NF-κB inhibitory and cytotoxic activities of hexacyclic triterpene acid constituents from Glechoma longituba

Phytomedicine 63 (2019) 153037

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NF-κB inhibitory and cytotoxic activities of hexacyclic triterpene acid constituents from Glechoma longituba

T

Xi-Lin Ouyanga,b,1, Feng Qina,1, Ri-Zhen Huangc, Dong Lianga, Chun-Gu Wangc, ⁎ ⁎⁎ Heng-Shan Wanga, , Zhi-Xin Liaoc, a

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Science, Guangxi Normal University, Guilin, Guangxi, People's Republic of China b College of Public Health and Management, Youjiang Medical University for Nationalities, Baise, Guangxi, People's Republic of China c Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, People's Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Lung cancer Glechoma longituba Hexacyclic triterpene acid Anticancer Nuclear factor-κB

Background: Non-Small-Cell Lung Cancer (NSCLC) is the most-frequent cause of cancer death, and novel chemotherapeutic drugs for treating NSCLC are urgently needed. 2α, 3α, 23-trihydroxy-13α, 27-cyclours-11-en-28oic acid (euscaphic acid G) is a new hexacyclic triterpene acid isolated by our group from Glechoma longituba (Nakai) Kupr. However, the underlying mechanisms responsible for the anticancer effects of hexacyclic triterpene acid have not been elucidated. Purpose: In the present work, we evaluated growth inhibitory effect of the new isolated hexacyclic triterpene acid and explored the underlying molecular mechanisms. Methods/study designs: Herbs were extracted and constituents were purified by chromatographic separation, including silica gel, ODS, MCI, Sephadex LH-20 and preparative HPLC. The compound structures were elucidated by the use of UV, NMR and MS spectral data. The anticancer activity of euscaphic acid G was evaluated by MTT assay. Cell cycle, apoptosis, reactive oxygen species and mitochondrial membrane potential were determined by flow cytometry. To display the possible mechanism of euscaphic acid G on NCI-H460 cells, RT-PCR, immunofluorescence and Western blot analysis were carried out. Results: A new hexacyclic triterpene acid, euscaphic acid G, together with fifteen known triterpenoids, was isolated from the aerial parts of G. longituba. Our results showed that euscaphic acid G exerted strong antiproliferative activity against NCI-H460 cells in a concentration- and time-dependent manner. Flow cytometry demonstrated euscaphic acid G arrested the cell cycle at G1 phase, induced cellular apoptosis, accompanied by ROS generation and mitochondrial membrane potential reduction. Mechanistic studies revealed that euscaphic acid G treatment inhibited IKKα/β phosphorylation and IκBα phosphorylation, which subsequently caused the blockage of NF-κB p65 phosphorylation and nuclear translocation. Conclusion: In conclusion, these results suggested that euscaphic acid G from G. longituba showed potential anticancer effects against lung cancer cells via inducing cell cycle arrest and apoptosis, at least partly, through NF-κB signaling pathways.

Abbreviations: COSY, (homonuclear chemical shift) correlation spectroscopy; DCFH-DA, dichlorofluorescin diacetate; DMEM, dulbecco's modified eagle medium; DMSO, dimethyl sulfoxide; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear single quantum coherence; HPLC, high performance liquid chromatography; IC50, 50% inhibition concentration; NCI-H460, Human lung cancer cells; NF-κB, nuclear factor-κB; NSCLC, non-small-cell lung cancer; PMSF, phenylmethyl sulphonylfluoride; RIPA, radio immunoprecipitation assay; ROS, reactive oxygen species; TLC, thin layer chromatography ⁎ Corresponding author at: State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Science, Guangxi Normal University, 15 Yucai Road, Guilin 541004, People's Republic of China. ⁎⁎ Co-corresponding author. E-mail addresses: [email protected] (H.-S. Wang), [email protected] (Z.-X. Liao). 1 These authors contributed equally. https://doi.org/10.1016/j.phymed.2019.153037 Received 12 April 2019; Received in revised form 13 July 2019; Accepted 19 July 2019 0944-7113/ © 2019 Published by Elsevier GmbH.

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illustrated. Our result revealed that EAG could be the strong candidate to induce apoptosis in NCI-H460 cancer cells by the blockage of NF-κB signaling pathway.

