Activation of PI3K/Akt/IKK-α/NF-κB signaling pathway is required for ...

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Apr 13, 2010 - Keywords. Quercetin Adenoid cystic carcinoma Apoptosis Akt NF-κB. Zhi-Jun Sun and Gang Chen contributed equally to this article.
Apoptosis (2010) 15:850–863 DOI 10.1007/s10495-010-0497-5

ORIGINAL PAPER

Activation of PI3K/Akt/IKK-a/NF-jB signaling pathway is required for the apoptosis-evasion in human salivary adenoid cystic carcinoma: its inhibition by quercetin Zhi-Jun Sun • Gang Chen • Xiang Hu • Wei Zhang • Yang Liu • Ling-Xin Zhu • Qian Zhou • Yi-Fang Zhao

Published online: 13 April 2010 Ó Springer Science+Business Media, LLC 2010

Abstract Quercetin, one of the most common natural flavonoids, has been reported to possess significant antitumor activities both in vitro and in vivo. The present study was to investigate the effects of quercetin on growth and apoptosis in human salivary adenoid cystic carcinoma (ACC). The result from MTT assay showed that quercetin decreased cell viability of both low metastatic cell line ACC-2 and high metastatic cell line ACC-M in a concentration- and time-dependent manner. Moreover, treatment with quercetin resulted in significantly increased apoptosis in ACC cells. Our data also revealed that the apoptosis induced by quercetin treatment was through a mitochondria-dependent pathway which showed close correlation with the down-regulation of the PI3K/Akt/IKK-a/NF-jB pathway. Most importantly, quercetin significantly prevented in vivo growth of ACC xenografts in nude mice, accompanied by induction of tumor cell apoptosis, suppression of NF-jB nuclear translocation, as well as downZhi-Jun Sun and Gang Chen contributed equally to this article.

Electronic supplementary material The online version of this article (doi:10.1007/s10495-010-0497-5) contains supplementary material, which is available to authorized users. Z.-J. Sun (&)  G. Chen  X. Hu  W. Zhang  Y. Liu  L.-X. Zhu  Q. Zhou  Y.-F. Zhao (&) The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China e-mail: [email protected] Y.-F. Zhao e-mail: [email protected] Z.-J. Sun  G. Chen  X. Hu  W. Zhang  Y.-F. Zhao Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, Wuhan, China

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regulation of Akt and IKK-a activation. In addition, we explored the clinical significance of the PI3K/Akt/IKK-a/ NF-jB signaling axis in ACC by immunohistochemical analysis of tissue specimens followed by the clustering analyses. We determined that the PI3K/Akt/IKK-a/NF-jB pathway is ubiquitously activated in ACC and plays an essential role in the evasion of apoptosis. Taken together, the results from our study implicated that quercetin would be a promising chemotherapeutic agent against ACC through its function of down-regulating the PI3K/Akt/IKKa/NF-jB signaling pathway. Keywords Quercetin  Adenoid cystic carcinoma  Apoptosis  Akt  NF-jB

Introduction Adenoid cystic carcinoma (ACC) is one of the most common malignancies of the salivary glands, but may also arise in the breast, cervix, vulva, as well as tracheobronchial tree [1]. It is a histologically distinctive neoplasm with a long-term poor prognosis, especially the high incidence of distant metastasis [2]. Most ACC patients (80–90%) die within 10–15 years after diagnosis [1]. Curative surgery has failed to reduce the overall mortality rate over the past several decades [3, 4]. Radiation and chemotherapy are not beneficial for patients with advanced disease [5, 6] and may potentially increase the risk of recurrence [7]. As to the root reason for the unfavorable prognosis of ACC, it might be the poor understanding of its molecular pathogenesis. It is therefore essential to discovery novel molecular events underlying the malignant development of this disease, which may allow for early detection and more importantly to develop new strategies of therapeutics.

