And Chemoresistant MDA-MB-231 Human Breast Cancer Cells Through Up-Regulation of IFIT2

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Article Baicalein Suppresses Stem Cell-Like Characteristics in Radio- and Chemoresistant MDA-MB-231 Human Breast Cancer Cells through Up-Regulation of IFIT2

So Yae Koh 1, Jeong Yong Moon 2, Tatsuya Unno 2,3 and Somi Kim Cho 1,2,3,* 1 Interdisciplinary Graduate Program in Advanced Convergence Technology and Science, Jeju National University, Jeju 63243, Korea; [email protected] 2 Subtropical/Tropical Organism Gene Bank, Jeju National University, Jeju 63243, Korea; [email protected] (J.Y.M.); [email protected] (T.U.) 3 Faculty of Biotechnology, College of Applied Life Sciences, SARI, Jeju National University, Jeju 63243, Korea * Correspondence: [email protected]; Tel.: +82-64-754-3348

Received: 18 February 2019; Accepted: 11 March 2019; Published: 14 March 2019

Abstract: Resistance to both chemotherapy and radiation therapy is frequent in triple-negative breast cancer (TNBC) patients. We established treatment-resistant TNBC MDA-MB-231/IR cells by irradiating the parental MDA-MB-231 cells 25 times with 2 Gy irradiation and investigated the molecular mechanisms of acquired resistance. The resistant MDA-MB-231/IR cells were enhanced in migration, invasion, and stem cell-like characteristics. Pathway analysis by the Database for Annotation, Visualization and Integrated Discovery revealed that the NF-κB pathway, TNF signaling pathway, and Toll-like receptor pathway were enriched in MDA-MB-231/IR cells. Among 77 differentially expressed genes revealed by transcriptome analysis, 12 genes involved in drug and radiation resistance, including interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), were identiﬁed. We found that baicalein effectively reversed the expression of IFIT2, which is reported to be associated with metastasis, recurrence, and poor prognosis in TNBC patients. Baicalein sensitized radio- and chemoresistant cells and induced apoptosis, while suppressing stem cell-like characteristics, such as mammosphere formation, side population, expression of Oct3/4 and ABCG2, and CD44highCD24low population in MDA-MB-231/IR cells. These ﬁndings improve our understanding of the genes implicated in radio- and chemoresistance in breast cancer, and indicate that baicalein can serve as a sensitizer that overcomes treatment resistance.

Keywords: baicalein; cancer stem cell; chemoresistance; IFIT2; radioresistance; triple-negative breast cancer

1. Introduction Breast cancer is the most prevalent cancer type worldwide and the second leading cause of cancer death for women, and over 60% of breast cancer patients receive radiation therapy [1]. Triple-negative breast cancers (TNBCs) are breast cancers that are negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). While only 15–20% of breast cancer patients are classiﬁed as TNBCs, TNBC patients present with factors related to poor prognosis and unfavorable features in histologic grade, tumor size, and metastasis [2,3]. Despite advances in breast cancer treatment, TNBC patients still rely on chemotherapy and radiotherapy for disease management. In particular, radiation therapy plays an important role in the management of invasive breast cancer and can improve the survival rate by preventing the spread of metastases [4]. Previously, signaling pathways, including Wnt/β-catenin signaling and androgen receptor signaling, were investigated as mechanisms of radioresistance in TNBC cells [5–7]. Adenosine triphosphate-binding

Nutrients 2019, 11, 624; doi:10.3390/nu11030624 www.mdpi.com/journal/nutrients Nutrients 2019, 11, 624 2 of 19 cassette (ABC) transporters, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling, and the epithelial–mesenchymal transition (EMT)-related pathway were reported as primary mechanisms for chemoresistance in TNBC [8,9]. Chemoresistance may occur simultaneously with radioresistance in cancer patients [10], and resistance to both chemotherapy and radiotherapy remains a major obstacle in achieving a durable response in TNBC patients. However, the underlying mechanisms for both radio- and chemoresistance have not been clarified. Therefore, it is necessary to improve our knowledge and the efficacy of chemotherapy and radiotherapy to prevent metastasis and to produce a continuous therapeutic effect. Cancer stem cells (CSCs), or tumor initiating cells (TICs), are a particular subpopulation of cells in cancers that are capable of self-renewal and differentiation into the heterogeneous lineages of cancer cells that comprise the tumor [11]. Human TNBC MDA-MB-231 cells are known to have higher CSC populations than other breast cancer cell lines [12,13]. CSCs are depicted at the top of the tumor hierarchy, developing chemo- and radioresistance, recurrence, and metastasis [14,15]. Cell surface markers or specific membrane transporters are used to identify CSCs. Cells expressing CD44highCD24low and CD133high on their surfaces have been suggested as breast CSCs [16,17]. Furthermore, ABC transporters such as multidrug-resistance-associated protein 1 (MRP1), multidrug-resistance protein 1 (MDR1), and ATP-binding cassette super-family G member 2 (ABCG2) are overexpressed in the CSCs of breast cancer, pumping out chemicals such as anti-cancer drugs or Hoechst 33342 [18]. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is an active compound of the roots of Scutellaria baicalensis Georgi, a traditional medicinal herb [19]. It is known for its biological benefits in reducing inflammation, tumor progression, and fibrosis, as well as targeting the tumor microenvironment [20–22]. Baicalein targets TNBC cells by inducing endoplasmic reticulum stress or changing mitochondrial membrane potentials by inducing intra-cellular reactive oxygen species (ROS) in the caspase-dependent pathway [23] or down-regulating special AT-rich sequence binding protein 1 (SATB1) and the Wnt/β-catenin pathway [24]. In resistant cancer cells, baicalein induced apoptosis by increasing death receptor 5 (DR5) in colon cancer expression [25]. However, the effect of baicalein on treatment-resistant breast cancer cells has not been studied. In this study, to identify the genes involved in the treatment resistance of TNBC cells and to assess the efficacy of phytochemicals that can overcome treatment resistance, we established and investigated the radio- and chemoresistant TNBC MDA-MB-231/IR cell line. We explored the mechanism underlying baicalein’s inhibition of the viability of treatment-resistant TNBC MDA-MB-231/IR cells and the possibility that baicalein can be a sensitizer to radiation and drugs for TNBC patients with therapy resistance.

2. Materials and Methods

2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), insulin, bovine serum albumin (BSA), βFGF, EGF, B-27 supplement, 100 × trypsin/EDTA, 10 × streptomycin/antibiotics, and TRIzol were purchased from Gibco (Gaithersburg, MD, USA), except for TRIzol (Ambion, Austin, TX, USA). Baicalein, luteolin, myricetin, kaempferol, rutin, quercetin, HEPES, Adriamycin (doxorubicin), 0 0 propidium iodide (PI), Hoechst 33342 dye, 2 ,7 -dichlorofluorescin diacetate (H2DCF-DA), and RNase A were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The reverse transcription system kit was purchased from Promega (Madison, WI, USA). TOPreal qPCR preMIX was obtained from Enzynomics (Daejeon, Korea). Annexin V-FITC apoptosis detection kit, MitoScreen (JC-1) kit, and Matrigel Matrix were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Cisplatin was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Amresco (Solon, OH, USA). The BCA protein assay kit was purchased from Thermo Fisher Scientific, Pierce Nutrients 2019, 11, 624 3 of 19

Protein Biology (Rockford, IL, USA). Primary antibodies were purchased from Cell Signaling (Danvers, MA, USA), except for IFIT2 (Santa Cruz Biotechnology, Dallas, TX, USA) and actin (Sigma Aldrich, St. Louis, MO, USA). Secondary antibodies were obtained from Vector Laboratories (Burlingame, CA, USA). PVDF membrane was obtained from Millipore (Billerica, MA, USA). The BS ECL Plus kit and 10× phosphate-buffered saline (PBS) were purchased from Biosesang (Seongnam, Gyeonggi, Korea).

2.2. Cell Culture and Generation of Resistant Cells MDA-MB-231 cells and the derived MDA-MB-231/IR cells were cultured in DMEM supplemented with 10% FBS and 1% streptomycin/antibiotics, and incubated at 37 ◦C in a humidified incubator (HERAcell 150i, Thermo Fisher, Rockford, IL, USA) with 5% CO2. After subculture, when cell confluency reached 70–80%, irradiations were performed. Irradiation was performed at the Applied Radiological Science Institute in Jeju National University using a 60CO Theratron-780 teletherapy (MDS Nordion, Ottawa, ON, Canada) unit at a dose rate of 1.52 Gy per minute. Twenty-five cycles of 2 Gy irradiation were performed over five weeks, and the surviving cells were named MDA-MB-231/IR cells.

