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leading to the promotion of cell proliferation&#44; survival and metastasis&#46;<a class="elsevierStyleCrossRefs" href="#bib0025"><span class="elsevierStyleSup">5&#44;6</span></a> Overexpression of <span class="elsevierStyleItalic">Wnt</span> genes&#44; such as <span class="elsevierStyleItalic">Wnt5a</span>&#44; is predictive of aggressive non-small-cell lung cancer &#40;NSCLC&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a> In fact&#44; Wnt&#47;&#946;-catenin signaling is not only highly activated in lung cancer but also in other cancers such as colon cancer&#46; It is an important oncogenic factor in tumorigenesis&#44; progression and metastasis&#46; Accumulation of nuclear &#946;-catenin caused by oncogenic Wnt signaling leads to its nuclear translocation and the formation of a complex with transcription factors&#44; such as T-cell factor 4 &#40;TCF-4&#41;&#44; to initiation the transcription of its downstream targets such as <span class="elsevierStyleItalic">c-Myc</span> and <span class="elsevierStyleItalic">cyclin D1</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Esculetin &#40;6&#44;7-dihydroxycoumarin&#41; is a derivative of coumarin found in <span class="elsevierStyleItalic">Artemisia capillaries&#44; Citrus limonia</span> and a number of other natural plants&#46; It has been evidenced to inhibit cell proliferation and induce apoptosis or cell cycle arrest in several cancer cell lines including human malignant melanoma and colon cancer cells&#44; and cancers including hepatocellular carcinoma&#46;<a class="elsevierStyleCrossRefs" href="#bib0045"><span class="elsevierStyleSup">9&#8211;13</span></a> The interaction between esculetin and &#946;-catenin disrupts the formation of &#946;-catenin-Tcf complex in colon cancer&#46;<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> Nuclear factor kappa-light-chain-enhancer of activated B cells &#40;NF-&#954;B&#41; activation has been documented to play an important role in the progression of epidermal growth factor receptor &#40;EGFR&#41; mediated lung tumor&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a> Previous studies have evidenced the inhibitory effect of esculetin on NF-&#954;B in pancreatic cancer cells and vascular smooth muscle cells&#46;<a class="elsevierStyleCrossRefs" href="#bib0080"><span class="elsevierStyleSup">16&#44;17</span></a> Additionally&#44; it was reported that esculetin may protect mice from lipopolysaccharide-induced acute lung injury via suppressing NF-&#954;B&#46;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">18</span></a> Thus&#44; in this study&#44; we investigated the effects of esculetin on lung cancer and the involvement of Wnt and NF-&#954;B signaling pathways&#46; We found that esculetin treatment exhibited inhibitory effects on the growth of lung cancer cells&#46; Esculetin downregulated Wnt targeted genes and suppressed NF-&#954;B&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Compound</span><p id="par0020" class="elsevierStylePara elsevierViewall">Esculetin &#40;6&#44;7-dihydroxycoumarin&#44; 98&#37; purity&#41; was purchased from Sigma-Aldrich &#40;St&#46; Louis&#44; MO&#41; and dissolved in dimethyl sulfoxide &#40;DMSO&#44; 0&#46;1&#37;&#41;&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Cell Culture</span><p id="par0025" class="elsevierStylePara elsevierViewall">Murine Lewis lung carcinoma &#40;LLC&#41; cells were cultured in Dulbecco&#39;s modified Eagles medium &#40;DMEM&#44; Life Technology&#44; Pleasanton&#44; CA&#44; USA&#41; supplemented with and penicillin &#40;60<span class="elsevierStyleHsp" style=""></span>IU&#47;ml&#41;&#47;streptomycin &#40;50<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml&#41; and 10&#37; fetal bovine serum &#40;Gibco&#44; Grand Island&#44; NY&#44; USA&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in a 5&#37; CO<span class="elsevierStyleInf">2</span> humidified incubator&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Measurement of Cell Proliferation</span><p id="par0030" class="elsevierStylePara elsevierViewall">Cell proliferation was measured using 3-&#40;4&#44;5-dimethylthiazol-2yl&#41;-2&#44;5-diphenyl-2H-tetrazolium bromide &#40;MTT&#41; assay&#46; LLC cells &#40;6&#215;10<span class="elsevierStyleSup">3</span>&#47;well&#41; were plated in 96-well plates&#46; After 24<span class="elsevierStyleHsp" style=""></span>h&#44; phosphate buffered saline &#40;PBS&#41; or esculetin at various concentrations &#40;20&#44; 40 or 80<span class="elsevierStyleHsp" style=""></span>&#956;mol&#47;l&#41; was added into each well&#46; MTT assay was performed every 24<span class="elsevierStyleHsp" style=""></span>h of incubation for 5 days&#46; Briefly&#44; 20<span class="elsevierStyleHsp" style=""></span>&#956;l MTT &#40;Amresco&#44; USA&#41; was added to each well and incubated for 4<span class="elsevierStyleHsp" style=""></span>h&#46; The formazan precipitation was dissolved in 150<span class="elsevierStyleHsp" style=""></span>&#956;l DMSO and the results were observed using a microplate reader &#40;BioTek Instruments&#44; Inc&#46;&#44; Vermont&#44; USA&#41; at 490<span class="elsevierStyleHsp" style=""></span>nm&#46; Each test was repeated for three times&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Colony Formation Assay</span><p id="par0035" class="elsevierStylePara elsevierViewall">The colony formation assay was performed in 6-well plates covered with 1&#37; low melting agarose and 0&#46;25<span class="elsevierStyleHsp" style=""></span>ml 2&#215; RPMI with 20&#37; FBS&#46; Cells &#40;500<span class="elsevierStyleHsp" style=""></span>cells&#47;well&#41;&#41; were mixed 0&#46;7&#37; low melting agarose and 0&#46;25<span class="elsevierStyleHsp" style=""></span>ml 2&#215; RPMI with 20&#37; FBS and seeded onto the bottom layer of soft agar&#46; The plates were incubated for 21&#8211;28 days at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in a 5&#37; CO<span class="elsevierStyleInf">2</span> humidified incubator&#46; Colony numbers were counted after staining with crystal violet and photographed using a phase-contrast microscope &#40;100&#215;&#41;&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Mice Model</span><p id="par0040" class="elsevierStylePara elsevierViewall">The experimental protocols were approved by the Ethics Committee&#46; BALB&#47;c mice &#40;6&#8211;8-week-old female&#41; were purchased from SLAC &#40;Shanghai&#44; China&#41;&#46; LLC cells &#40;1&#215;10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>cells&#47;100<span class="elsevierStyleHsp" style=""></span>&#956;l PBS&#41; were subcutaneously injected in the right inguinal region of mice&#46; Mice were divided into two groups&#44; untreated vehicle group and esculetin &#40;100<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#41; treated group for 20 days&#46; At the end of the treatment&#44; body weight was measured and mice were sacrificed for obtaining tumor size and weight&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Transwell Assay</span><p id="par0045" class="elsevierStylePara elsevierViewall">Invasion assay was performed to test the effect of esculetin on cell invasive ability using transwell invasion chambers coated or non-coated with Matrigel &#40;Becton Dickinson Labware&#44; USA&#41; according to the manufacturer&#39;s instructions&#46; Cells &#40;5000<span class="elsevierStyleHsp" style=""></span>cells&#47;well&#41; were plated onto the chambers in 100<span class="elsevierStyleHsp" style=""></span>&#956;l medium containing 0&#46;1&#37; DMSO or esculetin &#40;20&#44; 40 or 80<span class="elsevierStyleHsp" style=""></span>&#956;mol&#47;l&#41;&#46; After 48<span class="elsevierStyleHsp" style=""></span>h incubation&#44; the cells on the bottom surface of the membrane were fixed in 90&#37; alcohol and stained with crystal violet staining for counting using a microscope&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Western Blotting</span><p id="par0050" class="elsevierStylePara elsevierViewall">Cells were treated with or without esculetin for 24<span class="elsevierStyleHsp" style=""></span>h&#46; Cell were lysed by Lysis Buffer containing PMSF on ice&#46; Cell lysate was separated by 10&#37; SDS-PAGE gels and transferred onto a NC membrane &#40;Amersham Pharmacia Biotech&#44; USA&#41;&#46; The membranes were blocked by 10&#37; non-fat milk dissolved in 0&#46;1&#37; Tween 20 in TBS &#40;TBST&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>h&#46; The membranes were incubated overnight with primary antibodies including c-myc &#40;&#35;ab32072&#44; Abcam&#44; Cambridge&#44; MA&#44; USA&#41;&#44; cyclin D1 &#40;&#35;ab134175&#44; Abcam&#44; USA&#41;&#44; &#946;-catenin &#40;&#35;ab16051&#44; Abcam&#44; USA&#41;&#44; NF-&#954;B &#40;&#35;ab16502&#44; Abcam&#44; USA&#41; and I-&#954;B &#40;&#35;9244&#44; Cell Signaling Technology Inc&#46;&#44; USA&#41;&#44; and &#946;-actin &#40;&#35;ab8227&#44; Abcam&#44; USA&#41; as a control for normalization&#46; Signals were detected using an ECL Western blotting kit &#40;Amersham Pharmacia Biotech&#44; USA&#41;&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Statistical Analysis</span><p id="par0055" class="elsevierStylePara elsevierViewall">Student&#39;s <span class="elsevierStyleItalic">t</span> test was used to determine the significant difference between the control and experimental groups&#46; Differences were considered to be significant at <span class="elsevierStyleItalic">P</span>&#60;&#46;05&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Results</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Esculetin Inhibited Cell Viability and Colony Formation of Lewis Lung Carcinoma</span><p id="par0060" class="elsevierStylePara elsevierViewall">Effect of esculetin on cell viability of murine LLC was first evaluated by MTT assay&#46; Different doses of esculetin &#40;0&#44; 20&#44; 40 and 80<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; were used to treat LLC cells&#46; Esculetin suppressed cell viability in a dose- and time-dependent manner&#46; As shown in <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>A&#44; 40<span class="elsevierStyleHsp" style=""></span>&#956;M significantly reduced cell viability on day 5&#44; while 80<span class="elsevierStyleHsp" style=""></span>&#956;M had remarkably decreased cell viability since day 3 &#40;<span class="elsevierStyleItalic">P</span>&#60;&#46;05&#41;&#46; We also observed that 40 and 80<span class="elsevierStyleHsp" style=""></span>&#956;M esculetin led to reduced colony formation of LLC cells in soft agar &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>B and C&#44; <span class="elsevierStyleItalic">P</span>&#60;&#46;05&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Esculetin Suppressed Tumor Growth <span class="elsevierStyleItalic">In Vivo</span></span><p id="par0065" class="elsevierStylePara elsevierViewall">To ascertain the effect of esculetin on tumor growth <span class="elsevierStyleItalic">in vivo</span>&#44; LLC cells &#40;1&#215;10<span class="elsevierStyleSup">6</span><span class="elsevierStyleHsp" style=""></span>cells&#47;100<span class="elsevierStyleHsp" style=""></span>&#956;l PBS&#41; were subcutaneously injected into the right inguinal region of BALB&#47;c mice&#46; Mice were treated with esculetin &#40;100<span class="elsevierStyleHsp" style=""></span>mg&#47;kg bw&#41; or PBS for 20 days&#46; Body weight was measured after the treatment and mice were then sacrificed to obtain tumor size and weight&#46; We found markedly reduced size and weight of tumors in mice with esculetin treatment compared to those control mice &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A and B&#44; <span class="elsevierStyleItalic">P</span>&#60;&#46;05&#41;&#46; No significant change in body weight was observed between the two groups&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Esculetin had no Effect on the Invasion of LLC Cells</span><p id="par0070" class="elsevierStylePara elsevierViewall">After confirming the effect of esculetin on tumor growth&#44; we further tested whether esculetin affected cell invasion&#46; Evaluation of cell invasion under different doses of esculetin treatment for 48<span class="elsevierStyleHsp" style=""></span>h demonstrated that&#44; esculetin had no influence on the invasive activity of LLC cells &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>A&#41;&#46; Quantitative analysis was shown in <a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>B&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Esculetin Inhibited the Protein Expressions of c-myc and Cyclin D1</span><p