Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Chemical Constituents of Thai Citrus hystrix and Their Antiausterity Activity against the PANC‑1 Human Pancreatic Cancer Cell Line Sijia Sun,† Ampai Phrutivorapongkul,§ Dya Fita Dibwe,† Chandrasekar Balachandran,† and Suresh Awale*,† †
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Division of Natural Drug Discovery, Department of Translational Research, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan § Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand W Web-Enhanced Feature * S Supporting Information *
ABSTRACT: Human pancreatic cancer cells have an extreme tolerance to nutrition starvation, enabling them to survive in a hypovascular tumor microenvironment. Searching for agents that preferentially inhibit cancer cell viability under nutrition starvation conditions is a novel antiausterity strategy in anticancer drug discovery. In the present study, a hexane extract of the peels of Citrus hystrix fruits showed preferential cytotoxicity against PANC-1 human pancreatic cancer cells using a nutrient-deprived medium. Phytochemical investigation of this bioactive extract led to the isolation of 10 coumarins (1−10) including a new furanocoumarin (1). The isolated compounds were tested for their preferential cytotoxic activity against three different human pancreatic cancer cell lines [PANC-1, MIA PaCa-2, and PSN-1]. Among these, bergamottin (7) was identified as the most active constituent. In real-time live imaging, 7 was found to induce cell shrinkage, membrane blebbing, and disintegration of organelles in PANC-1 cells. Bergamottin (7) was also found to inhibit PANC-1 cell migration and colony formation. Mechanistically, 7 inhibited key survival proteins in the Akt/mTOR signaling pathway. Bergamottin (7) and related compounds are potential antiausterity candidates for drug development against pancreatic cancer. is commonly termed “austerity”.6 A search for agents that inhibit cancer cell tolerance to nutrition starvation (an antiausterity strategy) is a unique approach in anticancer drug discovery.7,8 Previous studies on this strategy have led to the discovery of some potent antiausterity agents of medicinal plants traditionally used, including those from Japan,7,8 Southeast Asia, 9−17 and the Democratic Republic of Congo.18−21 Some of these compounds also exhibited potent in vivo antitumor activity in mouse xenograft models.7,22−24 Recently, there has been a growing interest in the antiausterity strategy in anticancer drug discovery and has also inspired the efforts of synthetic chemists.24−30 Arctigenin, which was one of the most important antiausterity agents in this field, has successfully advanced to early phase II human clinical trial in the National Cancer Center Hospital East (Japan), and showed a significant survival benefit among advanced pancreatic cancer patients with no toxic effects.31 In a continuing effort, we recently screened the antiausterity activities of several Thai indigenous medicinal plants from Chiang Mai Province against the PANC-1 human pancreatic cancer cell line. We herein report the isolation and identification of the active constituents of Citrus hystrix DC.
P
ancreatic cancer is one of the most critical forms of cancer in having the lowest five-year survival rate of less than 5%.1−3 It is also one of the leading causes of deaths due to cancer in most developed countries, including Japan. Each year, an estimated 30 000 patients die due to pancreatic cancer in Japan, ranking, in turn, as fourth and fifth in terms of fatalities among female and male cancer patients. The incidence rate is equivalent to the mortality rate for this type of malignancy, and there has been no improvement in the survival rate despite the advances in the treatment modalities. Several chemotherapeutic agents such as gemcitabine, 5fluorouracil, and combination regimens such as S-1 (tegafur + gimeracil + oteracil potassium) and FOLFIRINOX (folinic acid +5-FU + irinotecan + oxaliplatin) are clinically used chemotherapeutic agents for advanced pancreatic cancer.4 However, human pancreatic cancers, in general, are resistant to these chemotherapeutic regimens and have shown no survival improvements. Therefore, there is an urgent demand for the discovery of alternative agents to combat this disease. Pancreatic tumors are hypovascular in nature and therefore lack a sufficient nutrient supply to aggressively growing tumor cells.5 Nevertheless, pancreatic cancer cells show an intrinsic tolerance to nutrition starvation and can survive for prolonged periods even in the complete absence of glucose, amino acids, and serum under physiological conditions. This survival capacity of cancer cells in the nutrition starvation condition © XXXX American Chemical Society and American Society of Pharmacognosy
Received: May 21, 2018
A
DOI: 10.1021/acs.jnatprod.8b00405 J. Nat. Prod. XXXX, XXX, XXX−XXX
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(Rutaceae) fruit peels together with the mechanism of action against PANC-1 cells.
