Biological and Pharmaceutical Bulletin
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Regular Articles
Identification and Characterization of a Novel NADPH Oxidase 1 (Nox1) Inhibitor That Suppresses Proliferation of Colon and Stomach Cancer Cells
Tsuyoshi YamamotoHirofumi NakanoKazuro ShiomiKiyofumi WanibuchiHisashi MasuiTakashi TakahashiYasuteru UranoTohru Kamata
Author information

2018 Volume 41 Issue 3 Pages 419-426


Reactive oxygen species (ROS) generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)1 mediate cellular signalings involved in normal physiological processes, and aberrant control of Nox1 has been implicated in the pathogenesis of various diseases. Therefore, Nox1 could have great potential as a therapeutic target. Here, we identified a novel Nox1 inhibitor, NOS31 secreted from Stretomyces sp. and analyzed its chemical structure. Furthermore, NOS31 was found to selectively inhibit Nox1-mediated ROS generation, with only a marginal effect on other Nox isoforms (Nox2–5) and no ROS scavenging activity. This compound blocked both Nox organizer 1 (NOXO1)/Nox activator 1 (NOXA1)-dependent and phorbol 12-myristate 13-acetate-stimulated Nox1-mediated ROS production in colon cancer cells. NOS31 inhibited the proliferation of several colon carcinoma and gastric cancer cell lines that upregulate the Nox1 system, whereas it had no appreciable effect on normal cells with low levels of Nox1. The finding suggests that NOS31 is a unique, potent Nox1 inhibitor of microbial origin and raises its possibility as a therapeutic agent for inhibiting gastrointestinal cancer cell growth.

There are accumulating evidences that reactive oxygen species (ROS), which catalyze reversible oxidation of target proteins, play an important role as second messenger-like molecules in a variety of physiological and pathophysiological processes. The predominant source for such intracellular ROS is represented by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs) and the related Dual oxidases (Duoxs). The Nox family members consist of five isoforms-Nox1–5.1) Noxs possess the conserved cytoplasmic NADPH/FAD-binding domain and the trans-membrane heme moiety and catalyze reduction of molecular oxygen (O2) to superoxide (O2) which is converted to H2O2. They differ in their tissue distribution, subcellular localization, and regulatory mode.1,2) Study over more than one decade revealed that Nox-dependent ROS elicit cellular signals in various cells in response to different stimuli including growth factors and cytokines. In addition, dysregulation of Nox-based oxidases has been recognized to be associated with diseases such as atherosclerosis, inflammation, diabetes and cancer.2,3) Among the Nox family proteins, Nox1 is regulated by its regulatory components, Nox organizer 1 (NOXO1) and Nox activator 1 (NOXA1). Nox1 is most abundantly expressed in the intestinal mucosa, and its normal role has recently been suggested to be involved in the maintenance of colon mucosal homeostasis4) and the restitution following colitis.5,6) With regard to the relationships with carcinogenesis, Nox1 is frequently upregulated in human colon cancer cell lines, and its expression increased along with the progression from adenoma to carcinoma.79) The constitutively activated Nox1 activity is thought to sustain the abnormal growth of colon cancer cells through increased production of signaling ROS.10,11) Furthermore, augmented Nox1-derived ROS production has been implicated in oncogenic K-Ras-induced transformation phenotype including anchorage-independent growth and Xenograft tumorigenicity,12) and the Nox1 expression was upregulted in colon cancer patient samples, correlating with hyperactivation mutation of K-Ras.8) Nox1 may also be involved in gastric cancer. The expression levels of Nox1 and NOXO1 were markedly increased in stomach cancer tissues as compared with their normal counterparts,13) and the study using the mouse model of gastric cancer demonstrated that Nox1-generated ROS are critical for development of tumor necrosis factor α (TNFα)-promoted stomach cancer.14) The available data suggest the causal relationships between oxidative stress by the dysregulated Nox1 system and progression of gastrointestinal cancer. Accordingly, antagonizing the activated Nox1 in some way may lead to an effective approach to suppress cancer phenotype, and Nox1 could be a potential molecular target for the therapeutic intervention of cancer.