2.3. Cell Viability The viability of MDA-MB-231 cells and MDA-MB-231/IR cells after sample treatment was determined by MTT assay. Brieﬂy, cells were cultured in 96-well plates at an initial density of 1 × 104 cells/mL in 200 µL per well. During radiation treatment, cells were directly irradiated in a 15-mL conical tube and seeded for 4 days. After the indicated time, the medium was removed, and 100 µL of MTT solution (1 mg/mL) was added; the formazan converted from MTT was dissolved in 150 µL of DMSO. Absorbance was detected by a microplate reader (Tecan, Männedorf, Zürich, Switzerland) at 570 nm.

2.4. Clonogenic Assay The colony formation assay was performed to calculate the surviving fraction. First, 1 × 104 cells were prepared for irradiation, which was performed at the Applied Radiological Science Institute in Jeju National University under the control of a radiologist. Cells (2 × 102) were irradiated by 0, 2, 4, 6, and 8 Gy of gamma rays and seeded on a 12-well plate. After 5 days of incubation, colonies on the plate were washed twice with PBS, ﬁxed, and dyed. Colonies were counted using the NIST integrated colony enumerator (NICE) software program (ftp://ftp.nist.gov/pub/physics/mlclarke/NICE).

2.5. Mammosphere Assay Mammosphere assay was performed according to a previous study [26]. For the mammosphere formation assay, serum-free DMEM supplemented with 1% BSA, 1 µM insulin, 10 ng/mL bFGF, 20 ng/mL EGF, and B-27 supplement was used. Cells were collected by trypsin and washed twice in PBS. Then, 2 × 102 cells were counted and seeded in a non-coated 24-well plate with the stem cell medium described above. Cells were incubated for 1 week to form mammospheres, and the number of mammospheres comprising ≥ 50 cells was counted.

2.6. Wound Healing Assay Cells were plated at 5 × 105 cells per well and incubated in a 6-well plate for 24 h to form a conﬂuent monolayer. A scratch in the shape of a cross was created using the tip of a sterilized 1000-µL pipette. Cells were washed twice with PBS, and the wells were reﬁlled with new DMEM supplemented with 5% FBS. The width of the gap created by the scratch was measured under a microscope immediately after scratching (0 h) and after 24 h. Nutrients 2019, 11, 624 4 of 19

2.7. Invasion Assay A transwell system (24-well plate, Corning, Cambridge, MA, USA) was used for the invasion assay. The upper inserts of the transwell were filled with 100 µL of 10% Matrigel solution diluted in cold serum-free DMEM. Cells were prepared at a density of 1 × 105 cells per well, washed twice in PBS, and transferred to the upper well of the transwell system in serum-free DMEM. The bottom well was filled with 600 µL of 2% FBS-DMEM. After 24 h of incubation, cells were removed and washed from the insert, except for the cells bound to the membrane. The membrane-bound cells were fixed with 1:1 acetone:methanol fixative and dyed with crystal violet. The stained invading cells were observed under a microscope and counted using the ImageJ program (National Institutes of Health, Bethesda, MD, USA), and the invasive rate of MDA-MB-231/IR cells was compared to that of MDA-MB-231 cells.

2.8. Flow Cytometry A FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used for flow cytometry analysis. The same number of cells (1 × 104) were detected by FACS. Cells (2 × 105) were detached by trypsin after incubated for 24 h. For cell cycle analysis, cells were washed twice with PBS and fixed in 70% ethanol. Two hours before analysis, cells were treated with RNase A (25 ng/mL) and PI (final concentration of 40 µg/mL) diluted in 500 µL of 2 mM EDTA-PBS and incubated at 37 ◦C to stain DNA. To detect apoptosis phenomena, the annexin V-FITC Apoptosis Detection Kit I was used following the manufacturer’s protocol. Briefly, cells were washed twice with PBS and mixed with both annexin V-FITC (1:20 dilution in 1× binding buffer) and propidium iodide (PI) (1:50 dilution in 1× binding buffer) for 15 min at room temperature. The analysis was performed within 30 min after the reaction. To detect cell surface markers, CD44-FITC and CD24-PE conjugated dyes were used for analysis. The conjugated dyes were diluted (1:10 dilution in 1× stain buffer) and incubated with cells at 4 ◦C for 15 min. The side population (SP) was observed with Hoechst 33342 dye. After being detached from the plate, cells were diluted in DMEM buffered with 1 mM HEPES, 2% FBS, and 5 mM Hoechst 33342 at 37 ◦C for 2 h. The MitoScreen (JC-1) kit was used following the manufacturer’s protocol to detect mitochondrial membrane potential. Briefly, cells were incubated with JC-1 dye (1:100 dilution in 1× assay buffer) at 37 ◦C for 15 min. After labeling, cells were washed, centrifuged, and resuspended in 500 µL of PBS.

2.9. Western Blot Assay Cells were prepared at a density of 1 × 105 on a 60-mm plate. After incubation or treatment, cells were lysed with RIPA lysis buffer. Most primary antibodies were used at 1:1000 dilutions, except GAPDH (1:10,000), and secondary antibodies were used at 1:5000 dilutions. Protein bands were detected using the BS ECL Plus kit.

2.10. Transcriptomic Analysis Transcriptomic analysis was performed according to a previous study [27]. Total RNA was extracted from human breast cancer cells using TRIzol. RNA (1 µg) was used to construct a library using the Illumina TruSeq mRNA Sample Prep kit (Illumina, San Diego, CA, USA). Poly-T oligo-attached magnetic beads were used to purify mRNA molecules. RNA sequencing (RNA-seq) was performed by Macrogen (Seoul, Korea) according to the manufacturer’s instructions. Prior to the transcriptome assembly, duplicate sequences were removed from raw reads using FastUniq, and the human genome GRCh38 was indexed using Spliced Transcripts Alignment to a Reference (STAR). Trinity was used to assemble reads into transcriptomes. Abundance was calculated using RNA sequencing by expectation maximization (RSEM) to determine statistically signiﬁcant DEGs (p < 0.001 and ≥ 2-fold change) with EdgeR. These DEGs were annotated using Trinotate [28] and uploaded to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS) to investigate the involved metabolic pathways. Nutrients 2019, 11, 624 5 of 19

2.11. Pathway Analysis We used the DAVID tool (Version 6.8 [29]) to predict pathways and gene ontology (GO). Functional analysis of DEGs revealed their associated GO molecular processes, biological processes, and cellular components.

2.12. Gene Expression Analysis Gene expression analyses and RFS were analyzed using TCGA, UCSC Xena browser [30] and the Kaplan–Meier plotter [31]. Patients negative for expression of ER and PR and ampliﬁcation of HER2 were considered TNBC patients. Gene expression was calculated using fragments per kilobase of transcript per million mapped reads (FPKM). Correlations and p-values were analyzed using the R program.

2.13. Statistical Analysis Statistical analyses were carried out by one-way ANOVA and t-test using SPSS (IBM Corp., NY, USA); p-values < 0.05 were considered to indicate statistical signiﬁcance and are denoted with an asterisk (*).