Table 1. NMR Spectroscopic Data (500 MHz, CDCl3) for Compound 1
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position
RESULTS AND DISCUSSION Following the above-mentioned antiausterity strategy,21 Thai indigenous plants were screened against the PANC-1 human pancreatic cancer cell line in nutrient-deprived medium (NDM). Among these, a hexane extract of C. hystrix fruit peel showed a potent preferential cytotoxic (PC) activity in NDM with a PC50 value of 1.1 μg/mL, without causing toxicity in normal nutrient-rich conditions. A phytochemical investigation led to the isolation of a new prenylated furanocoumarin-type compound (1) together with (R)-(+)-oxypeucedanin methanolate (2),32 (R)-(+)-oxypeucedanin hydrate (3),33 isoimperatorin (4),34 (S)-(−)-oxypeucedanin (5),35 (R)(+)-pabulenol (6),36 bergamottin (7),37 (R)-(+)-6′-hydroxy7′-methoxybergamottin (8),35 (R)-(+)-6′, 7′-dihydroxybergamottin (9),37 and 7-hydroxycoumarin (10).38
δC, type
1 2 3 4 5 6 7 8 9 10 11 12 1′
161.2, C 113.0, CH 139.4, CH 148.8, C 114.0, C 158.2, C 94.6, CH 152.6, C 107.4, C 105.0, CH 145.1, CH 74.3, CH2
2′ 3′ 4′ 5′ OCH3
76.2, 75.9, 20.7, 20.7, 49.3,
CH C CH3 CH3 CH3
δH (J in Hz)
6.29, d (10.3) 8.23, d (10.3)
7.18, s
7.01, 7.60, 4.57, 4.39, 3.95,
d (2.3) d (2.3) dd (10.0, 3.2) dd (10.0, 7.7) dd (7.7, 3.2)
1.27, s 1.24, s 3.27, s
was determined from the combination of the 1H−1H COSY, HMQC, and HMBC spectra. The connectivity between C-1′− C-2′ was determined by the correlations between H2-1′ and H2′ in the 1H−1H COSY spectrum. In the HMBC spectrum (Figure 1), correlations between H3-4′/C-2′, H3-5′/C-2′, and
Compound 1 was isolated as a yellow, amorphous solid. Its molecular formula was determined by HRFABMS to be C17H18O6 [m/z 319.1228 (M + H)+]. The UV spectra of 1 showed characteristic absorptions of furanocoumarin.39 The IR spectrum of 1 showed the absorptions due to a hydroxy group (3417 cm−1) and α,β-unsaturated lactone (1698, 1654 cm−1) group. The 1H NMR spectrum of 1 displayed the signals (Table 1) due to two tertiary methyl groups (δH 1.24, 1.27), a methoxy (δH 3.27), an oxymethine (δH 3.95, dd, J = 7.7, 3.2 Hz), and an oxymethylene (δH 4.57, 4.39, each dd, J = 10.0, 7.7 Hz), together with two pairs of aromatic doublets in the downfield region (δH 8.23, 6.29, each J = 10.3 Hz; and δH 7.01, 7.60, each J = 2.3 Hz), and an aromatic singlet (δH 7.18). The 13 C NMR (Table 1) and DEPT spectra of 1 exhibited signals of 17 carbons including those for a characteristic furanocoumarin unit [δC 161.2, 158.1, 152.6, 148.8, 114.0, 107.4 (all qC), 145.1, 139.4, 113.0, 105.0, 94.6 (all CH)] and those for two tertiary methyl groups (both δC 20.7), an oxymethylene (δC 74.3), an oxymethine (δC 76.2), and a quaternary oxygenated carbon (δC 75.9). These 1H and 13C NMR data were similar to those of an oxypeucedanin hydrate (3),33 except for the appearance of additional signals due to a methoxy group (δH 3.27, δC 49.3). Therefore, compound 1 was assumed to have a methoxy group at either C-2′ or C-3′, which
Figure 1. Connectivity (bold lines) deduced by the COSY and HMQC spectra and significant HMBC correlations (solid arrows) of compound 1.
−OCH3/C-2′ suggested the location of a methoxy group at C2′ (δC 76.2) in the side chain. Similarly, correlations between H2-1′/C-5 of the furanocoumarin unit suggested the location of the 3-hydroxy-2-methoxy-3-methylbutoxy group side chain at C-5 in the furanocoumarin nucleus. Therefore, the planar structure of 1 was deduced as 2′-methoxyoxypeucedanin hydrate (1) (Figure 1). Finally, the absolute configuration of 1 was determined by comparing the [α]D and electronic circular dichroism (ECD) spectral data with those of (R)-(+)-oxypeucedanin hydrate (3). Compound 1 showed a negative specific optical rotation ([α]23D −39.8), which is opposite those of 3 with known absolute configuration40 ([α]25D +51). Moreover, the Cotton effect of 1 ([θ]238 − 2310, [θ]256 +1105) was found to be opposite those of 3 ([θ]235 +2288, [θ]253 −1273) (Figure 2), suggesting the absolute configuration at C-2′ in 1 to be opposite that of 3. Therefore, 1 was concluded as (S)-(−)-2′-methoxyoxypeucedanin hydrate (1). B
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Figure 3. Preferential cytotoxic activity of bergamottin (7) against the PANC-1 human pancreatic cancer cell line in NDM and DMEM.