In this setting, the compounds to inhibit Nox1-based oxidase could be quite useful in not only its functional study but also treating diseases involving the Nox1 enzyme. Several compounds have been registered and widely used as NADPH oxidase inhibitors, but most of them are non-specific and not unique for Nox isoforms.15) For example, diphenylen iodonium (DPI) acts as a general flavoprotein inhibitor that inhibits not only all the Nox isoforms but also endothelial nitric oxide synthase (eNOS) and Xanthine oxidase (XO).16) Apocynin displays the ROS scavenging activity, i.e., the antioxidant activity rather than suppressing the NADPH oxidase activity.17) The ideal NADPH oxidase inhibitor is desired to have Nox isoform selectivity and no ROS scavenging activity. Recently, several new NADPH oxidase small molecule inhibitors that meet this standard have been identified from rational drug screening. For example, GKT137831 (pyrazolopyridine dione derivative) is the currently best-characterized selective inhibitor of Nox4/Nox1 and underwent clinical trials for the potential treatment of Nox4-related idiopathic pulmonary fibrosis.18) This dual inhibitor also attenuates cardiac fibrosis in Nox4-transgenic mice with strong oxidative stress in cardiomyocites, which suggests its therapeutic potential in hypertensive heart disease.19) ML171 (2-acetylphenothiazine) is Nox1 selective and likely a potent Nox1 inhibitor that antagonizes Nox1-dependent invasion of cancer cells, although not tested for preclinical animal models.20)

Contrasting to these studies focused on rationally designed chemicals, we took an alternative strategy by screening microbial metabolites. Thus far, numerous natural compounds have been reported as NADPH oxidase modulators, but only a few seem to directly affect the NADPH oxidase activity.21) For example, PG-L-1, a prodigiosin derivative from sea-living microorganism is best characterized as a Nox2 inhibitor that inhibits ROS generation in macrophage cells.22) Here, we report the identification of a novel NADPH oxidase inhibitor NOS31 produced from Streptomyces sp. and its structural and biochemical analyses. NOS31 inhibits Nox1-dependent ROS production with high Nox1 selectivity and possesses no antioxidant activity. NOS31 treatment also specifically blocks proliferation of Nox1-upregulating cancer cells such as colon and stomach cancer cells. Thus, NOS31 represents a unique naturally occurring, bioactive Nox1 inhibitor.



DPI was purchased from Calbiochem (La Jolla, CA, U.S.A.), and XO, superoxide dismutase (SOD), horseradish peroxidase (HRP), hypoxanthine, phorbol 12-myristate 13-acetate (PMA), and luminol from Sigma-Aldrich (St. Louis, MO, U.S.A.). L012 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Diacetated 5-benzoyl carbonylfluoresceins (NBzFDA), a membrane-permeable precursor that is hydrolyzed to NBzF by intracellular esterases, was prepared as described previously.23)

Isolation of NOS31 and NOS35

The crude extract of a culture filtrate of Streptomyces sp. was provided by Kyowa Hakko Co., Ltd. (Tokyo, Japan). The crude extract was adjusted at pH 2 by adding 1 N H2SO4 and the sample (0.8 L) was concentrated under reduced pressure to remove methanol, followed by extraction with ethyl acetate. The ethyl acetate extract (10.0 g) was chromatographed on a silica gel column (40×160 mm, Silica gel 60, Merck: Kenilworth, NJ, U.S.A.) by eluting with a chloroform and methanol gradient solvent system. The fraction (2.4 g) eluted with chloroform/methanol (10/1) was concentrated, applied to a HP20 column (40×130 mm), and eluted with stepwise gradient of H2O/MeOH. The 100% MeOH fraction (403 mg) was applied to a reversed-phase HPLC column (20×250 mm, Pegasil ODS SP100, Sensyu Scientific Co., Ltd.: Tokyo, Japan). Elution with 55% MeOH, monitored at UV 210 nm, yielded three compounds, 1 (56.2 mg), 2 (41.3 mg) and 3 (20.7 mg). Major products 1 and 2 (hereafter referred to NOS31 and NOS35, respectively) were further subjected to structural analyses. Structural analyses were performed according to our published method.24)

Cell Culture

HeLa human epithelial carcinoma, Panc-1 pancreatic cancer, MCF-7 breast cancer, normal rat kidney (NRK), and HEK293 cells were purchased from American Type Culture Collection (ATC C: Manasssas, VA, U.S.A.). Colon cancer CaCO2, DLD-1, RKO, HT-29 and HCT-116 cells were also purchased from ATC C. Stomach cancer NUGC4, MKN7, MKN45, MKN74, and AGS cells were obtained from Dr. J. Nakayama. Cells were maintained at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen: Carlsbad, CA, U.S.A.) supplemented with 10% fetal bovine serum (Invitrogen), 100 unit/mL penicillin, and 100 g/mL streptomycin.