3. Results

3.1. MDA-MB-231/IR Cells Exhibited Increased Radio- and Chemoresistance Compared to Parental Cells To better understand the molecular mechanism of acquired therapy resistance in breast cancer, we generated resistant cancer cells after repetitive 2 Gy irradiations up to a total of 50 Gy. The cells surviving following irradiation were named MDA-MB-231/IR cells. The survival of MDA-MB-231/IR cells after irradiation was significantly higher than that of MDA-MB-231 parental cells, indicating that radiation resistance was obtained in MDA-MB-231/IR cells (Figure1A). The survival rate of MDA-MB-231/IR cells after irradiation was 1.93, 9.17, and 4.18 times higher than that of MDA-MB-231 parental cells, at 4, 6, and 8 Gy, respectively (Figure1B). The higher survival of MDA-MB-231/IR cells compared to MDA-MB-231 cells against irradiation was also observed by MTT assay (Supplementary Figure S1A). Taking into account a previous report that chemoresistance may occur simultaneously with radioresistance in cancer patients [10], we also measured the chemoresistance of MDA-MB-231/IR against both Adriamycin (doxorubicin) and cisplatin, which were chosen for their common use in breast cancer therapy. Adriamycin significantly reduced the viability of MDA-MB-231 cells, with an IC50 of 393.33 ± 9.43 nM, whereas the IC50 in MDA-MB-231/IR cells was > 800 nM (Figure1C). Cisplatin also reduced viability of MDA-MB-231 cells with an IC50 of 22.39 ± 1.61 µM, twofold lower than its IC50 in MDA-MB-231/IR cells (46.44 ± 2.82 µM) (Figure1D). Moreover, both Adriamycin and cisplatin treatment increased the population of sub-G1 cells (Supplementary Figure S1B,C). The population of MDA-MB-231 cells in sub-G1 increased from 2.43 ± 0.52% to 11.87 ± 1.23% after treatment with 100 nM of Adriamycin, whereas in MDA-MB-231/IR cells, the population in sub-G1 increased only from 2.16 ± 0.11% to 3.81 ± 1.39% (Supplementary Figure S1B). Cisplatin treatment increased the sub-G1 population from 4.01 ± 0.28% to 10.91 ± 0.56% in MDA-MB-231 cells, whereas the sub-G1 population did not change in MDA-MB-231/IR cells (Supplementary Figure S1C). This indicates that Adriamycin and cisplatin treatment induced cell death in MDA-MB-231 cells by increasing sub-G1 and apoptosis. These phenomena were also confirmed by annexin V/PI staining (Supplementary Figure S1D,E). In conclusion, these results demonstrate that MDA-MB-231/IR cells were more resistant to Adriamycin and cisplatin treatment than were MDA-MB-231 cells. Nutrients 2019, 11, 624 6 of 19 Nutrients 2019, 11, x FOR PEER REVIEW 6 of 18

Figure 1.1.Comparison Comparison of parental of parental MDA-MB-231 MDA-MB-231 cells with radio-cells andwith chemoresistant radio- and MDA-MB-231/IRchemoresistant MDA-MB-231/IRcells. (A) Representative cells. ( imagesA) Representative of the clonogenic images assay of andthe (clonogenicB) the surviving assay fraction and (B of) the MDA-MB-231 surviving fractionand MDA-MB-231/IR of MDA-MB-231 cells and after MDA-MB-231/IR ﬁve days of cells irradiation. after five MTTdays assayof irradiation. of MDA-MB-231 MTT assay and of MDA-MB-231MDA-MB-231/IR and cells MDA-MB-231/IR after (C) Adriamycin cells after and (C (D) )Adriamycin cisplatin treatment and (D) for cisplatin 24 h. Asterisks treatment (*) for indicate 24 h. Asteriskssigniﬁcant (*) differences indicate significant at p < 0.05. differences at p < 0.05.

3.2. Stem Cell Characteristics Were More ProminentProminent in MDA-MB-231/IR CellsCells thanthan ParentParent CellsCells Microscopic observation observation revealed revealed that the parentalthat the MDA-MB-231 parental cellsMDA-MB-231 and the MDA-MB-231/IR cells and cellsthe wereMDA-MB-231/IR clearly differentiated cells were in termsclearly of differentiated morphology. MDA-MB-231/IR in terms of morphology. cells had anMDA-MB-231/IR especially elongated, cells hadspindle-like an especially cell morphology elongated, with spindle-like increased cell intercellular morphology distance. with Theincreased morphology intercellular of MDA-MB-231/IR distance. The morphologycells was altered of comparedMDA-MB-231/IR to the parental cells cells,was exhibitingaltered compared mesenchymal to spindle-shapedthe parental cells, cells (Figureexhibiting2A). Wemesenchymal next examined spindle-shaped whether the change cells in(Figure morphology 2A). inWe MDA-MB-231/IR next examined cells whether was related the tochange the EMT. in Themorphology wound healing in MDA-MB-231/IR assay revealed cells that was 39.55 related± 2.81% to ofthe the EMT. wound Thewas wound filled healing in MDA-MB-231 assay revealed cells, whilethat 39.55 65.85 ±± 2.81%3.11% of of the the wound gap was was filled filled in MDA-MB-231/IR in MDA-MB-231cells cells, (Figure while2 65.85B,C). In± 3.11% addition, of the an invasiongap was filledassay in using MDA-MB-231/IR a transwell system cells (Figure showed 2B,C). that the In addi numbertion, of an MDA-MB-231/IR invasion assay using cells a that transwell went through system showedthe membrane that the was number 1.48 ± of0.13 MDA-MB-231/IR times greater than cells that that of MDA-MB-231went through cellsthe membrane (Figure2D,E). was Moreover, 1.48 ± 0.13 in Westerntimes greater blot analysis, than that MDA-MB-231/IR of MDA-MB-231 cells cells exhibited (Figure higher 2D,E). expression Moreover, levels in ofWestern EMT markers blot analysis, such as vimentin,MDA-MB-231/IR slug, and cells snail exhibited compared higher to MDA-MB-231 expression cells levels (Figure of EMT2F,G). markers Numerous such studies as vimentin, have reported slug, andthat activationsnail compared of the EMTto MDA-MB-231 program confers cells CSC (Figure properties 2F,G). [32Numerous–34], therefore, studies we have examined reported the CSCthat activationcharacteristics of the of MDA-MB-231/IR EMT program co cells.nfers The CSC number properties of mammosphere [32–34], therefore, formations we inexamined MDA-MB-231/IR the CSC characteristicscells was 1.44-fold of greater MDA-MB-231/IR than that of MDA-MB-231 cells. The cellsnumber when theof same mammosphere number of individual formations cells wasin MDA-MB-231/IRseeded (Figure2H,I). cells To was scrutinize 1.44-fold one greater aspect ofthan stem that cell of properties, MDA-MB-231 fluorescence-activated cells when the same cell number sorting (FACS)of individual was utilized cells towas detect seeded the expression(Figure 2H,I). of cell To surface scrutinize markers. one Theaspect FACS of analysisstem cell demonstrated properties, high low fluorescence-activatedthat the CD44 CD24 cellcell sorting population (FACS) was was higher utilized in MDA-MB-231/IR to detect the expression cells (90.05 of± cell1.35%) surface than markers.in MDA-MB-231 The FACS cells analysis (84.98 ± demonstrated2.51%) (Figure that2J,K). the It CD44 is alsohigh showedCD24low that cell cells population in the side was population higher in (SP)MDA-MB-231/IR could be regarded cells (90.05 as stem ± 1.35%) cells, so than we measuredin MDA-MB the-231 percentage cells (84.98 of SP ± 2.51%) (shown (Figure in blue) 2J,K). from It the is alsomain showed population that (MP;cells in shown the side in purple) population by Hoechst (SP) could 33342 be stainingregarded (Figure as stem2L). cells, The so %SP we valuesmeasured for theMDA-MB-231 percentage and of SP MDA-MB-231/IR (shown in blue) cells from were the 1.52 main± 0.43%population and 2.62 (MP;± 0.38%,shown respectively in purple) (Figureby Hoechst2M), 33342 staining (Figure 2L). The %SP values for MDA-MB-231 and MDA-MB-231/IR cells were 1.52 ± 0.43% and 2.62 ± 0.38%, respectively (Figure 2M), indicating that the %SP in MDA-MB-231/IR cells

Nutrients 2019, 11, 624 7 of 19 Nutrients 2019, 11, x FOR PEER REVIEW 7 of 18 indicatingincreased thatby 1.72 the %SP± 1.13 in fold MDA-MB-231/IR compared to parent cells increasedal MDA-MB-231 by 1.72 ± cells.1.13 foldIn addition, compared Western to parental blot MDA-MB-231analysis also demonstrated cells. In addition, that the Western levels blotof stem analysis cell markers, also demonstrated including CD44, that the Oct3/4, levels ABCG2, of stem and cell markers,MDR1, were including increased CD44, Oct3/4,in MDA-MB-231/IR ABCG2, and MDR1, (Figure were 2N,O). increased In conclusion, in MDA-MB-231/IR in addition (Figure to2 N,O).drug Inresistance conclusion, and in radiation addition toresistance, drug resistance MDA-MB-231/IR and radiation cells resistance, had stronger MDA-MB-231/IR stem cell characteristics cells had stronger than stemdid the cell parent characteristics cells. than did the parent cells.