Figure 2. ECD spectrum of compounds 1 and 3.
and form tumor colonies by invading the normal tissues. Therefore, compounds inhibiting such cancer cell migration and colony formation would have therapeutic benefit against tumor metastasis. To investigate the efficacy of bergamottin (7) against PANC-1 cell migration, a wound-healing assay was performed in standard DMEM medium. As shown in Figure 5, control cells showed significant migration compared to the wound gap at T0 (95% migration). However, treatment with 10 μM bergamottin caused significant inhibition in migration (30% migration). These results suggest that 7 has antimetastatic potential. The potential of 7 was further investigated for its effect against the PANC-1 colony formation. PANC-1 cells (500 cells/well) were plated in a 24-well plate and treated with 7 at concentrations of 12.5 and 25 μM. The cells were then allowed to grow colonies for 10 days in a humified CO2 incubator at 37 °C. Notably, 7 at the concentration of 12.5 μM significantly inhibited colony formation of PANC-1 cells with total inhibition at 25 μM (Figure 6). Therefore, bergamottin (7) not only showed preferential cytotoxicity against pancreatic cancer cells in nutrient-deprived condition but also prevented cell proliferation and colony formation in normal nutrient-rich conditions. To understand the mechanism of action of bergamottin (7), Western blot analysis was carried out to investigate its effect on the key survival proteins that are known to be overexpressed in pancreatic cancer cells during nutrient starvation. In particular, a serine/threonine kinase Akt is activated in the majority of human tumors and induced cell proliferation, survival, angiogenesis, invasion, and migration. Akt is also known to activate during nutrition starvation and acquire a tolerance to nutrition starvation.6 Antiausterity agents such as arctigenin, pyrvinium pamoate, kigamicin D, and grandifloracin have been found to be potent inhibitors of Akt.7,11,22,23 PANC-1 cells cultured in nutrient-deprived medium showed a significant increment in Akt phosphorylation with respect to time (Figure 7). Treatment with 7, however, inhibited p-Akt in a time- and concentration-dependent manner. It also decreased the expression of total Akt and the phosphorylation of downstream
All the isolated compounds (1−10) were tested against three different human pancreatic cancer cell lines [PANC-1, MIA PaCa-2, and PSN-1] in standard nutrient-rich medium (Dulbecco’s modified Eagle’s medium, DMEM) and nutrientdeprived medium. Arctigenin, an antiausterity agent, was used as positive control.7 The data are presented as preferential cytotoxicity (PC50) values, which are representative of 50% cancer cell death in NDM without toxicity in DMEM (Table 2). Among the tested compounds, bergamottin (7) displayed a strong preferential cytotoxicity in NDM in a concentrationdependent manner against all the tested cell lines (Figure 3) [PC50 4.6 μM (PANC-1), 2.2 μM (MIA PaCa-2), and 9.4 μM (PSN-1)]. However, other isolated coumarins with an oxidized olefin or those derived from a prenyl group showed only moderate activity, suggesting the requirement of a geranyl group for this type of activity (Table 2). Bergamottin (7) as the most active constituent found was investigated for its effect on cell morphology and apoptosis by ethidium bromide−acridine orange (EB-AO) double staining assay. As shown in Figure 4, the control PANC-1 cells displayed exclusive green fluorescence with intact cell morphology. Treatment with bergamottin led to a concentration-dependent increase in the cells emitting red fluorescence and altered cellular morphology, indicative of dead cells. Treatment with bergamottin (7) at 25 μM caused 100% cell death and emitted exclusive red fluorescence. Bergamottin (7) was further investigated for its real-time effects against PANC-1 cells in NDM using a live-cell imaging system.15 PANC-1 cells were treated with 10 μM bergamottin (7) in NDM and incubated in a stage-top CO2 incubator at 37 °C on a digital cell imaging station; images were captured every 10 min under the phase-contrast mode for 24 h. Bergamottin (7) caused shrinkage of PANC-1 cells within 6 h of treatment and plasma blebbing from 17 h leading to total cell death within 24 h (movie 1). Pancreatic cancer cells are highly metastatic. During the metastasis process, tumor cells migrate into the distant location
Table 2. Preferential Cytotoxicity (PC50)a of Compounds 1−10 against the PANC-1, MIA PaCa-2, and PSN-1 Human Pancreatic Cancer Cell Lines in Nutrient-Deprived Medium cell line
1
2
3
4
5
6
7
8
9
10
arctigeninb
PANC-1 MIA PaCa-2 PSN-1
18.8 51.1 40.6
89.6 87.0 89.7
41.2 79.4 89.7
41.2 37.2 41.7
76.5 38.0 74.6
86.6 >100 >100
4.6 2.2 9.4
37.8 37.1 37.8
76.5 49.4 76.1
>100 >100 >100
0.7 1.9 0.7
a
Concentration at which 50% of cells were killed preferentially in nutrient-deprived medium without causing toxicity in nutrient-rich medium (DMEM). bPositive control used in this study. C
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Figure 4. Morphological changes of PANC-1 cells induced by bergamottin (7) in nutrient-deprived medium (NDM). PANC-1 tumor cells were treated with 7 at the indicated concentrations in NDM in a 24-well plate and incubated for 24 h. Cells were stained with ethidium bromide (EB) and acridine orange (AO) and photographed under fluorescence (red and green) and phase-contrast modes using an EVOS FL digital microscope (20× objective).