Vector Construction

Human Nox3 and Nox5 cDNAs were cloned into pcDNA3.0 (Invitrogen). pcDNA3.0-Nox1, pcDNA3.0-Nox2 and pcDNA3.3-Nox4 were previously described.25,26) pEFBOS-HA-NOXO1, pEFBOS-HA-NOXO1, pEFBOS-p67phox, pEFBOS-p47phox, and pEFBOS-p22phox were previously described.25)

Cytotoxity Assay

Cells were treated by NOS31 for 1 h, and cytotoxity assay was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Biotium: Hayward, CA, U.S.A.) according to the company’s protocol.

Cell Growth Assay

Live cells that were not stained by 0.5% trypan blue were counted.

Measurement of ROS Production

1. Fluorometric Analysis

Cells were trypsinized, suspended in Hanks’ Balanced Salt Solution (HBSS, Invitrogen), and mixed with 10 µM compounds or dimethyl sulfoxide (DMSO) as a control. The mixture (1–5×104 cells in 100 µL buffer per well) was dispensed to the 96-well plate and incubated in the CO2 incubator at 37°C for 30 min. A florescence probe NBzFDA (5 µM) was then added and incubated for additional 20 min. Florescence intensity at 520 nm with excitation at 490 nm was quantified by CytoFluor Multi-well Plate Reader Series 4000 (Thermo Fisher Scientific: San Jose, CA, U.S.A.).23)

2. Chemiluminescent Analysis

The cell suspension was treated with various chemical compounds and dispensed to the 96-well plate as described above and incubated in the CO2 incubator at 37°C for 45 min. HRP (4 U/mL) and 0.5 mM luminol or L012 were added and incubated at 37°C for 5 min in the CO2 incubator. Luminescence was quantified by Lmax (Molecular Devices: San Diego, CA, U.S.A.).

XO Assay

XO (0.05 U) with DMSO, NOS31 (10 µM), SOD (30 U), or DPI (2 µM) in 100 µL of HBSS was dispensed to the 96-well plate and preincubated at room temperature for 10 min. 0.5 mM hypoxanthine, 0.5 mM L012,26) and HRP (4 U/mL) were added and incubated at 37°C for 5 min. Luminescence was quantified as described above.

Assay of the NOS31 Selectivity to Nox1

For the assay of the selectivity to Nox1, HEK293 cells were co-transfected with equimolar pcDNA3.0-Nox1, pEFBOS-HA-NOXO1, pEFBOS-HA-NOXA1, and pEFBOS-p22phox. In the Nox2, Nox3, Nox4 and Nox5 selectivity assays, HEK293 cells were transfected with the appropriate expression vectors for each isoform. Nox2: pcDNA3.0-Nox2, pEFBOS-p67phox, pEFBOS-p47phox, and pEFBOS-p22phox. Nox3: pcDNA3.0-Nox3 and pEFBOS-p22phox. Nox4: pcDNA3.3-Nox4 and pEFBOS-p22phox. Nox5: pcDNA3.0-Nox5 (Nox5 does not require p22phox for the NADPH oxidase activity). Four micrograms of total DNA was transfected into 105 cells with Ca-Phosphate method (ProMega: Fitchburg, WI, U.S.A.). After 48 h, the cells were trypsinized, resuspended in 100 µL of HBSS, mixed with various concentrations of NOS31, dispensed to the 96-well plate and incubated in the CO2 incubator at 37°C for 45 min. A mixture (100 µL) of luminol (final 0.5 mM) and HRP (final 4 U/mL) was added and the sample plate was further incubated at 37°C for 5 min. Chemiluminescence was quantified by a luminometer. Data were fit in Graph Pad Prism5.

Statistical Analysis

Statistical analysis of the results from the two groups was performed using Student’s t-test. One-way ANOVA was performed with two or more groups, followed by Dunnett’s multiple-comparison test. Differences with p values of <0.05 were considered to be statistically significant. All statistical analyses were performed with IBM SPSS.