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Tumor progression is a multi-step process of somatic cell evolution which includes uncontrolled proliferation, impaired apoptosis, loss of differentiation, immortalization, neovascularization, invasion, and metastatic spread [8]. During this evolutionary transformation, the intrinsic or acquired resistance to apoptosis is widely regarded as one of the most important steps since evasion of apoptosis may largely contribute to carcinogenesis, malignant development, as well as chemo-, radio- and immuno-therapy resistance in most of the human cancers, including ACC [9]. A better understanding of the anti-apoptotic signaling pathways in ACC is particularly expected to provide a basis for a rational approach to develop molecular targeted therapies. Phosphatidylinositol 3-kinase (PI3K) and its direct downstream effector, Akt/PKB, play a pivotal role in cellular growth, proliferation, and survival through regulation of multiple downstream signals, among which the nuclear factor-jB (NF-jB) pathway is one of the most frequently mentioned targets. It is now well recognized that the PI3K/ Akt pathway-regulated NF-jB activation, through a p38/ IjBa kinase (IKK)-b-dependent or an IKK-a-dependent mechanism, is intensively involved in keeping tumor cells alive by blocking apoptosis. Actually, high activity of Akt and NF-jB in ACC has been previously confirmed, however they were respectively reported by independent investigators in different studies unrelated to each other or as to the anti-apoptotic events [10–13]. Meanwhile, the involvement of PI3K/Akt signaling pathway in NF-jB activation is indeed a cell-specific event [14, 15]. Thus, the precise functional relationship between Akt and NF-jB activation, as well as their clinical significance in ACC, especially in the evasion of apoptosis, is still far from clear. Epidemiological studies have shown that dietary uptake of flavonoids, a large group of polyphenolic compounds present in most fruits, vegetables, and beverages, is associated with a low risk of cancer [16]. Quercetin, as one of the most common flavonoids in nature, has been studied extensively as a chemoprevention agent in several cancer models due to its potent antioxidant, anti-tumor, and antiinflammatory properties [16, 17]. This natural flavonoid is capable of inhibiting the growth of a wide array of cancers such as prostate, breast, lung, colon, and ovarian [18–21]. Moreover, its pro-drug QC12 has entered in phase-I clinical studies for certain human cancers [17, 35]. Recent studies have revealed that the anti-growth activity of quercetin is associated closely with its ability to inhibit cell proliferation, induce cell apoptosis, and prevent tumorinduced angiogenesis [20, 22], probably through downregulation of growth factors, elevation of stress proteins, as well as modulation of phosphorylated signal transducers and transcription factors [16, 23, 24]. Previous studies have reported that quercetin was able to inhibit NF-jB

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activation in several cell lines, however the precise molecular mechanisms by which quercetin down-regulated NF-jB have not been well clarified. Most importantly, the anti-tumor activities of quercetin in ACC have not been reported yet. In the present study, we explored the anti-tumor activity of quercetin in human salivary adenoid cystic carcinoma, and showed that quercetin efficiently inhibited growth and induced apoptosis in ACC both in vitro and in vivo. These anti-tumor activities of quercetin were related to the suppression of PI3K/Akt/IKK-a/NF-jB pathway. Additionally, we determined the clinical significance of the activation of the PI3K/Akt/IKK-a/NF-jB signaling axis in the evasion of apoptosis in ACC by immunohistochemical analysis of tissue specimens followed by the clustering analyses. Taken together, the results from our study implicated that the PI3K/Akt/IKK-a/NF-jB signaling axis promotes apoptosis-evasion in ACC, and quercetin can specifically target this signaling pathway and significantly induce apoptosis in ACC. Thus, we are tempted to speculate that quercetin would be a promising chemotherapeutic agent against ACC through its function of down-regulating the PI3K/Akt/IKK-a/NF-jB signaling pathway.

Materials and methods Chemicals and antibodies Quercetin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-imidacarbocyanine iodide (JC-1), Hoechst 33258, propidium iodide (PI), 40 ,6diamidino-2-phenylindole (DAPI), LY294002, and ribonuclease (RNase) were purchased from Sigma. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GIBCO. All the used primary antibodies were purchased from Cell Signaling, except for Histone H2A, NF-jB (p65), PTEN, and p-IKKa antibodies from Santa Cruz. The expression vectors encoding constitutively active Akt (CA-Akt) as well as the corresponding empty vectors (pcDNA3.1) were kindly provided by Dr. Michael Robinson (University of Pennsylvania, USA). Clinical samples Fifty primary ACC specimens, diagnosed in accordance with the latest WHO (World Health Organization) classification of salivary gland tumors (23 cribriform, 18 tubular, 9 solid ACC), were retrieved from the Department of Oral Pathology, Stomatology School of Wuhan University. Six pericancerous normal salivary gland (NSG) tissues were

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used as the control. All specimens were fixed in buffered paraformaldehyde and embedded in paraffin. The histopathological diagnosis and classification were confirmed independently by two pathologists before immunostaining. Immunohistochemistry and double-labeling immunofluorescence histochemistry The immunohistochemical and double-labeling immunofluorescence histochemical analyses were performed according to our previous procedures [25], as described in Supplementary Materials and Methods. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay Apoptotic cells in tumor tissues were quantified by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, as summarized in Supplementary Materials and Methods. Cell culture and transient transfection The low (ACC-2) and high (ACC-M) metastatic cell lines of human salivary ACC [26] were obtained from the China Center for Type Culture Collection, and were maintained in DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 lg/ml streptomycin. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For transient transfection, ACC-M cells were seeded in 6 cm culture dishes at a density of 106 cells/dish. And then they were transfected with pUSE-CA-Akt and pUSE vector plasmids using the LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions. The expression of p-Akt after transfection was confirmed by Western blotting. Cell viability assay The effect of quercetin on cell viability was estimated by means of the MTT assay as described [27]. Briefly, ACC-2 and ACC-M cells at 2 9 104/well were plated into 96-well plates and exposed to quercetin at various concentrations (0–200 lM) for 24, 48 and 72 h. Then, 20 ll of MTT (5 mg/ml) was added to each well and incubated for another 4 h. After the supernatant was discarded, 150 ll of DMSO was added to each well, and absorbance was assessed at 490 nm using a 96-well microplate reader (Bio-Tek). Data are reported as a percentage of the control.

