancer that cells ZAKβ or normal might cells.play a Here, novel we role further in the hypothesize regulation thatof ZAKα ZAKβ-dependentplays a leading molecular role to triggermechanisms cell apoptosis in cancer following cells or 3-HF normal treatment cells. in Here human, we OS further cells. Tohypothesize confirm this that hypothesis, ZAKβ plays we transiently a leading role transfected to trigger siZAK cell βapoptosisin stable following clone cells 3 with-HF treatment ZAK, which in wehuman set up OS in cells. our To previous confirm research this hypothesis, [5]. In thewe Westerntransiently blot transfected analysis, siZAKβ we found in thatstable knockdown clone cells ZAKwithβ ZAKeliminated, which thewe downstreamset up in our signalingprevious eventsresearch mediated [5]. In the by W ZAKestern in stableblot analysis, clone cells we (Figurefound that4a). Subsequently,knockdown ZAKβ we transiently eliminated transfected the downstream siZAKβ followingsignaling 3-HFevents treatment mediated in humanby ZAK OS in cells. stable Further, clone wecells found (Figure that 4a).knockdown Subsequently, ZAKβ wecan transientattenuately the transfected downstream siZAKβ signaling following events 3 mediated-HF treatment by 3-HF in inhuman human OS OS cells. cells Further, (Figure we4b). found Interestingly, that knockdown we found ZAKβ that therecan attenuate was no significant the downstream e ffect onsignaling ZAKα proteinevents expressionmediated by (Figure 3-HF 4ina,b). human Taken OS together, cells (Figure these 4b results). Interestingly, indicate that we ZAKfoundβ thatmay there play awas major no rolesignificant in the regulation effect on ZAKα of molecular protein mechanisms expression (Figure in cancer 4a, cells 4b). or Taken normal together, cells. these results indicate that ZAKβ may play a major role in the regulation of molecular mechanisms in cancer cells or normal cells.

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Figure 4. Role of ZAK transcriptional variants and the effect of 3-HF on ZAKβ and ZAKα.(a) Western blot analysis of the expression of ZAK, pJNK, p-c-Jun, apoptotic protein C-Caspase 3, and survival Figureprotein 4. Bcl-xLRole of with ZAK siZAK transcriptionalβ by transient variants transfection and the for 24effect h in of a dose-dependent3-HF on ZAKβ mannerand ZAKα. (0–1.0 (aµ) gWestern/mL) blotin aanalysis stable cloneof the cell expression line expressing of ZAK, ZAK pJNK,α.( pb)-c Western-Jun, apoptotic blot analysis protein of C the-Caspase expression 3, and of survival ZAK, proteinpJNK, Bcl p-c-Jun,-xL with apoptotic siZAKβ protein by transient C-Caspase transfection 3, and survival for 24 proteinhours in Bcl-xL a dose with-dependent siZAKβ bymanner transient (0–1.0 μg/mtransfectionL) in a stable for 24 hclone in a dose-dependentcell line expressing manner ZAKα. (0–2.0 (bµ) gWestern/mL) following blot analysis 3-HF treatment of the expression in human of ZAK,OS cells. pJNK, p-c-Jun, apoptotic protein C-Caspase 3, and survival protein Bcl-xL with siZAKβ by 2.5.transient 3-HF Can transfecti Induce Cellon Apoptosisfor 24 hours by ZAK in aβ in dose Human-dependent OS Cells manner (0–2.0 μg/mL) following 3-HF treatment in human OS cells. To further understand the mechanism of ZAKβ following 3-HF treatment in human OS cells, we 2.5.used 3-HF the C TUNELan Induce assay Cell to A detectpoptosis apoptosis by ZAKβ following in Human 3-HF OS with Cells pre-transient transfection of shZAK and siZAKβ in human osteosarcoma cells. Based on the fluorescence microscopy results, we determined thatTo apoptosis further increasedunderstand with the 3-HF mechanism treatment of and ZAKβ decreased following under 3- ZAKHF treatment knockdown, in human especially OS ZAKcells,β we usknockdowned the TUNEL (Figure assay5a). to Wedetect also apoptosis used a JC-1 following mitochondrial 3-HF with membrane pre-transient potential transfection assay to monitor of shZAK andmitochondrial siZAKβ in health. human The osteosarcoma fluorescence microscopy cells. Based results on showed the fluorescence a loss of mitochondrial microscopy membrane results, we determinedpotential, indicating that apoptosis increased increased apoptosis with under 3-HF 3-HF treatment treatment. and However, decreased the un effectsder wereZAK attenuatedknockdown, especiallyin shZAK ZAKβ transfected knockdown cells, especially (Figure siZAK 5a). Weβ in also human used OS a cells JC- (Figure1 mitochondrial5b). Moreover, membrane the proportion potential assayof apoptotic to monitor cells following mitochondrial 3-HF treatment health. The increased fluorescence significantly, microscopy and the results proportion show ofed apoptotic a loss of mitochondrialcells following membrane shZAK or siZAKpotentialβ transfection, indicating of increase cells underd apoptosis 3-HF treatment under decreased3-HF treatment. significantly However, as thecompared effects were with attenuated the control groupin shZAK (Figure transfected5c). Taken cells, together, especially these results siZAKβ indicate in human that 3-HF OS treatmentcells (Figure 5b).in humanMoreover, OS cellsthe proportion can induce of cell apoptotic apoptosis cells via following the activation 3-HF of treatment ZAKβ. increased significantly, and the proportion of apoptotic cells following shZAK or siZAKβ transfection of cells under 3-HF treatment decreased significantly as compared with the control group (Figure 5c). Taken together, these results indicate that 3-HF treatment in human OS cells can induce cell apoptosis via the activation of ZAKβ.

