Rutin induces autophagy in cancer cells

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http://dx.doi.org/10.11620/IJOB.2016.41.1.045 pISSN 1226-7155, eISSN 2287-6618

Rutin induces autophagy in cancer cells

Mi Hee Park

¹

, Seyeon Kim

¹

, Yu-ri Song

¹

, Sumi Kim

¹

, Hyung-Joon Kim

²

, Hee Sam Na

¹

and Jin Chung

^1*

1

Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, Republic of Korea

2

Department of Oral Physiology, School of Dentistry, Pusan National University, Yangsan, Korea (received February 20, 2016; revised February 26, 2016; accepted February 27, 2016)

*Correspondence to: J. Chung, Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan-si, Gyeongsangnam-do, 626-810, South Korea.

E-mail: [email protected] ORCID: 0000-0002-6859-615X

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

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.

Key words: Rutin, autophagy, cancer cell

Introduction

Autophagy is a conserved intracellular catabolic pathway to eliminate 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 (autophagy- related 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

45

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46 Mi Hee Park, Seyeon Kim, Yu-ri Song, Sumi Kim, Hyung-Joon Kim, Hee Sam Na and Jin Chung

(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 of anticancer 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 RPMI 1640 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% CO

2

/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 signaling Technologies) 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 and A549 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 the culture 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 autophagy

induction, 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

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Figure 1. 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 of 3-MA for 30 min prior to treatment with 20 μM rutin for 24 h. The cell viability was then measured using the MTT assay.

(Figure 1B).

Next, to examine whether rutin-induced autophagy is involved 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 autophagy induction 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 of

inflammatory 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

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48 Mi Hee Park, Seyeon Kim, Yu-ri Song, Sumi Kim, Hyung-Joon Kim, Hee Sam Na and Jin Chung

Figure 2. 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.

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 pathway underlying rutin-induced autophagy. NF-κB (p65) is critical transcriptional factor in regulating pro-inflammatiory signal.

[15]. Furthermore, autophagy involves in inflammation and

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Figure 3. 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

Figure 4. 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.

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

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50 Mi Hee Park, Seyeon Kim, Yu-ri Song, Sumi Kim, Hyung-Joon Kim, Hee Sam Na and Jin Chung

inhibition of NF-κB expression is related with induction of autophagy. Inhibition of autophagy by 3-MA recovered rutin- induced 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 autophagy in 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 in several 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 [19-21]. 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 nutrient starvation, 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 by an 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 both autophagy 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.

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References

1. Gomes LC, Dikic I. Autophagy in antimicrobial immunity.

Mol Cell. 2014; 54:224-233. doi: 10.1016/j.molcel.

2014.03.009.

2. Deretic V. Autophagy: An Emerging Immunological Paradigm. J Immunol 2012; 189:15-20.

3. Deretic V, Saitoh T, Akira S. Autophagy in infection, inflammation and immunity. Nat Rev Immunol. 2013;

13:722-737. doi: 10.4049/jimmunol.1102108.

4. Yuk JM, Jo EK. Crosstalk between autophagy and inflammasomes. Mol Cells. 2013; 36:393-399. doi: 10.1007/

s10059-013-0298-0.

5. Mizushima N, Levine B, Cuervo AM, Klionsky DJ.

Autophagy fights disease through cellular self-digestion.

Nature. 2008; 451:1069–1075. doi: 10.1038/nature06639.

6. Poillet-Perez L, Despouy G, Delage-Mourroux R, Boyer- Guittaut M. Interplay between ROS and autophagy in cancer cells, from tumor initiation to cancer therapy. Redox Biol.

2015; 4:184-192. doi: 10.1016/j.redox.2014.12.003.

7. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, Englert JA, Rabinovitch M, Cernadas M, Kim HP, Fitzgerald KA, Ryter SW, Choi AM. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

Nat Immunol 2011; 12:222-230. doi: 10.1038/ni.1980.

8. Sharma S, Ali A, Ali J, Sahni JK, Baboota S. Rutin : therapeutic potential and recent advances in drug delivery.

Expert Opin Investig Drugs. 2013; 22:1063-1079. doi:

10.1517/13543784.2013.805744.

9. Fabjan N, Rode J, Kosir IJ, Wang Z, Zhang Z, Kreft I. Tartary buckwheat (Fagopyrum tataricum Gaertn.) as a source of dietary rutin and quercitrin. J Agric Food Chem 2003;

51:6452-6455.