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Determination of apoptosis Induction of apoptosis by quercetin in ACC-2 and ACC-M cells was determined as followed: (a) morphological evaluation by Hoechst staining; (b) quantitation of cells in sub-G1 DNA content by flow cytometry following staining with PI; (c) quantitation of cytoplasmic histone-associated DNA fragments with Cell Death Detection ELISAPLUS assay; (d) quantitative assessment of early and late apoptosis using the Annexin V-FITC/PI double staining assay; (e) detection of mitochondrial membrane potential change using the potentiometric dye JC-1; (f) quantitative measurement of cytochrome c release with Cytochrome c ELISA assay; (g) caspase activity measurement by Caspase Activity ELISA assay; and (h) Western blot analysis for Bax/Bcl-2 ratio and PARP cleavage. See Supplementary Materials and Methods for more details. Immunofluorescence analysis for cells The nuclear translocation of NF-jB was detected by indirect immunofluorescence analysis. For details, see Supplementary Materials and Methods. Western blotting ACC-2 and ACC-M cells that treated with indicated concentrations of quercetin were collected, and the extracts of nuclear and cytosol were prepared according to the method of Levites et al. [28]. The concentration of protein in the supernatants was estimated by the BCA assay (Pierce). After that, 50 mg of protein were separated on 10% SDSpolyacrylamide gels and electroblotted on nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk and probed with primary antibody at dilutions recommended by the suppliers. Immunoblots were detected by horseradish peroxidase-conjugated secondary antibody (Pierce) using chemiluminescence kit of Millipore and visualized by Versa Doc Imaging System (Bio-Rad). Nude mouse xenograft model Male athymic nu/nu mice (4 week old) were provided by the Experimental Animal Center of Wuhan University. The nude mice were housed in laminar flow cabinets under pathogen-free condition. All experimental procedures carried out in this study were performed in accordance with ‘‘Principles of laboratory animal care’’ (NIH publication No. 85-23, revised 1985). See Supplementary Materials and Methods for more details.

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Hierarchical clustering, data visualization and statistical analysis The staining scores that resulted from immunohistochemistry and TUNEL assay were converted into scaled values centered on zero in Microsoft excel. The hierarchical analysis was done using Cluster 3.0 (http://bonsai.ims. u-tokyo.ac.jp/*mdehoon/software/cluster/) with average linkage based on Pearson’s correlation coefficient as the selection variable on the unsupervised approach [29]. The results were visualized using the Java TreeView 1.0.5 (http://jtreeview.sourceforge.net/) as previously described [30]. The clustered data were arranged with markers on the horizontal axis and tissue samples on the vertical axis. Two biomarkers with a close relationship are located next to each other. All data were expressed as mean ± SEM of three independent experiments. One-way analysis of variance (ANOVA), Student–Newman–Keuls, and Spearman rank correlation test were used for statistical analysis. P \ 0.05 was considered significantly different.

Results

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dependent in both ACC-2 and ACC-M cells (Fig. 1d). These results were further confirmed by cell cycle analysis in quercetin-treated ACC cells, stained with PI. The typical hypoploid population or sub-G1 peak, corresponding to cells with low DNA content, was quantified. In ACC-2 cells (Supplementary Fig. 1a), sub-G1 DNA contents were increased from 4.7 to 15.56, 28.03, and 43.35% at concentrations of 25, 50, and 100 lM of quercetin, respectively. While in ACC-M cells (Fig. 1e), the percentage of this sub-G1 peak was shifted from 3.8 to 19.45, 35.48, and 56.66% when exposed to 25, 50, and 100 lM quercetin, respectively. To verify our data on the apoptosis induction by quercetin, a further measurement of apoptosis was determined by Annexin V-FITC and PI double-staining flow cytometry, which can quantitatively assess the early- and late-apoptotic cell population. As the results, quercetin produced a concentration-dependent increase in the apoptotic cell population. The basal apoptotic population in untreated ACC-2 cells was 3.17%, which increased to 64.57% when treated with 100 lM quercetin (Supplementary Fig. 1b). In untreated ACC-M cells the basal apoptotic population was 3.00%, and increased to 75.81% after 100 lM quercetin treatment (Fig. 1f). Apoptosis thus appeared to be the primary mode of cell death induced by quercetin.

Quercetin exerts cytotoxicity against ACC cells Quercetin stimulates caspases activation We examined the effect of quercetin on ACC cell viability using MTT assay. As shown in Fig. 1a, b, quercetin treatment decreased the cell viability of both ACC-2 and ACC-M cells in a concentration- and time-dependent manner. The 50% growth inhibition concentration (IC50) of quercetin in ACC-2 cells, obtained after 24, 48 and 72 h of incubation were 77, 49 and 30 lM, respectively. While in ACC-M cells, those values were 59, 37 and 28 lM, respectively. The concentrations of 25, 50, and 100 lM incubating for 24 h were chosen for the subsequent studies. Quercetin induces apoptosis in ACC cells To determine whether the cytotoxicity exerted by quercetin was due to apoptosis induction, morphological observation was firstly performed under microscope. As shown in Fig. 1c, after exposure to 50 lM quercetin for 24 h, several apoptotic morphological features such as apoptotic bodies, cell shrinkage and chromatin condensation were observed in quercetin-treated ACC-2 and ACC-M cells by Hoechst 33258 staining assays. To confirm quercetin-induced apoptosis in ACC cells, we quantitatively assessed the DNA fragmentation using the Cell Death Detection ELISAPLUS kit. Likewise, the results revealed that the formation of DNA fragments elicited by quercetin treatment was concentration-