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Figure 5. 3-HF activates ZAKα to initiate a synergistic effect by both ZAKβ and ZAKα, resulting in Figure 5. 3-HF activates ZAKα to initiate a synergistic effect by both ZAKβ and ZAKα, resulting in aggravated apoptosis in HOS cells. (a,b) Cells were seeded on 6-well plates and pre-transfected with aggravated apoptosis in HOS cells. (a), (b) Cells were seeded on 6-well plates and pre-transfected shZAK and siZAKβ for 24 h following 3-HF treatment in human OS cells. (a) The apoptotic effect with shZAK and siZAKβ for 24 h following 3-HF treatment in human OS cells. (a) The apoptotic effect was detected by TUNEL assay and DAPI staining in human OS cells. (b) Photograph showing JC-1 was detected by TUNEL assay and DAPI staining in human OS cells. (b) Photograph showing JC-1 red, JC-1 green merged image. The JC-1 green fluorescence indicates a decrease in mitochondrial red, JC-1 green merged image. The JC-1 green fluorescence indicates a decrease in mitochondrial membrane potential, an event in apoptosis. Increased concentrations of 3-HF enhanced the loss membrane potential, an event in apoptosis. Increased concentrations of 3-HF enhanced the loss of of mitochondrial membrane potential. Transiently transfected shZAK and siZAKβ attenuated the mitochondrial membrane potential. Transiently transfected shZAK and siZAKβ attenuated the loss loss of mitochondrial membrane potential following 3-HF treatment. Scale bars indicate 50 µm at of mitochondrial membrane potential following 3-HF treatment. Scale bars indicate 50 µm at 20× 20 magnification. (c) The percentage of apoptotic cells in 3-HF groups increased significantly compared magnification.× (c) The percentage of apoptotic cells in 3-HF groups increased significantly compared with the control group (parental cells) but decreased by transient transfection of shZAK and siZAKβ with the control group (parental cells) but decreased by transient transfection of shZAK and siZAKβ compared with the 3-HF treatment group. Flow charts: Q4, annexin V-positive and PI-negative cells compared with the 3-HF treatment group. Flow charts: Q4, annexin V-positive and PI-negative cells indicate early apoptotic cells; Q2, annexin V- and PI-positive cells represent late apoptotic cells. indicate early apoptotic cells; Q2, annexin V- and PI-positive cells represent late apoptotic cells. 3. Discussion 3. Discussion Osteosarcoma is an aggressive tumor that accounts for about 5% of pediatric malignancies. The majorityOsteosarcoma of patients is an aggressive are curable tumor withcombined that accounts treatment for about of chemotherapy5% of pediatric and malignancies. surgery [17 The,18]. mTheajority standard of patients chemotherapy are curable treatment with combined for osteosarcoma treatment is based of chemotherapy upon pre-operative and surgery and post-operative [17,18]. The standardchemotherapy chemotherapy with doxorubicin, treatment cisplatin, for osteosarcoma and methotrexate is based [19 up].on Osteosarcoma pre-operative is generally and post considered-operative chemotherapyas a highly invasive with metastatic doxorubicin cancer, cisplatin, with poor and prognosis. methotrexate The metastatic[19]. Osteosarcoma onset occurs is during generally the consideredearly stages as and a highly in post-surgery, invasive metastatic and therefore cancer 85% with of patients poor prognosis. with osteosarcoma The metastatic experience onset occurs cancer duringmetastasis the [20 early–22 ]. stages Combinational and in post treatment-surgery with, and surgery, therefore chemotherapy, 85% of patients and radiation with osteosarcoma therapy is experiencegenerally considered cancer metastasis clinically. [20– High-dose22]. Combinational chemotherapy treatment has shown with surgery, improvements chemotherapy with overall, and radiationthree-year therapy survival is rates generally of over 79%,considered and combinational clinically. High therapy-dose improves chemotherapy it to over has 85% shown [23]. improvementsHowever, the multifactorial with overall drugthree resistance-year survival in osteosarcoma rates of over hampers 79%, and the treatment combinational efficiency therapy and improveaffects prognosis,s it to over therefore 85% [23].the However long-term, the survivalmultifactorial of osteosarcoma drug resistance patients in osteosarcoma with recurrence hampers and themetastasis treatment is still efficiency poor [ 24 and]. In affects this context, prognosis strategies, therefore to enhance the long the-term effi survivalciencies of of chemotherapy osteosarcoma patientsregimens with with recu novelrrence combinatorial and metastasis therapy is still are desirable.poor [24]. In this context, strategies to enhance the efficienciesThe previous of chemotherapy studies on regimens U2OS and with 143B novel show combinatorial that 3-HF inhibits therapy tumor are desirable. growth and metastasis in vivoThe[ 8].previous In addition, studies 3-HF on can U2OS inhibit and endogenous 143B show Aurora that B 3 and-HF induce inhibit growths tumor inhibition growth of and the metastasiscancer cell in line vivo [7 ].[8 However,]. In addition, the mechanism 3-HF can in inhibit human endogenous OS cells remains Aurora unclear. B and In induce our previous growth inhibition of the cancer cell line [7]. However, the mechanism in human OS cells remains unclear. In