Introduction Non-Small-Cell Lung Cancer (NSCLC) comprises about 85% of lung cancers case, which is the most-frequent cause of cancer-related deaths worldwide (Chien et al., 2019; Siegel et al., 2016). NSCLC patients are often diagnosed at an advanced stage and have poor prognosis. Despite availability of more treatment options with chemotherapy and targettherapies, the efficacy of these remedies remains unsatisfactory due to intrinsic or acquired resistance (Wagner and Yang, 2010; Thress et al., 2015). Therefore, new drugs or improved strategies are urgently needed for NSCLC. Recently, compounds from traditional medicine have raised increasing interest due to their high efficiency and low toxicity (Reker et al., 2014). Glechoma longituba (Nakai) Kupr., as one species of Glechoma, which belongs to the Lamiaceae family, is distributed extensively in most part of China (Yang et al., 2006). This plant has been used as a common folk medicine in China for the treatment of urinary tract infection, infantile malnutrition, jaundice, cold, cough, pulmonary abscess, fracture, and rheumarthritis (Zhu et al., 2013). The crude extract of G. longituba revealed various bioactivities, such as anti-inflammatory, antibacterial, antioxidant and antitumor activities (Bozin et al., 2006; An et al., 2006; Majendra et al., 2004; Wang et al., 2018). Previous phytochemical investigations of G. longituba have demonstrated the presence of triterpenoids, sesquiterpenoids, flavonoids, organic acids and its derivatives in this plant (Zhu et al., 2013, 2008; Yuan et al., 2005; Jung et al., 2003). During our ongoing research on the chemical composition of traditional folk medicines, sixteen triterpenoids, including one novel ursane-type triterpenoid acid with a 13α, 27-cyclopropane ring, euscaphic acid G (EAG), were isolated from the aerial parts of G. longituba. Triterpenoids are highly multifunctional agents owing to their ability to interact with multiple biological targets and has been reported to block NF-κB activation (Huang et al., 2018a; Ren et al., 2018). Besides, Cheng et al. have reported that hexacyclic triterpene acids showed significant cytotoxicity against several cancer cell lines (Cheng et al., 2010). However, the underlying mechanism of hexacyclic triterpene acids has not been elucidated. Therefore, our present study aims to understand the anticancer potency of the new isolated hexacyclic triterpene acid (EAG, Fig. 1) against lung cancer cell lines (NCI-H460), along with the isolation and structural elucidation. The possible mechanism of EAG to induce apoptosis was also

Materials and methods Instruments and reagents Silica gel (200–300 mesh), MCI (CHP20, 75–150 μm), Sephadex LH20, and RP C18 (50 μm) for column chromatography were supplied by the Qingdao Marine Chemical Factory (Qingdao, China), Pharmacia Biotech (Sweden), Mitsubishi Chemical Corporation and YMC Corporation (Japan), respectively. HPLC analysis was performed on a Shimadzu LC-20A pump and a YMC-C18 column (4.6 × 250 mm, 5 µm, YMC, Japan) with a photodiode array detector at 206 nm. Preparative HPLC was carried out with a Shimadzu LC-6AD instrument (Shimadzu, Japan) with a YMC-C18 column (20 × 250 mm, 5 µm, YMC, Japan) with wavelength detector at 206 nm. Optical rotations were measured with a PerkinElmer polarimeter (Model 341), and UV spectra recorded on a PerkinElmer spectrophotometer (Model 650, PerkinElmer, USA). IR spectra were taken on a PerkinElmer FT-IR spectrometer. NMR spectra were obtained using an Avance III 500 MHz FT-NMR spectrometer (Bruker, Karlsruhe, Germany) with the residual solvent signals as reference, and the chemical shifts are indicated as δ values (ppm). LCHR/MS data were measured on an Agilent 6545B Q-TOF LC/MS (Agilent technologies, Quebec, Canada). Plant material G. longituba was collected in June 2014 from Jinxiu Yao Autonomous County of Guangxi, China, and was identified as a G. longituba (Nakai) Kupr by Professor Shaoqing Tang, College of Life Sciences of Guangxi Normal University. The sample (GL 201401) was preserved in the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources of Guangxi Normal University. Isolation and purification of EAG Whole dried grass of G. longituba (20 kg) was cut into small pieces, and then extracted three times with 75% ethanol for 96 h. After

Fig. 1. Structures of sixteen triterpenes isolated from G. longituba. 2

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filtration, the combined filtrate was evaporated under reduced pressure to dryness in a rotary evaporator to get a crude extract. Subsequently, the crude was suspended in refined water and partitioned successively with petroleum ether, ethyl acetate, and n-butanol to gather the corresponding fractions. The ethyl acetate fraction (Fr-EA) was separated on a silica gel column (petroleum ether/ethyl acetate as eluents) to afford 14 sub-fractions (EAs 1 to 14). EA 6 was further separated on a silica gel column to obtain ten sub-fractions by combining in accordance to their similarity in behavior on TLC. EA 6-7 were further purified by means of C-18 column chromatography eluting with MeOHeH2O to obtain three parts (EAs 6-7-1 to 3). Compounds 15 (15 mg) and 16 (12 mg) were isolated from EA 6-7-1 and EA 6-7-3 by preparative HPLC, respectively. The EA 9 was also further separated on a silica gel column to obtain nine parts by combining the same components. The EA 9-7 was separated by Sephadex LH-20 column chromatography eluted with MeOH to afford three parts (EA 9-7-1 to 3). EA 9-7-1 was further purified by preparative HPLC eluted with 70% methanol to obtain compound 6 (20 mg). EA 9-7-2 was further purified by preparative HPLC to obtain compounds 1 (EAG, 6 mg), 2 (36 mg), 5 (8.6 mg), 7 (8.6 mg), 8 (15 mg), and 9 (43 mg). EA 9-7-3 were chromatographed by C-18 column chromatography eluted with MeOHeH2O and further to remove impurities to purified by preparative HPLC finally obtain compounds 3 (6.6 mg), 4 (11 mg), 10 (10.5 mg), and 14 (13 mg). Compounds 11 to 13 were obtained from EA 9-9 by preparative HPLC eluted with 82% methanol. The purity of all compounds was checked by HPLC and NMR.

