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Journal of Ethnopharmacology

journal homepage: www.elsevier.com/locate/jep

Pharbitis Nil (PN) induces apoptosis and autophagy in lung cancer cells andautophagy inhibition enhances PN-induced apoptosis

Hyun Jin Junga, Ju-Hee Kangb, Seungho Choic, Youn Kyoung Sond, Kang Ro Leee,Je Kyung Seonga, Sun Yeou Kimb, Seung Hyun Ohb,⁎

a Korea Mouse Phenotyping Center, College of Veterinary Medicine, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Koreab College of Pharmacy, Gachon University, 191, Hambangmoe-ro, Yeonsu-gu, Incheon 21936, Republic of Koreac College of Veterinary Medicine, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Koread Biological and Genetic Resources Assessment Division, National Institute of Biological Resources, Incheon 22689, Republic of Koreae Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419-16, Republic of Korea

A R T I C L E I N F O

Keyword:Pharbitis NilApoptosisAutophagySTAT3ERK1/2

Compounds:Wortmannin (PubChem CID: 312145)U0126 (PubChem CID: 3006531)STX-0119 (PubChem CID: 4253236)SB202190 (PubChem CID: 5353940)

A B S T R A C T

Ethnopharmacological relevance: Pharbitis Nil (PN) is used as a main component of the existing drug, DA-9701, which was developed to treat functional dyspepsia (FD) in Korea. PN extracts isolated from its seeds havebeen reported to have anticancer effects.Aim of the study: The purpose of this study was to investigate the underlying mechanism of thechemotherapeutic effects of PN in lung cancer cells.Materials and methods: We performed MTT assays, colony formation assays, flow cytometry assays, Westernblot analysis, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence analysis, and cellcounting assays to study the molecular mechanism of chemotherapeutic effects of PN in lung cancer cells.Results: Our results indicate that PN induced autophagy as well as apoptosis. PN inhibited cell proliferationand survival by inducing apoptosis in several lung cancer cell lines. PN-treated cells also exhibited induction ofautophagy, as evidenced by increased protein expression levels and punctuate patterns of LC3 II. Moreover,activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which plays an important role inautophagy activation, was shown to be related with PN-induced autophagy. Interestingly, pharmacologicalblockade of autophagy activation with wortmannin and inhibition of ERK1/2 phosphorylation by U0126markedly enhanced PN-induced apoptosis and reduced cell viability, suggesting that autophagy induced by PNmay have a cytoprotective effect by suppressing apoptosis. PN- induced apoptosis was regulated by signaltransducer and activator of transcription 3 (STAT3) deactivation. Moreover, decrease of STAT3 activation inPN-treated cells was associated with reduced survivin expression, further demonstrating that PN-inducedapoptosis was regulated by STAT3 deactivation.Conclusion: We believe that PN, which is already proven to treat human patients with FD, might be a potentialanticancer drug for human lung cancer. In addition, our data suggest that the combination of PN treatment withan autophagy inhibitor or traditional anticancer agents may be an effective anticancer therapy.

1. Introduction

Lung cancer is a major cause of cancer-related deaths worldwide inboth men and women. Although lung cancers are treated with manytherapeutic agents such as Erlotinib and Gefitinib, intrinsic or acquiredresistance is a frequent occurrence (Paez et al., 2004; Pao et al., 2004).Moreover, most lung cancer patients are diagnosed in late stage, and

metastasis is observed in many cases (Klabunde et al., 2007). Thesecharacteristics have made it difficult to treat lung cancer as well as todevelop accurate chemotherapy drugs. Thus, it is important to identifynew effective chemotherapeutic agents to treat lung cancer. In recentyears, alternative medicines based on natural product extracts andtheir derivatives have received increased attention as new sources oftreatment (Mann, 2002; Reddy et al., 2003)

http://dx.doi.org/10.1016/j.jep.2017.07.020Received 2 May 2017; Received in revised form 10 July 2017; Accepted 15 July 2017

⁎ Corresponding author.E-mail addresses: eyeball@hanmail.net, eyeball@gachon.ac.kr (S.H. Oh).