id="par0075" class="elsevierStylePara elsevierViewall">As esculetin was identified as a potential inhibitor of Wnt&#47;&#946;-catenin signaling pathway&#44; we tested the protein expressions of c-myc and cyclin D1 as the target genes of Wnt&#47;&#946;-catenin signaling pathway&#46; There were significant decreases in the protein levels of c-myc and cyclin D1 in LLC cells after 24-h treatment of esculetin &#40;40 and 80<span class="elsevierStyleHsp" style=""></span>&#956;M&#41; compared to untreated cells &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>A and B&#44; <span class="elsevierStyleItalic">P</span>&#60;&#46;05&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Esculetin Reduced NF-kB Protein Level in LLC Cells</span><p id="par0080" class="elsevierStylePara elsevierViewall">As previously reported that esculetin inhibited NF-&#954;B pathway&#44; we tested the protein levels of NF-&#954;B and I-&#954;B protein levels after 24-h of 80<span class="elsevierStyleHsp" style=""></span>&#956;M esculetin treatment in LLC cells&#46; Compared to untreated LLC cells&#44; esculetin significantly downregulated protein level of NF-&#954;B &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>A and B&#44; <span class="elsevierStyleItalic">P</span>&#60;&#46;05&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Discussion</span><p id="par0085" class="elsevierStylePara elsevierViewall">The high incidence and mortality rate of lung cancer requires improvements in both early detection and therapeutic methods&#46; Despite the development of earlier detection using low-dose computed tomographic&#44;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">19</span></a> the overall survival rate was only slight increased&#46; Thus&#44; it is essential to identify new therapeutic compounds for effective treatment&#46; As previously reported&#44; esculetin is a natural compound inhibiting cell proliferation and inducing cell apoptosis in several cell lines&#46;<a class="elsevierStyleCrossRef" href="#bib0100"><span class="elsevierStyleSup">20</span></a> In this study&#44; we identified its anti-proliferative effect on a lung cancer cell line&#44; LLC&#46; We observed that esculetin decreased cell proliferation and colony formation in a dose- and time-dependent manner&#44; where 80<span class="elsevierStyleHsp" style=""></span>&#956;M esculetin treatment was determined to significantly affect cell proliferation and colony formation on day 3&#46; However&#44; the doses of esculetin varied in different cell lines&#46; For example&#44; 50<span class="elsevierStyleHsp" style=""></span>&#956;M of esculetin was enough to elicit a significant decrease of cell proliferation in human normal gastric epithelial cell line GES-1 on day 3&#44; while for other cells such as human gastric cancer cell lines MGC-803 and human hepatocellular carcinoma cell line SMMC-7721&#44; the doses were 12&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;M and 0&#46;3<span class="elsevierStyleHsp" style=""></span>mM&#44; respectively&#46;<a class="elsevierStyleCrossRefs" href="#bib0105"><span class="elsevierStyleSup">21&#44;12</span></a></p><p id="par0090" class="elsevierStylePara elsevierViewall">To confirm whether esculetin also acts as a tumor suppressor in lung cancer <span class="elsevierStyleItalic">in vivo</span>&#44; we treated mice with esculetin and found significantly reduced tumor size and weight in treated mice compared to control&#46; The anti-proliferative role was not only shown in lung cancer but also demonstrated in other cancers such as metastatic osteosarcoma<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> and hepatocellular carcinoma&#46;<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">12</span></a> Of note&#44; there was no difference in the body weight between treated and untreated mice&#44; suggesting that this dose of esculetin did not exhibit toxicity to mice&#46; It was suggested that injection of 700<span class="elsevierStyleHsp" style=""></span>mg&#47;kg esculetin per day showed no adverse effects on the body weight of mice&#46;<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">12</span></a> The same conclusion was drawn by another study using 50<span class="elsevierStyleHsp" style=""></span>mg&#47;kg of esculetin on rats&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">23</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Degradation of extracellular matrix is an essential step of tumor metastasis&#46; The inhibitory effect of esculetin on MMP regulation has been reported in several cell lines including human gastric cancer cells MGC-803&#44;<a class="elsevierStyleCrossRef" href="#bib0105"><span class="elsevierStyleSup">21</span></a> vascular smooth muscle cells<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a> and osteosarcoma LM8 cells&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> These results suggest that esculetin may suppress tumor invasion and metastasis <span class="elsevierStyleItalic">in vivo</span>&#44; which was also observed in previous studies&#46; An <span class="elsevierStyleItalic">in vivo</span> study on LM8-bearing mice demonstrated that less metastasis to lung and liver was found in mice treated with &#8764;70&#8211;140<span class="elsevierStyleHsp" style=""></span>&#956;M esculetin compared to that of untreated mice&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> However&#44; we did not observe the change in invaded cell number in LLC cells treated with esculetin&#46; It may be caused by differential effects of esculetin in different cell lines&#46; Further research is needed to reveal the detailed mechanism&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">In 2014&#44; Lee SY et al&#46; found that esculetin disrupted the formation of the &#946;-catenin&#47;Tcf transcriptional complex by binding with &#946;-catenin to suppress proliferation of colon cancer cells&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">25</span></a> The present study further evidenced that esculetin also inhibited Wnt signaling pathway in lung cancer cells&#44; where the expression of c-myc and Cyclin D1 was remarkably reduced by esculetin in a dose-dependent manner&#46; A recent study showed a similar result demonstrating that esculetin supplementation effectively suppressed tumor growth by targeting to c-myc induced energy metabolism&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">23</span></a> As a key transcription factor participating in the progress of tumor&#44; c-myc has been regarded as a potential target for tumor therapy&#46; The current results provide a possibility for the clinical use of esculetin in cancer treatment&#46; Additionally&#44; the inhibitory effect of esculetin on Cyclin D1 has also been reported in several previous studies&#46;<a class="elsevierStyleCrossRefs" href="#bib0130"><span class="elsevierStyleSup">26&#8211;28</span></a> In fact&#44; the role of esculetin in regulating Cyclin D1 was first reported in 2002&#44; where esculetin achieved anti-proliferative function by blocking phosphorylation of pRb to arrest human leukemia HL-60 cells at G1 phase&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> In addition to Wnt signaling&#44; esculetin treatment also decreased the expression level of NF-&#954;B in human pancreatic carcinoma cells PANC-1&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">30</span></a> It was reported that esculetin attenuated NF-&#954;B activation without affecting its inhibitor&#44; instead it was suggested that the reduced interaction between Nuclear Factor-Erythroid 2-related factor 2 &#40;Nrf2&#41; and its inhibitor Kelch-like ECH-associated protein1 &#40;KEAP1&#41; after esculetin treatment led to abrogated NF-&#954;B activity&#46;<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">17</span></a> Nrf2 exerts a protective function against reactive oxygen species by elevating the transcription of numerous anti-oxidant enzymes&#46;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">31</span></a> Giving the result of unchanged I-&#954;B in this study&#44; the inhibitory effect of esculetin on NF-&#954;B may be also due to the direct binding of esculetin to KEAP1&#46; Additionally&#44; it is well-known that activation of NF-&#954;B is associated with promotion of cell survival and proliferation by several mechanisms including upregulating anti-apoptotic proteins&#44; c-myc and Cyclin D1&#46;<a class="elsevierStyleCrossRefs" href="#bib0160"><span class="elsevierStyleSup">32&#44;33</span></a> Thus&#44; in this study&#44; down-regulated NF-&#954;B by esculetin may also be the cause of reduced expressions of c-myc and Cyclin D1&#46; In conclusion&#44; we identified that esculetin could be a potential agent for treatment against lung cancer by suppressing NF-&#954;B&#44; c-myc and Cyclin D1&#46;</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Conflicts of Interest</span><p id="par0105" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflict of interest&#46;</p></span></span>"
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              "titulo" => "Esculetin Suppressed Tumor Growth In Vivo"
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      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Esculetin was identified to inhibit cell proliferation and induce apoptosis or cell cycle arrest in several cancer cell lines&#46; However&#44; the effect of esculetin on lung cancer remains elusive&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">The anti-proliferative role of esculetin in murine Lewis lung carcinoma &#40;LLC&#41; cells was evaluated by 3-&#40;4&#44;5-dimethylthiazol-2yl&#41;-2&#44;5-diphenyl-2H-tetrazolium bromide &#40;MTT&#41; and colony formation assays&#46; BALB&#47;c mice were subcutaneously injected with LLC cells to investigate the inhibitory effect of esculetin on the growth of lung cancer xenograft&#46; Invasive ability was detected in esculetin treated and untreated LLC cells by transwell assay&#46; The association between esculetin and Wnt&#47;&#946;-catenin signaling&#44; as well as nuclear factor kappa-light-chain-enhancer of activated B cells &#40;NF-&#954;B&#41;&#44; was confirmed by testing the expression of c-myc&#44; Cyclin D1 and NF-&#954;B using Western blot&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Esculetin treatment in LLC cells led to significant decrease of cell proliferation in a time- and dose-dependent manner&#46; After injection of LLC cells into mice&#44; reduced size and weight of tumors were observed in esculetin treated mice compared to untreated mice&#46; However&#44; no difference in cell invasion was observed between the treated and untreated LLC cells&#46; Notably decreased expression of c-myc&#44; Cyclin D1 and NF-&#954;B were observed in LLC cells with esculetin treatment compared to untreated cells&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Esculetin plays an inhibitory role in the growth of lung cancer by down-regulating c-myc&#44; Cyclin D1 and NF-&#954;B&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Introduction"
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          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
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          2 => array:2 [
            "identificador" => "abst0015"
            "titulo" => "Results"
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          3 => array:2 [
            "identificador" => "abst0020"
            "titulo" => "Conclusion"
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      "es" => array:3 [
