Mol Cell Biochem DOI 10.1007/s11010-014-2317-7

Quercetin inhibits proliferation and invasion acts by up-regulating miR-146a in human breast cancer cells Si-feng Tao • Hai-fei He • Qiang Chen

Received: 22 June 2014 / Accepted: 23 December 2014 Ó Springer Science+Business Media New York 2015

Abstract Breast cancer is the most common female malignancies in the world which seriously impacts the female health. In recent years, various studies have been reported to determine the relevance of miRNAs to human cancer. One of these miRNAs, miR-146a has been downregulated in multiple human cancer types, but up-regulation showed inducing apoptosis. To determine the role of quercetin treated on breast cancer, we investigated the effect of quercetin on cell proliferation in human breast cancer cell lines MCF-7 and MDA-MB-231 with/without transfection of miR-146a mimic or anti-miR-146a. Furthermore, the expressions of bax and cleaved-caspase-3, mainly were increased in control and overexpression miR-146a groups, however, the expression of EGFR was inverse. All the results demonstrated that quercetin exhibited excellent effect on inhibiting cell proliferation in human breast cancer cells, which was performed by up-regulating miR-146a expression, then via inducing apoptosis through caspase-3 activation and mitochondrial-dependent pathways, and inhibiting invasion through down-regulating the expression of EGFR. Keywords Quercetin  miRNAs  miR-146a  EGFR  Breast cancer

Introduction Breast cancer is a malignant breast neoplasm originating from breast tissues, which is the most common female

S. Tao (&)  H. He  Q. Chen Department of Surgical Oncology, School of Medicine, Second Affiliated Hospital, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China e-mail: [email protected]

malignancy in the world. The incidence rate of breast cancer is about 10.4 %, which ranks first among all cancers in women with increases at an annual rate of 1–2 %. According to the World Health Organization, about 1.3 million women are diagnosed with breast cancer each year [1–3]. Due to the high morbidity and high mortality of breast cancer in recent years, the development of an effective method of treatment is urgently required. Quercetin (3,30 ,40 ,5,7-pentahydroxyflavone), easily extracted, isolated, and detected, is shown to be abundant in various plants such as fruits, vegetables, seeds, nuts, olive oil, tea, and red wine, thus it is widely present in human diet [2, 4, 5]. Numerous studies have shown that the quercetin is capable of inducing cytotoxic effects including inhibition of cell proliferation and apoptosis in various cancer cells [1, 4–7]. For example, the quercetin induced apoptosis through induction of Bax with concomitant inhibition of Bcl-2 in human breast cancer cells [2], and mitochondrial- and caspase-3-dependent pathways in human breast cancer MDA-MB-231 cells [4]. These diverse anti-tumor activities of quercetin make it a new agent with effective cancer prevention or therapy. MicroRNAs (miRNAs) are an abundant class of 18–25 nucleotides evolutionarily highly conserved single-stranded non-coding small RNAs [8, 9], which regulate gene expression by suppressing mRNA translation or cleaving target mRNAs to induce their degradation by binding to the 30 -UTR of target mRNAs [3, 10]. To date, more than 1,424 human miRNAs have been identified, and they are believed to collectively regulate the translation of more than 60 % of protein coding genes [11], and about 50 % of miRNA genes are located in tumor-related genes or fragile sites, suggesting that the miRNAs maybe connected with the human cancer [9]. Recent studies indicated that miRNAs play important roles in a wide variety of biological processes, involving cell