The involvement of caspase pathway in apoptosis has been established in many studies. To examine whether the apoptotic effects induced by quercetin were associated with caspase enzymes activation, the activities of caspase-8, -9 and -3 were investigated in quercetin-treated ACC-2 and ACC-M cells. The data showed that the activities of caspase-9 and caspase-3 increased approximately 3.4- and 3.9fold in 100 lM quercetin-treated ACC-2 cells, almost 3.7and 4.1-fold in 100 lM quercetin-treated ACC-M cells, respectively (Fig. 2a, b). But the activity of caspase-8 in ACC cells was not obviously changed by quercetin up to 100 lM (data not shown). These findings promoted us to hypothesize that the apoptosis induced by quercetin is probably through a mitochondria-dependent pathway. Quercetin induces mitochondrial membrane depolarization To validate our hypotheses, the effect of quercetin on mitochondrial membrane depolarization was investigated in ACC cells by employing the potentiometric dye JC-1. The percentages of cells with green fluorescence were scored as depolarized cells. As shown in Fig. 2c, quercetin treatment concentration-dependently increased the number of cells with green fluorescence in both ACC-2 and

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Fig. 1 Quercetin exerts cytotoxicity and induces apoptosis in ACC cells. a, b ACC-2 and ACC-M cell viability was measured by MTT assay in 96-well plates after treatment with increasing concentrations of quercetin, as indicated, for 24 h (filled circle), 48 h (filled square), and 72 h (filled triangle). The results were represented as a percentage of the control group. c Both ACC-2 and ACC-M cells were treated with 50 lM quercetin for 24 h, and then morphologic changes were captured using fluorescence microscopy with Hoechst 33258 staining. d Both ACC-2 and ACC-M cells were treated with indicated concentrations of quercetin for 24 h, and then DNA fragmentation was quantified using Cell Death Detection ELISAPLUS assay.

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e ACC-M cells were treated with indicated concentrations of quercetin for 24 h, and then cell cycle analysis was performed to show the DNA histograms using flow cytometry with PI staining. f ACC-M cells were treated with indicated concentrations of quercetin for 24 h, and then early- and late-apoptotic cells were determined by Annexin V-FITC/PI double-staining assay. Criteria were set to distinguish between viable (bottom left), early apoptotic (bottom right), late apoptotic (top right) and necrotic (top left) cells. All data were presented as means ± SEM from three different experiments with duplicate. * P \ 0.05; ** P \ 0.01 versus the control group

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ACC-M cells, further indicating that quercetin may induce cell apoptosis through a mitochondria-dependent pathway. Quercetin increases the release of cytochrome c from mitochondria The essential role of mitochondria in apoptosis is usually induced by chemical triggers, among which cytochrome c is generally regarded as the most important one [31]. Cytochrome c released from mitochondria can bind to Apaf-1 in the cytosol, allowing the recruitment of caspase9 and the formation of an apoptosome complex, resulting in caspase-3 activation and execution of cell death [31]. To access the involvement of mitochondrial release of cytochrome c in quercetin-induced apoptosis, the cytoplasmic extracts from quercetin-treated ACC cells were prepared and analyzed with Cytochrome c ELISA kit. It could be inferred from Fig. 2d that quercetin concentrationdependently increased the release of cytochrome c from mitochondria in both ACC-2 and ACC-M cells. The cytoplasmic content of cytochrome c in ACC-2 and ACC-M cells increased almost 3.8 and 9.6 times respectively, after treatment with 100 lM quercetin.

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investigated its effect on the nuclear translocation of NF-jB, an inducible transcription factor that intensively associated with the malignant progression of ACC [12, 32, 33]. The data revealed that quercetin treatment inhibited the nuclear translocation of NF-jB in both ACC-2 (Supplementary Fig. 2a) and ACC-M (Fig. 3a) cells in a concentration-dependent manner. Concurrently, quercetin decreased the phosphorylation of IKK-a but not IKK-b, as well as increased the expression of IjB-a, an inhibitor of NF-jB activation. Additionally, immunofluorescence analysis also confirmed the decreased nuclear translocation of NF-jB (p65) after exposure to 50 lM quercetin for 24 h (Supplementary Fig. 2b and Fig. 3b). Quercetin-induced cell apoptosis and NF-jB downregulation correlated with the suppression of PI3K/Akt/ IKK-a signaling axis

Because of the observed reduction in DWm and release of cytochrome c from mitochondria in quercetin-treated ACC cells, it was reasonable for us to study the pro-apoptotic and anti-apoptotic members that closely linked to mitochondrial control during apoptosis, such as Bax and Bcl-2. The expression levels of Bax and Bcl-2 protein were detected and analyzed by Western blotting. As shown in Fig. 2e, quercetin treatment increased the expression level of pro-apoptotic protein Bax, and meanwhile decreased the expression level of anti-apoptotic protein Bcl-2 in a concentration-dependent manner in both ACC-2 and ACC-M cells, which strongly suggested that quercetin-induced apoptosis in ACC cells was due, at least in part, to its alteration of the Bax/Bcl-2 ratio. Another significant feature of cell apoptosis is the increased level of PARP cleavage stimulated by caspase-3 activation. To determine whether quercetin treatment could result in PARP cleavage, the expression level of cleaved PARP was also detected and semi-quantitated by Western blotting. As expected, quercetin concentration-dependently increased the expression level of cleaved PARP in both ACC-2 and ACC-M cells (Fig. 2e).