Structural Analysis of NOS31 and NOS35

NOS31 was obtained as colorless solid. The molecular formula of NOS31 was established as C28H28N2O5 according to high-resolution (HR) ESI MS (electrospray ionization mass spectrometry) analysis, implying 16 degrees of unsaturation. The IR (infrared absorption) indicated the presence of aromatic ring, hydroxy and ketone functionalities. The physico-chemical properties and molecular formula are shown in Table 1. The 1H- and 13C-NMR data for NOS31 are summarized in Table 2. Analysis of the 1H-NMR and hetero-nuclear single quantum coherence (HSQC) spectra indicated the presence of two singlet methyl protons (δH 3.80 and 1.85), two methylenes (δH 3.38, 3.30 and δH 2.75, 2.55), three methine protons (δH 5.60, 5.01 and 4.02) as well as nine aromatic protons (δH, 7.54, 7.25, 7.21, 7.04, 6.98, 6.83, 6.76, 6.51 and 6.47). The presence of a 1, 4-disubstituted benzene ring, a 1,2-disubstituted benzene ring and a 1,2,4-trisubstituted benzene ring was suggested on the basis of the coupling patterns of aromatic protons. The 1,4-disubstituted benzene ring was readily identified from the signals at H-2′ (2H, J=8.1) and H-3′ (2H, J=8.1). The heteronuclear multiple bond coherence (HMBC) correlations from H-2′ to C-2′ (δC 128.8) and C-4′ (δC 158.8) and from H-3′ to C-3′ (δC 116.2), C-4′ and C-1′ (δC 132.2) also confirmed the presence of C-4′ oxygenated phenyl moiety. The correlation spectroscopy (COSY) correlation of H-4 (δΗ 6.83)/H-5 (δΗ 6.47) and the HMBC correlations from H-4 to C-3a (δC 122.2), C-6 (δC 162.6) and C-7 (δC 97.1), from H-5 to C-7 (δC 97.1) and C-3a, and from H-7 (δC 6.51) to C-3a, C-5, C-6 and C-7a (δC 162.4) revealed the presence of 1,2,4 tri-substituted phenyl moiety. The correlations from H3-8 (δΗ 3.80) to C-6 (δC 162.4) proved the presence of a methyl ester group at C-6 position. The COSY correlations of H-4″ (δΗ.54)/H-5″ (δΗ 6.98)/H-6″ (δΗ 7.04)/H-7″ (δΗ7.25) and the HMBC correlations from H-4″ to C-3″ (δC 111.5) and C-7″a (δC 138.1) and from H-5″ to C-3″a (δC 129.7) and remaining aromatic carbon C-2″ (δC 135.3) were suggested the presence of indole ring. Moreover, the COSY correlations of H-8″ (δΗ 2.75 and δΗ 2.55)/H-9″ (δΗ 4.02)/H-10″ (δΗ 3.30 and δΗ 3.38) indicated the CH2–CH–CH2 partial structure. The HMBC correlations from H-8″ to C-2″, C-3″ and C-3″a indicated that the connectivity of C-3″/C-8″. The HMBC correlations from H-9″ to C-11″ (δC 173.1) and from H-12″ (δΗ 1.85) to C-11″ indicated the acetyl amino substitution at C-9″ (δC 54.6). From these analyses, the indole fragment might be N-acetyl tryptophanol moiety. The NMR data of this fragment was identical to that of a previously reported N-acetyl tryptophanol.27) These three aromatic fragments were connected as described below. First, the COSY correlation of H-2 (δΗ 5.60)/H-3 (δΗ 5.01) indicated connection of C-2 (δC 93.1) to C-3 (δC 50.0). The HMBC correlations from H-3 to C-2″ and C-3″ indicated the connection of C-3/C-2″. And the HMBC correlations from H-2 to C-2′ and from H-2′ to C-2 proved the connectivity of C-2 and 1,4-disubstituted phenyl fragment. Finally, from the HMBC correlation from H-3 to C-3a and the degree of unsaturation, 2,3-dihydrobenzofuran was formed. Based on these data, NOS31 was determined to be a new 2,3-disubstituted-2,3-dihydrobenzofuran compound, which has a p-hydroxyphenyl and an N-acetyl tryptophanol moieties (Fig. 1).