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proliferation, differentiation, growth, apoptosis, and stress resistance [9, 12–14], even took part in human carcinogenesis as tumor suppressors or oncogenes through regulating the expression of their target genes [9, 15–18]. The aberrant expressions of miRNAs have been observed in various cancer types, including breast [8, 10], kidney [19], lung [20], colon [21], gastric [22], and prostate [23] cancers. Since miRNAs play important roles in tumorigenesis and cancer progression, miRNAs may be a potential target for cancer treatment. Quercetin, a flavone with effective cancer prevention and therapy, has been reported to regulate the expression of miRNAs, for example, quercetin could inhibit cell growth through inhibition of miR-27a in renal cancer cells [24] and in colon cancer cells [25]. The epidermal growth factor receptor (EGFR) is a member of transmembrane tyrosine kinase receptor. It has been demonstrated that EGFR was overexpressed in many cancers, including breast cancer [26]. Breast cancer patients with EGFR up-regulation have poor prognosis [27]. It has been reported that miR-24 can enhance tumor growth, local invasion, and metastasis by activating EGFR phosphorylation [27]. By now, EGFR has been one of the most successful rational agent targets in clinical cancer therapy [28], thus we investigated the expression of EGFR after treatment with quercetin and the relationship of miR146a and EGFR. Decrease of miR-146a expression has been reported in multiple human cancer types, involving prostate [23], lung [20, 29], gastric [30], and breast [31] cancers. As a result, we evaluated the up-regulation effect of quercetin on miR146a expression in MCF-7 and MDA-MB-231 human breast cancer calls and human breast cancer xenograft model.

Materials and methods Cell culture The human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Cell Resource Center (Shanghai, China). The medium and serum for the cell cultures were used as the American type culture collection (ATCC) recommended. MTT assay. MCF-7 and MDA-MB-231 cells dispersed in culture medium were seeded in 96-well plates with a density of 2 9 105 cells/well, then treated with 0, 25, 50, 80, and 100 lm/mL quercetin and incubated for 48 h. MTT was added to each well, and the plate was incubated at 37 °C for 4 h, then the DMSO was added to dissolve the crystal and placed on a shaker for 10 min. The OD value was measured with a microplate reader at 490 nm.

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Measurement of miR-146a expression Total RNA was isolated using a miRNeasy kit (Qiagen Inc., Valencia, CA, USA), and the content and purity was measured using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The miR-146a level was measured by real-time PCR using TaqMan miRNA assays (Ambion) according to the manufacturer’s instructions of Reverse Transcription Kit and TaqMan MicroRNA Assays Kit (Applied Biosystems, Carlsbad, CA, USA), and with U6 snRNA, a small nuclear RNA (snRNA) as endogenous control for miR-146a analysis. Relative expression was calculated by the comparative threshold cycle method. RNA oligonucleotide and cell transfection The mir-146a expression vector pSIF-GFP-miR-146a was derived from pSIF-GFP vector (System Biosciences). miR-146a mimics and anti-miR-146a were purchased from Ambion. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For transfection of the RNA oligonucleotide, 100 nmol/L miRNA mimics or antisense oligonucleotides were used. The transfection efficiency was estimated to be *80 % for MCF-7 and MDA-MB-231 cells using Cy3 dye-labeled RNA oligonucleotides (Ambion). For plasmid, 4 lg DNA was used in a six-well plate. G418 (200 lg/mL) was added 24 h after transfection. Xenograft assays in nude mice 6- to 8-week-old female BALB/c athymic nude mouse were purchased from Experiment Animal Center of Nanjing Medical University (Nanjing, China) and maintained in cage housing in a specifically designed pathogen-free isolation facility with a 12/24-h light/dark cycle. Twenty-four hours after transfection of RNA oligonucleotide and/or plasmid DNA, *2 9 106 cells were suspended in 100 lL PBS and then injected orthotopically into the third mammary gland on either side of the mouse. Six mice were included in one experimental group. The tumor volumes were calculated according to a standard formula: p 9 length 9 width2/6 [32]. The mice were sacrificed at the end of the experiment, and the tumors were removed and weighed. Western blot Cells were washed in PBS and lysed in 1 9 SDS loading buffer. Then the lysates were clarified by centrifugation (15, 0009g, 10 min, 4 °C) and collected. 20–50 lg of protein were fractionated on SDS-PAGE gels and electroblotted onto PVDF membrane (Cell Signaling Technology, Beverly, MA, USA). Antibodies were used against bax, cleaved-caspase-3, and b-actin (Cell Signaling Technology, Beverly, MA, USA).