Figure 5. Bergamottin (7) suppresses the migration of PANC-1 cells in a 24 h wound healing assay. (A) The broken white line indicates the wound areas at the start of the experiment. The assay was repeated three times, and sections of the representative images are shown. For the full image, see Supporting Information Figure S10. (B) Quantification of relative wound length. Data are mean ± SD. ***p < 0.001, vs control.
effector mTOR within 6 h of treatment, suggesting the inhibition of the Akt/mTOR signaling pathway by 7 leading to preferential cytotoxicity in nutrient starvation conditions. Bergamottin is one of the common constituents found in citrus fruits and juices. It is also known to interfere with the drug-metabolizing enzyme cytochrome P450 3A (CYP3A) in the intestine.41−44 Therefore, the combination of bergamottin with other drugs may cause adverse effects. However, bergamottin and other furanocoumarins are also main constituents of grapefruit juice, which is consumed worldwide and possesses beneficial biological activities such as antioxidative, anti-inflammatory, and antiosteoporosis activity.45
Bergamottin has been reported to suppress the proliferation of U266 multiple myeloma cells46 and NCI-87 gastric carcinoma cells.47 It also exhibits antitumor activity in lung adenocarcinoma in vivo through the induction of apoptosis, cell cycle arrest, and inhibition of cell migration and invasion.48 However, detailed studies on the anticancer activities of bergamottin against pancreatic cancer cell lines is still limited. In the present study, bergamottin (7) has been identified as a potent antiausterity agent capable of inducing preferential cytotoxicity against human pancreatic cancer cell lines under nutrition starvation conditions. Bergamottin (7) inhibited PANC-1 cell migration and colony formation and significantly D
DOI: 10.1021/acs.jnatprod.8b00405 J. Nat. Prod. XXXX, XXX, XXX−XXX
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ical Sciences, Faculty of Pharmacy, Chiang Mai University. A voucher specimen (APCH-2016) has been deposited at the Plant Herbarium Museum, Faculty of Pharmacy, Chiang Mai University, Thailand. Extraction and Isolation. The fresh fruits of Citrus hystix (40 kg) were collected and peeled to obtain 1.6 kg of fresh peels. These were dried and ground to obtain 336 g of powdered material, which was then extracted with 70% EtOH to give a hydroalcoholic extract (57.0 g, PC50 8.94 μg/mL). Next, 9.93 g of the extract was further partitioned with hexane and EtOAc to obtain a hexane extract (0.81 g, PC50 1.19 μg/mL), an EtOAc extract (3.23 g, PC50 6.7 μg/mL), and an aqueous extract (4.92 g, PC50 191 μg/mL). The most active hexane extract was chromatographed on silica gel by MPLC (Buchi MPLC, C-601/C-605 dual pump) using an n-hexane−EtOAc solvent system to give six fractions [fr. 1: n-hexane−EtOAc (95:5) eluate, 121 mg; fr. 2: n-hexane−EtOAc (85:15) eluate, 81.9 mg; fr. 3, n-hexane− EtOAc (75:25) eluate, 67.9 mg; fr. 4: n-hexane−EtOAc (70:30) eluate, 25.7 mg; fr. 5: n-hexane−EtOAc (50:50) eluate, 26.1 mg; fr. 6: EtOAc eluate, 13.5 mg]. Fraction 1 was subjected to normal-phase MPLC using an n-hexane−EtOAc gradient system to afford four subfractions [fr. 1-1: n-hexane eluate, 42.0 mg; fr. 1-2: n-hexane− EtOAc (98:02) eluate, 3.4 mg; fr. 1-3: n-hexane−EtOAc (96:04) eluate, 36.0 mg; fr. 1-4: n-hexane−EtOAc (94:06) eluate, 40.0 mg]. Subfraction 1-3 was purified using normal-phase preparative TLC with n-hexane−EtOAc (2:1) to give bergamottin37 (7, 16.0 mg), while subfraction 1-4 was purified using normal-phase preparative TLC with n-hexane−EtOAc (2:1) to give an additional amount of bergamottin37 (7, 15.0 mg) and isoimperatorin34 (4, 3.8 mg). Fraction 2 was subjected to normal-phase MPLC with an n-hexane− EtOAc gradient system to afford three subfractions [fr. 2-1: nhexane−EtOAc (94:06) eluate, 29.0 mg; fr. 2-2: n-hexane−EtOAc (90:10) eluate, 29.0 mg; fr. 2-3: n-hexane−EtOAc (85:15) eluate, 25.0 mg]. Subfraction 2-1 was purified using normal-phase preparative TLC with n-hexane−EtOAc (2:1) to give isoimperatorin34 (4, 12.0 mg). Subfraction 2-2 was subjected to TLC with H2O−CH3CN (2:1) and showed a mixture of two compounds; the mixture was further purified by HPLC on a ODS column (30 × 250 mm, 5 μm, Tosoh) using an eluent system consisting of solvent A (H2O) and solvent B (CH3CN), with a gradient (5 min 0% B, 30 min 30% B, 80 min 95% B), at a flow rate of 5 mL min−1, providing 0.6 mg of (S)-(−)-2′methoxyoxypeucedanin hydrate (1, 0.6 mg, tR 70.7 min) and (R)(+)-pabulenol36 (6, 0.5 mg, tR 71.3 