FigureFigure 2.2. StemStem cell-likecell-like characteristicscharacteristics ofof MDA-MB-231/IR MDA-MB-231/IR cells.cells. ((A)) MorphologiesMorphologies ofof MDA-MB-231MDA-MB-231 cellscells andand MDA-MB-231/IRMDA-MB-231/IR cells. ( B,C) Migration and (D,E) invasion werewere analyzedanalyzed onon samesame numbernumber ofof MDA-MB-231MDA-MB-231 cellscells andand MDA-MB-231/IRMDA-MB-231/IR cells for 24 h. (F,G) Western blotblot assayassay forfor detectiondetection ofof EMTEMT markersmarkers expressedexpressed inin MDA-MB-231MDA-MB-231 cellscells andand MDA-MB-231/IRMDA-MB-231/IR cells.cells. GAPDH was usedused asas aa control;control; bandband intensitiesintensities werewere quantiﬁedquantified usingusing ImageJ.ImageJ. ((HH,I,I)) MammosphereMammosphere formationformation overover sevenseven days,days, ((JJ,,KK)) expressionexpression ofof cellcell surfacesurface markers,markers, andand ((LL,,MM)) sideside populationpopulation (SP)(SP) werewere detecteddetected byby FACSFACS analyses.analyses. ((NN,,OO)) WesternWestern blot blot assay assay for for stem stem cell cell markers markers on on MDA-MB-231 MDA-MB-231 cells cells and and MDA-MB-231/IR MDA-MB-231/IR cells.cells. AsterisksAsterisks (*)(*) indicateindicate signiﬁcantsignificant differencesdifferences atatp p< < 0.05.0.05. 3.3. Transcriptomic Analysis of MDA-MB-231/IR Cells 3.3. Transcriptomic Analysis of MDA-MB-231/IR Cells To better understand chemo- and radioresistance, RNA-seq was performed to identify differentially To better understand chemo- and radioresistance, RNA-seq was performed to identify expressed genes (DEGs). A total of 31,498 transcripts were identified, including 39 up-regulated DEGs differentially expressed genes (DEGs). A total of 31,498 transcripts were identified, including 39 up-regulated DEGs and 38 down-regulated genes in MDA-MB-231/IR cells compared to MDA-MB-231 cells (p < 0.001) (Figure 3A). After identifying the DEGs, pathways and gene ontology

Nutrients 2019, 11, 624 8 of 19 and 38 down-regulated genes in MDA-MB-231/IR cells compared to MDA-MB-231 cells (p < 0.001) (Figure3A). After identifying the DEGs, pathways and gene ontology (GO) were analyzed using the DatabaseNutrients for 2019 Annotation,, 11, x FOR PEER Visualization REVIEW and Integrated Discovery (DAVID) software to visualize8 of 18 the gene regulatory network and examine how radiation affected transcription in MDA-MB-231 cell lines. (GO) were analyzed using the Database for Annotation, Visualization and Integrated Discovery Our analysis revealed that MDA-MB-231/IR cells were enriched in the NF-κB, tumor necrosis factor (DAVID) software to visualize the gene regulatory network and examine how radiation affected (TNF),transcription and Toll-like in MDA-MB-231 receptor (TLR) cell signaling lines. Our pathways analysis comparedrevealed that to MDA-MB-231MDA-MB-231/IR cells cells (Figure were 3B). GO biologicalenriched in process the NF-κ analysisB, tumor revealednecrosis factor the activation(TNF), and ofToll processes-like receptor including (TLR) signaling positive pathways regulation of cell division,compared regulation to MDA-MB-231 of transcription cells (Figure from 3B). RNA GO biological polymerase process II promoter, analysis revealed and regulation the activation of protein phosphorylationof processes including (Figure3C). positive GO cellularregulation component of cell division, analysis regula revealedtion of changestranscription infocal from adhesion,RNA nucleoplasm,polymerase cytoplasm, II promoter, and membraneand regulation activation of prot inein experimental phosphorylation groups (Figure (Figure 3C).3D). GO GO cellular molecular processcomponent terms indicated analysis thatrevealed ATP binding,changes in chromatin focal adhesion, binding, nucleoplasm, and interleukin-1 cytoplasm, (IL-1) and receptor membrane binding wereactivation highly regulated in experimental in MDA-MB-231/IR groups (Figure cells3D). (FigureGO molecular3E). Using process the terms results indicated of theDEG that ATP and GO binding, chromatin binding, and interleukin-1 (IL-1) receptor binding were highly regulated in analyses, we identified the enriched pathways to reveal the biological functions and molecular processes MDA-MB-231/IR cells (Figure 3E). Using the results of the DEG and GO analyses, we identified the involving the up- and down-regulated genes. Literature searches were performed to select genes that enriched pathways to reveal the biological functions and molecular processes involving the up- and play adown-regulated role in cancer resistance,genes. Literature and 12 searches genes were performed identified to based select on genes their that expression play a role levels in cancer and roles in cancer.resistance, The and expression 12 genes levels were identified of the 12 based selected on their genes expression are described levels byand difference roles in cancer. and log2The fold changeexpression in Table levels1. Ten of genes, the 12 selected aldo-keto genes reductase are descri 1C1bed (AKR1C1), by difference aldo-keto and log2 reductasefold change 1C2 in Table (AKR1C2), 1. aldo-ketoTen genes, reductase aldo-keto 1C3 (AKR1C3),reductase 1C1 coiled-coil (AKR1C1), domain-containing aldo-keto reductase 69 (CCDC69),1C2 (AKR1C2), ferritin aldo-keto light chain (FTL),reductase glutamine-fructose-6-phosphate 1C3 (AKR1C3), coiled-coil transaminasedomain-containing 2 (GFPT2), 69 (CCDC69), galectin-3-binding ferritin light proteinchain (FTL), (LG3BP), midkineglutamine-fructose-6-phosphate (MK), transforming growth transaminase factor beta induced 2 (GFPT2), (TGFBI), galectin-3-binding and twinfilin actinprotein binding (LG3BP), protein 1 (TWF1),midkine were (MK), up-regulated transforming in MDA-MB-231/IR growth factor beta cells induced by up (TGFBI), to 6.83 and log2 twinfilin fold, while actin two binding genes, protein IFIT2 and 1 (TWF1), were up-regulated in MDA-MB-231/IR cells by up to 6.83 log2 fold, while two genes, IFIT2 plakophilin 3 (PKP3), were down-regulated. Of the 12 selected genes, six genes, AKR1C1, MK, TGFBI, and plakophilin 3 (PKP3), were down-regulated. Of the 12 selected genes, six genes, AKR1C1, MK, AKR1C2, TWF1, and PKP3, are reported to function in metastasis, migration, and invasion. Three genes, TGFBI, AKR1C2, TWF1, and PKP3, are reported to function in metastasis, migration, and invasion. FTL, AKR1C3,Three genes, and FTL, CCDC69, AKR1C3, are and reported CCDC69, to be are involved reported in to drug be involved resistance, in drug while resistance, the remaining while the selected genesremaining are involved selected in stem genes cell-like are involved functions in stem (anti-differentiation), cell-like functions (anti-differentiation), metabolism, and apoptosis.metabolism, These resultsand imply apoptosis. that theThese 12 results selected imply genes that are the likely12 selected to be genes involved are likely in determining to be involved the in characteristicsdetermining of MDA-MB-231/IRthe characteristics cells. of MDA-MB-231/IR cells.

FigureFigure 3. Analyses 3. Analyses of differentiallyof differentially expressed expressed genesgenes (DEGs) in in MDA-MB-231/IR MDA-MB-231/IR cells cells compared compared to to parentalparental MDA-MB-231 MDA-MB-231 cells. cells. (A) Heatmap(A) Heatmap of DEG of expression.DEG expression. (B) Pathway (B) Pathway analysis analysis and (Cand) biological, (C) (D) cellular,biological, and (D ()E cellular,) molecular and ( geneE) molecular ontology gene (GO) ontology analyses (GO) of anal DEGsyses in of MDA-MB-231/IRDEGs in MDA-MB-231/IR cells. cells.

Nutrients 2019, 11, 624 9 of 19

Table 1. Differentially expressed genes (DEGs) in MDA-MB-231/IR cells related to cancer resistance. DEGs are expressed in differences, fold changes (log2), with their functions.