To unmask the upstream mechanisms behind the downregulation of NF-jB, we initially investigated the effect of quercetin on PI3K/Akt signaling pathway, a major upstream regulator of NF-jB, either through p38/IKK-bdependent or IKK-a-dependent mechanism [34]. Our results showed here that quercetin treatment concentrationdependently decreased the phosphorylation levels of PI3K (p85) and Akt but not p-38 (data not shown) in both ACC-2 (Supplementary Fig. 2c) and ACC-M (Fig. 3c) cells, which was consistent with its effects on IKK-a and IKK-b phosphorylation status. The above findings indicated the involvement of a PI3K/Akt/IKK-a signaling axis, and meanwhile excluded the possibility of a PI3K/Akt/p38/ IKK-b-dependent mechanism. To further determine the functional significance of PI3K/Akt/IKK-a signaling axis in the inhibitory activities of quercetin, we evaluate the effect of CA-Akt expression on quercetin-mediated IKK-a inactivation, NF-jB down-regulation, Bax/Bcl-2 ratio increase, PARP cleavage, and apoptosis induction in ACC cells. As shown in Fig. 3d, transient transfection of ACC cells with CA-Akt plasmids significantly increased the phosphorylation level of Akt compared with the empty vector-transfected control cells. Quercetin-mediated inactivation of IKK-a, down-regulation of NF-jB, cleavage of PARP, as well as increase in Bax/Bcl-2 ratio were obviously reversed by CA-Akt transfection (Fig. 3d). Moreover, over-expression of CA-Akt also partially but efficiently rescued quercetin-induced apoptosis in ACC-M cells, as represented by the Cell Death Detection ELISAPLUS (Fig. 3e) assay.

Quercetin down-regulates NF-jB

Effects on nude mice xenografts model

To explore the molecular mechanisms behind the mitochondria-dependent apoptosis induced by quercetin, we

To evaluate the anti-tumor potential of quercetin against ACC in vivo, we established the nude mouse xenograft

Quercetin increases Bax/Bcl-2 ratio and cleaved PARP level

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Fig. 2 Quercetin induces apoptosis via mitochondria-dependent pathway. Both ACC-2 and ACC-M cells were treated with indicated concentrations of quercetin for 24 h. a, b The activities of caspase-9 and caspase-3 were assessed by Caspase Activity Assay kit. c The change of DWm was detected using the potentiometric dye JC-1. The number of cells with green fluorescence representing mitochondrial membrane depolarization was expressed as a percentage of total cell

number. d Release of cytochrome c from mitochondria was quantitatively measured with Cytochrome c ELISA kit. e The expression levels of cleaved PARP, Bax, and Bcl-2 were determined by Western blotting. All values were presented as means ± SEM from three different experiments with duplicate. * P \ 0.05; ** P \ 0.01 versus the control group

model with ACC-M cells. As shown in Fig. 4a, b and c, quercetin treatment effectively prevented the tumor growth of ACC-M cells. To further correlate this in vivo tumorpreventive effect to the mechanisms identified in vitro, we then assessed the expression level of some intratumoral biomarkers in quercetin-treated ACC-M tumor tissues by

immunohistochemical analysis. Consistent with the findings in vitro, treatment with quercetin significantly induced cell apoptosis in tumor tissues as evidenced by the TUNEL assay, associating with the suppression of NF-jB nuclear translocation as well as the down-regulation of Akt and IKK-a activation (Fig. 4d).

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Fig. 3 Quercetin-induced cell apoptosis and NF-jB down-regulation correlates with the suppression of PI3K/Akt signaling pathway in ACC-M cell line. ACC-M cells were treated with indicated concentrations of quercetin for 24 h. a The expression levels of p-IKK-a/b, IjB-a, as well as NF-jB in both cytosolic and nuclear extract were determined by Western blotting. b The NF-jB localization in the nuclei was detected by indirect immunofluorescence analysis. c The phosphorylation level of PI3K and Akt determined by Western blotting. d The expression level of pAkt, p-IKK-a/b, nuclear NF-jB, as well as Bcl-2, Bax and cleaved PARP in lysates from ACC-M cells

transiently transfected with CA-Akt or empty vector plasmids and treated with or without 100 lM quercetin for 24 h. e Cell Death Detection ELISAPLUS assay for quantitation of apoptosis induction in ACC-M cells transiently transfected with CA-Akt or empty vector plasmids and treated with or without 100 lM quercetin for 24 h. Data were expressed as means ± SEM from three independent experiments with duplicate. * P \ 0.05 and ** P \ 0.01 versus DMSOtreated control cells, and # P \ 0.05 versus quercetin-treated empty vector-transfected cells