min). Subfraction 2-3 was purified using normal-phase preparative TLC with n-hexane−EtOAc (2:1) to give (S)-(−)-oxypeucedanin35 (5, 7.0 mg). Fraction 3 was subjected to normal-phase MPLC with an n-hexane−EtOAc gradient system to afford two subfractions [fr. 3-1: n-hexane−EtOAc (82:18) eluate, 25.0 mg; fr. 3-2: n-hexane−EtOAc (79:21) eluate, 43.0 mg]. Subfraction 31 was purified by normal-phase preparative TLC with n-hexane− EtOAc (2:1) to give (S)-(−)-oxypeucedanin35 (5, 3.8 mg). Subfraction 3-2 was subjected to preparative HPLC on a ODS column (30 × 250 mm, 5 μm, Tosoh) using an eluent system consisting of solvent A (H2O) and solvent B (CH3CN), with a gradient (5 min 0% B, 30 min 30% B, 80 min 95% B), at a flow rate of 5 mL min−1, providing 0.6 mg of 7-hydroxycoumarin38 (10, 0.5 mg, tR 50.0 min). Fraction 4 was subjected to normal-phase MPLC with an n-hexane−EtOAc gradient system to afford three subfractions [fr. 4-1: n-hexane−EtOAc (76:24) eluate, 13.0 mg; fr. 4-2: n-hexane−EtOAc (73:27) eluate, 10.0 mg; fr. 4-3: n-hexane−EtOAc (70:30) eluate, 2.6 mg]. Subfraction 4-1 was purified using normal-phase preparative TLC with n-hexane−EtOAc (2:1) to give (R)-(+)-oxypeucedanin methnolate32 (2, 1.3 mg) and (R)-(+)-6′-hydroxy-7′-methoxybergamottin35 (8, 1.8 mg). Fraction 5 was subjected to normal-phase MPLC with an n-hexane−EtOAc gradient system to afford three subfractions [fr. 5-1: n-hexane−EtOAc (65:35) eluate, 4.6 mg; fr. 5-2: n-hexane−EtOAc (55:45) eluate, 15.0 mg; fr. 5-3: n-hexane−EtOAc (50:50) eluate, 6.7 mg]. Subfraction 5-2 was purified using reversephase preparative TLC with CH3CN−MeOH−H2O (2:2:1) to give (R)-(+)-oxypeucedanin hydrate33 (3, 1.5 mg) and (R)-(+)-6′,7′dihydroxybergamottin37 (9, 2.6 mg). 2′-Methoxyoxypeucedanin hydrate (1): yellow, amorphous powder; [α]23D −40 (c 0.05, CHCl3); UV (MeOH) λmax (log ε)
Figure 6. Effect of bergamottin (7) on colony formation by PANC-1 cells. (A) Representative wells showing PANC-1 cell colonies. See Supporting Information Figure S11 for three replication well images. (B) Graph showing mean values of the area occupied by PANC-1 cell colonies (three replications). **** p < 0.0001 when compared with the untreated control group.
Figure 7. Effect of bergamottin (7) against the expression key survival proteins p-Akt, Akt, p-mTOR, and mTOR under NDM in PANC-1 cells. GAPDH was used as a loading control.
inhibited key proteins in the Akt/mTOR signaling pathway; therefore, it represents an attractive candidate for drug development against pancreatic cancer based on the antiausterity strategy.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P2100 digital polarimeter. UV/vis spectra were taken on a U-5100 UV−vis ratio-beam spectrophotometer (Hitachi). CD measurements were carried out on a JASCO J-805 spectropolarimeter. IR spectra were measured with a JASCO FT/IR460 Plus spectrophotometer. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded using a JEOL ECA500II Delta spectrometer with tetramethylsilane as an internal standard, and chemical shifts are expressed in δ values. HRFABMS measurements were carried out on a JEOL JMS-AX505HAD mass spectrometer, and glycerol was used as the matrix. Medium-pressure liquid chromatography (MPLC) was performed with a Büchi MPLC C-605 binary gradient pump system with normal-phase silica gel (silica gel 60N, spherical, neutral, 40−50 μm, Kanto Chemical). Analytical and preparative TLC was carried out on precoated silica gel 60F254 and RP-18F254 plates (Merck, 0.25 or 0.50 mm thickness). Semipreparative HPLC separation was performed on an Agilent 1260 infinity quaternary LC VL instrument with a TSKgel ODS-100 V column (250 × 30 mm i.d.; 5 μm, Tosoh). Plant Material. The fresh fruits of C. hystrix (40 kg) were purchased from an organic farmer’s market, Muang district, Chiang Mai Province, Thailand, in June 2016. The specimens were identified by Dr. Wannaree Charoensup, Botanist, Department of PharmaceutE