Abbreviations: PN, Pharbitis Nil; ERK1/2, Extracellular signal-regulated kinases 1 and 2; STAT3, Signal transducer and activator of transcription 3, RT-PCR, Reverse transcriptase-polymerase chain reaction; siRNA, Small interfering RNA; PARP, poly ADP-ribose polymerase; Wort, Wortmannin; LC3, Microtubule-associated protein 1 light chain 3; PI, Propidiumiodide; MTT, 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide

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Available online 18 July 20170378-8741/ © 2017 Elsevier B.V. All rights reserved.

MARK

Pharbitis Nil is one of the representative medicinal herbs for cancertreatment in East Asia (Ko et al., 2004). Extracts of its seeds and rootsare known to have antitumor effects. A previous study showed that PNextracts have apoptotic effects in AGS gastric cancer cells by regulatingtumor suppressor genes (Ko et al., 2004). Another group reported thatPN inhibits proliferation and increases apoptosis in breast cancer cells(Ju et al., 2011). Recently, lignans isolated from PN seeds (pharbi-lignans) were shown to have anti-inflammatory activity and cytotoxi-city in cancer cells (Kim et al., 2014). However, the underlyingmechanisms of growth inhibition and induction of cell death havenot been precisely elucidated. Moreover, PN is one of the main sourcesof an existing drug in Korea. DA-9701 (Motilitone) is formulated fromextracts of PN and Corydalis Tuber and is a newly launched herbal drugfor treating patients with functional dyspepsia (Lee et al., 2008). DA-9701 has prokinetic effects on mice, rats, and humans without sideeffects and is undergoing phase II clinical trials for approval by the USFDA (Choi et al., 2009; Lee et al., 2017). It represents that extracts ofPN are safe to treat human patients.

Autophagy is induced and functions as a cytoprotective processagainst apoptosis in response to cellular stresses such as hypoxia,nutrient deprivation, and chemotherapeutic agents (Kondo et al., 2005;Levine, 2007; Song et al., 2009). Numerous compounds from plantextracts have been shown to trigger both autophagy and apoptosis,

whereas induced autophagy exerts a protective role to suppressapoptosis in treated cells. Aristolochic acid I and dendropanoxideinduce autophagy via the ERK1/2 pathway, which attenuates apoptoticeffects (Lee et al., 2013; Zeng et al., 2012). Riccardin D and paclitaxelalso activate cytoprotective autophagy and apoptosis (Kim et al., 2013;Wang et al., 2013). The protective role of autophagy helps cancer cellsevade cell death and develop resistance during chemotherapy. Inpaclitaxel-resistant cervical cancer cells, autophagy induction has beenobserved, and inhibition of autophagy increases sensitivity of resistantcells to paclitaxel (Peng et al., 2014). These results demonstrate thattargeting autophagy in cancer cells has great potential as a therapeuticstrategy in apoptosis-resistant cells. However, it remains highly con-troversial whether or not autophagy exerts cytoprotective effects oncancer cells since it is known to trigger cell death under certain stressconditions. Resveratrol activates cytotoxic autophagy and subsequentlyapoptosis in ovarian cancer cells (Lang et al., 2015). Thus, it is essentialto determine whether or not activation of autophagy by chemother-apeutic agents has a protective or cytotoxic effect in cancer cells.

Autophagy and apoptosis are interconnected in their control of cellfates. The ERK1/2 pathway has been shown to control both mechan-isms (Sridharan et al., 2011). Activation level of ERK is important forgrowth, proliferation, and survival of cancer cells. Recent studies haveevaluated that ERK activation is involved in induction of autophagy.

Fig. 1. Effects of PN on cell viability of various human lung cancer cells. (A) Hop62, H460, and A549 cells were incubated with various concentrations of PN for 48 h, after which cellproliferation rate was measured by MTT assay. (B) Anchorage-dependent colony formation assay for cancer cells was performed following treatment with PN for 2 weeks. The valuesshown are means + SD. *P < 0.05; **P < 0.01; ***P < 0.005 compared with the untreated group.