The function-related PI3K/Akt/IKK-a/NF-jB signaling axis is activated in ACC and correlated with the apoptosis-evasion

ACC tissues. Examples of immunohistochemical results of the selected cases are shown in Fig. 5a. In the selected ACC tissues, the cytoplasmic immunostaining for PTEN protein was nearly not detectable in contrast to NSG tissues. However p-Akt (Ser473) showed intense staining mostly in the cytoplasm of most ACC cells, accompanied by a strong nuclear staining of phosphorylated-IKK-a. Concurrently, the nuclear staining of NF-jB p65 was also detected in some ACC cells in the same area concomitant

To unmask the nature of the deregulated molecular mechanism behind the anti-apoptotic process of ACC cells, and also to make our present study more clinically significant, we next explored the expression level and/or activation status of a number of signaling molecules in 50

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Fig. 4 Quercetin prevents tumor growth in vivo. a Tumor regression observed in ACC xenografts treated with quercetin. ACC-M cells were used to establish xenografts in athymic nu/nu mice, and tumorbearing animals were given with quercetin (1 g/kg, p.o., daily; n = 8) or corn oil (Control, 100 ll, p.o., daily; n = 8) for 32 consecutive days. An example of tumor regression in quercetin-treated animals is depicted. b Lesions dissected from ACC-M xenografts after treatment with quercetin or vehicle control for 32 days. c Tumor volume from ACC-M xenograft in both control and quercetin-treated groups was assessed daily, as indicated. d Immunohistochemical analysis of the indicated biomarkers in both control and quercetin-treated ACC-M tumor tissues

with low apoptotic index revealed by the TUNEL staining. Moreover, the results of double-labeling immunofluorescence histochemistry also confirmed the concurrence of Akt activation and NF-jB p65 nuclear translocation in ACC, for the observation that the ACC cells with high expression of p-Akt was almost positive for NF-jB nuclear location (Fig. 5b). As shown in Fig. 5c, when the staining scores of the tested markers in the ACC tissues were evaluated against NSG tissues, all of them were significantly different between NSG and ACC cases across all the three histologic types. Furthermore, the Student–Newman–Keuls test also showed that the staining scores of NF-jB p65, p-IKK-a, and p-IKK-b were significantly higher in solid type than those in cribriform and tubular types (P \ 0.01), whereas no significant difference was found among these three types for PTEN, p-PI3K, p-Akt, as well as TUNEL. Most importantly, the Spearman rank test showed significant correlation between the staining of p-PI3K, p-Akt (Ser473), p-IKK-a, NF-jB p65 as well as the TUNEL (Table 1), and these results were then nicely reflected by the cluster analysis (Fig. 6a), which further implicated the existence of a function-related PI3K/Akt/IKK-a/NF-jB

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pathway and its role in the apoptosis-evasion in ACC. In general, a high proportion of ACC cases were concurrently negative for PTEN and positive for p-PI3K, p-Akt, p-IKK-a, as well as NF-jB p65, and correlated well with the low apoptotic index (Cluster 1). However, we also found that a certain proportion of the ACC tissues were positive for PTEN, but still showed high phosphorylation level of Akt. Notably, IKK-b probably behaved as an independent marker and did not show any correlation with the other markers except for NF-jB p65. In the heat-map shown in Fig. 6a, the length and the subdivision of the branches reflect the correlation among the tested cases (left) as well as the markers (top). As expected, p-PI3K, p-Akt, p-IKK-a, NF-jB p65, as well as TUNEL clustered together. PTEN was clustered closely to them, but p-IKK-b was distantly clustered. Of interest, using this clustering analysis, most of the solid ACC (Cluster 2) and all of the NSG (Cluster 3) cases were clustered together respectively, reflecting the significant differences. To further determine the functional relationship among this PI3K/Akt/IKK-a/NF-jB pathway as well as its significance in the apoptosis-evasion of ACC, we then tested

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Fig. 5 Immunoreactivities of tested markers in ACC. a Immunohistochemical staining of PETN, p-Akt, p-IKK-a, NF-jB (p65), as well as TUNEL in both NSG and ACC tissues. b Double-labeling immunofluorescence histochemistry in both NSG and ACC tissues. The arrows indicate the concurrence of Akt activation and NF-jB

(p65) nuclear translocation. c Immunostaining scores for indicated markers in NSG (N), as well as cribriform (C), tubular (T) and solid (S) ACC tissues. * P \ 0.05 versus NSG, and # P \ 0.05 versus solid ACC

Table 1 Spearman rank test analyses of immunostaining Marker

p-PI3K

PTEN

n = 56 (P \ 0.005) n = 56 (P \ 0.001) n = 56 (P \ 0.005) n = 56 (P \ 0.001) n = 56 (NS)

p-PI3K p-Akt

p-Akt

p-IKK-a

NF-jB

p-IKK-b

n = 56 (P \ 0.001) n = 56 (P \ 0.001) n = 56 (P \ 0.001) n = 56 (NS) n = 56 (P \ 0.001) n = 56 (P \ 0.001) n = 56 (NS)

p-IKK-a

n = 56 (P \ 0.001) n = 56 (NS)