No. Difference Fold Gene Full Name Role in Cancer 1 97.57 6.83 AKR1C1 Aldo-keto reductase 1C1 Metastasis 2 79.67 6.64 FTL Ferritin light chain Drug resistance 3 77.43 2.92 MK Midkine Metastasis 4 62.20 6.01 LG3BP Galectin-3-binding protein Anti-differentiation 5 46.92 3.04 GFPT2 Glutamine-fructose-6-phosphate Transaminase 2 Metabolism 6 42.64 3.34 TGFBI Transforming growth factor beta induced Metastasis 7 42.35 3.87 AKR1C3 Aldo-keto reductase 1C3 Drug resistance 8 37.59 2.96 CCDC69 Coiled-coil domain-containing 69 Drug resistance 9 14.51 4.13 AKR1C2 Aldo-keto reductase 1C2 Metastasis 10 12.55 +Inf TWF1 Twinﬁlin actin binding protein 1 Migration 11 −4.33 −Inf PKP3 Plakophilin 3 Invasion Interferon induced protein with tetratricopeptide Apoptosis 12 −85.90 −2.84 IFIT2 repeats 2 mediator Inf, inﬁnity: expression of the indicated gene was not determined.

3.4. Baicalein Treatment Reversed the Level of IFIT2 Expression in MDA-MB-231/IR Cells There have been compelling reports of polyphenols, flavones, and flavonols that are effective against breast cancer [35,36]. Therefore, we conducted a cell viability test on MDA-MB-231/IR cells against 10 representative anti-cancer phytochemicals: three phenols, three flavones, and four flavonols. Among the compounds tested, the cell-killing effect of baicalein was shown in both MDA-MB-231 cells and the treatment-resistant MDA-MB-231/IR cells, notably showing a lower IC50 value in MDA-MB-231/IR cells (Supplementary Table S1). MTT assay revealed that, baicalein effectively induced the death of MDA-MB-231/IR cells in a time- and dose-dependent manner, with an IC50 of 38.58 ± 2.86 µM at 24 h (Figure4A). Baicalein was, therefore, selected for further experiments. We hypothesized that baicalein may target MDA-MB-231/IR cells by regulating the expression of the 12 selected genes identified by transcriptomics. Expression of the 12 genes was analyzed by real-time PCR, and the results showed that their expression levels in MDA-MB-231/IR cells were consistent with the transcriptome analysis, except for TWF1 (Figure4B). Interestingly, only IFIT2 appeared to be affected by baicalein treatment, and the expression of IFIT2 was reversed in a dose-dependent manner (Figure4C). To determine the role of IFIT2, we used Xena browser and the Kaplan-Meier plotter to analyze the relationships between IFIT2 expression levels and the progression of The Cancer Genome Atlas (TCGA) breast cancer cohort and the survival rate of TNBC patients. IFIT2 expression was decreased in metastatic breast cancer compared to primary tumor or normal tissue (n = 1247, one-way analysis of variance [ANOVA], p-value = 0.0014, f = 6.593) (Figure4D). The relapse-free survival (RFS) graph analyzed using the Kaplan–Meier plotter revealed that lower IFIT2 expression levels were associated with poor prognosis (lower RFS) compared to TNBC patients expressing higher IFIT2 levels (n = 618, log-rank p-value = 0.014, hazard ratio [HR] = 0.72, probe id: 217502-at) (Figure4E). Taken together, these results indicate that baicalein can target MDA-MB-231/IR cells by reversing IFIT2 expression, which is known to be associated with metastasis and recurrence. Nutrients 2019, 11, 624 10 of 19 Nutrients 2019, 11, x FOR PEER REVIEW 10 of 18

FigureFigure 4.4. BaicaleinBaicalein treatment treatment reversed reversed levels levels of IFIT2, of IFIT2, which which is known is known to be associated to be associated with metastasis with metastasisand recurrence. and recurrence. (A) MTT assay (A) ofMTT baicalein assay onof MDA-MB-231/IRbaicalein on MDA-MB-231/IR cells for 6, 12, cells and 24for h. 6, ( B12,) Expression and 24 h. (levelsB) Expression of DEGs involved levels of in DEGs resistance involved in MDA-MB-231/IR in resistance cells.in MDA-MB-231/IR (C) IFIT2 gene expression cells. (C after) IFIT2 baicalein gene expressiontreatment for after 24 h.baicalein (D) IFIT2 treatment expression for in24 primaryh. (D) IFIT2 tumor, expression metastatic, in andprimary solid tumor, normal metastatic, tissue of breast and solidcancer normal patients tissue based of onbreast Xena cancer browser. patients (E) Relapse-free based on Xena survival browser. (RFS) ( plotE) Relapse-free of TNBC patients survival analyzed (RFS) plotby IFIT2 of TNBC expression patients with analyzed the Kaplan–Meier by IFIT2 expression plotter. Asterisks with the (*)Kapl indicatean–Meier signiﬁcant plotter. differencesAsterisks (*) at indicatep < 0.05. significant differences at p < 0.05. 3.5. Baicalein Suppressed the Stem Cell-Like Characteristics of MDA-MB-231/IR Cells 3.5. Baicalein Suppressed the Stem Cell-Like Characteristics of MDA-MB-231/IR Cells We tested whether the stem cell-like properties of MDA-MB-231/IR cells could be reversed We tested whether the stem cell-like properties of MDA-MB-231/IR cells could be reversed with with increased IFIT2 expression following treatment with baicalein. Western blot, migration, and increased IFIT2 expression following treatment with baicalein. Western blot, migration, and invasion assays were performed to confirm changes in the EMT phenotype of MDA-MB-231/IR cells. invasion assays were performed to confirm changes in the EMT phenotype of MDA-MB-231/IR cells. Baicalein treatment decreased migration (0.61 fold) and the expression of slug in MDA-MB-231/IR cells Baicalein treatment decreased migration (0.61 fold) and the expression of slug in MDA-MB-231/IR (Figure5A–D). Invasion ability also decreased (0.43-fold) after baicalein treatment (Figure5E,F). To further cells (Figure 5A–D). Invasion ability also decreased (0.43-fold) after baicalein treatment (Figure examine the changes in stem cell-like characteristics, mammosphere formation, CD44highCD24low, and 5E,F). To further examine the changes in stem cell-like characteristics, mammosphere formation, SP population analyses were performed. The size and number of mammospheres and the expression CD44highCD24low, and SP population analyses were performed. The size and number of levels of stem cell-like markers were significantly decreased after baicalein treatment (Figure5G,H). mammospheres and the expression levels of stem cell-like markers were significantly decreased The expression levels of stem cell markers Oct3/4 and ABCG2 decreased following baicalein treatment after baicalein treatment (Figure 5G,H). The expression levels of stem cell markers Oct3/4 and (Figure5I,J). In addition, compared to control (baicalein 0 µM), the percentage of CD44highCD24low cell ABCG2 decreased following baicalein treatment (Figure 5I,J). In addition, compared to control (baicalein 0 μM), the percentage of CD44highCD24low cell population and SP were decreased following

Nutrients 2019, 11, 624 11 of 19 Nutrients 2019, 11, x FOR PEER REVIEW 11 of 18 baicaleinpopulation treatment and SP were (Figure decreased 5K–N following). In summary, baicalein baicalein treatment treatm (Figureent5K–N). decreased In summary, stem baicaleincell-like propertiestreatment decreasedin MDA-MB-231/IR stem cell-like cells. properties in MDA-MB-231/IR cells.

FigureFigure 5. 5. EffectsEffects of of baicalein baicalein on on characteristics of of MDA-MB-231/IR cells. cells. (A (A,B,B) )Migration Migration assay assay and and (C(C,D,D)) Western Western blot blot analysis analysis of of EMT EMT proteins proteins in in MD MDA-MB-231/IRA-MB-231/IR cells cells were were pe performedrformed after after baicalein baicalein treatmenttreatment for for 24 24 h. h. GAPDH GAPDH was was used used as as a acontrol; control; band band intensities intensities were were qu quantiﬁedantified using using ImageJ. ImageJ. EffectsEffects of of baicalein baicalein on on stem stem cell-like cell-like characteristics characteristics were were determined determined by by (E (E,F,F) )invasion invasion assay assay for for 24 24 h h andand ( (G,H)) mammospheremammosphere formationformation assay assay for for one one week. week. (I,J )( WesternI,J) Western blot assayblot forassay stem for cell stem markers cell markersin MDA-MB-231 in MDA-MB-231 cells after cells baicalein after baicalein treatment treatment for 24 h.for ( K24,L h.) The(K,L percentage) The percentage of expression of expression of cell ofsurface cell surface markers markers and ( Mand,N )(M side,N) population side population (SP) on (SP) MDA-MB-231/IR on MDA-MB-231/IR cells werecells were detected detected by FACS by FACSafter baicaleinafter baicalein treatment treatment for 24 for h. Asterisks24 h. Aste (*)risks indicate (*) indicate signiﬁcant significant differences differences at p < at 0.05. p < 0.05.