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Antitumor agents such as soyasaponins, capsaisin, and dendropan-oxide induce autophagy through ERK1/2 phosphorylation in humancancer cells (Choi et al., 2010; Ellington et al., 2006; Lee et al., 2013).

STAT3 is another regulator of both autophagy and apoptosis. As atranscriptional activator, STAT3 increases expression of downstreamproteins, which leads to increase survival and proliferation of cancercells. Recent studies revealed that human cancer cells acquiredresistant to anticancer drugs such as cisplatin via upregulation ofSTAT3 activation (Zhang et al., 2013). Thus, STAT3 has been known asa therapeutic target to treat human cancers. Several compounds thatare included in natural products like isocryptotanshineone and benzo-furan have been studied to have antitumor effects through STAT3inactivation (Guo et al., 2016; Kang et al., 2017)

In this study, we examined the effects of PN extracts on viability oflung cancer cells as well as the possible biological mechanisms of PNextracts. We observed that PN induced autophagy through an ERK1/2-dependent mechanism, and inhibition of autophagy using an ERK1/2blocker or autophagy inhibitor in PN-treated cells increased itsapoptotic effects. Collectively, these results suggest that activatedautophagy is a cytoprotective process to suppress apoptosis.

2. Material and methods

2.1. Cells and materials

Human lung cancer cell lines Hop62, H460, and A549 wereobtained from the American Type Culture Collection (Manassas, VA,USA). All cell lines were cultured in RPMI1640 medium containing10% fetal bovine serum and penicillin/streptomycin (GIBCO, GrandIsland, NY, USA). Antibodies against PARP, capase-3, pAKT, AKT,pERK1/2, ERK1/2, pP38, p38, pSTAT3, STAT3, and LC3 werepurchased from Cell Signaling Technology (Danvers, MA, USA), andthose against GAPDH and tubulin were purchased from Millipore(Schwalbach, Germany). p62 antibody was purchased from R&Dsystem (Minneapolis, MN, USA). Wortmannin and STX-0119 were

purchased from Calbiochem (La Jolla, CA, USA). U0126 and SB202190were purchased from Chemicon (Temecula, CA, USA) and Tocris(Bristol, UK), respectively.

2.2. Preparation of PN

PN extracts were prepared as indicated previously (Kim et al.,2014). Briefly, seeds of PN were air-dried and were then extractedusing 50% EtOH three times at room temperature. After filtration, thefiltrate was concentrated under reduced pressure using a rotavapor.The quality of PN was evaluated by checking chlorogenic acid usingHPLC as it was determined for PN in DA-9701 (Lee et al., 2008). Andthe plant name of PN was checked with the website (http://www.theplantlist.org).

2.3. MTT assay

To investigate the inhibitory effects of PN on proliferation of humanlung cancer cells, 3-(4.5-dimethylthiazol-2-yl)−2.5-diphenyltetrazo-lium bromide (MTT, Sigma-Aldrich, St Louis, MO) assay was per-formed. Lung cancer cells were cultured in 96 well-plates and treatedwith various concentrations (5–50 μg/ml) of PN. Cells were incubatedfor 48 h at 37 °C. Subsequently, 20 μl of MTT (2.5 mg/ml) was appliedto each well, followed by incubation at 37 °C for 2 h. After thesupernatant was removed, 100 μl of DMSO was added to wells. Theabsorbance was detected at 570 nm using a microplate spectrometer.

2.4. Colony formation assay

The effect of PN on survival of lung cancer cells was detected usingcolony formation assay. Cells were plated in 12-well-plates at a densityof 300 cells per well and treated with the indicated concentration of PNfor 11 days. Then, methanol and crystal violet were applied to fix andstain the cells, respectively.

Fig. 2. Apoptotic effect of PN in lung cancer cells. (A) H460 cells were treated with 10 and 20 μg/ml of PN and the percentage of apoptotic cells was detected by flow cytometry withAnnexin V/PI staining. (B) Hop62, H460, and A549 cells were incubated with the indicated concentrations of PN, and PARP and caspase 3 were detected by immunoblotting.

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2.5. Western blot analysis

Western blot analysis was conducted as described previously (Kanget al., 2015). Total cell lysates were prepared in modified RIPA buffer.