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our previous publication, we demonstrated that doxorubicin can induce cell apoptosis by

Int.overexpression J. Mol. Sci. 2020, of21 ,ZAK 3366 in human OS cells [5]. Furthermore, we have also demonstrated that ZAKβ9 of 12 can enhance ZAKα expression in human OS cells, resulting in a synergistic apoptotic effect [16]. However, due to the adverse side effects associated with doxorubicin, various alternative drugs are publication,under consideration we demonstrated [25,26]. Moreover, that doxorubicin about 40% can–45% induce of patients cell apoptosis with high by- overexpressiongrade osteosarcoma of ZAK are ineither human only OS partially cells [5 ].responsive Furthermore, or completely we have also unresponsive demonstrated to that doxorubicin ZAKβ can (Dox), enhance due ZAK to theα expressionincreased drug in human efflux OS by cells, the resultingtransporter in aprotein synergistic ABCB1 apoptotic [19]. effect [16]. However, due to the adverse side eInffects this associated study, we with provided doxorubicin, evidence various that 3- alternativeHF can induce drugs cell are apoptosis under consideration by upregulation [25,26 of]. Moreover,ZAKβ (Figure about 6) 40%–45%. In correlation of patients with our with previous high-grade report, osteosarcoma 3-HF induced are either an increase only partially in ZAKα, responsive β levels, orresult completelying in reduced unresponsive cell viability to doxorubicin and an increase (Dox), duein cell to apoptosis the increased in human drug e fflOSux cells. by the ZAK transporter has been proteinreported ABCB1 as a tumor [19]. suppressor protein in cancers of other origin [10,27]. Similar to our present results,In this overexpression study, we provided of ZAK evidence has been that previously 3-HF can reported induce cell to elevate apoptosis apoptosis by upregulation in hepatoma of ZAK cellsβ (Figure[10]. Kinase6). In activity correlation of ZAK with has our also previous been report, demonstrated 3-HF induced to trigger an increase G2 arrest in, thereby ZAK α, reducingβ levels, resultingproliferation in reduced in ZAK cell-expressing viability cells and an[13 increase]. Therefore in cell, ZAK apoptosis is an ideal in human target OSto enhance cells. ZAK the has effect been of reportedchemotherapy as a tumor against suppressor osteosarcoma protein, and in cancerssince 3- ofHF other selectively origin [promotes10,27]. Similar ZAKβ to expression our present, it results, causes overexpressiona domino effect ofon ZAKapoptosis has been via ZAKα previously. Consequently, reported toit is elevate a potential apoptosis candidate in hepatoma for further cells studies [10]. Kinasein this activitydirection. of ZAK has also been demonstrated to trigger G2 arrest, thereby reducing proliferation in ZAK-expressingIn conclusion, cellsour results [13]. Therefore, confirm the ZAK hypothesis is an ideal that target 3-HF to can enhance induce the human effect OS of chemotherapycell apoptosis, againstmainly osteosarcoma,via ZAKβ, not and ZAKα. since According 3-HF selectively to this promotes finding, ZAKβ ZAKβ mayexpression, play a novel it causes role a dominoin the clinical effect ontherapy apoptosis for osteosarcoma via ZAKα. Consequently,. it is a potential candidate for further studies in this direction.