Mol Cell Biochem

Proteins were detected using HRP-conjugated secondary antibodies followed by enhanced chemiluminescence (ECL). All antibodies were used at a dilution of 1:2,000. Blots were then exposed to radiographic film to visualize immunoreactive signals. Signals were quantified using Multi Gauge Image Analysis software (FujiFilm, Japan). Statistical analysis All the data are expressed as mean ± SEM, and AVOVA analysis was performed. Statistical analyses were performed using SPSS Version 11.0 (Chicago, IL, USA) with statistical significance set at P B 0.05.

Results Growth-inhibitory effects of quercetin on MCF-7 and MDA-MB-231 cells The effects of quercetin on the growth of human breast cancer cell line MCF-7 and MDA-MB-231 cells were evaluated by MTT assay. As indicated in Fig. 1, all doses of quercetin significantly inhibited the growth of MCF-7 and MDA-MB-231 cells, and the inhibition displayed both dose-and time-dependent relationships with quercetin treatment. In addition, the IC50 of quercetin was 80 lm/mL for MCF-7 cells and 50 lm/mL for MDA-MB-231 cells, respectively. Quercetin can inhibit cell growth via up-regulation of miR-146a expression To further identify the mechanism of growth inhibition effects of quercetin on MCF-7 and MDA-MB-231 cells, we

Fig. 1 Inhibitory effect of quercetin on the growth of human breast cancer cell lines MCF-7 and MDA-MB-231 in a dose- and timedependent manner. a Inhibitory effect of quercetin on the growth of MCF-7 human breast cancer cells. b Inhibitory effect of quercetin on

performed real-time PCR to detect miR-146a expression after treatment with four doses of quercetin for 48 h. As shown in Fig. 2a, the levels of the miR-146a expression in MCF-7 and MDA-MB-231 were significantly increased after treatment with quercetin for 48 h in a dose-dependent manner. To determine whether quercetin inhibits cell growth through regulating the expression of miR-146a in human breast cancer cell lines MCF-7 and MDA-MB-231, we investigated the cell proliferation in cells transfected miR146a mimic by MTT assay. As shown in Fig. 2b, c, transfection of miR-146a mimic significantly inhibited the growth of both cell lines after treatment with quercetin for 48 h at dose of 80 lm/mL. Instead, there was no significant change after transfecting with anti-miR-146a. In addition, overexpression of miR-146a enhanced the proliferation inhibition of quercetin. Quercetin stimulates the expression of bax and caspase3 and inhibits the expression of EGFR via up-regulation of the miR-146a expression As indicated in Fig. 3, the expressions of bax and cleavedcaspase-3 were increased after transfection with miR-146a mimics but had no change with the transfection of anti-miR146a after treatment with quercetin for 24 h at dose of 80 lm/mL. Meanwhile, induction of miR-146a expression could enhance this effect. Metastasis is the primary cause of breast cancer mortality, and EGFR plays an important role in tumor invasion and metastasis, thus we detected the expression of EGFR. As indicated in Fig. 4, the up-regulation of the EGFR expression treated with quercetin was enhanced after transfecting with miR-146a mimics, but had no change with the transfection of anti-miR-146a.

the growth of MDA-MB-231 human breast cancer cells. *P \ 0.05, **P \ 0.01, ***P \ 0.001 indicate significant differences from the respective control groups

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Fig. 2 Quercetin can inhibit cell growth via up-regulation of miR146a expression. a Quercetin induced the expression of miR-146a in human breast cancer cell lines MCF-7 and MDA-MB-231. b Upregulation of miR-146a expression can inhibit cell growth in MCF-7

human breast cancer cells. c Up-regulation of miR-146a expression can inhibit cell growth in MDA-MB-231 human breast cancer cells. *P \ 0.05, **P \ 0.01, ***P \ 0.001 indicate significant differences from the respective control groups