Protein concentrations were measured using BCA assay (PierceBiotechnology, Rockford, IL, USA). Equivalent amounts of proteinwere resolved in sodium dodecyl sulfate-polyacrylamide gel electro-phoresis gels and electrotransferred onto PVDF membrane. The blot

Fig. 3. PN induces autophagy. (A) Hop62, H460, and A549 cells were exposed to the indicated concentrations of PN, and LC3 expression levels were measured by western blot analysis.(B) p62 expression was detected by western blot analysis in PN-treated lung cancer cells. (C) H460 and A549 cells were treated with 20 μg/ml of PN in the presence or absence of 1 μMwortmannin (Wort), and levels of LC3 were detected by western blot analysis. (D) LC3 distribution were viewed under confocal microscopy after nuclei and endogenous LC3 proteinswere stained with DAPI (blue) and anti-LC3 antibody (FITC, green), respectively. Arrowheads indicate apoptotic nuclei.

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was then incubated with primary antibodies and washed, followed byincubation with HRP-conjugated secondary antibodies. The protein-antibody complexes were detected using chemiluminescence westernblotting detection reagents (GE Healthcare, Chalfont St. Giles, UK).

2.6. Flow cytometry analysis

Cells were seeded in 6-well-plates, and PN was treated to lung cancercells. Cells were harvested by EDTA-trypsin, washed with PBS, and stainedwith Annexin V-FITC and propidium iodide (PI) according to the manufac-

turer's instructions (Cell Signaling Technology). Stained cells were analyzedwith a flow cytometer (FACSan, BD Bioscience, San Jose, CA, USA).

2.7. Reverse transcription-polymerase chain reaction (RT-PCR)

Reverse transcriptase (RT)-polymerase chain reaction (PCR) wasperformed to evaluate changes in mRNA expression. Total RNA wasisolated from lung cancer cells using TRIzol reagent (Invitrogen,Carlsbad, CA). RNA extracted from cells was used as a template forreverse-transcription using a cDNA synthesis kit according to the

Fig. 4. Induction of autophagy by PN through ERK1/2 activation. (A) Western blot of indicated proteins was performed after 48 h of treatment with different concentrations of PN inHop62, H460, and A549 cancer cells. (B) Lung cancer cells were treated with 20 μg/ml of PN in the presence or absence of 10 μM U0126, and LC3 and pERK1/2 expression levels weredetected by western blotting analysis. (C) A549 cells were exposed to 20 μg/ml of PN in the presence or absence of 20 μM of SB202190 and then analyzed by western blotting. (D)Endogenous LC3 (green) and the nuclei (blue) were stained and observed after A549 cells received 20 μg/ml of PN in the presence or absence of 10 μM U0126. Arrowheads indicatednuclei undergoing apoptosis.

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manufacturer's instructions (Takara, Kyoto, Japan). RT-PCR wasconducted with gene-specific primers for GAPDH (forward,5′CCACCCATGGCAAATTCCATGGCA-3; reverse, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′), Survivin (forward, 5′-GCATGGGTG

CCCCGACGTTG-3′; reverse, 5′-GCTCCGGCCAGAGGCCTCAA-3′).The PCR products were separated on a 1.5% agarose gel in trisace-tate/ethylenediaminetetraacetic acid buffer.

Fig. 5. Inhibition of autophagy increases apoptotic effects of PN. (A) The percentage of apoptotic cell was evaluated by flow cytometry after cells were incubated with 20 μg/ml of PN inthe presence or absence of 1 μM wortmannin. (B) Amounts of PARP and caspase 3 were analyzed by immunoblotting. (C) ATG7 protein levels were detected by immunoblotting afterA549 cells were treated with scrambled siRNA (SC) and ATG7 siRNA. (D) A549 cells were incubated with PN for 48 h after scrambled siRNA (SC) and ATG7 siRNA were applied to A549cells. A549 cell proliferation rate was measured by MTT assay. **P < 0.01 compared with the siSC and PN treated control group.