        "titulo" => "Resumen"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introducci&#243;n</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Se ha determinado que la esculetina inhibe la proliferaci&#243;n celular e induce la apoptosis o la detenci&#243;n del ciclo celular en varias l&#237;neas celulares de c&#225;ncer&#46; Sin embargo&#44; su efecto sobre el c&#225;ncer de pulm&#243;n es todav&#237;a desconocido&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Se estudi&#243; el papel antiproliferativo de la esculetina en c&#233;lulas murinas de carcinoma pulmonar de Lewis &#40;LLC&#41; mediante ensayos de bromuro de 3-&#40;4&#44;5-dimetil-2-tiazolil&#41;-2&#44;5-difenil-2H-tetrazolio &#40;MTT&#41; y de formaci&#243;n de colonias&#46; Se inyectaron subcut&#225;neamente c&#233;lulas LLC a ratones BALB&#47;c para investigar el efecto inhibidor de la esculetina sobre el crecimiento del xenoinjerto de c&#225;ncer de pulm&#243;n&#46; La capacidad invasiva en c&#233;lulas LLC tratadas o no tratadas con esculetina se evalu&#243; mediante el ensayo transwell&#46; La asociaci&#243;n entre la se&#241;alizaci&#243;n de la esculetina y la de Wnt&#47;&#946;-catenina&#44; y con el factor nuclear potenciador de las cadenas ligeras kappa de c&#233;lulas B activadas &#40;NF-&#954;B&#41; se confirm&#243; midiendo la expresi&#243;n de c-myc&#44; de ciclina D1 y NF- &#954;B usando Western blot&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">El tratamiento con esculetina de las c&#233;lulas LLC disminuy&#243; significativamente la proliferaci&#243;n celular de una manera dependiente del tiempo y de la dosis&#46; Tras la inyecci&#243;n de c&#233;lulas LLC en ratones&#44; se observ&#243; que los tumores de los ratones tratados con esculetina eran de menor tama&#241;o y peso que los de los ratones no tratados&#46; Sin embargo&#44; no se observ&#243; diferencia en la invasividad celular entre las c&#233;lulas LLC tratadas y las no tratadas&#46; Se destac&#243; la disminuci&#243;n de la expresi&#243;n de c-myc&#44; ciclina D1 y NF-&#954;B en c&#233;lulas LLC tratadas con esculetina en comparaci&#243;n con c&#233;lulas no tratadas&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusi&#243;n</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">La esculetina desempe&#241;a un papel inhibitorio en el crecimiento del c&#225;ncer de pulm&#243;n a trav&#233;s de la regulaci&#243;n de c-myc&#44; ciclina D1 y NF-&#954; B&#46;</p></span>"
        "secciones" => array:4 [
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            "titulo" => "Introducci&#243;n"
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            "identificador" => "abst0030"
            "titulo" => "M&#233;todos"
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          2 => array:2 [
            "identificador" => "abst0035"
            "titulo" => "Resultados"
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          3 => array:2 [
            "identificador" => "abst0040"
            "titulo" => "Conclusi&#243;n"
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        "etiqueta" => "Fig&#46; 1"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Esculetin treatment of LLC cells impaired its proliferation in liquid and semi-soft cultures&#46; &#40;A&#41; Effect of esculetin treatment on LLC cell growth in liquid culture&#46; MTT assay was performed in 96 wells plate&#46; Mean&#177;SD of 6 wells&#46; &#40;B&#41; Effect of esculetin treatment on colony formation of LLC cells&#46; Cells were seeded into soft agar in triplicate&#44; and colonies were counted after 21&#8211;28 days of culturing&#46; Mean&#177;SD &#40;3 wells&#41; are expressed as percent variation relative to vehicle treated cells &#40;control&#41;&#46; &#42;<span class="elsevierStyleItalic">P</span>&#60;&#46;05&#46; &#40;C&#41; Representative pictures of colonies of LLC treated with esculetin&#46;</p>"
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        "etiqueta" => "Fig&#46; 2"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Esculetin treatment suppressed the growth of LLC-derived tumors&#46; LLC cells were subcutaneously inoculated in the right inguinal region of BALB&#47;c mice at the number of 1&#215;10<span class="elsevierStyleSup">6</span> each&#44; which was followed by daily Es treatment at the dose of 100<span class="elsevierStyleHsp" style=""></span>mg&#47;kg bw or PBS vehicle &#40;VC&#41; treatment for 20 days&#46; Tumors were dissected at the end of the experiments &#40;A&#41; and the tumor weight was measured &#40;B&#41;&#46; Body weight was measured prior to tumor dissection &#40;B&#41;&#46; Data were expressed as mean&#177;S&#46;E&#46;M&#46; &#40;<span class="elsevierStyleItalic">n</span>&#61;6 in each group&#41;&#46; &#42;Compared to that from PBS vehicle-treated LLC cells&#44; &#42;<span class="elsevierStyleItalic">P</span>&#60;&#46;05&#46;</p>"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Esculetin at indicated concentrations does not alter invasiveness of LLC cell significantly&#46; &#40;A&#41; Cell invasion ability was assessed by transwell assay after a 48-h treatment with esculetin&#46; &#40;B&#41; The percentages of cells that could invade via the membrane relative to control are shown as a histogram&#46; Data were expressed as mean&#177;S&#46;E&#46;M&#46;</p>"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">Esculetin inhibits the expression of target proteins&#44; c-Myc and cyclin D1&#44; of &#946;-catenin pathway in LLC cells&#44; not altering the level of &#946;-catenin itself&#46; Proteins expression in LLC cells with or without Esculetin treatment &#40;24<span class="elsevierStyleHsp" style=""></span>h&#41; was measured by Western blot &#40;A&#41;&#46; The relative protein level was normalized with &#946;-actin and the quantitative data were represented as bar graph &#40;B&#41;&#46; Data were expressed as mean&#177;S&#46;E&#46;M&#46;</p>"
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        "etiqueta" => "Fig&#46; 5"
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          "en" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">Esculetin attenuates NF-&#954;B pathway in LLC cells&#46; Western blot analysis shows decrease in protein levels of transcription factor NK-&#954;B and unaltered levels of its inhibitor I-&#954;B in LLC cells&#46; Proteins expression in LLC cells with or without Esculetin treatment &#40;24<span class="elsevierStyleHsp" style=""></span>h&#41; was measured by Western blot &#40;A&#41;&#46; The relative protein level was normalized with &#946;-actin and the quantitative data were represented as bar graph &#40;B&#41;&#46; Data were expressed as mean&#177;S&#46;E&#46;M&#46; &#42;<span class="elsevierStyleItalic">P</span>&#60;&#46;05&#46;</p>"
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      "titulo" => "References"
      "seccion" => array:1 [
        0 => array:2 [
          "identificador" => "bibs0015"
          "bibliografiaReferencia" => array:33 [
            0 => array:3 [
              "identificador" => "bib0005"
              "etiqueta" => "1"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Cancer statistics&#44; 2016"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:3 [
                            0 => "R&#46;L&#46; Siegel"
                            1 => "K&#46;D&#46; Miller"
                            2 => "A&#46; Jemal"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.3322/caac.21332"
                      "Revista" => array:6 [
                        "tituloSerie" => "CA Cancer J Clin"
                        "fecha" => "2016"
                        "volumen" => "66"
                        "paginaInicial" => "7"
                        "paginaFinal" => "30"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26742998"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            1 => array:3 [
              "identificador" => "bib0010"
              "etiqueta" => "2"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer &#40;Adjuvant Navelbine International Trialist Association &#91;ANITA&#93;&#41;&#58; a randomised controlled trial"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "J&#46;Y&#46; Douillard"
                            1 => "R&#46; Rosell"
                            2 => "M&#46; De Lena"
                            3 => "F&#46; Carpagnano"
                            4 => "R&#46; Ramlau"
                            5 => "J&#46;L&#46; Gonzales-Larriba"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/S1470-2045(06)70804-X"
                      "Revista" => array:6 [
                        "tituloSerie" => "Lancet Oncol"
                        "fecha" => "2006"
                        "volumen" => "7"
                        "paginaInicial" => "719"
                        "paginaFinal" => "727"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/16945766"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            2 => array:3 [
              "identificador" => "bib0015"
              "etiqueta" => "3"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Lung adjuvant cisplatin evaluation&#58; a pooled analysis by the LACE Collaborative Group"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "J&#46;P&#46; Pignon"
                            1 => "H&#46; Tribodet"
                            2 => "G&#46;V&#46; Scagliotti"
                            3 => "J&#46;Y&#46; Douillard"
                            4 => "F&#46;A&#46; Shepherd"
                            5 => "R&#46;J&#46; Stephens"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1200/JCO.2007.13.9030"
                      "Revista" => array:7 [
                        "tituloSerie" => "J Clin Oncol"
                        "fecha" => "2008"
                        "volumen" => "26"
                        "paginaInicial" => "3552"
                        "paginaFinal" => "3559"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/18506026"
                            "web" => "Medline"
                          ]
                        ]
                        "itemHostRev" => array:3 [
                          "pii" => "S1054139X16303214"
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                          "issn" => "1054139X"
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                    ]
                  ]
                ]
              ]
            ]
            3 => array:3 [
              "identificador" => "bib0020"
              "etiqueta" => "4"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Vinorelbine plus cisplatin vs&#46; observation in resected non-small-cell lung cancer"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "T&#46; Winton"
                            1 => "R&#46; Livingston"
                            2 => "D&#46; Johnson"
                            3 => "J&#46; Rigas"
                            4 => "M&#46; Johnston"
                            5 => "C&#46; Butts"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1056/NEJMoa043623"
                      "Revista" => array:6 [
                        "tituloSerie" => "N Engl J Med"
                        "fecha" => "2005"
                        "volumen" => "352"
                        "paginaInicial" => "2589"
                        "paginaFinal" => "2597"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/15972865"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            4 => array:3 [
              "identificador" => "bib0025"
              "etiqueta" => "5"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Fibulin-3 suppresses Wnt&#47;beta-catenin signaling and lung cancer invasion"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "X&#46; Chen"
                            1 => "J&#46; Meng"
                            2 => "W&#46; Yue"
                            3 => "J&#46; Yu"
                            4 => "J&#46; Yang"
                            5 => "Z&#46; Yao"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1093/carcin/bgu023"
                      "Revista" => array:6 [
                        "tituloSerie" => "Carcinogenesis"
                        "fecha" => "2014"
                        "volumen" => "35"
                        "paginaInicial" => "1707"
                        "paginaFinal" => "1716"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/24480807"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            5 => array:3 [
              "identificador" => "bib0030"
              "etiqueta" => "6"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Human arrest defective 1 acetylates and activates beta-catenin&#44; promoting lung cancer cell proliferation"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:3 [
                            0 => "J&#46;H&#46; Lim"
                            1 => "J&#46;W&#46; Park"
                            2 => "Y&#46;S&#46; Chun"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1158/0008-5472.CAN-06-3171"
                      "Revista" => array:6 [
                        "tituloSerie" => "Cancer Res"
                        "fecha" => "2006"
                        "volumen" => "66"
                        "paginaInicial" => "10677"
                        "paginaFinal" => "10682"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/17108104"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            6 => array:3 [
              "identificador" => "bib0035"
              "etiqueta" => "7"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor &#8211; an expression in non-small-cell lung cancer"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "C&#46;L&#46; Huang"
                            1 => "D&#46; Liu"
                            2 => "J&#46; Nakano"