Tumor growth inhibition effect and miR-146a expression in nude mouse

in many human cancer cell lines [6, 7, 33]. Our results showed that quercetin inhibited cell proliferation in human breast cancer cells in a dose- and time-dependent manner, which is in agreement with other reports showing that quercetin inhibits the cell growth in human breast cancer cells [1, 4–6]. Simultaneously, the tumor growth was suppressed and the miR-146a expression was up-regulated in orthotopic xenograft nude model by treating with quercetin. Cao et al., fund that quercetin could inhibit tumor growth in melanoma xenografted mice model through inhibiting STAT3 signaling [34], and the study of Du et al. showed that quercetin suppressed tumor growth by suppressing intratumoral HIF-1a in BALB/c mice bearing 4T1 breast cancer [35]. Our study indicated that quercetin induced cell apoptosis probability through up-regulating miR-146a expression, which was confirmed by cell transfection experiments. The results showed that the inhibition of cell growth by treating with quercetin was enhanced after

To evaluate the anti-tumor effects of quercetin, we performed an in vivo study using a nude mouse orthotopic xenograft model. As shown in Fig. 5a, tumor volume was affected by quercetin compared with tumors in control mice. Figure 5b shows that the expression of miR-146a was significantly enhanced after treatment with quercetin for 8 weeks.

Discussion Breast cancer is the most common female malignancies in the world and it has a serious impact on female health [1]. Quercetin, a bioactive plant flavonoid widely distributed in a various fruits, vegetables, and plants, has been reported to effectively inhibit cell proliferation and cycle progression

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Mol Cell Biochem Fig. 3 Quercetin stimulates the expressions of bax and cleavedcaspase-3 via up-regulation of the miR-146a expression. a Quercetin stimulates the expressions of bax and cleavedcaspase-3 via up-regulation of the miR-146a expression in MCF-7 human breast cancer cells. b Quercetin stimulates the expressions of bax and cleavedcaspase-3 via up-regulation of the miR-146a expression in MDA-MB-231 human breast cancer cells

Fig. 4 Quercetin inhibits the expression of EGFR via upregulation of the miR-146a expression. a Quercetin inhibits the expression of EGFR via upregulation of the miR-146a expression in MCF-7 human breast cancer cells. b Quercetin inhibits the expression of EGFR via up-regulation of the miR-146a expression in MDA-MB-231 human breast cancer cells

transfecting with miR-146a mimic, but reversed by antimiR-146a. MicroRNAs (miRNAs) aberrant expression is involved in many human diseases, including cancer. In recent years, various studies including case–control researches and meta-analyses have been reported to determine the relevance of miRNAs with human cancer [3, 6, 8, 9, 20, 21, 36]. One of these miRNAs, miR-146a has been down-

regulated in multiple human cancer types [20, 22, 23, 37], accordingly, up-regulation of miR-146a was showed to induce apoptosis [30, 38]. Taken together, miR-146a may function as a tumor-suppressor gene in human cancer. It was also reported that quercetin could inhibit cell growth through inhibition of miR-27a in renal cancer cells [24] and in colon cancer cells [25]. Taken together, the results suggest that quercetin induces apoptosis through up-

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Fig. 5 a Tumor growth inhibition effect of quercetin on nude mouse orthotopic xenograft model. b miR-146a expression in nude mouse. **P \ 0.01 indicate significant differences from the respective control groups

family is known to promote cell proliferation, migration, invasion, and angiogenesis [40]. The expression of EGFR can be regulated by different miRNAs, which is involved in the development of cancer. For example, it has been reported that miR-24 could enhance tumor growth, local invasion, and metastasis by activating EGFR phosphorylation [27]. However, miR-146a can inhibit the invasion of pancreatic cancer cells, hepatocellular carcinoma cells, prostate cancer, and non-small cell lung cancer cells via suppressing EGFR pathway [41–44]. Furthermore, only one study revealed that miR-146a repressed the expression of EGFR through binding to its 30 -untranslated region in castration-resistant prostate cancer. And then the exact mechanism of how miR-146a regulated EGFR needs more studies. In our data, the induction of miR-146a expression enhanced the effect of quercetin on down-regulating the expression of EGFR. In conclusion, quercetin exhibited excellent effect on inhibiting cell proliferation in human breast cancer cells, which was performed by up-regulating miR-146a expression, and the miR-146a induced apoptosis through cleavedcaspase-3 activation and mitochondrial-dependent pathways, and inhibited invasion through down-regulating the expression of EGFR. However, the mechanism of quercetin modulating miR-146a needs further clarification.