TUNEL n = 56 (P \ 0.01) n = 56 (P \ 0.001) n = 56 (P \ 0.001) n = 56 (P \ 0.05)

n = 56 (P \ 0.005) n = 56 (P \ 0.005)

NF-jB p-IKK-b

n = 56 (NS)

The values represent number of cases analyzed (n) and P values. NS not significant

the effect of LY294002, a specific PI3K inhibitor, on the activation of Akt, IKK-a and NF-jB, as well as the survival of ACC cells. As expected, LY294002 (20 lM) concurrently suppressed the phosphorylation of both Akt and IKK-a as well as inhibited the nuclear translocation of NF-jB p65, while it showed no effect on the activation status of IKK-b (Fig. 6b). In addition, LY294002 treatment also significantly induced the apoptosis of ACC cells concomitant with the increase of both Bax/Bcl-2 ratio and PARP cleavage (Fig. 6b). Taken together, all the above data strongly suggested that a function-related PI3K/Akt/ IKK-a/NF-jB signaling axis is ubiquitously activated in ACC and intensely correlated with the process of apoptosis-evasion.

Discussion Epidemiological studies have shown that dietary uptake of flavonoids is associated with a low risk of cancer [16]. As one of the most common flavonoids in nature, quercetin has attracted considerable interest on account of its potent antioxidant, anti-tumor, and anti-inflammatory properties [17]. This natural flavonoid has been extensively studied as a chemoprevention agent and shown to possess anti-growth effect in various tumor cells [16]. Moreover, the pro-drug of quercetin, QC12, has entered in phase-I clinical studies for certain human cancers [17, 35]. In the present study, however, quercetin was reported for the first time to exert growth inhibitory effect in ACC. MTT assay showed that

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Fig. 6 Activation of the function-related PI3K/Akt/IKK-a/NF-jB signaling pathway is essential for the apoptosis-evasion in ACC. a Clustering analyses in which the closeness of the columns directly indicates correlation. b, c Effect of LY294002 on ACC cell apoptosis

as well as the expression of apoptosis-related proteins and the activation of p-Akt, p-IKK-a, and NF-jB (p65). Data were expressed as means ± SEM of three independent experiments. ** P \ 0.01 versus control cells

quercetin decreased the viability in both ACC-2 and ACC-M cells lines in a concentration- and time-dependent manner. Consistently, apoptotic features were also significantly exhibited in quercetin-treated ACC cells as demonstrated by Hoechst 33258 staining, Cell Death Detection ELISAPLUS assay, cell cycle analysis, and Annexin V-FITC/PI double-labeling, confirming the cytotoxicity exerted by quercetin was mainly through apoptosis induction. Generally, apoptosis-related signaling is demonstrated to comprise two classical pathways. One is directly from death receptor ligation that triggers recruitment of the precursor form of caspase-8 to a death-inducing complex, while the other is initiated in mitochondria [36]. In the latter pathway, cytochrome c is released from mitochondria into the cytosol, followed by a binding to Apaf1 and ATP,

and then it activates caspase-9 and subsequently activates caspase-3, which consequently leads to the cleavage of the DNA repair associated nuclear enzyme PARP to form a 89 kDa fragment [36]. In the present study, we found that quercetin effectively attenuated DWm, released cytochrome c, activated caspase-3, and cleaved PARP in both ACC-2 and ACC-M cells. Also, the Bax/Bcl-2 ratio was significantly increased by quercetin treatment. All these data strongly suggest that quercetin induces apoptosis in ACC cells through a mitochondria-dependent pathway. It is commonly known that the transcription factor NF-jB plays a pivotal role in carcinogenesis of various cancers, serving as one of the key elements in apoptotic pathways, through regulation of its target gene products including survivin, Bcl-2, Bcl-xl, and IAP1/2 [37]. In addition, current studies have implicated that NF-jB is