RT-PCR RT-PCR assay was performed as described previously Huang et al. (2018b). Total cellular RNA was extracted using the RNA pure Kit (Aidlab, RN0302, China) following the step-by-step protocol provided by manufacturer. The cDNA was prepared after reverse transcription of cellular RNA samples for 30 min at 42 °C with the High Capacity cDNA Reverse Transcription Kit (TaKaRa, Biotechnology, Dalian). The SYBR® Green PCR Master Mix (Fermentas, K0251, Lithuania) and specific primer pairs were used for selected genes, and GAPDH was used as the reference gene. RT-PCR was performed according to the following conditions: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C using 0.5 μl of complementary (c) DNA, 2 × SYBR Green PCR Master Mix, and 500 nM of the forward and reverse primers on a 7500 real-time PCR System (Applied Biosysterms). The real-time RT-PCR was performed in triplicate for each experimental group. The primer pairs for NF-κB and GAPDH were used as follows: NF-κB, forward: 5′-ATGTGGAGATCATTGAGCAGC-3′ and reverse: 5′- CCTGGTC CTGTGTAGCCATT-3′; GAPDH, forward: 5′-CCCACTCCTCCACCTTT GAC-3′ and reverse: 5′-TCTTCCTCTTGTGCTCTTGC-3′. Apoptosis analysis NCI-H460 cells were seeded at the density of 2 × 106 cells/ml of the DMEM medium with 10% FBS on 6-well plates to the final volume of 2 ml. The plates were incubated for overnight and then treated with different concentrations EAG for 24 h. Briefly, after treatment with EAG for 24 h, cells were collected and washed with PBS twice, and then resuspend cells in 1 × Binding Buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration of 1 × 106 cells /ml. The cells were subjected to 5 μl of FITC Annexin V and 5 μl propidium iodide (PI) staining using annexin-V FITC apoptosis kit (BD, Pharmingen) followed the 100 μl of the solution was transfer to a 5 ml culture tube and incubate for 30 min at RT (25 °C) in the dark. The apoptosis ratio was quantified by system software (CellQuest; BD Biosciences).

Cell lines and cell cultures NCI-H460, HepG2, T24, SKOV3, MGC-803, HL-7702 cells were all obtained from the Institute of Biochemistry and Cell Biology, China Academy of Sciences. NCI-H460, HepG2, T24, SKOV3, MGC-803, HL7702 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37 °C in a humidified atmosphere with 5% CO2.

ROS assay

Cytotoxicity assay

NCI-H460 cells were seeded into six-well plates, and following treatment, were incubated with 10 mM DCFH-DA (Beyotime, Haimen, China) dissolved in cell-free medium for 30 min at 37 °C and in the dark. They were then washed three times with PBS. Cellular fluorescence was measured with a Nikon ECLIPSETE2000-S fluorescence microscope at 485 nm excitation and 538 nm emission.

NCI-H460, HepG2, T24, SKOV3, MGC-803, HL-7702 cell lines were grown on 96-well microtitre plates at a cell density of 10 × 105 cells/ well in DMEM medium with 10% FBS. DMEM and FBS were obtained from Gibco-Thermo (BRL Co. Ltd., USA). The plates were incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air for overnight. Therewith, the cells were exposed to different concentrations of target compounds and HCPT, and incubated for another 48 h. The cells were stained with 10 μl of MTT at incubator for about 4 h. The medium was thrown away and replaced by 100 μl DMSO. The O. D. Value was read at 570/630 nm enzyme labeling instrument.

Mitochondrial membrane potential staining Mitochondrial depolarization was assayed in NCI-H460 cells using a JC-1 probe (Beyotime, Haimen, China). Briefly, cells cultured in sixwell plates after the indicated treatment were incubated with an equal volume of JC-1 staining solution (5 μg/ml) at 37 °C for 20 min and rinsed twice with PBS. Mitochondrial membrane potentials were monitored by determining the relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates using a Nikon ECLIPSETE2000-S fluorescent microscope. Mitochondrial depolarization was indicated by an increase in the green/red fluorescence intensity ratio.