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2.8. Immunofluorescence analysis

A549 cells were seeded on sterile coverslips and treated with PN.After discarding the media, cells were incubated with 100% MeOH for20 min at −20 °C and blocked with blocking solution (Life technolo-gies, MD, USA) for 1 h at RT. Fixed cells were then incubated with anti-LC3 antibody overnight at 4 °C. FITC-conjugated secondary antibody(Santa Cruz Biotechnology, Santa Cruz, CA, USA) was then applied to

cells for 2 h at RT in the dark. The nucleus was stained and mountedwith Vectashiled mounting medium (Vector Laboratories, CA, USA).

2.9. Cell counting assay

A549 cells were seeded in 12-well-plates and treated with PN. Cellwere collected using EDTA-trypsin and mixed with trypan blue. 10 μlof each mixture was loaded onto a hemocytometer. The number of

Fig. 6. Suppression of ERK1/2 increases PN-induced apoptotic cell death. (A) After treatment with 20 μg/ml of PN in the presence or absence of 10 μM U0126, apoptotic cells weredetected by flow cytometry. (B) Immunoblotting was performed to detect protein levels of PARP, caspase 3, and pERK1/2. (C) The number of A549 cells was counted under amicroscope. (D) Anchorage-dependent colony formation assay for cancer cells was conducted following treatment with PN and U0126 for 2 weeks. The values shown are means + SD. *P< 0.05 compared with the untreated group. ###P < 0.005 or #P < 0.05 compared with the single U0126 or PN treatment group.

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unstained cells (live cells) was counted out of the total number of cellsunder a microscope.

2.10. RNA interference

Small interfering RNA (siRNA) against human ATG7 and STAT3were synthesized by Bioneer (Daejeon, Korea). For siRNA treatment,duplexed siRNA was introduced into A549 cells using LipofectamineRNAiMAX (Invitrogen). Cells were lysed for western blotting analysis48 h after transfection.

2.11. Statistical analysis

Data are shown as means ± standard deviations. For statisticalanalysis of comparative data, a two-tailed student's t-test was utilizedusing Microsoft Excel software. Values of *p < 0.05; **P < 0.01; ***P <0.005 were considered significant difference and indicated by asterisksin the figures.

3. Results

3.1. PN reduces cell viability in various human lung cancer cells

We performed MTT assay to determine whether or not PN exerts acytotoxic effect on human lung cancer cells. Hop62, H460, and A549cells were treated with various concentrations of PN. Fig. 1A shows thatPN significantly inhibited proliferation of lung cancer cells in a dose-dependent manner. PN treatment at a concentration of 20 μg/mleffectively inhibited proliferation of all treated cell lines. We furtherinvestigated if PN suppresses colony formation in lung cancer cells. Asshown in Fig. 1B, long-term treatment with PN dramatically reducedcolony formation in all treated cell lines. These results suggest that PN

significantly reduced proliferation and survival of lung cancer cells.

3.2. Apoptosis is induced by PN

To determine if PN reduces cell viability by inducing apoptosis, weanalyzed the apoptotic effects of PN by flow cytometry analysis. H460cells were stained with Annexin V- PI after 72 h of PN treatment.Fig. 2A shows that PN significantly increased the fraction of apoptoticcells as the percentage of apoptotic cells increased from 5.71% to42.69% in cells treated with 20 μg/ml of PN compared to the controlgroup. It is well known that poly ADP-ribose polymerase (PARP) andcaspase 3 are cleaved and activated during cell apoptosis (Boulareset al., 1999). Therefore, we performed western blot analysis toinvestigate the effects of PN on cleavage of PARP and caspase 3.Increased amount of cleaved caspase 3 and PARP as well as reductionof uncleaved caspase 3 were observed upon PN treatment in a dose-dependent manner (Fig. 2B). These data indicate that PN inducedapoptosis via caspase 3 and PARP in lung cancer cell lines.