3.6.3.6. Baicalein Baicalein Induced Induced Apoptosis Apoptosis and and Reversed Reversed Ra Radio-dio- and and Chemoresistance Chemoresistance in in MDA-MB-231/IR MDA-MB-231/IR Cells Cells AfterAfter identifying identifying IFIT2 IFIT2 induction induction and and the the decrease decreasedd stem stem cell-like cell-like properties, properties, we we then then examined examined thethe effect effect of of baicalein baicalein on on apoptosis apoptosis in in MDA-MB-231/IR MDA-MB-231/IR cells. cells. Western Western blot blot results results indicated indicated that that the the inductioninduction of of apoptosis apoptosis by by baicalein baicalein treatment treatment was was accompanied accompanied by by up-regulation up-regulation of of IFIT2. IFIT2. After After ± treatmenttreatment with with baicalein, baicalein, the the level level of of IFIT2 IFIT2 increased increased markedly, markedly, by 28.24 ± 0.900.90 times, times, while while the the levels levels γ ± ± ofof γ-H2AX-H2AX increased increased 5.47 5.47 ± 0.910.91 fold, fold, Bax Bax increased increased 4.20 4.20 ± 0.67 0.67fold,fold, cleaved cleaved caspase-3 caspase-3 increased increased 4.20 ± 0.90 fold, and cleaved PARP increased 5.23 ± 0.95 fold (Figure 6A,B). Cell cycle and annexin V/PI

Nutrients 2019, 11, 624 12 of 19

NutrientsNutrientsNutrients 2019 2019, 11 2019, ,x 11 FOR, 11,Nutrients x, FOR xPEER FOR PEER 2019REVIEWPEER REVIEW, 11 REVIEW, x FOR PEER REVIEW 12 of12 1812 of of18 18 12 of 18 4.20 ± 0.90 fold, and cleaved PARP increased 5.23 ± 0.95 fold (Figure6A,B). Cell cycle and annexin stainingstainingstaining also also alsoshowed V/PIstaining showed showed stainingan analsoincrease an increase showed alsoincrease in showed ap in an in optotic ap apincrease anoptoticoptotic increase cells cellsin cells after apoptotic in after apoptotic after baicalein baicalein baicaleincells cells treatment after treatment after treatment baicalein baicalein (Figure (Figure (Figure treatment treatment 6C – 6CE). 6C– E). In– E).(Figure (Figure In In 66C–E).C–E). InIn particular,particular,particular, JC- 1JC stainingJC-particular,1particular,- 1staining staining revealed revealed JC-1JC-1 revealed stainingstainingthat, that, that,as the revealedrevealedas as theconcentration the concentration concentration that,that, asas thetheof baicalein concentrationconcentrationof of baicalein baicalein increased, increased, increased, ofof baicaleinbaicalein a decrease a adecrease increased,decrease in red in in red a red decrease in redred fluorescence,fluorescence,fluorescence, from ﬂuorescence, fromfluorescence, from 98.9 98.9 ±98.9 3.51% ± ± 3.51% from 3.51% to 79.898.9 to98.9 to 79.8 ± 79.8± 2.21%, 3.51%± ±2.21%, 2.21%, towas 79.879.8 was observed,was ±± observed, observed,2.21%, indicating was indicating indicating observed, depolarization depolarization depolarization indicating of depolarization th of ofe th the e of thethe mitochondrialmitochondrialmitochondrial membranemitochondrial membrane membrane potential potential potential membranemembrane (Figure (Figure (Figure potential potential 6F,G). 6F,G). 6F,G). In (Figure (Figure addition, In In addition, 6 addition,F,G). 6F,G). combination In combination addition, In combination addition, combinationtreatment treatment combination treatment of treatment baicalein of of baicalein treatment baicalein of baicalein of baicalein with withwith with radiation radiation radiation decreasedradiationwith decreased decreased radiation decreasedcolony colony colonydecreased formation colony formation formation colony formation and and theand formation the andsurvival the survival the survival survivaland fraction fraction the fraction fractionsurvival of MDAof of MDA of MDAfraction- MDA-MB-231/IRMB-MB-231/IR-MB -231/IRof-231/IR MDA-MB-231/IR cells cells cells cells compared cells comparedcomparedcompared to baicalein to to tocompared baicalein baicalein baicalein treatment treatment to treatment treatment baicalein alone alone alone (Figuretreatment (Figure (Figure (Figure 6H,I). alone 6H,I).6 H,I). 6H,I). We (Figure We also We also also alsotested6H,I tested tested tested). theWe the the effectthealso effect effect effect tested of of combination of combination of combinationthe combination effect treatmentof combination with treatmenttreatmenttreatment with with with severalseveraltreatment several several anti anti-cancer anti-with cancer anti-cancer -severalcancer drugs drugs drugs drugsanti-cancer on on oncell on cell viability. viability. cell drugs viability. viability. Theon The Thecell viability viability The viability. viability viability of of MDA-MB-231/IR MDA ofThe of MDA MDAviability-MB-MB-231/IR-MB- 231/IRof-231/IR cellsMDA-MB-231/IR cells cells treated cells with bothcells treatedtreatedtreated with with withboth bothbaicalein treated bothbaicalein baicalein baicalein with and and both Adriamycinand Adriamycinand baicaleinAdriamycin Adriamycin (with (withand (with concentration Adriamycin(with concentration concentration concentration (with of 0,of 12.5, 0,concentrationof of12.5, 0, 25,0, 12.5, 50,12.5,25, and25,50, 25, 50,ofand 100 50, 0,and nM) 100and12.5, 100 nM) or 10025, baicaleinnM) nM)or50, orand or and 100 cisplatin nM) or baicaleinbaicaleinbaicalein and and cisplatinand (withcisplatinbaicalein cisplatin (with concentration (withand (with conc cisplatin conc entrationconcentration ofentration 0,(with 10,of 0, 20,concentrationof 10,of 0, 30, 0, 10,20, 40,10, 20,30, and20, 30,40, 30, of 5040,and 0,40,µ andM)10, 50and was20,μM)50 50 30,μM) decreasedμM)was 40, was decreasedwasand decreased 50decreased compared μM) compared was compared compared to decreased negative controlcompared or to negativetoto negative negative control control controlcellsto or negative cells treatedor or cells treatedcells control withtreated treated with baicalein or with cellswithbaicalein baicalein treate alone,baicalein alone,d or withalone, anti-canceralone, or baicalein anti or or anti- canceranti- drug canceralone,-cancer drug alone, ordrug drug alone,anti-cancer with alone, alone, with combination with withcombinationdrug combination combination alone, index with (CI) combination values < 1 indexindexindex (CI) (CI) values (CI) values values (Figureindex< 1 <(Figure <1 (CI) 61(FigureJ,K, (Figure values 6J,K, and 6J,K, Supplementary6J,K,and < 1and Supplementary (Figureand Supplementary Supplementary 6J,K, Table and Table S2). Supplementary Table TheseTable S2). S2). These resultsS2). These These results Table suggest results results S2).suggest that suggest These suggest baicalein that result that baicalein that functions sbaicalein suggestbaicalein as that a sensitizer baicalein functionsfunctionsfunctions as a as sensitizer as a tofunctionsasensitizer sensitizer anti-cancer to anti asto to aanti- cancer drugssensitizeranti-cancer-cancer anddrugs drugsto radiation drugs anti-cancerand and radiation and inradiation radiation resistant drugs in resistant inand TNBCin resistant resistantradiation MDA-MB-231/IRTNBC TNBC TNBC in MDA resistant MDA MDA-MB-MB- 231/IR-TNBC cells.MB-231/IR-231/IR MDA-MB-231/IRcells. cells. cells. cells.