FigureFigure 6. 6.Schematic Schematic diagram diagram showing showing the eﬀ ectthe of effect 3-HF onof human3-HF on OS cellshuman apoptosis. OS cells Arrow apoptosis. represents Arrow representsproteins proteins modulated modulated by 3-HF or by shZAK 3-HF andor shZAK siZAKβ and. siZAKβ.

4. MaterialsIn conclusion, and Methods our results conﬁrm the hypothesis that 3-HF can induce human OS cell apoptosis, mainly via ZAKβ, not ZAKα. According to this ﬁnding, ZAKβ may play a novel role in the clinical therapy4.1. Cell forCulture osteosarcoma.

4. MaterialsThe human and OS Methods cell line was purchased from the American Type Culture Collection (ATCC, CRL- 1543) (Rockville, MD, USA). Human OS cells were grown in Eagle’s Minimum Essential Medium 4.1.(Sigma, Cell CultureSt. Louis, MO, USA) containing 10% fetal bovine serum (Clontech, Mountain View, CA, USA) and 1% Pen-Strep Ampho (CORNING, Flintshire, UK) in humidified air (5% CO2) at 37 °C. The human OS cell line was purchased from the American Type Culture Collection (ATCC, CRL-1543) (Rockville, MD, USA). Human OS cells were grown in Eagle’s Minimum Essential Medium 4.2. Whole Cell Extraction (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (Clontech, Mountain View, CA, USA) and 1%Cultured Pen-Strep human Ampho osteosarcoma (CORNING, cells Flintshire, were trypsinized UK) in humidiﬁed and washed air once (5% COwith2) PBS. at 37 Then,◦C. the cell pellet was collected and lysed in lysis buffer (50 mM Tris (pH 7.5), 0.5 M NaCl, 1.0 mM EDTA (pH 4.2.7.5), Whole 10% glycerol, Cell Extraction 1 mM β-ME, 1% IGEPAL-630 and proteinase inhibitor) and centrifuged at 12,000 rpm Culturedfor 30 min. human Then, osteosarcoma the supernatant cells was were collected trypsinized in a new and 1.5 washed mL Eppendorf once with tube PBS. and Then, stored the cellat - pellet20 °C. was collected and lysed in lysis buﬀer (50 mM Tris (pH 7.5), 0.5 M NaCl, 1.0 mM EDTA (pH 7.5), 10% glycerol, 1 mM β-ME, 1% IGEPAL-630 and proteinase inhibitor) and centrifuged at 12,000 rpm for 30 min. Then, the supernatant was collected in a new 1.5 mL Eppendorf tube and stored at 20 C. − ◦ Int. J. Mol. Sci. 2020, 21, 3366 10 of 12

4.3. MTT Assay Cell viability was examined by the use of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Human osteosarcoma cells (1 104) were seeded on 24-well plates with MEM medium. × The following day, the cells were treated with diﬀerent concentrations of 3-HF and cultured for 24 h. Then, cell viability was assessed using the MTT reagent at a concentration of 5 mg/mL. Following incubation at 37 ◦C for 3 h, the reaction was stopped by adding 200 µL of dimethyl sulfoxide (DMSO). After the crystals were dissolved, the absorbance of each sample was determined at O.D. 570 nm.