3.3. PN significantly induces autophagy in lung cancer cells

It has been recently revealed that natural products induce bothapoptosis and autophagy in human cancer cells. However, regulation ofautophagy by PN has not been studied yet. To investigate whether ornot PN regulates autophagy, we examined changes in microtubule-associated protein 1 light chain 3 (LC3) protein levels. Conversion ofLC3 I into its lipidated form at the C-terminal end (LC3 II) is regardedas a key marker of autophagy (Mizushima et al., 2010). Lipidation ofLC3 to form LC3 II aids the elongation and maturation of autophago-somes (Mizushima et al., 2010). In our results, PN triggered conversionof LC3 I into its lapidated LC3 II form in a dose-dependent manner inall treated cells (Fig. 3A). Another autophagy marker, p62, is recruited

Fig. 7. Regulation of PN-induced cell death by STAT3 deactivation. (A) A549 cells were treated with STX-0119 for 48 h at various concentrations, and whole cell lysates were examinedto detect STAT3 and LC3 protein levels. (B) Scrambled siRNA (SC) and STAT3 siRNA were applied to A549 cells, and STAT3 and LC3 protein levels were detected by immunoblotting.(C) Lung cancer cells were treated with PN for 48 h, and RNA levels of survivin were detected using RT-PCR. GAPDH was detected as a loading control. (D) Hop62 and A549 cells weretreated with PN, and survivin expression was detected by western blotting.

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to autophagosome and degraded as autophagy flux is increased(Itakura and Mizushima, 2011). Fig. 3B also showed that PN inducedautophagy in lung cancer cells. Upregulation of LC3 II levels by PNtreatment was abrogated when a specific autophagy inhibitor, wort-mannin (wort), was applied (Fig. 3C). LC3 I is located in the cytoplasmwhile its lipidated form mainly aggregates in the autophagosomemembranes as autophagy increases, as evidenced by punctuate pat-terns in the cytoplasm using immunoreactivity assays (Lee et al., 2013).As shown in Fig. 3D, PN treatment increased punctuate patterns ofLC3, whereas co-treatment with wort reduced this pattern. Theseobservations show that PN significantly increased autophagy.

3.4. PN induces autophagy via activation of the ERK pathway

Autophagy is regulated by several molecular pathways. Recentstudies have demonstrated that activated mTOR-AKT, ERK1/2, p38,and STAT3 are involved in regulation of autophagy (Sridharan et al.,2011; You et al., 2015b). As shown in Fig. 4A, PN treatment resulted inincreased levels of phosphorylated ERK1/2 and p38 in all treated celllines, whereas no alteration of phosphorylated AKT levels was detectedunder the same experimental conditions. To determine which kinasecontributes to PN-induced autophagy, the ERK1/2 inhibitor U0126and p38 blocker SB202190 were used to inactivate these kinases. Co-treatment of U0126 with PN notably inhibited induction of autophagy(Fig. 4B). However, pharmacological inhibition of p38 failed tosuppress autophagy induction in PN-treated cells (Fig. 4C). Theseresults show that PN-induced autophagy was specifically regulated bythe ERK1/2 pathway, and this was further confirmed by immunofluor-escence staining. Single treatment with U0126 did not alter LC3 IIaccumulation, whereas U0126 combined with PN significantly reducedLC3 II puncta (Fig. 4D). Taken together, these observations provideevidence that PN activated ERK1/2 signaling followed by autophagy.

3.5. Inhibition of autophagy enhances apoptosis by PN

Autophagy remains a controversial research topic since it canfunction as a cytotoxic or cytoprotective factor in response to antic-ancer agents. As indicated by the white arrowheads in Figs. 3D and 4D,cells treated with both PN and wort or U0126 showed an increase inapoptotic nuclei as visualized by DAPI. This result suggests that PN-induced autophagy may have a cytoprotective effect on lung cancercells. To investigate the effects of PN-induced autophagy on cellapoptosis, flow cytometry analysis was performed using Annexin V-PI double staining. Pharmacological inhibition of autophagy by wort inPN-treated cells dramatically enhanced the percentage of apoptoticcells compared with those treated with PN alone (Fig. 5A). Specifically,the fraction of apoptotic cells increased from 31.52% in PN-treatedH460 cells to 69.81% in co-treated cells and the apoptotic cellpercentage increased from 11.45% to 21.81% in A549 cells. To confirmthe above results, cleavage of PARP and caspase 3 was investigated byimmunoblotting and the apoptotic effects of PN were elevated in thepresence of wort (Fig. 5B). In addition to pharmacological inhibitionwith wort, we used siRNA against autophagy related gene 7(ATG7) toreduce autophagy in A549 cells (Fig. 5C). ATG7 is known as anautophagy promoting gene, which regulates autophagosome formation(Komatsu et al., 2005). ATG7 siRNA-transfected cells showed de-creased proliferation rate compared to scramble (control) group whenPN was applied (Fig. 5D). These results show that autophagy inhibitionincreased PN-induced apoptosis in lung cancer cells.