Immunofluorescence staining Studies were performed as previously described (Lu et al., 2016). Briefly, NCI-H460 cells were cultured in six-well plates (2 × 105 cells per well) for 24 h and then pretreated with EAG (4 and 8 μM) for 24 h, followed by TNF-α for another 30 min. The cells were then washed and fixed with anhydrous methanol for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 1% BSA for 10 min at room temperature. Next, cells were incubated with primary NF-κB antibody (Abcam, USA, cat: ab183559) in PBS overnight at 4 °C, followed by FITC-conjugated antibody secondary antibodies. After being washed with PBS three times, cells were incubated in 10 μM DAPI for 15 min in the dark. The fluorescence of the NF-κB protein and nucleus were green and blue, respectively, and live cell imaging was simultaneously accomplished using a fluorescent microscope (Olympus BX-51, Toyko, Japan).

Hoechst 33258 staining NCI-H460 cells were seeded at the density of 2 × 106 cells/ml of the DMEM medium with 10% FBS on 6-well plates to the final volume of 2 ml. The culture medium containing the compound was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. After washing twice with phosphate buffered saline (PBS), the cells were stained with 0.5 ml of Hoechst 33258 (Beyotime) for 5 min and again washed twice with PBS. Nuclear staining was observed with a Nikon 3

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The IR spectrum showed absorption peaks of hydroxyl (3377 cm–1), carbonyl (1694 cm–1), and olefin (1633 cm–1) groups. A total of 30 carbons in 13C NMR data combined with DEPT 135 suggested that EAG consists of five methyl, nine methylene, nine methine, and seven quaternary carbons. In the 1H NMR spectrum, five methyl groups were found in with chemical shift at δH 1.33 (3H, s), 1.07 (3H, d, J = 6.0 Hz), 0.93 (3H, s), 0.85 (3H, d, J = 5.8 Hz), and 0.81 (3H, s). Among them, two methyl groups are split into two peaks, which suggested that EAG is an ursane-type triterpenoid. Furthermore, the 1H and 13C NMR data of EAG displayed a double bond (δC 141.0 and 120.0) with two olefinic protons at δH 6.11 (dd, J = 10.1, 2.8 Hz, 1H) and 5.35 (dd, J = 10.1, 1.9 Hz, 1H). The 13C NMR data also showed three hydroxyl groups (δC 79.5, 71.7, and 66.6) and a carbonyl carbon (δC 181.2). Correlations from the protons at C-23 (δH 3.87, 3.72) to carbons at δC 79.5 (CH-3), 42.4 (C-4), and δC 17.5 (CH3-24) confirmed the attachment of this hydroxymethyl group by the HMBC spectrum. In addition, HMBC correlations observed from the protons at δH 6.12 to 53.8 (C-9), 29.8 (C13) and δH 5.35 to 29.8 (C-13), 36.0 (C-14) were located the olefinic protons occurring to C-11 and C-12. HMBC also provided the following cross-peaks of H-27 (δH 0.39)/C-13 (δC 30.2), C-8 (δC 34.5), C-18 (δC 46.3), and C-12 (δC 141.0), indicating the formation of in-plane cyclopropane among C-13, C-14, and C-27. Eight unsaturations, including five rings of ursane-type triterpenoid, one carbonyl, and a double bond by valence bond calculation, suggested the presence of a cyclopropane. These NMR data suggested that EAG is a hexacyclic triterpene skeleton with the 13, 27-cyclopropane ring structure (Cheng et al., 2010). The purity of EAG was higher than 95% analyzed by HPLC, see supporting information (Fig. S1.7). Configurational determination of 2,3-dihydroxy can be unambiguously carried out by analyzing signal peaks of the protons on oxygen bearing carbon atoms. The magnitudes of JH‑2/H‑3 (1.56 Hz) and JH-2/H-1 (10.9 Hz) indicated that these protons are in axial positions. The NOESY correlations of 13, 27-cyclopropane ring with H-16 was observed, indicating that the configuration was 13α, 27-cyclopropane ring. Spatial location of the two alkene hydrogens, that is, Z configuration, was deduced according to the coupling constant between the two olefins (J = 10.1 Hz). Thus, EAG was inferred to be 2α, 3α, 23trihydroxy-13α, 27-cyclours-11-en-28-oic acid. The 1H NMR and 13C NMR data of EAG are shown in Table 1. The COSY and key observed HMBC correlations are shown in Fig. 2.