Disclosure

regulating miR-146a expression in human breast cancer cell lines MCF-7 and MDA-MB-231. Furthermore, miR146a induced apoptosis via inhibiting the expressions of IRAK1 and TRAF-6 which are the upstream molecules of NF-jB [22, 23, 30, 31], thus, the inhibition of NF-jB activity followed by up-regulation of miR-146a may be associated with the induction of quercetin on apoptosis. Apoptosis is a systematically regulated process involving the expression of many gene products. One of the major genes regulating apoptosis, the pro-apoptosis Bax is of particular interest; Bax protein translocates to mitochondria upon exposure to stimuli of apoptosis, and induces the release of cytochrome c and activates the caspase [39]. Caspase-3 belongs to the family of cysteine aspartyl proteases (caspase) whose activation is required during apoptosis and the cleaved-caspase-3 is the activated caspase-3 which a better indicator of apoptosis [4, 39]. It has been reported that quercetin induced apoptosis through activating caspase-3 and Bax [2, 6]. As showed in our results, the induction of miR-146a expression enhanced the effect of quercetin on increasing the expressions of bax and cleaved-caspase-3. EGFR is one of the excellent rational drug targets in clinical cancer therapy, including breast cancer [28]. EGFR

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The authors have nothing to disclose.

References 1. Deng XH, Song HY, Zhou YF, Yuan GY, Zheng FJ (2013) Effects of quercetin on the proliferation of breast cancer cells and expression of survivin in vitro. Exp Ther Med 6:1155–1158 2. Duo J, Ying GG, Wang GW, Zhang L (2012) Quercetin inhibits human breast cancer cell proliferation and induces apoptosis via Bcl-2 and Bax regulation. Mol Med Rep 5:1453–1456 3. Vimalraj S, Miranda PJ, Ramyakrishna B, Selvamurugan N (2013) Regulation of breast cancer and bone metastasis by microRNAs. Dis Markers 35:369–387 4. Chien SY, Wu YC, Chung JG, Yang JS, Lu HF, Tsou MF, Wood WG, Kuo SJ, Chen DR (2009) Quercetin-induced apoptosis acts through mitochondrial- and caspase-3-dependent pathways in human breast cancer MDA-MB-231 cells. Hum Exp Toxicol 28:493–503 5. Zhang H, Zhang M, Yu L, Zhao Y, He N, Yang X (2012) Antitumor activities of quercetin and quercetin-50 ,8-disulfonate in human colon and breast cancer cell lines. Food Chem Toxicol 50:1589–1599 6. Chou CC, Yang JS, Lu HF, Ip SW, Lo C, Wu CC, Lin JP, Tang NY, Chung JG, Chou MJ, Teng YH, Chen DR (2010) Quercetinmediated cell cycle arrest and apoptosis involving activation of a caspase cascade through the mitochondrial pathway in human breast cancer MCF-7 cells. Arch Pharm Res 33:1181–1191 7. Lee YK, Park OJ (2010) Regulation of mutual inhibitory activities between AMPK and Akt with quercetin in MCF-7 breast cancer cells. Oncol Rep 24:1493–1497

