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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.41 No.1 pp.45-51
DOI : https://doi.org/10.11620/IJOB.2016.41.1.045

Rutin induces autophagy in cancer cells

Mi Hee Park1, Seyeon Kim1, Yu-ri Song1, Sumi Kim1, Hyung-Joon Kim2, Hee Sam Na1, Jin Chung1
1Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, Republic of Korea
2Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan, Korea
J. Chung, Department of Oral Microbiology,School of Dentistry, Pusan National University, Yangsan-si,Gyeongsangnam-do, 626-810, South Korea. E-mail: jchung@pusan.ac.kr ORCID: 0000-0002-6859-615X
February 20, 2016 February 26, 2016 February 27, 2016

Abstract

Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside)is a bioactive flavonoid from the plant kingdom. Rutin has been studied as potential anticancer agent due to its wide range of pharmacological properties including antioxidative, anti-inflammatory and anticancer. Autophagy is a conserved intracellular catabolic pathway to maintain cell homeostasis by formation of autophagosome. Processing of autophagy involves various molecules including ULK1 protein kinase complex, Beclin-1–Vps34 lipid kinase complex, ATG5, ATG12, and LC3 (light chain 3). Cargo-carried autophagosomes fuse with lysosomes resulting in autophagolysosome to eliminate vesicles and degrade cargo. However, the actions of rutin on autophagy are not clearly understood. In this study, we analyzed the effect of rutin on autophagy and inflammation in cancer cell lines. Interestingly, rutin induced autophagy in leukemia (THP-1), oral (CA9-22), and lung (A549) cell lines. TNF-α, key modulator of inflammation, was upregulated by inhibition of rutin-induced autophagy. Taken together, these data indicated that rutin induced autophagy and consequently suppressed TNF-α production.

Rutin , autophagy , cancer cell

초록

    Pusan National University
    International Journal of Oral Biology

    This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Autophagy is a conserved intracellular catabolic pathway toeliminate cytosolic debris, including damaged organelles, protein aggregates, and long-lived proteins, by formation of double-membrane vesicles, named autophagosome [1]. Furthermore, autophagy involves in the removal of invading microbes and immunological mediators [2]. Autophagosome fuses with lysosomes to deliver the contents for degradation. In the initiation step of the autophagy process, ULK1 protein kinase complex and Beclin-1–Vps34 lipid kinase complex are essential for induction of autophagy [3]. During elongation reaction, two ubiquitin-like conjugation systems in which one system (ATG12-5 conjugate) is formed by ATG5 (autophagyrelated 5) and ATG12 and the other (LC3-II) is formed by LC3 (light chain 3), which are required for the formation of the autophagosome. In the maturation step of autophagy, the autophagosomes fuse with lysosomes for breakdown of vesicle and degradation of cargo by formation autophagolysome [4]. Several reports showed that dysregulation of autophagy is involved in many human disease including cancer and inflammatory diseases [5]. Autohagy is considered as a tumor suppressor by regulating ROS production [6]. Furthermore, autophagy inhibits excessive inflammatory response [3, 7]

    Recently, research of natural compound such as flavonoid,preventing cancer and other chronic diseases such as chronic inflammation has dramatically increased. Intervention with these natural compounds is referred as chemoprevention and cancer chemoprevention is a main treatment method for cancer. Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) is an active ingredient and a flavonoid of the flavonol-type [8]. Rutin is present in many typical plants, such as curry leaf (Murraya koenigii), buckwheat seed, passion flower, apple, orange, grapefruit, lemon, and tea, and also known as quercetin-3- rutinoside or sophorin [9]. Rutin has a several pharmacological properties including antioxidative, antiallergic, anti-inflammatory and vasoactive, antitumor, antibacterial, antiviral, and antiprotozoal activity [8, 10]. In breast cancer MAD-MB-231 cells, rutin has bioactivity and anticancer activity [11]. Furthermore, rutin protects DNA damage from pro-carcinogens in HTC hepatic cells [12]. Several studies reported that rutin induced cell death in several cancer cells [13]. Although these studies shed some light on the anti-cancer actions of rutin via apoptosis, the exact targets and precise molecular mechanism of action of rutin on autophagy are poorly understood.