ECLIPSETE2000-S fluorescence microscope at 350 nm excitation and 460 nm emission wavelengths. Western blot assay Total cell lysates from cultured NCI-H460 cells after EAG treatments as mentioned earlier were obtained by lysing the cells in ice-cold RIPA buffer with protease and phosphatase inhibitor and stored at −20 °C for future use. The protein concentrations were quantified by Bradford method (BIO-RAD) using Multimode varioscan instrument (Thermo Fischer Scientifics). Equal amounts of protein per lane was applied in 12% SDS polyacrylamide gel for electrophoresis and transferred to polyvinylidine difluoride (PVDF) membrane (Amersham Biosciences). After the membrane was blocked at room temperature for 2 h in blocking solution, primary antibody was added and incubated at 4 °C overnight. P-IκBα, p-IKKβ and NF-κB antibodies were purchased from Abcam, USA. After three TBST washes, the membrane was incubated with corresponding horseradish peroxidase-labeled secondary antibody (1:2000) (Santa Cruz) at room temperature for 1 h. Membranes were washed with TBST three times for 15 min and the protein blots were detected with chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with developer and fixed with fixer solution. In vitro cell migration assay/wound healing assay NCI-H460 cells (5 × 105 cells/well) were cultured in 6 well plates as confluent monolayers for 24 h. Then artificial scratch on the monolayers were created with 200 ml sterile pipette tip and washed twice with PBS to remove non-adherent cells. The media containing 0, 4, 8 and 12 μM of EAG were added to each well. The migration of cells across the scratched area were photographed by using phase contrast microscope (Nikon) at 0 h (immediately) and 24 h time interval after treatment in three or more randomly selected fields. Statistics The data were processed by the Student's t -test with the significance level p ≤ 0.05 using SPSS. Results and discussion Structure identification of EAG

Cytotoxic effects of EAG

The EtOAc-soluble fraction of aqueous ethanol (75%) extract from the whole grass of G. longituba was separated by silica gel column chromatography, RP-C18 column chromatography, MCI, Sephadex LH20 and preparative HPLC to obtain sixteen triterpene compounds (Fig. 1). Their structures were identified as 2α, 3α, 23-trihydroxy-13α, 27-cyclours-11-en-28-oic acid (1, EAG), 2α,3α,23-trihydroxyurs-12-en28-oic acid (2) (Sashida et al., 1992), 2α,3β,24-trihydroxy-olean-12-en28-oic acid (3) (García-Granados et al., 2007), 2α,3β,24-trihydroxyurs12-en-28-oic acid (4) (Sashida et al., 1992), 2α,3α,23- trihydroxyursa12,20(30)-dien-28-oic acid (5) (Sashida et al., 1992), 2α,3α-dihydroxyurs-12-en-28-oic acid (6) (Kojima and Ogura, 1986), 2α,3α,24-trihydroxyurs-12-en-28-oic acid (7) (Kojima et al., 1989), 2α,3β-dihydroxyolean-12-en-28-oic acid (8) (Sommerwerk et al., 2015), corosolic acid (9) (Wang et al., 2012), 2α,3β-dihydroxy-13α,27-cyclours-11-en-28-oic acid (10) (Cheng et al., 2010), ursolic acid (11) (Seebacher et al., 2003), 3α-hydroxyurs-12-en-28-oic acid (12), oleanolic acid (13) (Seebacher et al., 2003), 2α,3α,24-trihydroxyolean-12-en-28-oic acid (14) (Lee et al., 2008), betulinic acid (15) (Rosas et al., 2007), and betulin (16) (Sholichin et al., 2008). EAG was obtained as white powdery solid, [α]20D = +24.4 (c = 0.10, CH3OH). The molecular formula C30H46O5 was determined by HRESIMS spectrum (m/z 487.3417, [M + H]+) and 13C NMR data.

Since the antitumor activity of pentacyclic triterpene has been extensively studied, in the present work, we only evaluated the antitumor activity of the newly isolated hexacyclic triterpenoid. To start with, we explored the cytotoxic activity of EAG against a panel of human tumor cell lines including NCI-H460 (human lung cancer), T24 (human bladder cancer), SKOV3 (human ovarian cancer), HepG2 (human liver cancer) cells and HL-7702 (human hepatocytic). 10-hydroxycamptothecin (HCPT) was used as positive control. As depicted in Fig. 3A, EAG markedly inhibited the growth of all tested cancer cell lines, with IC50 value ranged from 4 to 11 µM. The NCI-H460 cell line was the most sensitive cell line toward EAG (Fig. 3A). Accordingly, we further tested the cytotoxicity of EAG in NCI-H460 cell line for different time. Fig. 3B clearly showed that EAG had a cytotoxic effect against NCI-H460 cells in a time- and dose-dependent manner. EAG had IC50 values of 13.41 ± 1.45 µM (24 h), 4.11 ± 0.49 µM (48 h) and 2.28 ± 0.17 µM (72 h) against NCI-H460 cells, respectively. In addition, the apoptotic effects of EAG on HL-7702 normal cells, compared with HCPT, were further detected by flow cytometer. The data showed that HCPT induced remarkable apoptosis on HL-7702 cells, while EAG had little effects on apoptosis (Fig. S1). Hence, NCI-H460 cells were used as models for the subsequent studies.