Table 1. Physico-chemical Properties of NOS31 and NOS35
Appearance[α]D26 (MeOH)Molecular formulaHR ESI-MS (m/z)UV λmax (MeOH) nm (ε)IR ν(KBr) cm−1
NOS31Colorless solid−69.88 (c 0.1)C28H28N2O5Found: 495.1895 Calcd: 495.1896 [M+Na]+294 (15810), 285 (17429), 226 (49333), 205 (64857)3395, 2935, 1619, 1596, 1519, 1496, 1457, 1145, 833, 748
NOS35Colorless solid−48.84 (c 0.1)C28H28N2O5Found: 495.1897 Calcd: 495.1896 [M+Na]+293 (11048), 285 (12429), 224 (33286), 204 (47667)3421, 2931, 1643, 1623, 1515, 1496, 1145, 748
Table 2. 1H- and 13C-NMR Data for NOS31 and NOS35
PositionδCδH (J, Hz)δCδH (J, Hz)
293.15.60 (d, 10.0)88.05.78 (d, 10.0)
350.05.01 (d, 10.0)55.34.77 (d, 10.0)
4125.96.83 (d, 8.0)126.16.86 (d, 8.0)
5107.66.47 (dd, 8.0, 2.2)107.76.50 (dd, 8.0, 2.4)
797.16.51 (d, 2.2)97.16.49 (d, 2.4)
855.83.80 (s)55.93.79 (s)
2′128.87.21 (d, 8.1)130.57.01 (d, 8.6)
3′116.26.76 (d, 8.1)116.56.72 (d, 8.6)
4″119.27.54 (d, 8.0)119.97.60 (d, 8.0)
5″119.76.98 (ddd, 8.0, 8.0, 1.2)119.96.99 (dd, 8.0, 8.0)
6″122.77.04 (ddd, 8.0, 8.0, 1.2)123.27.10 (dd, 8.0, 8.0)
7″111.97.25 (d, 8.0)112.37.30 (d, 8.0)
8″26.62.75 (dd, 14.6, 7.2), 2.55 (dd, 14.6, 6.4)26.62.78 (dd, 14.4, 7.0), 2.58 (dd, 14.4, 6.8)
9″54.64.02 (m)54.24.02 (m)
10″64.33.30 (m), 3.38 (dd, 10.8, 4.8)64.23.37 (dd, 11.0, 6.4), 3.27 (dd, 11.0, 4.6)
12″22.91.85 (s)22.91.88 (s)

1H-NMR: 400 MHz in CD3OD (ref. 3.31 ppm). 13C-NMR: 100 MHz in CD3OD (ref. 49.0 ppm).

Fig. 1. Structures of NOS31 and NOS35

NOS35 was obtained as colorless powder. HR-FAB MS analysis demonstrated that its molecular formula was the same as that of NOS31 (C28H28N2O5). The 1H- and 13C-NMR data of NOS35 were quite similar to that of NOS31 (Table 2). Analysis of the two-dimensional (2D) NMR spectra also indicated the presence of three aromatic fragments, 6-methoxy-2,3-dihydrobenzofuran, 1,4-disubstituted phenyl moiety and N-acetyl tryptophanol, as in NOS31. The 13C-NMR chemical shifts of C-2 (δC 88.0) and C-3 (δC 55.3) were slightly different from that of NOS31. The connectivity of three fragments was determined by detailed analysis of the HMBC spectrum. The HMBC correlation from H-2 (δΗ 5.78) to C-3″ (δC 112.6) proved the connection of C-2 and C-2″ of N-acetyl tryptophanol fragments. Moreover, the HMBC correlations of H-3 (δΗ 4.77) to C-2′ (δC 130.5) and H-2′ (δΗ 7.01) to C-3 (δC 55.3) indicated the connectivity of C-3 and 1, 4-disubstituted benzene ring. Based on these data, NOS35 was determined to be a 2,3-disubstituted-2,3-dihydrobenzofuran compound, in which the substitution pattern is opposite to that of NOS31 (Fig. 1). Taken together, these results indicate that NOS35 is a structural isomer of NOS31 although determination of their exact configuration awaits further study.