FigureFigureFigure 6. Baicalein 6. 6.Baicalein BaicaleinFigure induced induced 6. induced6.Baicalein Baicalein apoptosis apoptosis apoptosis induced induced and apoptosisand sensitized and apoptosis sensitized sensitized and resistantsensitized and resistant resistant sensit MDA resistant ized MDA MDA-MB -resistant MDA-MB-231/IR-MB231/IR-MB-231/IR-231/IR cells.MDA-MB-231/IR cells. cells. (A , cells.B ( A) (,AB (,)BA ), B )cells. Expression (A,B) ExpressionExpressionExpression levels levels levels oflevelsExpression IFIT2 of of IFIT2 of IFIT2(left IFIT2 (left-levelshand (left- (left-handhand- handyof-axis) IFIT2 y- yaxis) -andaxis)y (left-hand-axis) and apoptosis and apoptosis and apoptosis y apoptosis-axis) marker marker and marker proteins markerapoptosis proteins proteins proteins(right marker (right -(handright (right-hand- handproteins- handy-axis) y- yaxis) -after(right-handaxis)y -axis)after after after y-axis) baicalein after baicaleinbaicaleinbaicalein treatment treatment treatmenttreatment baicalein for for24 for h. 24 treatment for 24 (C h. ) 24h. ( CellC h.()C Cell) ( C cyclefor Cell) Cell cycle24 cycle analysis, cycleh. analysis,( C analysis,) analysis, Cell (D, E (cycleD) ( annexinD, (ED,)E ,Eanalysis, annexin) ) annexin annexin V/PI V/PI( D staining, V/PI V/PI,E ) staining, annexin staining, staining, and andV/PI (F and and,G ( )F staining, (, JCFG,,G)- 1 JC) JC-1 JC-1 - and1 staining (F,G) wereJC-1 stainingstainingstaining were were wereperformed performedperformed stainingperformed to wereobserve to to to observe performedobserve induction induction induction to ofobserve apoptosis ofof of apoptosisapoptosis apoptosisinduction by by baicalein.by baicalein.by ofbaicalein. baicalein.apoptosis (H (,H I()H, I(Baicaleinby)H,I Baicalein), Ibaicalein.Baicalein) Baicalein sensitized sensitized sensitized(H sensitized,I) Baicalein MDA-MB-231/IR sensitized MDAMDAMDA-MB--M231/IR-MB-B231/IR-231/IR cellscellsMDA-MB-231/IR cells tocells to irradiation. to irradiation. to irradiation. irradiation. cells(J,K (J (,) KJ toBaicalein,(K)J ,irradiation. Baicalein)K Baicalein) Baicalein sensitized sensitized sensitized ( Jsensitized,K) Baicalein MDA MDA-MB-231/IR MDA MDA-MB sensitized--MB231/IR-MB-231/IR-231/IR cells MDA-MB-231/IR cells tocells Adriamycin to to Adriamycin Adriamycin cells (0,to Adriamycin 12.5, 25, 50, (0, 12.5,(0,(0, 12.5, 25,12.5, 50,25, 25, and50, 50, and and(0,100 and 12.5, 100nM) 100 100 nM)25, nM)and nM) 50, and andcisplatin and and cisplatin cisplatin cisplatin 100 (0, nM) 10, (0, (0, (0, and 20,10, 10, 10, 30,20,cisplatin 20,20, and30, 30,30, and 40 andand (0, μM) 40 10, 4040 μM) treatmentµ20,μM)M) treatment30, treatmenttreatment and (▬ 40 ( ▬μ (( :M)▬ Adriamycin : treatment: Adriamycin Adriamycin: Adriamycin or(▬ oror :or cisplatinAdriamycin alone, or cisplatincisplatincisplatin alone, alone, alone, ▬ cisplatin▬ :▬ :Adriamycin Adriamycin : Adriamycin: Adriamycin alone, or▬ or cisplatin cisplatinor: or Adriamycincisplatin cisplatin with with with baicaleinwith baicalein or baicalein cisplatin baicalein 10 10 µμM, 10M,with 10 μM, ▬ μM, baicalein : ▬ Adriamycin :▬ Adriamycin : Adriamycin: 10Adriamycin μM, or cisplatinor▬ cisplatin or :or Adriamycincisplatin cisplatin with baicalein or cisplatin 20 µM). withwith baicaleinwith baicalein baicalein 20 μM). 20Asteriskswith 20 μM). AsterisksμM). baicalein Asterisks Asterisks (*) indicate(*) 20 indicate (*)μ (*)M). indicate signiﬁcant indicate Asterisks significant significant significant differences (*) differencesindicate differences differences at significantp at< 0.05.p at< at0.05.p

4. Discussion4.4. Discussion Discussion 4. Discussion TreatingTreatingTreating breast breast breast Treating cancer cancer cancer remains breast remains remains cancer challenging challenging challenging remains despite despitechallenging despite the the various the variousdespite various advances advancesthe advances various in in surgery, in advances surgery, surgery, in surgery, chemotherapy,chemotherapy,chemotherapy, andchemotherapy, and radiotherapy and radiotherapy radiotherapy and [37] [37]radiotherapy. [37] TNBC. TNBC. TNBC is a is subtype [37].is a asubtype subtypeTNBC of breast ofis of breasta breastsubtype cancer cancer cancer distinguishedof breast distinguished distinguished cancer by distinguished bythe by the the by the absenceabsenceabsence of ER,of of ER, PR, ER,absence PR, and PR, and HER2,and of HER2, ER,HER2, which PR, which which and are areHER2,potential are potential potential which target target are target molecules potential molecules molecules intarget advancedin in advanced molecules advanced therapies therapies intherapies advanced, such, such, suchas therapies, as as such as HER2HER2HER2 antigen antigen antigen or hormoneHER2or or hormone hormone antigen treatment treatment ortreatment hormone [2,3] [2,3]. [2,3]Since treatment. Since. Since the theTNBC the [2,3]. TNBC TNBC subtype Since subtype subtype th ise TNBCthe is is themost the subtypemost mostmalignant malignant malignant is the subtype, most subtype, subtype, malignant subtype,