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Effects of EAG on NF-кB in NCI-H460 cells

Table 1 NMR data of compound 1 (δ in ppm, J in Hz, pyridine-d5). 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a

C

43.1 (t) 66.6 (d) 79.5 (d) 42.4 (s) 43.8 (d) 18.7 (t) 37.4 (t) 34.5 (s) 53.8 (d) 38.0 (s) 119.6 (d) 141.0 (d) 29.8 (s) 36.0 (s) 22.5 (t) 24.9 (t) 46.5 (s) 46.8 (d) 40.9 (d) 38.1 (d) 31.1 (t) 38.3 (t) 71.7 (t) 17.5 (q) 19.2 (q) 16.5 (q) 16.6 (t) 181.2 (s) 18.2 (q) 20.8 (q)

1

Recently, numbers of compounds have been found to inhibit NF-κB through interacting with key molecules in the signaling pathway (Rana et al., 2016). Triterpenoids exhibited potent anticacncer effects and were reported to be promising inhibitors of NF-κB (Huang et al., 2017; Kong et al., 2016). To investigate the effects of EAG on NF-κB, NCI-H460 cells were exposed to different concentrations of EAG, and then harvested for Western blot analysis. As shown in Fig. 4A, EAG significantly reduced phosphorylation of NF-кB p65 in a concentrationdependent manner, whereas showed minor inhibitory effect on the expression of total NF-κB p65 in NCI-H460 cells. Likewise, quantitative real time-PCR (qRT-PCR) results further confirmed the NF-κB p65 mRNA levels were dose-dependently suppressed by EAG (Fig. 4B). Taken together, these data implied that EAG exerted its anticancer potency by inhibiting NF-кB signaling pathway.

H

2.12 (oa,1H), 1.75 (o,1H) 4.30 (d, J = 10.9 Hz, 1H) 4.16 (d, J = 1.56 Hz,, 1H) 2.04 (o, 1H) 1.64 (o, 1H), 1.39 (o, 1H) 1.80 (o, 1H), 1.38 (o, 1H) 2.00 (o, 1H) 5.35 (d, J = 10.1 Hz, 1H) 6.12 (d, J = 10.3 Hz, 1H)

2.43 (o, 1H), 1.56 (o, 1H) 1.78 (o, 1H), 1.45 (o, 1H) 2.51 2.55 1.02 1.37 1.91 3.87 0.81 0.93 1.33 0.39

EAG inhibited the nuclear translocation of NF-кB p65

(d, J = 11.0 Hz, 1H) (s, 1H) (o, 1H) (o, 1H), 1.07 (o, 1H) (o, 1H), 1.80 (o, 1H) (d, J = 10.7 Hz, 1H), 3.72 (d, J = 10.8 Hz, 1H) (s, 3H) (s, 3H) (s, 3H), (d, J = 4.7 Hz, 1H), 1.30 (o, 1H)

Translocation to nuclear is a symbol for NF-κB activation and is an important step for NF-кB to activate various target genes transcription (Rajitha et al., 2016). Mechanistically, NF-κB nuclear translocation was examined using immunofluorescence staining. As shown in Fig. 4C, in untreated cells (control), an intense nuclear fluorescence was observed, showing the nuclear translocation of NF-κB. Treatment with EAG for 24 h dramatically diminished the nuclear accumulation of NF-κB p65 in a dose-dependent manner. Therefore, these results further confirmed that EAG significantly blocked the nuclear translocation of NF-κB.

1.07 (d, J = 6.1 Hz, 3H), 0.85 (t, J = 6.7 Hz, 3H)

o: overlapped.

EAG inhibits the activity of IKKβ and IκBα phosphorylation The phosphorylation and activation of the IKKα/β, which subsequently phosphorylates of IκBα, result in the nuclear translocation and activation of NF-κB (Christian et al., 2016). To elucidate if EAG -mediated inactivation of NF-κB is caused by inhibition IκBα phosphorylation, the expression of phosphorylated IKKα/β and IκBα were evaluated by Western blotting assay. As depicted in Fig. 4D, EAG markedly inhibited the phosphorylation of IκBα in a concentrationdependent manner. Meanwhile, as illustrated in Fig. 4D, similar trend was also observed on p-IKKα/β and NF-κB. The results revealed that EAG blocked NF-κB activation via impairment of IκBα phosphorylation mediated by the IKKα/β phosphorylation.

Fig. 2. COSY and key observed HMBC correlations for compound 1(EAG).