Effects of NOS31 on ROS Production in Colon Cancer Cells

To identify selective inhibitors of Nox1, we first screened various natural products derived from microbial metabolites. Nox1 is highly expressed and responsible for the increased level of intracellular H2O2 in colon cancer cells.7,8) We therefore developed the novel screening method, in which the ability of compounds to inhibit ROS generation in colon cancer CaCO2 cells was measured by using a fluorescence probe, NBzFDA.23) NBzFDA is highly sensitive and selective for hydrogen peroxide, and its practical use was previously demonstrated in detection of intracellular ROS produced in activated macrophages and epidermoid A431 cells.23) Among 200 compounds tested in the primary assay, NOS31 secreted from Streptomyces sp. was found to have the highest inhibitory activity (a 60% reduction of the ROS level at 10 µM), and its comparison with some of other samples was shown in Fig. 2A. Notably, prodigiosin, an inhibitor of the phagocytic Nox2 oxidase28) exhibited only the marginal inhibitory activity on the ROS production as compared with NOS31. Furthermore, the alternative ROS assay using a chemiluminescence probe L-012 also confirmed that NOS31 indeed blocked SOD-inhibitable superoxide generation in CaCO2 cells (Fig. 2B). The results suggest that the observed inhibitory activity of NOS31 in the primary screening is attributed to suppression of cellular ROS production but not interference of the ROS production probe NBzFDA, validating the usefulness of NBzFDA-based fluorescence assay in screening of Nox1-catalyzed ROS production. NOS31 inhibited ROS production in CaCO2 cells in a dose-dependent manner, and its structural isomer NOS35 had no inhibitory activity (Fig. 2C). The possibility that NOS31 behaves as a false positive was excluded by performing the cell-free H2O2-scavenging test and the MTT-based cytotoxity test. NOS31 neither scavenged H2O2 through direct reaction nor exhibited the cytotoxic activity (Figs. 2D, E). We also tested NOS31 for the ability to block the formation of ROS by one of flavoenzymes, XO which is responsible for cellular superoxide production. While SOD showed superoxide scavenging effects, NOS31 did not inhibit superoxide production by XO (Fig. 2F). In contrast, DPI, a general inhibitor for Noxs and other flavoenzymes suppressed XO, although to a lesser extent as compared with SOD (Fig. 2F). Thus, the data suggest the selectivity of NOS31 in blocking Nox1-dependent ROS generation.

Fig. 2. Characterization of Effects of NOS31 on ROS Production

(A) Comparison of various compounds for the ability to inhibit ROS production in colon cancer cells. Compounds H0142, M2011, M2014, M3514, and M3518 were isolated from the acid-treated fraction containing naphtoquinone derived from culture broth of the mitomycin producing strain of Streptomyces sp. Prodigiosin was isolated as described.28) CaCO2 cells were pretreated with 10 µM of indicated compounds for 1 h and subjected H2O2 production assay by using NBzFDA as described in Materials and Methods. The data represent mean±standard deviation (S.D.) (n=4), in separate three experiments. (B) Inhibition of superoxide production in CaCO2 cells by NOS31. CaCO2 cells were pretreated with 10 µM NOS31, SOD (100 U/200 µL), or DMSO for 1 h and subsequently incubated with 0.5 mM L-012 and HRP (4 U/mL). Relative luminescence intensity (RLU) was measured. The data represent mean±S.D. (n=4), in three separate experiments. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparison t-test. p versus DMSO control <0.05. (C) Dose-response of ROS production inhibition by NOS31, and comparison of NOS31 with NOS35. CaCO2 cells were pretreated with increasing amounts of NOS31 or with 10 µM NOS35 and subjected to ROS production assay as described in (B). The data represent mean±S.D. (n=4), in separate three experiments. (D) H2O2 scavenging test. 50 µM H2O2 was incubated with 10 µM NOS31 or DMSO for 10 min at 25°C and subjected to NBzFDA-based fluorescence assay. The data represent relative fluorescence intensity [mean±S.D. (n=4)], in three separate experiments. p versus DMSO control >0.05 by Student’s t-test. (E) Cytotoxity test. CaCO2 cells were treated with 10 µM NOS31 or DMSO for 1h and subjected to MTT assay. The data represent mean±S.D. (n=4), in three separate experiments. p versus DMSO control>0.05 by Student’s t-test. (F) Scavenging effects for superoxide production by hypoxanthine/xanthine oxidase system. Xanthine oxidase (0.05 U/200 µL) was incubated with 10 µM NOS31, SOD (30 U/200 µL), 5 µM DPI, or DMSO for 10 min at 25°C. Superoxide production was determined by chemiluminescent L-012 assay. The data represent mean±S.D. (n=4), in three separate experiments. Statistical analysis was performed by one-way ANOVA with Dunnett’s t-test. P1 versus DMSO control >0.05. P2 versus DMSO control <0.05.