Mol Cell Biochem 8. Iorio MV, Ferracin M, Liu GG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Me´nard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65:7065–7070 9. Li YJ, Zhang ZY, Mao YY, Jin MJ, Jing FY, Ye ZH, Chen K (2014) A genetic variant in MiR-146a modifies digestive system cancer risk: a meta-analysis. Asian Pac J Cancer Prev 15:145–150 10. Luo D, Wilson JM, Harvel N, Liu J, Pei L, Huang S, Hawthorn L, Shi H (2013) A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells. J Transl Med 11:57 11. Elsarraj HS, Stecklein SR, Valdez K, Behbod F (2012) Emerging functions of microRNA-146a/b in development and breast cancer: microRNA-146a/b in development and breast cancer. J Mammary Gland Biol Neoplasia 17:79–87 12. Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11:597–610 13. Papagiannakopoulos T, Kosik KS (2008) MicroRNAs: regulators of oncogenesis and stemness. BMC Med 6:16 14. Winter J, Diederichs S (2011) MicroRNA biogenesis and cancer. Methods Mol Biol 676:3–22 15. Iorio MV, Croce CM (2009) MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 27:5848–5856 16. Kwak PB, Iwasaki S, Tomari Y (2010) The microRNA pathway and cancer. Cancer Sci 101:2309–2315 17. Ruan K, Fang X, Ouyang G (2009) MicroRNAs: novel regulators in the hallmarks of human cancer. Cancer Lett 285:116–126 18. Sumazin P, Yang X, Chiu HS, Chung WJ, Iyer A, Llobet-Navas D, Rajbhandari P, Bansal M, Guarnieri P, Silva J, Califano A (2011) An extensive microRNA-mediated network of RNA-RNA interactions regulates established oncogenic pathways in glioblastoma. Cell 147:370–381 19. Majid S, Saini S, Dar AA, Hirata H, Shahryari V, Tanaka Y, Yamamura S, Ueno K, Zaman MS, Singh K, Chang I, Deng G, Dahiya R (2011) MicroRNA-205 inhibits Src-mediated oncogenic pathways in renal cancer. Cancer Res 71:2611–2621 20. Wu C, Cao Y, He Z, He J, Hu C, Duan H, Jiang J (2014) Serum levels of miR-19b and miR-146a as prognostic biomarkers for non-small cell lung cancer. Tohoku J Exp Med 232:85–95 21. Du W, Ma XL, Zhao C, Liu T, Du YL, Kong WQ, Wei BL, Yu JY, Li YY, Huang JW, Li ZK, Liu L (2014) Associations of single nucleotide polymorphisms in miR-146a, miR-196a, miR149 and miR-499 with colorectal cancer susceptibility. Asian Pac J Cancer Prev 15:1047–1055 22. Crone SG, Jacobsen A, Federspiel B, Bardram L, Krogh A, Lund AH, Friis-Hansen L (2012) microRNA-146a inhibits G proteincoupled receptor-mediated activation of NF-jB by targeting CARD10 and COPS8 in gastric cancer. Mol Cancer Ther 11:71 23. Xu B, Wang N, Wang X, Tong N, Shao N, Tao J, Li P, Niu X, Feng N, Zhang L, Hua L, Wang Z, Chen M (2012) MiR-146a suppresses tumor growth and progression by targeting EGFR pathway and in a p-ERK-dependent manner in castration-resistant prostate cancer. Prostate 72:1171–1178 24. Li W, Liu M, Xu YF, Feng Y, Che JP, Wang GC, Zheng JH (2014) Combination of quercetin and hyperoside has anticancer effects on renal cancer cells through inhibition of oncogenic microRNA-27a. Oncol Rep 31:117–124 25. Del Follo-Martinez A, Banerjee N, Li X, Safe S, Mertens-Talcott S (2013) Resveratrol and quercetin in combination have anticancer activity in colon cancer cells and repress oncogenic microRNA-27a. Nutr Cancer 65:494–504 26. Huang L, Wong CC, Mackenzie GC, Sun Y, Cheng KW, Vrankova K, Alston N, Ouyang N, Rigas B (2014) Phospho-