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244 (1.30), 264 (0.43), 312 (0.43), 340 (0.24) nm; CD (EtOH) [θ]213 −1607, [θ]238 −2310, [θ]256 +1105; IR (KBr) νmax 3417, 2924, 2855, 2365, 1654, 1438, 1410, 1317, 1022, 954, 709, 674 cm−1; 1H NMR and 13C NMR see Table 1; HRFABMS m/z 319.1228 [M + H]+ (calcd for C17H19O6, 319.1182). Preferential Cytotoxicity Assay against Pancreatic Cancer Cells. The human pancreatic cancer cell lines PANC-1 (RBRCRCB2095) and MIA PaCa-2 (RBRC-RCB2094) from the Riken BRC cell bank and PSN-1 (ATCC CRM-CRL-3211) from the American Type Culture Collection were purchased and maintained in standard DMEM supplemented with 10% fetal bovine serum at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. Preferential cytotoxicity was determined as previously described.11 In brief, PANC-1 cells (1.5 × 104 cells/100 μL/well) in DMEM were seeded in 96-well plates and incubated for 24 h. The cells were then washed twice with Dulbecco’s phosphate-buffered saline (PBS) and replaced with either DMEM or NDM containing serially diluted test samples and incubated for 24 h. The medium was then replaced with 100 μL of DMEM containing 10% WST-8 cell counting kit solution. After 3 h of incubation, the absorbance was measured at 450 nm. Cell viability was calculated from the mean values for three wells using the following equation: Cell viability (%) =
Abs(test samples) − Abs(blank) Abs(control) − Abs(blank)
temperature with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulins as the secondary antibody. The bands were detected with an enhanced chemiluminescence solution (PerkinElmer).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00405. 1
H NMR, 13C NMR, HMQC, HMBC, COSY, UV, IR, and HRFABMS spectra for 1, HPLC profile and conditions for 1, 3, and 6, full image of wound healing assay and colony formation assay (PDF) Captures of live imaging (PDF)
W Web-Enhanced Feature *
Movie of live imaging of the effect of bergamottin (7, 10 μM) against PANC-1 cells is available in the HTML version of the paper.
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AUTHOR INFORMATION
Corresponding Author
The preferential cytotoxicity was expressed as the concentration at which 50% of cells died preferentially in NDM (PC50). Morphological Analysis. For the morphological investigation, 2 × 105 PANC-1 cells were seeded in 30 mm dishes and incubated overnight for attachment. The cells were then washed twice with PBS followed by treatment with bergamottin (7) in NDM and incubated for a further 24 h in a humified CO2 incubator. After a 24 h incubation period, cells were treated with 10 μL of AO and EB double staining reagent and incubated for 15 min in the dark. The images were then captured by an EVOS FL cell imaging system (20× objective). Live Cell Imaging. PANC-1 cells (2× 105) were plated in 35 mm cell culture dishes and allowed to attach overnight in complete medium. The cells were then washed twice with PBS, treated with 10 μM bergamottin (7) in NDM, and immediately placed inside a stagetop incubator (BLAST CP-089 AR) that was maintained at 37 °C and 5% CO2. Live images were captured under the phase contrast mode every 10 min on an EVOS FL cell imaging system for 24 h. Wound Healing Assay. PANC-1 cells (1.0 × 106) were plated in 35 mm cell culture dishes and incubated at 37 °C under humidified 5% CO2 for 12 h for cell attachment. The medium was then replaced with serum-free DMEM and allowed to incubate for a further 10 h. The cell monolayer was then scratched using 10 μL tips, washed twice with PBS, followed by treatment with bergamottin (7) (10 μM) in DMEM, and further incubated for 24 h. The images were captured under a Nikon-Eclipse TS100 microscope (10× objective). Colony Formation Assay. PANC-1 cells were plated in 24-well plates at a density of 500 cells/well in DMEM (1 mL/well) and incubated at 37 °C under humidified 5% CO2 for 24 h for cell attachment. The cells were treated with bergamottin (7) at noncytotoxic concentrations of 12.5 and 25 μM in DMEM and allowed to grow for 10 days. After this period, cells were washed with PBS, fixed with 4% formaldehyde, and stained with crystal violet for 15 min. Each experiment was repeated three times. Colony area measurement was carried out using the ImageJ plugin “Colony Area”.49 Western Blot Analysis. Proteins were separated by gel electrophoresis on a polyacrylamide gel containing 0.1% sodium dodecyl sulfate and transferred to polyvinylidene fluoride membranes. The membranes were blocked with Block Ace (DS Pharma Medical, Suita, Japan), washed with PBS containing 0.1% polyoxyethylene sorbitan monolaurate (Wako Pure Chemical), and incubated overnight with primary antibodies diluted in Can Get Signal (Toyobo, Osaka, Japan). After washing, the membranes were incubated for 45 min at room
*Tel: +81-76-434-7640. Fax: +81-76-434-7640. E-mail:
[email protected]. ORCID
Suresh Awale: 0000-0002-5299-193X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by a Grant in Aid for Scientific Research (JP16K08319) from the Japan Society for the Promotion of Science (JSPS) and by a President’s discretionary grant (# 011910) of the University of Toyama and a Director’s Leadership Grant from the Institute of Natural Medicine.