Characterization of the Nox1-Inhibitory Activity of NOS31

To further establish the inhibitory effects of NOS31 on the Nox1-dependent ROS generation, HEK 293 cells were exogenously transfected with Nox1 and its regulatory components NOXO1 and NOXA1 and subjected to the dose-response analysis. Nox1-dependent ROS production was inhibited by NOS31 in a dose-dependent manner, and the ICD50 value for NOS31 was 2.0 µM (Fig. 3). Of note, NOS31 was much less potent with other Nox enzymes Nox2, Nox3, Nox4, and Nox5 in HEK293 reconstitution systems. The ICD50 value of NOS31 to Nox1 differed from those to Nox2–5 isoforms by at least 14 fold (Table 3), indicating that NOS31 has the excellent Nox1 selectivity.

Fig. 3. Dose–Response of Nox1-Inhibition by NOS31 in HEK293 Cell Systems

HEK 293 cells were co-transfected with Nox1, NOXO1, NOXA1, and p22phox expression vectors as described in Materials and Methods and treated with increasing amounts of NOS31 for 1 h and subjected to luminol-based ROS production assay. The ROS level was normalized to that in the transfected cells untreated with NOS31. The data represent mean±S.D. (n=4), in three separate experiments.

Table 3. ICD50 Values of NOS31
NoxsICD50 (µM)

Cell-based assay of ROS production was performed in the HEK293 reconstitution system as described in Materials and Methods.

The specificity of NOS31 inhibition to Nox1-derived ROS generation was further assessed in other control experiments. Because ROS generation in Nox1-upregulated CaCO2 cells is enhanced by overexpression of its regulatory subunits NOXO1 and NOXA1, we determined whether NOS31 prevents their enhancing activity. Our data showed that NOS31 impaired the ability of overexpressed NOXO1 and NOXA1 to increase ROS synthesis in CaCO2 cells (Fig. 4A). PMA has recently been reported to stimulate the NOXO1 phosphorylation via protein kinase C, promoting assembly of Nox1/NOXO1/NOXA1 complexes and thereby optimize H2O2 production by Nox1.29,30) This led us to examine the inhibitory effect of NOS31 on PMA-induced, Nox1-derived ROS production in colon cancer HT-29 cells. NOS31 markedly down-regulated PMA-stimulated ROS production in HT-29 cells (Fig. 4B). Since PMA also enhances the Nox2 activity by modulating phosphorylation of its regulatory subunit p67phox,31) and Nox2 as well as Nox1 is expressed in HT-29 cells, the observed reduction in the ROS level might be attributed to inhibition of the PMA-stimulated Nox2 activity but not the Nox1 activity. However, this possibility is ruled out because HT-29 cells do not possess p67phox intrinsically.32) Taken together, the results further support the notion that NOS31 is a potent and specific inhibitor for Nox1.

Fig. 4. NOS31 Inhibits the NOXO1/NOXA1-Dependent and PMA-Stimulated Nox1 Activity.

(A) NOS31 inhibits NOXO1/NOXA1-induced ROS production. CaCO2 cells were co-transfected with pEF-Boss-HA-NOXO1, pEF-Boss-HA-NOXA1 or control vectors, treated with 10 µM NOS31 or DMSO for 1 h and subjected to luminol-based ROS production assay. (B) NOS31 suppresses the PMA-induced Nox1 activity. HT-29 cells were treated with 10 µM NOS31 or DMSO for 1 h, incubated with or without 200 nM PMA for 10 min and subjected to luminol-based ROS production assay. Through (A) and (B), the data represent mean±S.D. (n=4), in three separate experiments. p versus control <0.05 by Student’s t-test.