Fig. 3. Anticancer potency of EAG. (A) IC50 values of EAG and HCPT against different cancer cell lines, respectively. (B) Cell viability of EAG on NCI-H460 cells for the indicated times. Results are representative of at least three independent experiments and shown as the mean ± S.D. *p < 0.05, **p < 0.01 compared with control group. 5

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Fig. 4. Inhibition of NF-κB activation in NCIH460 cells by EAG. (A) Western blot analyses were conducted to evaluate the effects of EAG on the expression of NF-κB p65 and phosphorylated NF-κB p65. β-actin used as loading control. (B) Total mRNA were extracted and converted to cDNA and detected by qRT-PCR for NF-κB p65. (C) The effects of EAG on the nuclear translocation of the NF-κB protein. (D) The effect of EAG on the expression of p-IκBα and p-IKKα/β. (E) The effect of EAG on the expression of TAK1, TAB1 and AP-1. Results are representative of at least three independent experiments and shown as the mean ± S.D. *p < 0.05, **p < 0.01 compared with control group.

EAG inhibits TAK1–NF-κB and AP-1 signaling cascade in NCI-H460 cells

activate the intrinsic caspase pathway and lead to the apoptosis (Buglio et al., 2012). To determine whether TAK1–NF-κB signaling is involved in EAG–induced inhibition of NCI-H460 cells, we examined the TAK1 and TAB1 protein expressions in NCI-H460 cells. The effects of EAG on the constitutive levels of TAK1, TAB1 and c-jun in NCI-H460 cells are given in Fig. 4E. EAG treatment reduced the protein expressions of TAK1, TAB1 and c-jun in a concentration-dependent manner. These results suggested that EAG blocked the interaction of TAB1 with TAK1, thereby mediating the inactivation of TAK1 and downstream signaling.

As a mediator of the activated NF-κB or AP-1 signaling cascade, TAK1 (TGFβ–activated kinase 1) binds to the adaptor protein, TAB1, and subsequently activates downstream signaling kinases, such as inhibitor of IκB kinase (IKK)α/β, mitogen-activated protein kinase (MAPK), and c-jun NH2-kinase (JNK) and modulates the NFκB–dependent genes (Sato et al., 2005; Shim et al., 2005). Because disturbances in TAK1–NF-κB signaling are implicated in many types of cancer progression, inhibition of the TAK1–NF-κB cascade could 6

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Fig. 5. EAG triggered apoptosis through the mitochondrial death pathway. (A) Intracellular production of ROS by EAG following a 24 h incubation visualized by fluorescence microscope. (B) NCI-H460 cells were pretreated with or without NAC for 1 h, followed by 12 µM EAG for 24 h, and cells were subjected to flow cytometry analysis of apoptosis. (C) EAG decreased the mitochondrial membrane potential (MMP) of NCI-H460 cells, as measured via fluorescence microscope with JC-1 staining.

EAG triggers ROS generation

central to the process of death induced by EAG.

NF-κB is a well-known redox-sensitive transcription factor (Lee et al., 2009). Moreover, reactive oxygen species (ROS) have been reported to contribute to cell proliferation, apoptosis and cell death (Huang et al., 2018a). To determine whether EAG affects on ROS levels in NCI-H460 cells, the fluorescent staining was carried out to detect intracellular ROS production using DCFH-DA probe. No significant green fluorescence was observed in the control group cells, but in contrast, EAG significantly induced ROS production at the indicated concentrations as reflected by the increase green fluorescence (Fig. 5A). To identify the role of ROS in mediating EAG-induced anticancer effects in NCI-H460 cells, a common ROS scavenger, NAC was used to inhibit ROS production. As shown in Fig. 5B, co-treatment with NAC abolished EAG-induced apoptosis. These results suggested that anticancer effect of EAG in NCI-H460 cells was potentially mediated by increased ROS accumulation.

Effects of EAG morphological change of NCI-H460 cells To further validate cell apoptosis upon treatment of EAG, the morphological change NCI-H460 cells were detected by Hoechst 33258 staining after treatment with EAG for 24 h at concentrations of 4 μM, 8 μM and 12 μM. As shown in Fig. 6A, most of the cells exhibited the weak blue fluorescence; while for EAG treated group, over-brightened wizened nucleus can be observed. Remarkably, the morphologic change of apoptotic features were seen, such as cellular rounded, cell shrinkage, apoptosis bodies after NCI-H460 cells were treated with EAG for 24 h (Fig. 6A). These results indicated that apoptosis of NCI-H460 cells was induced by EAG in a concentration-dependent manner. EAG induces apoptosis in NCI-H460 cells The ability of EAG to induce apoptosis was further investigated by flow cytometry. NCI-H460 cells were allowed to grow in the presence or absence of EAG for 24 h, and co-stained with 7-AAD and Annexin-V/PE. Four quadrant images, Q1, Q2, Q3, and Q4, represented four different cell states: necrotic cells, late apoptotic or necrotic cells, early apoptotic cells and living cells, respectively. In the cells treated with EAG, we observed a dose-dependent increase in the percentage of apoptotic cells, at the concentrations of 4 μM, 8 μM and 12 μM for 24 h. As shown in Fig. 6B, few (1.21%) apoptotic cells were present in the control panel, in contrast, the percentage rose to 9.76% at the concentration of 4 μM after treatment with EAG for 24 h. In the presence of EAG with concentrations of 8 μM and 12 μM, the apoptotic cells were further increased to 15.56% and 23.71%, respectively. These data confirmed that