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REFERENCES
(1) Strong, K.; Mathers, C.; Epping-Jordan, J.; Resnikoff, S.; Ullrich, A. Eur. J. Cancer Prev. 2008, 17, 153−161. (2) Editorial Board of the Cancer Statistics in Japan. Cancer Statistics in Japan 2017; Foundation for Promotion of Cancer Research (FPCR), 2018. http://ganjoho.jp/en/professional/statistics/ brochure/index.html. (3) Li, D.; Xie, K.; Wolff, R.; Abbruzzese, J. L. Lancet 2004, 363, 1049−1057. (4) Van, C. E.; Vervenne, W. L.; Bennouna, J.; Humblet, Y.; Gill, S.; Laethem, J.-L V.; Verslype, C.; Scheithauer, W.; Shang, A.; Cosaert, J.; Moore, M. J. J. Clin. Oncol. 2009, 27, 2231−2237. (5) Chung, H. W.; Bang, S. M.; Park, S. W.; Chung, J. B.; Kang, J. K.; Kim, J. W.; Seong, J. S.; Lee, W. J.; Song, Y. S. Int. J. Radiat. Oncol., Biol., Phys. 2004, 60, 1494−1501. (6) Izuishi, K.; Kato, K.; Ogura, T.; Kinoshita, T.; Esumi, H. Cancer Res. 2000, 60, 6201−6207. (7) Awale, S.; Lu, J.; Kalauni, S. K.; Kurashima, Y.; Tezuka, Y.; Kadota, S.; Esumi, H. Cancer Res. 2006, 66, 1751−1757. (8) Awale, S.; Nakashima, E. M.; Kalauni, S. K.; Tezuka, Y.; Lu, J.; Kurashima, Y.; Esumi, H.; Kadota, S. Bioorg. Med. Chem. Lett. 2006, 16, 581−583. (9) Awale, S.; Ueda, J.; Athikomkulchai, S.; Abdelhamed, S.; Yokoyama, S.; Saiki, I.; Miyatake, R. J. Nat. Prod. 2012, 75, 1177− 1183.
F
DOI: 10.1021/acs.jnatprod.8b00405 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
(39) Najda, A.; Dyduch, J.; Świca, K.; Kapłan, M.; Papliński, R.; Sachadyn-Król, M.; Klimek, M. Food Sci. Technol. Res. 2015, 21, 67− 75. (40) Youkwan, J.; Sutthivaiyakit, S.; Sutthivaiyakit, P. J. Nat. Prod. 2010, 73, 1879−1883. (41) Bailey, D. G.; Malcolm, J.; Arnold, O.; Spence, J. D. Br. J. Clin. Pharmacol. 1998, 46, 101−110. (42) Paine, M. F.; Oberlies, N. H. Expert Opin. Drug Metab. Toxicol. 2007, 3, 67−80. (43) Paine, M. F.; Widmer, W. W.; Hart, H. L.; Pusek, S. N.; Beavers, K. L.; Criss, A. B.; Brown, S. S.; Thomas, B. F.; Watkins, P. B. Am. J. Clin. Nutr. 2006, 83, 1097−1105. (44) Hanley, M. J.; Cancalon, P.; Widmer, W. W.; Greenblatt, D. J. Expert Opin. Drug Metab. Toxicol. 2011, 7, 267−286. (45) Hung, W. L.; Suh, J. H.; Wang, Y. J. Food Drug Anal. 2017, 25, 71−83. (46) Kim, S. M.; Lee, J. H.; Sethi, G.; Kim, C.; Baek, S. H.; Nam, D.; Chung, W. S.; Kim, S. H.; Shim, B. S.; Ahn, K. S. Cancer Lett. 2014, 354, 153−163. (47) Sekiguchi, H.; Washida, K.; Murakami, A. J. Clin. Biochem. Nutr. 2008, 43, 109−117. (48) Wu, H. J.; Wu, H. B.; Zhao, Y. Q.; Chen, L. J.; Zou, H. Z. Oncol. Rep. 2016, 36, 324−332. (49) Guzmán, C.; Bagga, M.; Kaur, A.; Westermarck, J.; Abankwa, D. PLoS One 2014, 9, e92444.