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intensively associated with the malignant progression of ACC, and strategies specifically against NF-jB may mitigate the development of this aggressive neoplasm due to the influence on cell proliferation, apoptosis and invasion, as well as tumor-induced angiogenesis [12, 32, 33, 38]. Thus, it is reasonable for us to identify NF-jB as a target of quercetin for the mitochondria-dependent apoptosis induction in ACC. As expected, our results expressly showed that treatment with quercetin significantly up-regulated the expression of IjB-a and inhibited the nuclear translocation of NF-jB, suggesting that NF-jB is involved in the induction of mitochondria-dependent apoptosis in ACC cells by quercetin. Actually, quercetin has been previously reported to possess inhibitory effect on NF-jB activation [39], but the underlying mechanism remains poorly understood. PI3K, the direct upstream regulator of serine-threonine kinase Akt, is one of the most frequently mentioned targets in human cancers [40–45], including ACC [43–45]. In lots of studies, flavonoids have been reported to possess high antitumor potential involving blockade of PI3K/Akt signaling pathway [46]. Notably, a very recent research reported that quercetin, as one of the most common natural flavonoids, could also inhibit the activation of PI3K/Akt pathway through direct bounding to PI3K [47]. Previous studies have revealed that PI3K/Akt pathway-dependent activation of NF-jB, through a p38/IKKb-dependent or an IKKadependent mechanism, plays an important role in tumor progression including anti-apoptotic processes [41, 48–50]. However, the involvement of PI3K/Akt signaling pathway in NF-jB activation is a cell- and tissue-specific event [14, 15]. To obtain a better understanding of the molecular mechanisms by which quercetin functions as an anticancer agent, we try to assess the functional significance of PI3K/ Akt signaling pathway in quercetin-mediated inhibitory activities in ACC cells. The results initially showed that the quercetin-mediated inhibition of ACC is closely associated with suppression of PI3K/Akt pathway, based on the observations of down-regulated phosphorylation level of both PI3K (p85) and Akt (Ser473). Moreover, transient transfection of ACC cells with CA-Akt partially but significantly prevented quercetin-induced apoptosis, accompanied by obviously reversed inactivation of IKK-a, downregulation of NF-jB, cleavage of PARP, as well as increase in Bax/Bcl-2 ratio confirming the intensive involvement of an function-related PI3K/Akt/IKK-a/NFjB pathway. Thus, it might be concluded that the apoptosis induced by quercetin in ACC cells is due, at least in part, to the down-regulation of NF-jB through a PI3K/Akt/IKK-a signaling axis. Our in vivo studies further verified the in vitro effects of quercetin against ACC. Quercetin treatment effectively prevented the tumor growth of ACC-M cells in nude mouse

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xenograft model, associating with the induction of cell apoptosis in tumor tissues. Meanwhile, immunohistochemical analyses showed that quercetin significantly down-regulated the nuclear translocation of NF-jB, as well as the phosphorylation levels of IKK-a and Akt. These findings are consistent with the results in vitro. To make above findings more meaningful, we tried to explore the clinical significance of PI3K/Akt/IKK/NF-jB signaling pathway in ACC. Although the relatively high activities of Akt and NF-jB in ACC have been respectively reported by independent investigators in several different studies [10–13], they were never functionally related, and also the functional significance of activated Akt in ACC progression, especially in the steps of apoptosis-evasion, has not been reported yet. Meanwhile, as described above, the participance of PI3K/Akt signaling pathway in NF-jB activation is a cell-specific event. Thus, one major objective of this study is to determine if the activation of a function-related PI3K/Akt/IKK/NF-jB signaling pathway exists in ACC, and if this activated signaling axis is essential for the apoptosis-evasion of ACC. Immunohistochemical results showed that the activation status of PI3K, Akt, IKK-a and NF-jB p65 are significantly higher in ACC tissues when compared to NSG tissues. Furthermore, the double-labeling immunofluorescence histochemistry analysis also confirmed the concurrence of Akt activation and NF-jB nuclear translocation in ACC tissues. Most importantly, their activations are significantly correlated with each other and also with the low apoptotic index as demonstrated by the Spearman rank test and reflected by the cluster analysis. All these data expressly implicated the existence of a function-related PI3K/Akt/IKK/NF-jB signaling pathway as well as its essential role in the apoptosisevasion in ACC. To further validate the above conclusion, we then tested the effect of LY294002, a specific PI3K inhibitor, on the activation status of Akt, IKK and NF-jB, as well as the survival of ACC cells. As expected, LY294002 concurrently suppressed the phosphorylation of both Akt and IKK-a and inhibited the nuclear translocation of NF-jB p65. Moreover, LY294002 treatment also significantly increased the Bax/Bcl-2 ratio and PARP cleavage, and effectively induced the apoptosis of ACC cells as represented by the Cell Death Detection ELISAPLUS assay, which is a widely accepted technique for quantitation of apoptotic cell death. Thus, it seems to be a consequence that the function-related PI3K/Akt/IKK-a/NF-jB signaling pathway may be one of the most important molecular mechanisms which underlies the malignant progression especially the apoptosis-evasion of ACC. In summary, we reveal the existence of a functionrelated PI3K/Akt/IKK-a/NF-jB signaling pathway in ACC, which is ubiquitously activated and intensely correlated with the apoptosis-evasion. Furthermore, we also

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demonstrate here that quercetin can specifically target the PI3K/Akt/IKK-a/NF-jB pathway, and efficiently inhibits growth and induces apoptosis in ACC both in vitro and in vivo. We are tempted to speculate that quercetin would be a promising chemotherapeutic agent against ACC. Acknowledgments This work was supported by grants from National Natural Science Foundation of China (30600712) to Dr. Z.J. Sun and (30872894, 30973330) to Prof. Y.F. Zhao. We thank Prof. Ashok B. Kulkarni, Dr. Panomwat Amornphimoltham of National Institute of Dental and Crainofacial Research, and Dr. Yan-Song Bian of National Institute on Deafness and Other Communication Disorders for their critical reading and kind editing of the manuscript. Conflict of interest disclosed.

No potential conflicts of interest were

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