3.6. Inhibition of ERK1/2 pathway enhances apoptosis by PN

Inhibition of autophagy by wort was shown to increase thepercentage of PN-induced apoptotic cells. Based on our observationthat PN-induced autophagy was regulated by ERK1/2 signaling, wehypothesized that deactivation of ERK1/2 would increase the apoptotic

effect of PN. Using FACS analysis, we observed that U0126 itself didnot alter the fraction of apoptotic cells, but co-treatment with PNaccelerated PN-induced apoptotic death in A549 cells (Fig. 6A). Inaccordance with FACS analysis, cleavage of PARP increased in cellstreated with both U0126 and PN (Fig. 6B). Cell counting and colonyformation assays provided other evidence that confirms our hypothesis.ERK1/2 inhibition led to increased PN-induced apoptosis (Fig. 6C-D).Taken together, our data suggest that blockage of ERK phosphorylationresulted in acceleration of PN-induced apoptosis.

3.7. STAT3 deactivation contributes to apoptotic effects of PN

Contrary to ERK and p38 activation, PN treatment reduced STAT3activation in a dose-dependent manner (Fig. 4A). Reduction ofphospho-STAT3 is related to elevation of both autophagy and apoptosis(Xiong et al., 2008; You et al., 2015b). Therefore, we evaluated whetheror not reduced pSTAT3 activation could regulate the LC3 II proteinlevel. Pharmacological inhibition of STAT3 by STX-0119 and treatmentwith siRNA against STAT3 did not alter LC3 II protein expression(Fig. 7A-B). Next, we hypothesized that reduced STAT3 activation mayregulate the apoptotic effects of PN. Survivin is one of the STAT3downstream proteins and has been known as an apoptosis inhibitorprotein (Gritsko et al., 2006). In PN-treated cells, the transcriptexpression level of survivin was down-regulated in a dose-dependentmanner. (Fig. 7C). In agreement with the mRNA level, survivin proteinexpression was also reduced upon PN treatment (Fig. 7D). Theseobservations indicate that dephosphorylation of STAT3 regulated theapoptotic but not autophagy effects of PN.

4. Discussion

Many traditional herbal extracts have been used to treat cancer,including Pharbitis Nil as a representative anticancer medicinal herb(Ko et al., 2004). Extracts of PN have been reported to exhibitantitumor activities in several cancer cells (Ju et al., 2011; Kim et al.,2014). Especially, seeds of PN are known to contain antitumor lignansand increase cell death in breast cancer cells (Ju et al., 2011; Kim et al.,2014).

Identification of existing drugs for new use has gained popularitydue to the benefits of reduced costs and risks of developing new drugs.DA-9701 is formulated as 50% EtOH extract from PN, which is thesame as PN extracts used in this study, and has already been approvedto treat human patients with FD (Lee et al., 2008). Thus, treatmentwith PN extracts is safe for humans even though toxicity is a growingproblem for traditional herbal medicines. Moreover, it shows that evencrude herbal extracts can be defined as medicines.

In this study, we determined that PN effectively inhibited prolifera-tion and survival rate by inducing apoptotic cell death in lung cancercells. Moreover, our results demonstrated for the first time that PNinduced autophagy to delay apoptosis, as evidenced by increasedprotein levels of LC3 II and punctuate patterns of LC3.