(10) Awale, S.; Ueda, J.; Athikomkulchai, S.; Dibwe, D. F.; Abdelhamed, S.; Yokoyama, S.; Saiki, I.; Miyatake, R. J. Nat. Prod. 2012, 75, 1999−2002. (11) Ueda, J.; Athikomkulchai, S.; Miyatake, R.; Saiki, I.; Esumi, H.; Awale, S. Drug Des., Dev. Ther. 2014, 8, 39−47. (12) Awale, S.; Li, F.; Onozuka, H.; Esumi, H.; Tezuka, Y.; Kadota, S. Bioorg. Med. Chem. 2008, 16, 181−189. (13) Nguyen, N. T.; Nguyen, M. T.; Nguyen, H. X.; Dang, P. H.; Dibwe, D. F.; Esumi, H.; Awale, S. J. Nat. Prod. 2017, 80, 141−148. (14) Nguyen, H. X.; Nguyen, N. T.; Dang, P. H.; Thi, P. H.; Nguyen, M. T. T.; Can, M. V.; Dibwe, D. F.; Ueda, J.; Awale, S. Phytochemistry 2016, 122, 286−293. (15) Nguyen, H. X.; Do, T. N. V.; Le, T. H.; Nguyen, M. T. T.; Nguyen, N. T.; Esumi, H.; Awale, S. J. Nat. Prod. 2016, 79, 2053− 2059. (16) Nguyen, M. T. T.; Nguyen, N. T.; Nguyen, K. D. H.; Dau, H. T. T.; Nguyen, H. X.; Dang, P. H.; Le, T. M.; Phan, T. H. N.; Tran, A. H.; Nguyen, B. D.; Ueda, J.; Awale, S. Planta Med. 2014, 80, 193− 200. (17) Dibwe, D. F.; Awale, S.; Kadota, S.; Morita, H.; Tezuka, Y. J. Nat. Prod. 2014, 77, 1241−1244. (18) Li, J.; Seupel, R.; Bruhn, T.; Feineis, D.; Kaiser, M.; Brun, R.; Mudogo, V.; Awale, S. J. Nat. Prod. 2017, 80, 2807−2817. (19) Lombe, B. K.; Feineis, D.; Mudogo, V.; Brun, R.; Awale, S.; Bringmann, G. RSC Adv. 2018, 8, 5243−5254. (20) Fayez, S.; Feineis, D.; Mudogo, V.; Awale, S.; Bringmann, G. RSC Adv. 2017, 7, 53740−53751. (21) Kotoku, N.; Ishida, R.; Matsumoto, H.; Arai, M.; Toda, K.; Setiawan, A.; Muraoka, O.; Kobayashi, M. J. Org. Chem. 2017, 82, 1705−1718. (22) Lu, J.; Kunimoto, S.; Yamazaki, Y.; Kaminishi, M.; Esumi, H. Cancer Sci. 2004, 95, 547−552. (23) Esumi, H.; Lu, J.; Kurashima, Y.; Hanaoka, T. Cancer Sci. 2004, 95, 685−690. (24) Kudou, N.; Taniguchi, A.; Sugimoto, K.; Matsuya, Y.; Kawasaki, M.; Toyooka, N.; Miyoshi, C.; Awale, S.; Dibwe, D. F.; Esumi, H.; Kadota, S.; Tezuka, Y. Eur. J. Med. Chem. 2013, 60, 76−88. (25) Magolan, J.; Coster, M. J. Curr. Drug Delivery 2010, 7, 355− 369. (26) Magolan, J.; Adams, N. B. P.; Onozuka, H.; Hungerford, N. L.; Esumi, H.; Coster, M. J. ChemMedChem 2012, 7, 766−770. (27) Farley, C. M.; Dibwe, D. F.; Ueda, J.; Hall, E. A.; Awale, S.; Magolan, J. Bioorg. Med. Chem. Lett. 2016, 26, 1471−1474. (28) Turner, P. A.; Griffin, E. M.; Whatmore, J. L.; Shipman, M. Org. Lett. 2011, 13, 1056−1059. (29) Palframan, M. J.; Kociok-Köhn, G.; Lewis, S. E. Org. Lett. 2011, 13, 3150−3153. (30) Bergner, M.; Duquette, D. C.; Chio, L.; Stoltz, B. M. Org. Lett. 2015, 17, 3008−3010. (31) Ikeda, M.; Sato, A.; Mochizuki, N.; Toyosaki, K.; Miyoshi, C.; Fujioka, R.; Mitsunaga, S.; Ohno, I.; Hashimoto, Y.; Takahashi, H.; Hasegawa, H.; Nomura, S.; Takahashi, R.; Yomoda, S.; Tsuchihara, K.; Kishino, S.; Esumi, H. Cancer Sci. 2016, 107, 1818−1824. (32) Shokoohinia, Y.; Sajjadi, S. E.; Gholamzadeh, S.; Fattahi, A.; Behbahani, M. Pharm. Biol. 2014, 52, 1543−1549. (33) Gökay, O.; Kühner, D.; Los, M.; Götz, F.; Bertsche, U.; Albert, K. Anal. Bioanal. Chem. 2010, 398, 2039−2047. (34) Marumoto, S.; Miyazawa, M. Bioorg. Med. Chem. 2010, 18, 455−459. (35) Row, E. C.; Brown, S. A.; Stachulski, A. V.; Lennard, M. S. Org. Biomol. Chem. 2006, 4, 1604−1610. (36) Lee, Y. Y.; Lee, S.; Jin, J. L.; Yun-Choi, H. S. Arch. Pharmacal Res. 2003, 26, 723−726. (37) Girennavar, B.; Poulose, S. M.; Jayaprakasha, G. K.; Bhat, N. G.; Patil, B. S. Bioorg. Med. Chem. 2006, 14, 2606−2612. (38) Suzuki, H.; Ikeda, T.; Matsumoto, T.; Noguchi, M. Agric. Biol. Chem. 1977, 41, 205−206. G
DOI: 10.1021/acs.jnatprod.8b00405 J. Nat. Prod. XXXX, XXX, XXX−XXX