Inhibitory Effects of NOS31 on the Growth of Cancer Cells

We next addressed the effect of NOS31 on the proliferation of several colon cancer cell lines in vitro. Treatment with NOS31 suppressed the growth of CaCO2 and HT-29 cells in the time- and dose-dependent manner, respectively (Fig. 5A, B). A 72 h exposure to 10 µM NOS31 decreased the growth rate of CaCO2, HT-29, DLD-1, RKO, and HCT-116 cells by 49–74% as compared with vehicle control treatment (Table 4). This is consistent with our previous finding that ablation of Nox1 by Nox1 small interfering RNA (siRNAs) attenuated the proliferation of colon cancer cells.10)

Fig. 5. NOS31 Inhibits Proliferation of Colon Cancer Cells

(A) CaCO2 cells (104 per well) were inoculated into 24 well plate and treated with 10 µM NOS31 or DMSO for indicated intervals. The cell number was counted. p versus DMSO control <0.05. (B) HT-29, MCF-7 and Panc-1 cells were treated with various concentrations of NOS31 or DMSO for 72 h, and the cell number was counted. Values were normalized to DMSO control (5.7×104). p for HT-29 versus MCF-7 or Panc-1 <0.05. Through (A) and (B), the data represent mean±S.D. (n=5), in three separate experiments. Statistical analysis was performed by one-way ANOVA, followed by Dunnett’s multiple comparison t-test.

Table 4. Effects of NOS31 on the Growth of Various Human Cancer Cells
Cell lines (cell growth: % control)
Colon cancerStomach cancerOthers

Various human cancer cells were seeded in a 24-well plate and cultured in the presence of 10 mM NOS31 or DMSO for 72 h. The cell number was counted. NIH3T3 and NRK cells as normal controls were also tested. Values were normalized to DMSO control (5–7×105 cells). The data represent mean±S.D. (n=5). All experiments were performed in two separate experiments.

The augmented expression of Nox1 and NOXO1 was detected in association with carcinogenesis in the human stomach, including intestinal- and diffuse-type adenocarcinoma.13) Moreover, the study utilizing a mouse model of gastric cancer demonstrated that a pro-inflammatory cytokine TNF-α increased Nox1-derived ROS generation by inducing the expression of NOXO1, thereby contributing to progression of gastric cancer.14) These observations suggest the involvement of Nox1-derived oxygen radicals in the development of inflammation-related stomach cancer. We therefore determined whether NOS31 could block the proliferation of gastric cancer cell lines. The growth rate of five stomach cancer cell lines with different differentiation phenotypes was significantly reduced following the compound treatment (Table 4). In contrast, the compound failed to inhibit the growth of breast cancer MCF-7, cervical carcinoma HeLa and pancreatic cancer Panc-1 cells (Fig. 5B and Table 4). Likewise, NOS31 did not significantly inhibit the growth of NIH3T3 and NRK cells (Table 4). While Nox1 is upregulated in all of colon cancer and stomach cancer cell lines tested, little or no Nox1 is expressed in HeLa, Panc-1, MCF-7, NIH3T3 and NRK cells.12,33,34) Thus, the higher susceptibility of Nox1-upregulating cells to NOS31 further strengthens the possibility that the compound preferentially targets the Nox1 system. Collectively, these results suggest that NOS31 potently blocks the growth of colorectal cancer as well as gastric cancer cells.

In conclusion, using our screening method, we identified a novel Nox1 inhibitor NOS31 from the culture of Streptomyces sp. It is worthwhile to note that NOS31 is a natural compound of microbial origin, while previously identified Nox1 inhibitors such as GKT137831 and ML171 come from rational drug discovery. NOS31 specifically blocked Nox1-dependent ROS generation, having neither direct antioxidant activity nor cytotoxity. The compound displayed only a marginal effect on other Nox isoforms Nox2–5 and almost no effect on the XO activity. NOS31 also showed the anti-proliferative activity toward colon and stomach cancer cell lines that upregulate the Nox1 oxidase. The detailed mechanism of inhibition of the Nox1-multisubunit enzyme by NOS31 remains to be determined. Nevertheless, the evidence for Nox1-specific inhibition by NOS31 indicates that NOS31 may have therapeutic potential in diseases involving deregulated Nox1, including cancer.


We thank Y. Hasegawa, Y. Terasawa and N. Takehara for technical assistance. We thank Dr. J. Nakayama for stomach cancer cell lines and Drs. H. Sumimoto and T. Leto for providing Nox expression vectors. This work was supported by Grant-in-Aid for Adaptable and Seamless Technology transfer program through target-driven R & D (A-STEP), the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.K.).

Conflict of Interest

The authors declare no conflict of interest.

© 2018 The Pharmaceutical Society of Japan