10. Al-Dhabi NA, Arasu MV, Park CH, Park SU. An up-to-date review of rutin and its biological and pharmacological activities. EXCLI J. 2015; 14:59-63. doi: 10.17179/

excli2014-663.

11. Ghasemzadeh A, Jaafar HZ, Rahmat A, Devarajan T.

Evaluation of Bioactive Compounds, Pharmaceutical Quality, and Anticancer Activity of Curry Leaf (Murraya koenigii L.). Evid Based Complement Alternat Med 2014:873803. doi: 10.1155/2014/873803.

12. Cristina Marcarini J, Ferreira Tsuboy MS, Cabral Luiz R, Regina Ribeiro L, Beatriz Hoffmann-Campo C, Ségio Mantovani M. Investigation of cytotoxic, apoptosis- inducing, genotoxic and protective effects of the flavonoid rutin in HTC hepatic cells. Exp Toxicol Pathol 2011;

63:459-65. doi: 10.1016/j.etp.2010.03.005.

13. Perk AA, Shatynska-Mytsyk I, Gerçek YC, Boztaş K, Yazgan M, Fayyaz S, Farooqi AA. Rutin mediated targeting of signaling machinery in cancer cells. Cancer Cell Int. 2014;

14:124. doi: 10.1186/s12935-014-0124-6.

14. Levine B, Mizushima N, Virgin H. Autophagy in immunity and inflammation. Nature 2011; 469:323-335.

15. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009;

1:a001651. doi: 10.1101/cshperspect.a001651.

16. Harris J. Autophagy and cytokines. Cytokine. 2011;

56:140-144. doi: 10.1016/j.cyto.2011.08.022.

17. Surh YJ. Cancer chemoprevention with dietary phytochemicals.

Nat Rev Cancer. 2003; 3:768-780.

18. Alonso-Castro AJ, Domínguez F, García-Carrancá A. Rutin exerts antitumor effects on nude mice bearing SW480 tumor.

Arch Med Res. 2013; 44:346-351. doi: 10.1016/j.arcmed.

2013.06.002.

19. Shahid A, Ali R, Ali N, Kazim Hasan S, Rashid S, Majed F, Sultana S. Attenuation of genotoxicity, oxidative stress, apoptosis and inflammation by rutin in benzo(a)pyrene exposed lungs of mice: plausible role of NF-κB, TNF-α and Bcl-2. J Complement Integr Med. 2016; 13:17-29. doi:

10.1515/jcim-2015-0078.

20. Zhou YF, Guo B, Ye MJ, Liao RF, Li SL. Protective Effect of Rutin Against H2O2-Induced Oxidative Stress and Apoptosis in Human Lens Epithelial Cells. Curr Eye Res.

2015:1-10.

21. Kandemir FM, Ozkaraca M, Yildirim BA, Hanedan B, Kirbas A, Kilic K, Aktas E, Benzer F. Rutin attenuates gentamicin- induced renal damage by reducing oxidative stress, inflammation, apoptosis, and autophagy in rats. Ren Fail 2015; 37:518-525. doi: 10.3109/0886022X.2015.1006100.

22. Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 2001; 8:569-581.

23. White E, Mehnert JM, Chan CS. Autophagy, Metabolism, and Cancer. Clin Cancer Res 2015; 21:5037-5046. doi:

10.1158/1078-0432.

24. Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promoting chronic inflammation: a magic bullet?

Science 2013; 339:286-291. doi: 10.1126/science.1232227.

25. Harris J, Hartman M, Roche C, Zeng SG, O'Shea A, Sharp FA, Lambe EM, Creagh EM, Golenbock DT, Tschopp J, Kornfeld H, Fitzgerald KA, Lavelle EC. Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.

J Biol Chem. 2011; 286:9287-9597. doi: 10.1074/jbc.M110.

202911.

26. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG ST, Omori H NT, Yamamoto N, Komatsu M, Tanaka K, Kawai T, Tsujimura T, Takeuchi O, Yoshimori T, Akira S. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature. 2008; 456:264-268. doi:

10.1038/nature07383.

27. Park MH, Jung SY, Na HS, Chung J. Porphyromonas gingivalis induces autophagy in THP-1-derived macrophages.