    The present study aimed to elucidate the mechanism ofanticancer effect of rutin on cancer cells and to determine the molecular mechanism of rutin-induced autophagy in cancer cells. We observed that rutin induced autophagy without regulation of cell growth in various cancer cell lines including THP-1, CA9-22 and A549 cells. Furthermore, in THP-1 and CA9-22 cells, rutin suppressed the secretion of pro-inflammatory cytokine, TNFα. These finding provide compelling evidence that rutin induced autophagy and consequently inhibited inflammatory response of cancer cells.

    Materials and Methods

    Cell lines and compounds

    THP-1, CA9-22 and A549 cells were cultured in RPMI1640 medium with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U of penicillin/ml, and 100 μg of streptomycin/ml at 37°C in a 5% CO2/95% air incubator. THP-1 cells were differentiated into macrophage-like cells with 50 ng/ml of Phorbol 12-mystristate 13-acetate (PMA; Sigma). PMA-differentiated THP-1-derived macrophages, CA9-22 and A549 cells were pretreated with 1 mM of 3-methyladenine (3-MA; sigma) or 1 μM rapamycin (sigma) for 30 min and treated with rutin (20 μM or 40 μM) for 24 h. Anti-ATC5 and anti-LC3 were from Cell Signaling Technology (Beverly, MA). Anti-BECN (Beclin-1), anti-p65, anti-β-actin and anti- GAPDH antibodies were from Santa Cruz.

    Western blotting

    Cells were lysed in RIPA buffer (Cell signalingTechnologies) and total cell lysates were analyzed by immunoblot. Enhanced chemiluminescence was used for signal detection and the signals were visualized using Super Signal West Femto maximum sensitivity substrate (Pierce, Rockford, USA) with LAS-4000 FUJIFILM Luminescent Image Analyzer.

    Cell growth Assay

    PMA-differentiated THP-1-derived macrophages, CA9-22 andA549 were seeded and refreshed with new medium containing rutin with or without 3-MA or rapamycin. After 24 h of incubation, the medium was removed and changed with serum-free medium containing 500 μg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) for 1 h. The MTT-containing medium was removed after 1 h of incubation. The incorporated dye was dissolved in 300 μl/well DMSO, and the plates were read at a wavelength of 570 nm using an ELISA reader. Absorbance in the treated cells was expressed as percentage of the control.

    Measurement of TNF-α

    To determine the amount of TNF-α released into theculture media after rutin treatment, we analyzed the amount in accordance with the manufacturer’s instructions using an ELISA kit (eBioscience, San Diego, CA, USA).

    Results

    Rutin induces autophagy in PMA-induced differentiated THP-1 cells

    To investigate the role of rutin in regulating autophagyinduction, PMA-induced differentiated THP-1 cells were treated with rutin at a various concentration and cell lysates were examined by immune blot analysis. Figure 1A showed that treatment with rutin induced the expression of Beclin1 and increased the formation of ATG5/12 conjugation as analyzed by Western blot. Furthermore, rutin increased the expression of LC3-II form (marker of autophagic activity). To confirm rutin-induced autophagy, we used 3-MA, specific inhibitor of autophagy. 3-MA inhibited rutin-induced autophagy markers, LC3-II, ATG5/12 and slightly Beclin1 (Figure 1B).

    Next, to examine whether rutin-induced autophagy isinvolved in cell growth, we performed MTT assay. Rutin did not affect cell viability and inhibition of rutin-induced autophagy by 3-MA also did not affect cell viability (Figure 1C and D). Taken together, these results suggest that the rutin induced autophagy without inhibition of cell growth.

    Rutin induces autophagy in oral cancer and lung cancer cell lines

    To investigate the role of rutin in regulating autophagyinduction in other cancer cell lines, CA9-22 (oral cancer cell line) and A549 (lung cancer cell lines) cells were treated with rutin at a various concentration and cell lysates were examined by immune blot analysis. Rutin induced autophagy in CA9-22 cells and A549 cells (Figure 2A and B). Consistent to Figure 1C, treatment of rutin had no effect on the viability of CA9-22 cells and A549cells (Figure 2C and D). Furthermore inhibition of rutin-induced autophagy by 3-MA also did not affect cell viability in CA9-22 and A549 cells (Figure 2E and F). These results suggest that rutin also induces autophagy in other cancer cell lines.