EAG reduce mitochondrial membrane potential Mitochondria are the major source of ROS that are closely connected with apoptosis (Diebold and Chandel, 2016). Mitochondria participate in the induction of apoptosis, and loss of mitochondrial membrane potential (MMP) is an early event in apoptotic process. Therefore, the influence of EAG on the MMP of NCI-H460 cells was examined by JC-1 staining. Our results indicated that treatment with EAG led to the loss of MMP in NCI-H460 cells (Fig. 5C). When NCIH460 cells were exposed to EAG, we found a dose-dependent enhancement in the green (monomeric) fluorescence versus that of control (Fig. 5C), further suggesting that mitochondrial dysfunction may be 7

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Fig. 6. EAG induced apoptosis in NCI-H460 cells. (A) EAG induced apoptotic in NCI-H460 cells were determined by Hoechst 33,258 staining and were photographed via fluorescence microscopy. (B) Annexin V-FITC and PI staining to evaluate apoptosis in NCI-H460 cells following EAG treatment. NCI-H460 cells were treated with EAG (4, 8 and 12 μM, for 24 h), incubated with annexin V-FITC and PI and analyzed using flow cytometry. (C) Cell cycle analysis of NCI-H460 cells treated with various concentrations of EAG for 24 h by flow cytometry. (D) EAG changed the protein expression levels of apoptosis regulators in NCI-H460 cells. Results are representative of at least three independent experiments and shown as the mean ± S.D. *p < 0.05, **p < 0.01 compared with control group.

EAG effectively induced apoptosis in NCI-H460 cells.

cells.

EAG induced cell cycle arrest in NCI-H460 cells

EAG suppresses the expression of related proteins involved in apoptosis and cell-cycle

To determine whether the growth inhibition observed resulted due to cell cycle arrest, NCI-H460 cells were exposed to various concentrations of EAG for 24 h, and the distribution of cells in the cycle was determined by propidium iodide (PI) staining and flow-cytometric analysis. As seen in Fig. 6C, EAG treatment resulted in a significantly higher number of cells in the G1 phase (from 30.55% to 56.02%) with gradually decreased cell number at S phase (67.85% to 36.90%) by the highest concentration treatment. These results indicated that EAG might have more potential to induce G1 cell cycle arrest for NCI-H460

After activation, NF-κB is translocated to the nucleus and activates transcription of genes responsible for apoptosis and cell growth, such as Bcl-2, c-Myc and Cyclin D1, which are overexpressed and required for malignant transformation and growth of cancer cells. Western blotting showed that EAG treatment resulted in a reduction of Bcl-2 and c-Myc in a concentration-dependent manner (Fig. 6D). In contrast, exposure of NCI-H460 cells to EAG caused a dramatic increase in Bax compared with control cells. 8

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Fig. 7. In vitro cell migration assay. NCI-H460 cells were treated with EAG (4, 8 and 12 μM) and artificial scratches were done with sterile 200 ml pipette. The images were captured by using a Nikon Te2000 deconvolution microscope at 0 h and 24 h.

Foundation of China (21431001, 81760626), the Ministry of Education Innovation Team Fund (IRT_16R15, 2016GXNSFGA380005), and Natural Science Foundation of Guangxi Province (AB17292075, 2017GXNSFAA198034) and Guangxi Funds for Distinguished Experts.

The cell death pathways generally include apoptosis (Type I), autophagy (Type II), and necrosis (Type III). Expression patterns of autophagy-related proteins provide key information about the autophagic state of a cell. As shown in Fig. 6D, the Western blot data revealed that the autophagy-related proteins LC3-I/II were not markedly change following EAG treatment. These findings indicated that autophagy was associated with cell death induced by EAG.

Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2019.153037.

EAG inhibited the migration of NCI-H460 cell in vitro

References

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Conclusion In summary, sixteen triterpenoids were isolated from an ethanolic extract of Glechoma longituba, leading to identify a new hexacyclic triterpene acid, 2α, 3α, 23-trihydroxy-13α, 27-cyclours-11-en-28-oic acid, and sixteen known triterpenes. The new compound, EAG, exhibited potential anticancer activity against five cancer cell lines and lower toxicity towards normal human cells. The present study demonstrated that EAG suppressed lung cancer cells growth via inducing apoptosis and cell-cycle arrest, at least partially, dependent on the ROS level and the mitochondrial membrane potential. A mode of action study further revealed that the NF-κB/AP-1 signaling pathway represents an important molecular target for anticancer potency of EAG. These data suggested that hexacyclic triterpene acid from an ethanolic extract of G. longituba has potential as a lead in developing drugs for the treatment of lung cancer. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by the National Natural Science 9

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