                            3 => "S&#46; Ishikawa"
                            4 => "K&#46; Kontani"
                            5 => "H&#46; Yokomise"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1200/JCO.2005.02.2871"
                      "Revista" => array:7 [
                        "tituloSerie" => "J Clin Oncol"
                        "fecha" => "2005"
                        "volumen" => "23"
                        "paginaInicial" => "8765"
                        "paginaFinal" => "8773"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/16314637"
                            "web" => "Medline"
                          ]
                        ]
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                          "pii" => "S1054139X16304001"
                          "estado" => "S300"
                          "issn" => "1054139X"
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                    ]
                  ]
                ]
              ]
            ]
            7 => array:3 [
              "identificador" => "bib0040"
              "etiqueta" => "8"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Modulating the wnt signaling pathway with small molecules"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:2 [
                            0 => "F&#46;H&#46; Tran"
                            1 => "J&#46;J&#46; Zheng"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1002/pro.3122"
                      "Revista" => array:6 [
                        "tituloSerie" => "Protein Sci"
                        "fecha" => "2017"
                        "volumen" => "26"
                        "paginaInicial" => "650"
                        "paginaFinal" => "661"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/28120389"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            8 => array:3 [
              "identificador" => "bib0045"
              "etiqueta" => "9"
              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Induction of apoptosis by esculetin in human leukemia U937 cells through activation of JNK and ERK"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "C&#46; Park"
                            1 => "C&#46;Y&#46; Jin"
                            2 => "G&#46;Y&#46; Kim"
                            3 => "I&#46;W&#46; Choi"
                            4 => "T&#46;K&#46; Kwon"
                            5 => "B&#46;T&#46; Choi"
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                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:5 [
                        "tituloSerie" => "Toxicol Appl Pharm"
                        "fecha" => "2008"
                        "volumen" => "227"
                        "paginaInicial" => "219"
                        "paginaFinal" => "228"
                      ]
                    ]
                  ]
                ]
              ]
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                0 => array:2 [
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                    0 => array:2 [
                      "titulo" => "Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism"
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                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
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                            1 => "E&#46; Calvino"
                            2 => "A&#46; Garcia-Perez"
                            3 => "A&#46; Herraez"
                            4 => "J&#46;C&#46; Diez"
                          ]
                        ]
                      ]
                    ]
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                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.cbi.2014.06.021"
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                        "tituloSerie" => "Chem-Biol Interact"
                        "fecha" => "2014"
                        "volumen" => "220"
                        "paginaInicial" => "129"
                        "paginaFinal" => "139"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/24995577"
                            "web" => "Medline"
                          ]
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                      ]
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                  ]
                ]
              ]
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            10 => array:3 [
              "identificador" => "bib0055"
              "etiqueta" => "11"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Esculetin induces death of human colon cancer cells via the reactive oxygen species-mediated mitochondrial apoptosis pathway"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "A&#46;D&#46; Kim"
                            1 => "X&#46; Han"
                            2 => "M&#46;J&#46; Piao"
                            3 => "S&#46;R&#46; Hewage"
                            4 => "C&#46;L&#46; Hyun"
                            5 => "S&#46;J&#46; Cho"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.etap.2015.03.003"
                      "Revista" => array:6 [
                        "tituloSerie" => "Environ Toxicol Pharmacol"
                        "fecha" => "2015"
                        "volumen" => "39"
                        "paginaInicial" => "982"
                        "paginaFinal" => "989"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25818986"
                            "web" => "Medline"
                          ]
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                    ]
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                ]
              ]
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                0 => array:2 [
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                    0 => array:2 [
                      "titulo" => "Esculetin&#44; a coumarin derivative&#44; exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway"
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                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
                            0 => "J&#46; Wang"
                            1 => "M&#46;L&#46; Lu"
                            2 => "H&#46;L&#46; Dai"
                            3 => "S&#46;P&#46; Zhang"
                            4 => "H&#46;X&#46; Wang"
                            5 => "N&#46; Wei"
                          ]
                        ]
                      ]
                    ]
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                  "host" => array:1 [
                    0 => array:2 [
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                        "tituloSerie" => "Braz J Med Biol Res"
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                        "volumen" => "48"
                        "paginaInicial" => "245"
                        "paginaFinal" => "253"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25517918"
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                    0 => array:2 [
                      "titulo" => "Esculetin&#44; a coumarin derivative&#44; exhibits anti-proliferative and pro-apoptotic activity in G361 human malignant melanoma"
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                        0 => array:2 [
                          "etal" => false
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                            1 => "J&#46;Y&#46; Jang"
                            2 => "J&#46;H&#46; Shim"
                            3 => "P&#46;K&#46; Myung"
                            4 => "J&#46;I&#46; Chae"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.15430/JCP.2015.20.2.106"
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                        "tituloSerie" => "J Cancer Prev"
                        "fecha" => "2015"
                        "volumen" => "20"
                        "paginaInicial" => "106"
                        "paginaFinal" => "112"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26151043"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
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            13 => array:3 [
              "identificador" => "bib0070"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Esculetin suppresses proliferation of human colon cancer cells by directly targeting beta-catenin"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "S&#46;Y&#46; Lee"
                            1 => "T&#46;G&#46; Lim"
                            2 => "H&#46; Chen"
                            3 => "S&#46;K&#46; Jung"
                            4 => "H&#46;J&#46; Lee"
                            5 => "M&#46;H&#46; Lee"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:5 [
                        "tituloSerie" => "Cancer Prev Res &#40;Phila&#41;"
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                        "paginaInicial" => "1356"
                        "paginaFinal" => "1364"
                      ]
                    ]
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                ]
              ]
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            14 => array:3 [
              "identificador" => "bib0075"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "FAS and NF-kappaB signalling modulate dependence of lung cancers on mutant EGFR"
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                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "T&#46;G&#46; Bivona"
                            1 => "H&#46; Hieronymus"
                            2 => "J&#46; Parker"
                            3 => "K&#46; Chang"
                            4 => "M&#46; Taron"
                            5 => "R&#46; Rosell"
                          ]
                        ]
                      ]
                    ]
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                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1038/nature09870"
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                        "tituloSerie" => "Nature"
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                        "volumen" => "471"
                        "paginaInicial" => "523"
                        "paginaFinal" => "526"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21430781"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            15 => array:3 [
              "identificador" => "bib0080"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Inhibitory effect of esculetin on migration&#44; invasion and matrix metalloproteinase-9 expression in TNF-alpha-induced vascular smooth muscle cells"
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                        0 => array:2 [
                          "etal" => false
                          "autores" => array:4 [
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                            1 => "U&#46;S&#46; Lee"
                            2 => "W&#46;J&#46; Kim"
                            3 => "S&#46;K&#46; Moon"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.3892/mmr.2011.420"
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                        "tituloSerie" => "Mol Med Rep"
                        "fecha" => "2011"
                        "volumen" => "4"
                        "paginaInicial" => "337"
                        "paginaFinal" => "341"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21468574"
                            "web" => "Medline"
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              ]
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              "identificador" => "bib0085"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => false
                          "autores" => array:6 [
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                            2 => "V&#46; Saini"
                            3 => "C&#46; Steffi"
                            4 => "M&#46; Tiwari"
                            5 => "D&#46; Saluja"
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                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
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                        "tituloSerie" => "Mol Cancer"
                        "fecha" => "2016"
                        "volumen" => "15"
                        "paginaInicial" => "64"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/27756327"
                            "web" => "Medline"
                          ]
                        ]
                      ]