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

aspirin (MDC-22) inhibits breast cancer in preclinical animal models: an effect mediated by EGFR inhibition, p53 acetylation and oxidative stress. BMC Cancer 14:141 Du WW, Fang L, Li M, Yang X, Liang Y, Peng C, Qian W, O’Malley YQ, Askeland RW, Sugg SL, Qian J, Lin J, Jiang Z, Yee AJ, Sefton M, Deng Z, Shan SW, Wang CH, Yang BB (2013) MicroRNA miR-24 enhances tumor invasion and metastasis by targeting PTPN9 and PTPRF to promote EGF signaling. J Cell Sci 126:1440–1453 Flynn JF, Wong C, Wu JM (2009) Anti-EGFR therapy: mechanism and advances in clinical efficacy in breast cancer. J Oncol 2009:526963 Chen G, Umelo A, Lv S, Teugels E, Fostier K, Kronenberger P, Dewaele A, Sadones J, Geers C, Gre`ve JD (2013) miR-146a inhibits cell growth, cell migration and induces apoptosis in nonsmall cell lung cancer cells. PLoS One 8:e60317 Sha M, Ye J, Zhang LX, Luan ZY, Chen YB (2013) Celastrol induces apoptosis of gastric cancer cells by miR-146a inhibition of NF-jB activity. Cancer Cell Int 13:50 Bhaumik D, Scott GK, Schokrpur S, Patil CK, Campisi J, Benz CC (2008) Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells. Oncogene 27:5643–5647 Jiang S, Zhang HW, Lu MH, He XH, Li Y, Gu H, Liu MF, Wang ED (2010) MicroRNA-155 functions as an OncomiR in breast cancer by targeting the suppressor of cytokine signaling 1 gene. Cancer Res 70:3119–3127 Kim MC (2014) Quercetin induces apoptosis by inhibiting MAPKs and TRPM7 channels in AGS cells. Int J Mol Med 33:1657–1663 Cao HH, Tse AK, Kwan HY, Yu H, Cheng CY, Su T, Fong WF, Yu ZL (2014) Quercetin exerts anti-melanoma activities and inhibits STAT3 signaling. Biochem Pharmacol 87:424–434 Du G, Lin H, Wang M, Zhang S, Wu X, Lu L, Ji L, Yu L (2010) Quercetin greatly improved therapeutic index of doxorubicin against 4T1 breast cancer by its opposing effects on HIF-1a in tumor and normal cells. Cancer Chemother Pharmacol 65:277–287 Catucci I, Yang R, Verderio P, Pizzamiglio S, Heesen L, Hemminki K, Sutter C, Wappenschmidt B, Dick M, Arnold N, Bugert P, Niederacher D, Meindl A, Schmutzler RK, Bartram CC, Ficarazzi F, Tizzoni L, Zaffaroni D, Manoukian S, Barile M, Pierotti MA, Radice P, Burwinkel B, Peterlongo P (2010) Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as lowpenetrance alleles in German and Italian familial breast cancer cases. Hum Mutat 31:1052–1057 Li X, Xu B, Moran MS, Zhao Y, Su P, Haffty BG, Shao C, Yang Q (2012) 53BP1 functions as a tumor suppressor in breast cancer via the inhibition of NF-jB through miR-146a. Carcinogenesis 33:2593–2600 Xiao B, Zhu ED, Li N, Lu DS, Li W, Li BS, Zhao YL, Mao XH, Guo G, Yu PW, Zou QM (2012) Increased miR-146a in gastric cancer directly targets SMAD4 and is involved in modulating cell proliferation and apoptosis. Oncol Rep 27:559–566 Shin Y, Lee Y (2013) Cytotoxic activity from Curcuma zedoaria through mitochondrial activation on ovarian cancer cells. Toxicol Res 29:257–261 Mu Z, Klinowska T, Dong X, Foster E, Womack C, Fernandez SV, Cristofanilli M (2014) AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor (EGFR), HER2, and HER3: preclinical activity in HER2 nonamplified inflammatory breast cancer models. J Exp Clin Cancer Res 33:47 Li Y, VandenBoom TG, Wang Z, Kong D, Ali S, Philip PA, Sarkar FH (2010) Up-regulation of miR-146a contributes to the inhibition of invasion of pancreatic cancer cells. Cancer Res 70(8 Suppl):5703

123

Mol Cell Biochem 42. Xu B, Wang N, Wang X, Zhang L, Hua L, Wang Z, Chen M (2012) MiR-146a suppresses tumor growth and progression by targeting EGFR pathway and in a p-ERK-dependent manner in castration-resistant prostate cancer. Prostate 72(11):1171–1178 43. Chen G, Umelo IA, Lv S, Geers C, De Gre`ve J (2013) miR-146a inhibits cell growth, cell migration and induces apoptosis in nonsmall cell lung cancer cells. PLoS One 8(3):e60317

123

44. Huang S, He R, Rong M, Dang Y, Chen G (2014) Synergistic effect of MiR-146a mimic and cetuximab on hepatocellular carcinoma cells. Biomed Res Int 2014:384121