In eukaryotic cells, autophagy is evolutionarily conserved to destroydamaged organelles and recycle cellular proteins to protect againstseveral stimuli (Kondo et al., 2005; Levine, 2007; Song et al., 2009).However, it is debatable whether or not autophagy participates in celldeath or the survival mechanism of cancer. Many natural products withanticancer effects have been used to investigate if autophagy supportsor attenuates apoptosis. We demonstrated that PN-induced autophagyplays a role in repressing the apoptotic machinery of PN. Inhibition ofautophagy by wortmannin considerably potentiated PN-induced apop-tosis while wortmannin treatment alone did not affect cell fate, asconfirmed by reduction of LC3 puncta and LC3 II protein levels as wellas an increased number of Annexin V/PI -positive cells and cleavedPARP expression. These data are in accord with recent research.Dendropanoxide, paclitaxel, and dihydroptychantol A have been stu-died to induce both apoptosis and autophagy, whereas induced

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autophagy plays a protective role (Kim et al., 2013; Lee et al., 2013; Liet al., 2011)

Autophagy is controlled by several protein kinases. ActivatedmTORC1 inhibits autophagy induction (Zhou et al., 2013). ERK1/2and p38 phosphorylation regulate the balance between apoptosis andautophagy in response to therapeutic antitumor agents. (Sridharanet al., 2011) Activation of p38 increases autophagy induction under ERstress in yeasts as well as in the presence of chemotherapeutic agents inhuman cancer cells (Corcelle et al., 2007; Prick et al., 2006). Recentstudies have proposed that ERK1/2 phosphorylation is involved inautophagic vacuole maturation to form autophagosomes. ERK1/2 isassociated with the extra luminal face of autophagic vesicles byinteracting with several ATG proteins and LC3 II (Martinez-Lopezet al., 2013). We observed that PN mediated ERK1/2 and p38activation in a dose-dependent manner. Pharmacological inhibitionof ERK1/2 and p38 was utilized to determine whether or not the twokinases regulate PN-induced autophagy. SB202190, a p38 inhibitor,did not alter LC3 II protein expression, whereas the ERK inhibitorU0126 attenuated the PN-induced LC3 II protein level and puncta. Thecombination of PN and U0126 increased apoptotic cell death comparedto single PN treatment. In accordance with the results of PN andwortmannin treatment, PN-induced autophagy was shown to play acytoprotective role in lung cancer cells. Although p38 plays animportant role in autophagy induction, our results show that ERKactivation regulated PN-induced autophagy.

The JAK/STAT3 signaling pathway has recently been regarded asanother modulator of autophagy. Cytoplasmic STAT3 is known as anegative regulator of autophagy, and STAT3 inhibitors such as statichave been shown to reduce autophagic flux (Shen et al., 2012).Crizotinib, a chemotherapeutic drug that inactivates cytoplasmic andnuclear STAT3, induces cytoprotective autophagy in lung cancer cells(You et al., 2015a). The JAK/STAT3 inhibitor cucurbitacin I increasesautophagy, which plays a protective role in glioblastoma multiformecells (Yuan et al., 2014). In contrast, STAT3 activation in mitochondriaregulates autophagy in a positive feedback loop. IL6 activates STAT3and enhances mitochondrial localization of pSTAT3, which up-regu-lates autophagic flux in pancreatic cancer cells (Qin et al., 2015).However, our data show that STAT3 deactivation was not related toPN-induced autophagy. Reduction of pSTAT3 regulated transcriptionaland translational survivin expression, suggesting that PN increasedapoptosis by inhibiting survivin.

In conclusion, we demonstrated for the first time that PN mediatedboth apoptosis and autophagy, contributing to cell death. PN treatmentin lung cancer cells increased apoptosis by decreasing STAT3 activa-tion, followed by reduction of survivin expression. Autophagy wasinduced through the ERK pathway, which had cytoprotective effects forcell survival under PN-treated conditions. Our findings indicate thatPN extracts can be used to treat lung cancer, and suggest the possibilityof drug repurposing.

Acknowledgement

This work was supported by a grant from the National Institute ofBiological Resources (NIBR), funded by the Ministry of Environment(MOE) of the Republic of Korea (NIBR201725201).

This research was supported by the National Research Foundationof Korea (NRF) funded by the Ministry of Science, ICT & FuturePlanning (No. 2012M3A9D1054622)

Conflict of interest

The authors declare no conflict of interest.

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