                    ]
                  ]
                ]
              ]
            ]
            17 => array:3 [
              "identificador" => "bib0090"
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              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Effects of esculetin on lipopolysaccharide &#40;LPS&#41;-induced acute lung injury via regulation of RhoA&#47;Rho Kinase&#47;NF-small ka&#44; CyrillicB pathways in vivo and in vitro"
                      "autores" => array:1 [
                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
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                            1 => "Q&#46; Guo"
                            2 => "H&#46; Wang"
                            3 => "H&#46; Zhang"
                            4 => "C&#46; Wang"
                            5 => "P&#46; Zhang"
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                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.3109/10715762.2015.1087643"
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                        "tituloSerie" => "Free Radic Res"
                        "fecha" => "2015"
                        "volumen" => "49"
                        "paginaInicial" => "1459"
                        "paginaFinal" => "1468"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26514440"
                            "web" => "Medline"
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                    ]
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                ]
              ]
            ]
            18 => array:3 [
              "identificador" => "bib0095"
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              "referencia" => array:1 [
                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Reduced lung-cancer mortality with low-dose computed tomographic screening"
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                            3 => "W&#46;C&#46; Black"
                            4 => "J&#46;D&#46; Clapp"
                            5 => "R&#46;M&#46; Fagerstrom"
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                        ]
                      ]
                    ]
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                  "host" => array:1 [
                    0 => array:2 [
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                        "tituloSerie" => "New Engl J Med"
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                        "paginaInicial" => "395"
                        "paginaFinal" => "409"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21714641"
                            "web" => "Medline"
                          ]
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                    ]
                  ]
                ]
              ]
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            19 => array:3 [
              "identificador" => "bib0100"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Esculetin induces apoptosis through EGFR&#47;PI3K&#47;Akt signaling pathway and nucleophosmin relocalization"
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                          "etal" => true
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                            3 => "Y&#46;H&#46; Choi"
                            4 => "H&#46; Park"
                            5 => "S&#46; Jung"
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                  "host" => array:1 [
                    0 => array:2 [
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                        "tituloSerie" => "J Cell Biochem"
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                        "paginaInicial" => "1210"
                        "paginaFinal" => "1221"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26447856"
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              "identificador" => "bib0105"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS"
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                        0 => array:2 [
                          "etal" => false
                          "autores" => array:5 [
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                            2 => "W&#46; Lv"
                            3 => "Y&#46; Jiang"
                            4 => "L&#46; He"
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                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
                      "doi" => "10.1016/j.cbi.2015.09.015"
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                        "tituloSerie" => "Chem-Biol Interact"
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                        "paginaInicial" => "51"
                        "paginaFinal" => "60"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/26388407"
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                ]
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              "identificador" => "bib0110"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Antitumor and antimetastatic actions of dihydroxycoumarins &#40;esculetin or fraxetin&#41; through the inhibition of M2 macrophage differentiation in tumor-associated macrophages and&#47;or G 1 arrest in tumor cells"
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                          "etal" => false
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                      ]
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                  "host" => array:1 [
                    0 => array:2 [
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                        "tituloSerie" => "Eur J Pharmacol"
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                        "paginaInicial" => "115"
                        "paginaFinal" => "125"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/25445053"
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                          "issn" => "01406736"
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              "identificador" => "bib0115"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "Morin and Esculetin supplementation modulates c-myc induced energy metabolism and attenuates neoplastic changes in rats challenged with the procarcinogen 1&#44;2-dimethylhydrazine"
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                          "etal" => false
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                    0 => array:2 [
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                        "tituloSerie" => "Eur J Pharmacol"
                        "fecha" => "2017"
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                        "paginaInicial" => "20"
                        "paginaFinal" => "31"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/27989504"
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                0 => array:2 [
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                    0 => array:2 [
                      "titulo" => "Inhibitory effect of esculetin on migration&#44; invasion and matrix metalloproteinase-9 expression in TNF-&#945;-induced vascular smooth muscle cells"
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                        0 => array:2 [
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                    0 => array:2 [
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                        "tituloSerie" => "Mol Med Rep"
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                        "volumen" => "4"
                        "paginaInicial" => "337"
                        "paginaFinal" => "341"
                        "link" => array:1 [
                          0 => array:2 [
                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21468574"
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              "identificador" => "bib0125"
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                0 => array:2 [
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                    0 => array:2 [
                      "titulo" => "Esculetin suppresses proliferation of human colon cancer cells by directly targeting &#946;-catenin"
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                            4 => "H&#46;-J&#46; Lee"
                            5 => "M&#46;-H&#46; Lee"
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                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "Cancer Prevent Res"
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                        "paginaInicial" => "1356"
                        "paginaFinal" => "1364"
                        "itemHostRev" => array:3 [
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              "identificador" => "bib0130"
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "p38 MAPK activation is required for esculetin-induced inhibition of vascular smooth muscle cells proliferation"
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                          "etal" => false
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                            3 => "Y&#46;H&#46; Choi"
                            4 => "W&#46;J&#46; Kim"
                            5 => "S&#46;K&#46; Moon"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
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                  ]
                ]
              ]
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                            4 => "J&#46;-I&#46; Chae"
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                        "paginaInicial" => "106"
                      ]
                    ]
                  ]
                ]
              ]
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                      "titulo" => "Differential effects of esculetin and daphnetin on in vitro cell proliferation and in vivo estrogenicity"
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                          "etal" => true
                          "autores" => array:6 [
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                            3 => "M&#46;L&#46; Dominguez-Lopez"
                            4 => "M&#46;J&#46; Garcia-Mondragon"
                            5 => "A&#46; Maldonado-Espinoza"
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                    ]
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                        "paginaInicial" => "35"
                        "paginaFinal" => "41"
                        "link" => array:1 [
                          0 => array:2 [
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                            "web" => "Medline"
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                      ]
                    ]
                  ]
                ]
              ]
            ]
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                          "autores" => array:5 [
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                            3 => "Y&#46;L&#46; Lin"
                            4 => "T&#46;H&#46; Tseng"
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                      ]
                    ]
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                        "link" => array:1 [
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                            3 => "G&#46; Shen"
                            4 => "W&#46;-S&#46; Jeong"
                            5 => "S&#46; Yu"
                          ]
                        ]
                      ]
                    ]
                  ]
                  "host" => array:1 [
                    0 => array:2 [
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                        "paginaFinal" => "1489"
                        "link" => array:1 [
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Vol. 54. Issue 3.
Pages 128-133 (March 2018)
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Vol. 54. Issue 3.
Pages 128-133 (March 2018)
Original article
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Esculetin Attenuates the Growth of Lung Cancer by Downregulating Wnt Targeted Genes and Suppressing NF-κB
La esculetina atenúa el crecimiento del cáncer de pulmón por downregulation de los genes diana de Wnt y supresión de NF-κB
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Xiangyun Zhu, Jiaping Gu, Hongxian Qian
Corresponding author
qianhongxianwuxi@126.com