4.4. Transient Transfection ShRNA-mediated ZAKα plasmid DNA was kindly provided by Dr. J. J. Yang (Chung Shan medical university, Taichung, Taiwan). The cells were grown to 60% conﬂuence by the day of transfection. The shRNA-mediated ZAKα was transfected into human osteosarcoma cells using the PureFection transfection reagent according to the manufacturer’s guidelines (System Biosciences, Mountain View, CA, USA). After 24 h, the cells were harvested and extracted for analysis.

4.5. siRNA Transfection Cells were plated in growth medium without antibiotics for 24 h prior to transfection. Transient transfection of siZAKβ was done using the PureFection transfection reagent according to the manufacturer’s instructions (System Biosciences, Mountain View, CA, USA). The cells were harvested 24 h after transfection.

4.6. Western Blot The Western blot analysis was performed following previous reports [28]. Proteins were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes. Non-specific protein binding was blocked in blocking buffer (5% milk, 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20), and proteins were blotted with specific antibodies in the blocking buffer at 4 ◦C overnight. After incubation with the secondary antibody for 1 h at room temperature, densitometric analysis of the immunoblots was performed using the AlphaImager 2000 digital imaging system (Digital Imaging System, Commerce, CA, USA). For repeated blotting, PVDF membranes were stripped with Restore Western Blot Stripping Buffer at room temperature for 10 min. The protein levels, represented by their band intensity, were determined by normalizing with the corresponding intensity of the internal control. The intensity was measured using imageJ 1.52v (NIH, Bethesda, MD, USA).

4.7. TUNEL The APO-BrdU TUNEL Assay Kit (Roche, Basel, Switzerland) was used to perform the terminal transferase-mediated dUTP nick-end labeling of nuclei, following Wo et al. [19] and the manufacturer’s protocol.

4.8. JC-1 Staining The mitochondrial membrane potential was assessed using the fluorescent probe JC-1 dye purchased from Sigma. Briefly, human OS cells were pre-transfected with shZAK and siZAKβ individually, and then treated with 3-HF and incubated with JC-1 working solution for 20 min at 37 ◦C in the dark. Cells were washed with cold JC-1 staining buffer, placed on ice, and observed immediately by fluorescence microscopy.

4.9. Detection of Cell Apoptosis Using Flow Cytometry The detection of apoptosis in human OS cells, which were treated with 3-HF in a dose-dependent manner and transiently transfected with shZAK and siZAKβ following 3-HF treatment, was analyzed by determining the ratio of cells along with the nucleus concentration and fragment. Cells were Int. J. Mol. Sci. 2020, 21, 3366 11 of 12 collected after incubation and then suspended in the buﬀer. During the apoptosis assay, the cells were stained with propidium iodide and annexin V-FITC (BD Biosciences, San Jose, CA, USA) and determined by ﬂow cytometry.

4.10. Antibodies and Reagents The following antibodies were used in this study: anti-pJNK, anti-p-c-jun, anti-Bcl-xL, anti-β-gal, and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Anti-C-Caspase-3 was purchased from Cell Signalling Technology (Danvers, MA, USA). The ZAK monoclonal antibody (M02) was purchased from Abnova (Taipei, Taiwan). 3-HF was purchased from Sigma. siZAKβ was kindly provided by Dr. J. J. Yang (Chung Shan Medical University, Taichung, Taiwan).

4.11. Statistical Analysis The data shown are provided as the means standard deviation (SD) of three (3) independent ± experiments. For comparisons between multiple groups, statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test using GraphPad 5 statistical software (San Diego, CA, USA). p < 0.05 was considered as signiﬁcant.

Author Contributions: C.-Y.F. designed the experiments; I.-S.L. and Y.-S.T. performed the experiments; B.M., Y.-L.Y., W.-W.K., and C.-Y.H. analyzed the data; J.-J.Y. and M.A.S. contributed reagents/materials/analysis tools; M.A.S. and T.-F.W. revised the draft. All authors have read and agreed to the published version of the manuscript. Funding: This study was also supported in part by China Medical University and Asia University (CMU101-ASIA-08). Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

OS Osteosarcoma ZAK Zipper sterile-alpha-motif kinase ∆ΨM Mitochondrial membrane potential

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