    Rutin-induced autophagy inhibits production of TNFα

    Since autophagy is critical for the regulation ofinflammatory response [14] including TNFα. We investigated whether rutin-induced autophagy suppressed inflammatory response. Treatment with 20 μM rutin inhibited TNFα production in PMA-induced differentiated THP-1 cells (Figure 3A). Moreover, 3-MA suppressed rutin-mediated inhibition of TNFα production compared with that in rutin-treated cells. In oral cancer cell CA9-22 cells, treatment of 40 μM rutin inhibited TNFα production and rutin-mediated decrease of TNFα production was inhibited by 3-MA (Figure 3C and D). Taken together, these results suggest that the TNFα production was downregulated by rutin in THP-1 and CA9-22 cells.

    Rutin inhibits NF-κB signaling

    Next, we investigated the molecular signal pathwayunderlying rutin-induced autophagy. NF-κB (p65) is critical transcriptional factor in regulating pro-inflammatiory signal. [15]. Furthermore, autophagy involves in inflammation and cytokine production [16]. In order to whether or not NF-κB is involved in rutin-induced autophagy, we examined effect of rutin on NF-κB activation using Western blot analysis. Rutin decreased the expression of NF-κB in whole cell lysates in PMA-induced differentiated THP-1 cells (Figure 4A) and CA9-22 cells (Figure 4B). We next examined that rutin-induced inhibition of NF-κB expression is related with induction of autophagy. Inhibition of autophagy by 3-MA recovered rutininduced suppression of NF-κB expression in PMA-induced differentiated THP-1 cells (Figure 4C). These results demonstrate that rutin-induced autophagy is involved in regulating of NF-κ B activation.

    Discussion

    In this study, we demonstrate that rutin induces autophagyin several cancer cell lines such as THP-1, CA9-22, and A549 without modulation of cell death and inhibits TNFα production. Furthermore, rutin suppressed the expression of NF-κB in THP-1and CA9-22 cells. Our studies reveal that rutin suppressed the TNFα production via inducing autophagy and inhibiting NF-κB signaling.

    Recently, epidemiological an dietary intervention studies inseveral animals model and cancer cell lines have suggest that diet-derived natural compounds such as flavonoids could have potential uses in the chemopreventive agents in cancer [17]. Rutin, a flavonoid of the flavonol-type, has been reported to exert a wide range of pharmacological properties, including antioxidant, antimicrobial, antifungal and antiallergic activity, that have been exploited in human medicine and nutrition [10]. Furthermore, rutin has multispectrum pharmacological benefit for treatment of various chronic disease, such as diabetes, hypertension, and cancer [8, 10]. Rutin has a DNA protective effects in HTC hepatic cells [12] and reduces tumor growth in mice xenografted with SW480 colon cancer cells [18]. Many studies reported that the protective effect of rutin targeting apoptosis an inflammation marker [19, 20, 21]. Although the underlying mechanism of rutin of governing these effects is actively studied, the precise effects and mechanism of autophagy by rutin has not been clearly understood.

    Autophagy is required for cell survival during nutrientstarvation, development and differentiation [22]. Furthermore autophagy is essential for degradation and recycle of cellular components and these autophagic cargos are surrounded by a double-membrane called autophagosome for maintain of homeostasis [2]. Autophagy is tightly regulated by various signaling molecules and autophagic components including Beclin1 (ATG6), ATG5, ATG12, and LC3. In some cases, cancer is initiated by suppression of autophagy. Loss of function of the essential autophagy gene BECL1(ATG6) is associated with human breast, ovarian, and prostate cancer [23].