Corresponding author.
Department of Respiratory Medicine, The Second People's Hospital of Wuxi, No. 68 Zhongshan Road, Wuxi 214002, China
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Abstract
Introduction

Esculetin was identified to inhibit cell proliferation and induce apoptosis or cell cycle arrest in several cancer cell lines. However, the effect of esculetin on lung cancer remains elusive.

Methods

The anti-proliferative role of esculetin in murine Lewis lung carcinoma (LLC) cells was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and colony formation assays. BALB/c mice were subcutaneously injected with LLC cells to investigate the inhibitory effect of esculetin on the growth of lung cancer xenograft. Invasive ability was detected in esculetin treated and untreated LLC cells by transwell assay. The association between esculetin and Wnt/β-catenin signaling, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), was confirmed by testing the expression of c-myc, Cyclin D1 and NF-κB using Western blot.

Results

Esculetin treatment in LLC cells led to significant decrease of cell proliferation in a time- and dose-dependent manner. After injection of LLC cells into mice, reduced size and weight of tumors were observed in esculetin treated mice compared to untreated mice. However, no difference in cell invasion was observed between the treated and untreated LLC cells. Notably decreased expression of c-myc, Cyclin D1 and NF-κB were observed in LLC cells with esculetin treatment compared to untreated cells.

Conclusion

Esculetin plays an inhibitory role in the growth of lung cancer by down-regulating c-myc, Cyclin D1 and NF-κB.

Keywords:
Esculetin
Lung cancer
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)
C-myc
Cyclin D1
Resumen
Introducción

Se ha determinado que la esculetina inhibe la proliferación celular e induce la apoptosis o la detención del ciclo celular en varias líneas celulares de cáncer. Sin embargo, su efecto sobre el cáncer de pulmón es todavía desconocido.

Métodos

Se estudió el papel antiproliferativo de la esculetina en células murinas de carcinoma pulmonar de Lewis (LLC) mediante ensayos de bromuro de 3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2H-tetrazolio (MTT) y de formación de colonias. Se inyectaron subcutáneamente células LLC a ratones BALB/c para investigar el efecto inhibidor de la esculetina sobre el crecimiento del xenoinjerto de cáncer de pulmón. La capacidad invasiva en células LLC tratadas o no tratadas con esculetina se evaluó mediante el ensayo transwell. La asociación entre la señalización de la esculetina y la de Wnt/β-catenina, y con el factor nuclear potenciador de las cadenas ligeras kappa de células B activadas (NF-κB) se confirmó midiendo la expresión de c-myc, de ciclina D1 y NF- κB usando Western blot.

Resultados

El tratamiento con esculetina de las células LLC disminuyó significativamente la proliferación celular de una manera dependiente del tiempo y de la dosis. Tras la inyección de células LLC en ratones, se observó que los tumores de los ratones tratados con esculetina eran de menor tamaño y peso que los de los ratones no tratados. Sin embargo, no se observó diferencia en la invasividad celular entre las células LLC tratadas y las no tratadas. Se destacó la disminución de la expresión de c-myc, ciclina D1 y NF-κB en células LLC tratadas con esculetina en comparación con células no tratadas.

Conclusión

La esculetina desempeña un papel inhibitorio en el crecimiento del cáncer de pulmón a través de la regulación de c-myc, ciclina D1 y NF-κ B.

Palabras clave:
Esculetina
Cáncer de pulmón
Factor nuclear potenciador de las cadenas ligeras kappa de células B activadas (NF-κB)
C-myc
Ciclina D1
Full Text
Introduction

Lung cancer is the third most prevalent cancer in the United States. Although less tobacco use contributes a reduced mortality rate,1 lack of efficient treatment is still an obstruction to the survival of lung cancer patients. Surgery resection, chemotherapy and radiation therapy have been used as mainstay treatments, while recent advances in early detection and targeted therapy have improved the outcome and prognosis of lung cancer. However, several limits such as therapy resistance contribute to treatment failures and cancer recurrence.2–4

Previous evidence showed that Wnt/β-catenin signaling was frequently activated in lung cancer, leading to the promotion of cell proliferation, survival and metastasis.5,6 Overexpression of Wnt genes, such as Wnt5a, is predictive of aggressive non-small-cell lung cancer (NSCLC).7 In fact, Wnt/β-catenin signaling is not only highly activated in lung cancer but also in other cancers such as colon cancer. It is an important oncogenic factor in tumorigenesis, progression and metastasis. Accumulation of nuclear β-catenin caused by oncogenic Wnt signaling leads to its nuclear translocation and the formation of a complex with transcription factors, such as T-cell factor 4 (TCF-4), to initiation the transcription of its downstream targets such as c-Myc and cyclin D1.8

Esculetin (6,7-dihydroxycoumarin) is a derivative of coumarin found in Artemisia capillaries, Citrus limonia and a number of other natural plants. It has been evidenced to inhibit cell proliferation and induce apoptosis or cell cycle arrest in several cancer cell lines including human malignant melanoma and colon cancer cells, and cancers including hepatocellular carcinoma.9–13 The interaction between esculetin and β-catenin disrupts the formation of β-catenin-Tcf complex in colon cancer.14 Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation has been documented to play an important role in the progression of epidermal growth factor receptor (EGFR) mediated lung tumor.15 Previous studies have evidenced the inhibitory effect of esculetin on NF-κB in pancreatic cancer cells and vascular smooth muscle cells.16,17 Additionally, it was reported that esculetin may protect mice from lipopolysaccharide-induced acute lung injury via suppressing NF-κB.18 Thus, in this study, we investigated the effects of esculetin on lung cancer and the involvement of Wnt and NF-κB signaling pathways. We found that esculetin treatment exhibited inhibitory effects on the growth of lung cancer cells. Esculetin downregulated Wnt targeted genes and suppressed NF-κB.

MethodsCompound

Esculetin (6,7-dihydroxycoumarin, 98% purity) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in dimethyl sulfoxide (DMSO, 0.1%).

Cell Culture

Murine Lewis lung carcinoma (LLC) cells were cultured in Dulbecco's modified Eagles medium (DMEM, Life Technology, Pleasanton, CA, USA) supplemented with and penicillin (60IU/ml)/streptomycin (50μg/ml) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO2 humidified incubator.

Measurement of Cell Proliferation

Cell proliferation was measured using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. LLC cells (6×103/well) were plated in 96-well plates. After 24h, phosphate buffered saline (PBS) or esculetin at various concentrations (20, 40 or 80μmol/l) was added into each well. MTT assay was performed every 24h of incubation for 5 days. Briefly, 20μl MTT (Amresco, USA) was added to each well and incubated for 4h. The formazan precipitation was dissolved in 150μl DMSO and the results were observed using a microplate reader (BioTek Instruments, Inc., Vermont, USA) at 490nm. Each test was repeated for three times.

Colony Formation Assay

The colony formation assay was performed in 6-well plates covered with 1% low melting agarose and 0.25ml 2× RPMI with 20% FBS. Cells (500cells/well)) were mixed 0.7% low melting agarose and 0.25ml 2× RPMI with 20% FBS and seeded onto the bottom layer of soft agar. The plates were incubated for 21–28 days at 37°C in a 5% CO2 humidified incubator. Colony numbers were counted after staining with crystal violet and photographed using a phase-contrast microscope (100×).

Mice Model

The experimental protocols were approved by the Ethics Committee. BALB/c mice (6–8-week-old female) were purchased from SLAC (Shanghai, China). LLC cells (1×106cells/100μl PBS) were subcutaneously injected in the right inguinal region of mice. Mice were divided into two groups, untreated vehicle group and esculetin (100mg/kg) treated group for 20 days. At the end of the treatment, body weight was measured and mice were sacrificed for obtaining tumor size and weight.

Transwell Assay

Invasion assay was performed to test the effect of esculetin on cell invasive ability using transwell invasion chambers coated or non-coated with Matrigel (Becton Dickinson Labware, USA) according to the manufacturer's instructions. Cells (5000cells/well) were plated onto the chambers in 100μl medium containing 0.1% DMSO or esculetin (20, 40 or 80μmol/l). After 48h incubation, the cells on the bottom surface of the membrane were fixed in 90% alcohol and stained with crystal violet staining for counting using a microscope.