Nutrients 2019, 11, 624 13 of 19

4. Discussion Treating breast cancer remains challenging despite the various advances in surgery, chemotherapy, and radiotherapy [37]. TNBC is a subtype of breast cancer distinguished by the absence of ER, PR, and HER2, which are potential target molecules in advanced therapies, such as HER2 antigen or hormone treatment [2,3]. Since the TNBC subtype is the most malignant subtype, having many heterogeneous CSCs and limiting the application of advanced therapeutic agents, such as HER2 antigen, androgen receptor modulator, or hormone treatment, the need for the discovery and development of new treatments that can target TNBC is increasing [37–40]. Phytochemicals are natural compounds derived from plants or fruits. Flavonoids are a subclass of phytochemicals known to have beneficial effects for human health, including anti-cancer effects. The intake of flavonols and flavones, including baicalein, has been associated with a decreased risk of breast cancer [36]. Previous studies on baicalein were mainly focused on inhibiting adhesion, migration, invasion, and cell death in various cancer cells, including breast cancer. The mechanisms of baicalein in cancers involve activation of autophagy through AMP-activated protein kinase (AMPK)-unc-51-like kinase 1 (ULK), cell cycle arrest by CDC2/Cyclin B1, or induction of apoptosis by inhibition of the PI3K/Akt pathway, transforming growth factor (TGF) signaling, myeloid leukemia 1 (Mcl-1), and mammalian target of rapamycin (mTOR) signaling [41–43]. Baicalein also overcame TNF-related apoptosis-inducing ligand (TRAIL) resistance by enhancing TRAIL-2 promoter activity through induction of CCAAT/enhancer-binding protein homologous protein (CHOP) in human colon cancer SW480 cells or by increasing expression of TRAIL-2 through ROS induction [25]. However, no study has examined treatment-resistant breast cancer cells using analysis of overall gene expression patterns via transcriptomics, selection of candidate genes, and gene expression analysis before and after treatment with baicalein. In this study, we identified the effect of baicalein on resistant TNBC MDA-MB-231/IR cells generated by repetitive irradiation. Baicalein reversed chemo- and radioresistance, as well as migration, invasion, and stem cell-like properties of the resistant breast cancer cells. Transcriptomic analysis is a large-scale process using high-throughput techniques to examine RNA molecules of cells [44–46]. Comparative transcriptomic studies reveal the different expression patterns between two samples [47]. Analyses of DEGs of MDA-MB-231/IR cells compared to MDA-MB-231 cells revealed genes that were up- or down-regulated by repeated irradiation. DEGs that meet both difference change over four times and fold change over 2.5 times between MDA-MB-231/IR and MDA-MB-231 cells were selected, and 12 genes that are known to play a role in cancer were identified. GO analyses revealed that gene categories that might be associated with chemo- and radioresistance, such as innate immune response, focal adhesion, and the IL-1 receptor binding pathway, were highly activated in MDA-MB-231/IR cells [48–50]. Pathway analysis by DAVID revealed that the NF-κB pathway, TNF signaling pathway, and TLR pathway, which interact with signaling pathways that regulate IFIT2, were enriched in MDA-MB-231/IR cells. When we tested the hypothesis that baicalein could reverse the expression of the 12 DEGs, we surprisingly found that only IFIT2 expression was reversed in a dose-dependent manner after treatment with baicalein. Therefore, we propose that the IFIT2 gene is critical for treatment resistance in breast cancer and plays an important role in overcoming resistance with baicalein. Interestingly, when protein interactions were predicted using String 8.0, IFIT1, IFIT2, IFIT3, and TRAF3 were highly interactive, while only IFIT2 was associated with resistance in MDA-MB-231/IR cells (data not shown). IFIT2, also called interferon-stimulated gene 54 (ISG54), is one of four IFIT proteins, including IFIT1/ISG56, IFIT2/ISG54, IFIT3/ISG60, and IFIT5/ISG58, characterized by a helix-turn-helix motif [51]. It has been reported that IFIT1 and IFIT2 interact with eukaryotic initiation factor 3 (eIF3) to inhibit mRNA translation and recognize viral mRNA structure, which lacks O-methylation [52,53]. In addition to its anti-viral activity, IFIT2 expression has been reported to inhibit cancer cell growth and migration. It has been reported that IFIT2 can interact with cytoskeleton-associated proteins, such as beta-tubulin or cytokeratin 18, and regulate cell mitosis and cell motility while activating protein kinase C (PKC) signaling [54,55]. TNF-α secretion, leading to angiogenesis and metastasis of oral squamous Nutrients 2019, 11, 624 14 of 19 cell carcinoma, can be regulated by IFIT2 depletion [56]. The tumor suppressor promyelocytic leukemia zinc-finger protein (PLZF) was reported to increase expression of IFIT2 in gallbladder cancer [57]. Increased expression of IFIT2 has also been reported during induction of apoptosis by various stimuli, such as interferons, cisplatin, lncRNAs, and miRNAs, in oral cancer cells, osteosarcoma cells, gastric cancer cells, and colorectal cancer cells [56,58–61]. In human ovarian cancer cells, the down-regulation of IFIT2 was regulated by activation of the Ras/MEK signaling pathway [62]. Induction of IFIT2 has been reported to be involved in apoptosis signaling by disturbing mitochondrial membrane potential in HeLa cells [63]. Here, we confirmed previous reports by conducting JC-1, annexin V/PI, and cell cycle analyses after baicalein treatment. While inducing apoptosis in resistant TNBC MDA-MB-231/IR cells, baicalein increased the mRNA and protein expression IFIT2. In addition, we found that baicalein treatment altered the malignant features of MDA-MB-231/IR cells (e.g., migration, invasion, and stem cell-like properties), and these phenomena were accompanied by reversal of IFIT2 expression. Consistent with these results, TCGA data analysis also revealed that metastasis samples showed lower IFIT2 expression compared to primary tumor and normal tissue, and Kaplan-Meier plots in TNBC patients showed that patients with low IFIT2 expression levels had a higher possibility of relapse (Figure4D,E).

5. Conclusions In conclusion, we demonstrated for the ﬁrst time that the phytochemical baicalein reduced stem cell-like properties and induced apoptosis through up-regulation of IFIT2 in chemo- and radioresistant TNBC cells. Baicalein may be a novel phytochemical for inhibiting CSCs and metastasis in TNBC. Our data also suggest that combining conventional therapies with baicalein treatment could enhance therapeutic effects, and we suggest baicalein as a chemo- and radiosensitizer for TNBC patients. Further studies are needed to identify the underlying mechanisms regulated by IFIT2 in resistant breast cancer cells and to investigate the therapeutic effects of IFIT2 on a variety of cancer types possessing high CSCs and expressing resistance.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/3/624/s1, Figure S1. Cell viabilities and induction of apoptosis in MDA-MB-231 cells and MDA-MB-231/IR cells treated with irradiation or anti-cancer drugs, Table S1. IC50 of phytochemical treatments on MDA-MB-231 cells and MDA-MB-231/IR cells. Table S2. CI value of combination treatments on MDA-MB-231/IR cells. Author Contributions: Conceptualization: S.Y.K. and S.K.C.; methodology: S.Y.K. and J.Y.M.; software: T.U.; validation: S.K.C.; formal analysis: S.Y.K., J.Y.M., and T.U.; investigation: S.Y.K. and S.K.C.; resources: S.Y.K. and S.K.C.; data curation: S.Y.K. and S.K.C.; writing—original draft preparation: S.Y.K.; writing—review and editing: S.K.C.; visualization: J.Y.M.; supervision: S.K.C.; project administration: S.K.C; funding acquisition: S.K.C. Acknowledgments: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016R1A2B4016005) and by the 2019 scientific promotion program funded by Jeju National University. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ABC Adenosine triphosphate-binding cassette ABCG 2ATP-binding cassette super-family G member 2 AKR1C1 Aldo-keto reductase 1C1 AKR1C2 Aldo-keto reductase 1C2 AKR1C3 Aldo-keto reductase 1C3 Akt Protein kinase B AMPK AMP-activated protein kinase BSA Bovine serum albumin CCDC69 Coiled-coil domain-containing 69 Nutrients 2019, 11, 624 15 of 19

CHOP CCAAT/enhancer-binding protein homologous protein CI Combination index CSCs Cancer stem cells DAVID Database for Annotation, Visualization and Integrated Discovery DEGs Differentially expressed genes DMEM Dulbecco’s modiﬁed Eagle’s medium DMSO Dimethyl sulfoxide DR5 Death receptor 5 eIF3 Eukaryotic initiation factor 3 EMT Epithelial-mesenchymal transition ER Estrogen receptor FACS Fluorescence-activated cell sorting FBS Fetal bovine serum FPKM Fragments per kilobase of transcript per million mapped reads FTL Ferritin light chain GFPT2 Glutamine-fructose-6-phosphate transaminase 2 GO Gene ontology HER2 Human epidermal growth factor receptor 2 HR Hazard ratio IC50 Inhibitory concentration of 50 IFIT2 Interferon-induced protein with tetratricopeptide repeats 2 IL-1 Interleukin-1 IR Irradiation ISG54 Interferon-stimulated gene 54 KAAS Automatic Annotation Server KEGG Kyoto Encyclopedia of Genes and Genomes LG3BP Galectin-3-binding protein Mcl-1 Myeloid leukemia 1 MDR1 Multidrug-resistance protein 1 MK Midkine MP Main population MRP1 Multidrug-resistance-associated protein 1 mTOR Mammalian target of rapamycin MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide PBS Phosphate buffered saline PI Propidium iodide PI3K Phosphatidylinositol 3-kinase PKP3 Plakophilin 3 PLZF Promyelocytic leukemia zinc-ﬁnger protein PR Progesterone receptor RFS Relapse-free survival RIG-1 Retinoic acid-inducible gene-1 RNA-seq RNA-sequencing ROS Reactive oxygen species RSEM RNA sequencing by Expectation Maximization SATB1 Special AT-rich sequence binding protein 1 SP Side population STAR Spliced Transcripts Alignment to a Reference TCGA The Cancer Genome Atlas TGF Transforming growth factor Nutrients 2019, 11, 624 16 of 19

TGFBI Transforming growth factor beta induced TICs Tumor initiating cells TLR Toll-like receptor TNBCs Triple-negative breast cancers TNF Tumor necrosis factor TRAIL TNF-related apoptosis-inducing ligand TWF1 Twinﬁlin actin binding protein 1 ULK Unc-51-like kinase 1

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