    At least in some cases, tumor progression is stimulated byan inflammatory microenvironment, which contains high concentration of potentially genotoxic ROS as well as various mitogenic cytokines [24]. In addition, Growing evidence reveals that autophagy modulates inflammatory response. Atutophagy regulates IL-1β expression via degradation and modulates IL-1 family production [25, 26]. We previously reported that inhibition of P. gingivalis-induced autophagy decreased IL-1β secretion by modulating NLRP3 and AIM2 inflammasome [27]. Furthermore, autophagy induces ubiquitination of AIM2 and NLRP3 inflammasome to limit excess production of IL-1β [28]. Thus, identification of natural agents modulating autophagy and inflammation would be an important advance for cancer therapy. Until now, the precise effect of rutin on involvement in autophagy and inflammation in various cancer cell lines has not been clearly understood. Thus, we examined effect of rutin on autophagy and inflammatory response. Here, we showed first time that rutin induced autophagy in human leukemia (THP-1), oral (CA9-22), and lung (A549) cancer cell lines. Moreover, inhibition of rutin-induced autophagy increased TNFα production. Further study is needed to reveal the precise mechanism. Although the underlying mechanisms governing these effects are not yet fully understood, the available evidence collectively indicates that rutin may be of therapeutic benefit in clinical settings, suggesting its potential use as an anticancer agent or an adjunct to current cancer therapies.

    In conclusion, we have identified rutin targets for bothautophagy and inflammation. Our data demonstrate that rutin induced autophagy of cancer cells such as THP-1, CA9-22, and A549cells, but did not affective to the cell viability of cancer cells. Moreover, inhibition of rutin-induced autophagy resulted in increased of TNFα production. Rutin-induced autophagy is associated with NF-κB. The ability of rutin to induce autophagy and suppress the production of TNFα that acts as a regulator of tumorigensis, suggests that rutin could be an attractive anticancer drug.

    Acknowledgements

    This work was supported by a 2-Year Research Grant of Pusan National University.

    Figure

    327_F1.jpg

    Rutin induces autophagy in THP-1-derived macrophages (A) THP-1-derived macrophages were treated with the indicated concentrations of rutin for 24 h. The cell lysates were subjected to Western blot using anti-LC3, anti-Beclin1 and anti-ATG5 antibodies. GAPDH was used as loading control. (B) THP-1-derived macrophages were pretreated with 1 mM of 3-MA for 30 min prior to treatment with 20 μM rutin for 24 h. The cell lysates were subjected to Western blot using anti-LC3, anti-Beclin1 and anti-ATG5 antibodies. (C and D) U87 cells were treated with the indicated concentrations of rutin for 24 h or pretreated with 1 mM of3-MA for 30 min prior to treatment with 20 μM rutin for 24 h. The cell viability was then measured using the MTT assay.

    327_F2.jpg

    Rutin induces autophagy in CA9-22 cells and A549 cells (A and B) CA9-22 cells and A549 cells were treated with the indicated concentrations of rutin for 24 h. The cell lysates were subjected to Western blot using anti-LC3 and anti-Beclin1 antibodies. GAPDH was used as loading control. (C and D) CA9-22 cells and A549 cells were treated with the indicated concentrations of rutin for 24 h. The cell viability was then measured using the MTT assay. (E and F) CA9-22 cells and A549 cells were pretreated with 1 mM of 3-MA for 30 min prior to treatment with 40 μM rutin for 24 h. The cell viability was then measured using the MTT assay.

    327_F3.jpg

    Rutin-induced autophagy suppresses production of TNFα (A) THP-1-derived macrophages were treated with the indicated concentrations of rutin for 24 h. Cell culture supernatant were assayed for human TNFα using ELISA (B) THP-1-derived macrophages were pretreated with 1 mM of 3-MA for 30 min prior to treatment with 20 μM rutin for 24 h. Cell culture supernatant were assayed for human TNFα using ELISA. (C) CA9-22 cells were treated with the indicated concentrations of rutin for 24 h. Cell culture supernatant were assayed for human TNFα using ELISA. (D) CA9-22 cells were pretreated with 1 mM of 3-MA for 30 min prior to treatment with 40 μM rutin for 24 h. Cell culture supernatant were assayed for human TNFα using ELISA

    327_F4.jpg

    Rutin regulates NF-κB expression (A) THP-1-derived macrophages were treated with the indicated concentrations of rutin for 24 h. The cell lysates were subjected to Western blot using anti-p65 antibody. GAPDH was used as loading control. (B) CA9-22 cells were treated with the indicated concentrations of rutin for 24 h. The cell lysates were subjected to Western blot using anti-p65 antibody. (C) THP-1-derived macrophages were pretreated with 1 mM of 3-MA for 30 min prior to treatment with 20 μM rutin for 24 h. The cell lysates were subjected to Western blot using anti-p65 antibody.

    Table

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