Western Blotting

Cells were treated with or without esculetin for 24h. Cell were lysed by Lysis Buffer containing PMSF on ice. Cell lysate was separated by 10% SDS-PAGE gels and transferred onto a NC membrane (Amersham Pharmacia Biotech, USA). The membranes were blocked by 10% non-fat milk dissolved in 0.1% Tween 20 in TBS (TBST) at 37°C for 1h. The membranes were incubated overnight with primary antibodies including c-myc (#ab32072, Abcam, Cambridge, MA, USA), cyclin D1 (#ab134175, Abcam, USA), β-catenin (#ab16051, Abcam, USA), NF-κB (#ab16502, Abcam, USA) and I-κB (#9244, Cell Signaling Technology Inc., USA), and β-actin (#ab8227, Abcam, USA) as a control for normalization. Signals were detected using an ECL Western blotting kit (Amersham Pharmacia Biotech, USA).

Statistical Analysis

Student's t test was used to determine the significant difference between the control and experimental groups. Differences were considered to be significant at P<.05.

ResultsEsculetin Inhibited Cell Viability and Colony Formation of Lewis Lung Carcinoma

Effect of esculetin on cell viability of murine LLC was first evaluated by MTT assay. Different doses of esculetin (0, 20, 40 and 80μM) were used to treat LLC cells. Esculetin suppressed cell viability in a dose- and time-dependent manner. As shown in Fig. 1A, 40μM significantly reduced cell viability on day 5, while 80μM had remarkably decreased cell viability since day 3 (P<.05). We also observed that 40 and 80μM esculetin led to reduced colony formation of LLC cells in soft agar (Fig. 1B and C, P<.05).

Fig. 1.

Esculetin treatment of LLC cells impaired its proliferation in liquid and semi-soft cultures. (A) Effect of esculetin treatment on LLC cell growth in liquid culture. MTT assay was performed in 96 wells plate. Mean±SD of 6 wells. (B) Effect of esculetin treatment on colony formation of LLC cells. Cells were seeded into soft agar in triplicate, and colonies were counted after 21–28 days of culturing. Mean±SD (3 wells) are expressed as percent variation relative to vehicle treated cells (control). *P<.05. (C) Representative pictures of colonies of LLC treated with esculetin.

(0.2MB).
Esculetin Suppressed Tumor Growth In Vivo

To ascertain the effect of esculetin on tumor growth in vivo, LLC cells (1×106cells/100μl PBS) were subcutaneously injected into the right inguinal region of BALB/c mice. Mice were treated with esculetin (100mg/kg bw) or PBS for 20 days. Body weight was measured after the treatment and mice were then sacrificed to obtain tumor size and weight. We found markedly reduced size and weight of tumors in mice with esculetin treatment compared to those control mice (Fig. 2A and B, P<.05). No significant change in body weight was observed between the two groups.

Fig. 2.

Esculetin treatment suppressed the growth of LLC-derived tumors. LLC cells were subcutaneously inoculated in the right inguinal region of BALB/c mice at the number of 1×106 each, which was followed by daily Es treatment at the dose of 100mg/kg bw or PBS vehicle (VC) treatment for 20 days. Tumors were dissected at the end of the experiments (A) and the tumor weight was measured (B). Body weight was measured prior to tumor dissection (B). Data were expressed as mean±S.E.M. (n=6 in each group). *Compared to that from PBS vehicle-treated LLC cells, *P<.05.

(0.13MB).
Esculetin had no Effect on the Invasion of LLC Cells

After confirming the effect of esculetin on tumor growth, we further tested whether esculetin affected cell invasion. Evaluation of cell invasion under different doses of esculetin treatment for 48h demonstrated that, esculetin had no influence on the invasive activity of LLC cells (Fig. 3A). Quantitative analysis was shown in Fig. 3B.

Fig. 3.

Esculetin at indicated concentrations does not alter invasiveness of LLC cell significantly. (A) Cell invasion ability was assessed by transwell assay after a 48-h treatment with esculetin. (B) The percentages of cells that could invade via the membrane relative to control are shown as a histogram. Data were expressed as mean±S.E.M.

(0.16MB).
Esculetin Inhibited the Protein Expressions of c-myc and Cyclin D1

As esculetin was identified as a potential inhibitor of Wnt/β-catenin signaling pathway, we tested the protein expressions of c-myc and cyclin D1 as the target genes of Wnt/β-catenin signaling pathway. There were significant decreases in the protein levels of c-myc and cyclin D1 in LLC cells after 24-h treatment of esculetin (40 and 80μM) compared to untreated cells (Fig. 4A and B, P<.05).

Fig. 4.

Esculetin inhibits the expression of target proteins, c-Myc and cyclin D1, of β-catenin pathway in LLC cells, not altering the level of β-catenin itself. Proteins expression in LLC cells with or without Esculetin treatment (24h) was measured by Western blot (A). The relative protein level was normalized with β-actin and the quantitative data were represented as bar graph (B). Data were expressed as mean±S.E.M.

(0.12MB).
Esculetin Reduced NF-kB Protein Level in LLC Cells

As previously reported that esculetin inhibited NF-κB pathway, we tested the protein levels of NF-κB and I-κB protein levels after 24-h of 80μM esculetin treatment in LLC cells. Compared to untreated LLC cells, esculetin significantly downregulated protein level of NF-κB (Fig. 5A and B, P<.05).

Fig. 5.

Esculetin attenuates NF-κB pathway in LLC cells. Western blot analysis shows decrease in protein levels of transcription factor NK-κB and unaltered levels of its inhibitor I-κB in LLC cells. Proteins expression in LLC cells with or without Esculetin treatment (24h) was measured by Western blot (A). The relative protein level was normalized with β-actin and the quantitative data were represented as bar graph (B). Data were expressed as mean±S.E.M. *P<.05.

(0.1MB).
Discussion

The high incidence and mortality rate of lung cancer requires improvements in both early detection and therapeutic methods. Despite the development of earlier detection using low-dose computed tomographic,19 the overall survival rate was only slight increased. Thus, it is essential to identify new therapeutic compounds for effective treatment. As previously reported, esculetin is a natural compound inhibiting cell proliferation and inducing cell apoptosis in several cell lines.20 In this study, we identified its anti-proliferative effect on a lung cancer cell line, LLC. We observed that esculetin decreased cell proliferation and colony formation in a dose- and time-dependent manner, where 80μM esculetin treatment was determined to significantly affect cell proliferation and colony formation on day 3. However, the doses of esculetin varied in different cell lines. For example, 50μM of esculetin was enough to elicit a significant decrease of cell proliferation in human normal gastric epithelial cell line GES-1 on day 3, while for other cells such as human gastric cancer cell lines MGC-803 and human hepatocellular carcinoma cell line SMMC-7721, the doses were 12.5μM and 0.3mM, respectively.21,12

To confirm whether esculetin also acts as a tumor suppressor in lung cancer in vivo, we treated mice with esculetin and found significantly reduced tumor size and weight in treated mice compared to control. The anti-proliferative role was not only shown in lung cancer but also demonstrated in other cancers such as metastatic osteosarcoma22 and hepatocellular carcinoma.12 Of note, there was no difference in the body weight between treated and untreated mice, suggesting that this dose of esculetin did not exhibit toxicity to mice. It was suggested that injection of 700mg/kg esculetin per day showed no adverse effects on the body weight of mice.12 The same conclusion was drawn by another study using 50mg/kg of esculetin on rats.23

Degradation of extracellular matrix is an essential step of tumor metastasis. The inhibitory effect of esculetin on MMP regulation has been reported in several cell lines including human gastric cancer cells MGC-803,21 vascular smooth muscle cells24 and osteosarcoma LM8 cells.22 These results suggest that esculetin may suppress tumor invasion and metastasis in vivo, which was also observed in previous studies. An in vivo study on LM8-bearing mice demonstrated that less metastasis to lung and liver was found in mice treated with ∼70–140μM esculetin compared to that of untreated mice.22 However, we did not observe the change in invaded cell number in LLC cells treated with esculetin. It may be caused by differential effects of esculetin in different cell lines. Further research is needed to reveal the detailed mechanism.

In 2014, Lee SY et al. found that esculetin disrupted the formation of the β-catenin/Tcf transcriptional complex by binding with β-catenin to suppress proliferation of colon cancer cells.25 The present study further evidenced that esculetin also inhibited Wnt signaling pathway in lung cancer cells, where the expression of c-myc and Cyclin D1 was remarkably reduced by esculetin in a dose-dependent manner. A recent study showed a similar result demonstrating that esculetin supplementation effectively suppressed tumor growth by targeting to c-myc induced energy metabolism.23 As a key transcription factor participating in the progress of tumor, c-myc has been regarded as a potential target for tumor therapy. The current results provide a possibility for the clinical use of esculetin in cancer treatment. Additionally, the inhibitory effect of esculetin on Cyclin D1 has also been reported in several previous studies.26–28 In fact, the role of esculetin in regulating Cyclin D1 was first reported in 2002, where esculetin achieved anti-proliferative function by blocking phosphorylation of pRb to arrest human leukemia HL-60 cells at G1 phase.29 In addition to Wnt signaling, esculetin treatment also decreased the expression level of NF-κB in human pancreatic carcinoma cells PANC-1.30 It was reported that esculetin attenuated NF-κB activation without affecting its inhibitor, instead it was suggested that the reduced interaction between Nuclear Factor-Erythroid 2-related factor 2 (Nrf2) and its inhibitor Kelch-like ECH-associated protein1 (KEAP1) after esculetin treatment led to abrogated NF-κB activity.17 Nrf2 exerts a protective function against reactive oxygen species by elevating the transcription of numerous anti-oxidant enzymes.31 Giving the result of unchanged I-κB in this study, the inhibitory effect of esculetin on NF-κB may be also due to the direct binding of esculetin to KEAP1. Additionally, it is well-known that activation of NF-κB is associated with promotion of cell survival and proliferation by several mechanisms including upregulating anti-apoptotic proteins, c-myc and Cyclin D1.32,33 Thus, in this study, down-regulated NF-κB by esculetin may also be the cause of reduced expressions of c-myc and Cyclin D1. In conclusion, we identified that esculetin could be a potential agent for treatment against lung cancer by suppressing NF-κB, c-myc and Cyclin D1.

Conflicts of Interest

The authors declare that they have no conflict of interest.

References
[1]
R.L. Siegel, K.D. Miller, A. Jemal.
Cancer statistics, 2016.
CA Cancer J Clin, 66 (2016), pp. 7-30
[2]
J.Y. Douillard, R. Rosell, M. De Lena, F. Carpagnano, R. Ramlau, J.L. Gonzales-Larriba, et al.
Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomised controlled trial.
Lancet Oncol, 7 (2006), pp. 719-727
[3]
J.P. Pignon, H. Tribodet, G.V. Scagliotti, J.Y. Douillard, F.A. Shepherd, R.J. Stephens, et al.
Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group.
J Clin Oncol, 26 (2008), pp. 3552-3559
[4]
T. Winton, R. Livingston, D. Johnson, J. Rigas, M. Johnston, C. Butts, et al.
Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer.
N Engl J Med, 352 (2005), pp. 2589-2597
[5]
X. Chen, J. Meng, W. Yue, J. Yu, J. Yang, Z. Yao, et al.
Fibulin-3 suppresses Wnt/beta-catenin signaling and lung cancer invasion.
Carcinogenesis, 35 (2014), pp. 1707-1716
[6]
J.H. Lim, J.W. Park, Y.S. Chun.
Human arrest defective 1 acetylates and activates beta-catenin, promoting lung cancer cell proliferation.
Cancer Res, 66 (2006), pp. 10677-10682
[7]
C.L. Huang, D. Liu, J. Nakano, S. Ishikawa, K. Kontani, H. Yokomise, et al.
Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor – an expression in non-small-cell lung cancer.
J Clin Oncol, 23 (2005), pp. 8765-8773
[8]
F.H. Tran, J.J. Zheng.
Modulating the wnt signaling pathway with small molecules.
Protein Sci, 26 (2017), pp. 650-661
[9]
C. Park, C.Y. Jin, G.Y. Kim, I.W. Choi, T.K. Kwon, B.T. Choi, et al.
Induction of apoptosis by esculetin in human leukemia U937 cells through activation of JNK and ERK.
Toxicol Appl Pharm, 227 (2008), pp. 219-228
[10]
V. Rubio, E. Calvino, A. Garcia-Perez, A. Herraez, J.C. Diez.
Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism.
Chem-Biol Interact, 220 (2014), pp. 129-139
[11]
A.D. Kim, X. Han, M.J. Piao, S.R. Hewage, C.L. Hyun, S.J. Cho, et al.
Esculetin induces death of human colon cancer cells via the reactive oxygen species-mediated mitochondrial apoptosis pathway.
Environ Toxicol Pharmacol, 39 (2015), pp. 982-989
[12]
J. Wang, M.L. Lu, H.L. Dai, S.P. Zhang, H.X. Wang, N. Wei.
Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway.
Braz J Med Biol Res, 48 (2015), pp. 245-253
[13]
Y.J. Jeon, J.Y. Jang, J.H. Shim, P.K. Myung, J.I. Chae.
Esculetin, a coumarin derivative, exhibits anti-proliferative and pro-apoptotic activity in G361 human malignant melanoma.
J Cancer Prev, 20 (2015), pp. 106-112
[14]
S.Y. Lee, T.G. Lim, H. Chen, S.K. Jung, H.J. Lee, M.H. Lee, et al.
Esculetin suppresses proliferation of human colon cancer cells by directly targeting beta-catenin.
Cancer Prev Res (Phila), 6 (2013), pp. 1356-1364
[15]
T.G. Bivona, H. Hieronymus, J. Parker, K. Chang, M. Taron, R. Rosell, et al.
FAS and NF-kappaB signalling modulate dependence of lung cancers on mutant EGFR.
Nature, 471 (2011), pp. 523-526
[16]
S.J. Lee, U.S. Lee, W.J. Kim, S.K. Moon.
Inhibitory effect of esculetin on migration, invasion and matrix metalloproteinase-9 expression in TNF-alpha-induced vascular smooth muscle cells.
Mol Med Rep, 4 (2011), pp. 337-341
[17]
R. Arora, S. Sawney, V. Saini, C. Steffi, M. Tiwari, D. Saluja.
Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1.
[18]
T. Chen, Q. Guo, H. Wang, H. Zhang, C. Wang, P. Zhang, et al.
Effects of esculetin on lipopolysaccharide (LPS)-induced acute lung injury via regulation of RhoA/Rho Kinase/NF-small ka, CyrillicB pathways in vivo and in vitro.
Free Radic Res, 49 (2015), pp. 1459-1468
[19]
D.R. Aberle, A.M. Adams, C.D. Berg, W.C. Black, J.D. Clapp, R.M. Fagerstrom, et al.
Reduced lung-cancer mortality with low-dose computed tomographic screening.
New Engl J Med, 365 (2011), pp. 395-409
[20]
Y.J. Jeon, J.H. Cho, S.Y. Lee, Y.H. Choi, H. Park, S. Jung, et al.
Esculetin induces apoptosis through EGFR/PI3K/Akt signaling pathway and nucleophosmin relocalization.
J Cell Biochem, 117 (2016), pp. 1210-1221
[21]
H. Pan, B.H. Wang, W. Lv, Y. Jiang, L. He.
Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS.
Chem-Biol Interact, 242 (2015), pp. 51-60
[22]
Y. Kimura, M. Sumiyoshi.
Antitumor and antimetastatic actions of dihydroxycoumarins (esculetin or fraxetin) through the inhibition of M2 macrophage differentiation in tumor-associated macrophages and/or G 1 arrest in tumor cells.
Eur J Pharmacol, 746 (2015), pp. 115-125
[23]
S.H. Sharma, S. Thulasingam, D.R. Chellappan, P. Chinnaswamy, S. Nagarajan.
Morin and Esculetin supplementation modulates c-myc induced energy metabolism and attenuates neoplastic changes in rats challenged with the procarcinogen 1,2-dimethylhydrazine.
Eur J Pharmacol, 796 (2017), pp. 20-31
[24]
S.-J. Lee, U.-S. Lee, W.-J. Kim, S.-K. Moon.
Inhibitory effect of esculetin on migration, invasion and matrix metalloproteinase-9 expression in TNF-α-induced vascular smooth muscle cells.
Mol Med Rep, 4 (2011), pp. 337-341
[25]
S.-Y. Lee, T.-G. Lim, H. Chen, S.K. Jung, H.-J. Lee, M.-H. Lee, et al.
Esculetin suppresses proliferation of human colon cancer cells by directly targeting β-catenin.
Cancer Prevent Res, 6 (2013), pp. 1356-1364
[26]
E.S. Yun, S.S. Park, H.C. Shin, Y.H. Choi, W.J. Kim, S.K. Moon.
p38 MAPK activation is required for esculetin-induced inhibition of vascular smooth muscle cells proliferation.
Toxicol in Vitro, 25 (2011), pp. 1335-1342
[27]
Y.-J. Jeon, J.-Y. Jang, J.-H. Shim, P.K. Myung, J.-I. Chae.
Esculetin, a coumarin derivative, exhibits anti-proliferative and pro-apoptotic activity in G361 human malignant melanoma.
J Cancer Prevent, 20 (2015), pp. 106
[28]
F.A. Jimenez-Orozco, A.A.R. Rosales, A. Vega-Lopez, M.L. Dominguez-Lopez, M.J. Garcia-Mondragon, A. Maldonado-Espinoza, et al.
Differential effects of esculetin and daphnetin on in vitro cell proliferation and in vivo estrogenicity.
Eur J Pharmacol, 668 (2011), pp. 35-41
[29]
C.J. Wang, Y.J. Hsieh, C.Y. Chu, Y.L. Lin, T.H. Tseng.
Inhibition of cell cycle progression in human leukemia HL-60 cells by esculetin.
Cancer Lett, 183 (2002), pp. 163-168
[30]
R. Arora, S. Sawney, V. Saini, C. Steffi, M. Tiwari, D. Saluja.
Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1.
Mol Cancer, (2016), pp. 15
[31]
W. Li, T.O. Khor, C. Xu, G. Shen, W.-S. Jeong, S. Yu, et al.
Activation of Nrf2-antioxidant signaling attenuates NFκB-inflammatory response and elicits apoptosis.
Biochem Pharmacol, 76 (2008), pp. 1485-1489
[32]
R. Ruiz-Ramos, L. Lopez-Carrillo, A.D. Rios-Perez, A. De Vizcaya-Ruíz, M.E. Cebrian.
Sodium arsenite induces ROS generation, DNA oxidative damage, HO-1 and c-Myc proteins NF-κB activation and cell proliferation in human breast cancer MCF-7 cells.
Mutat Res/Genet Toxicol Environ Mutag, 674 (2009), pp. 109-115
[33]
S. Shishodia, H.M. Amin, R. Lai, B.B. Aggarwal.
Curcumin (diferuloylmethane) inhibits constitutive NF-κB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma.
Biochem Pharmacol, 70 (2005), pp. 700-713
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