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In vitro apoptotic effect of cassaine-type diterpene amides from Erythrophleum fordii on PC-3 prostate cancer cells
Bioorganic & Medicinal Chemistry Letters 24 (2014) 4989–4994
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
In vitro apoptotic effect of cassaine-type diterpene amides from Erythrophleum fordii on PC-3 prostate cancer cells Tran Manh Hung a, To Dao Cuong a, Jeong Ah Kim b, Jeong Hyung Lee c, Mi Hee Woo a, Byung Sun Min a,⇑ a
College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Republic of Korea College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea c College of Natural Science, Kangwon National University, Kangwon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 6 June 2014 Revised 26 August 2014 Accepted 9 September 2014 Available online 16 September 2014 Keywords: Erythrophleum fordii Leguminosae Cassaine diterpenoid amide Erythrophlesin H PC-3 apoptosis
a b s t r a c t Cytotoxic activity-guided fractionation of Erythrophleum fordii led to the isolation of two new cassaine diterpenoid–diterpenoid amide dimers, erythrophlesins H–I (1, 2). Spectral data indicated that they consist of asymmetrical dimeric structure via an ester bond between two cassaine diterpenoids. MTT assay confirmed that compound 1, erythrophlesin H, had the strongest cytotoxic effect toward the human prostate cancer cell line, PC-3. The molecular mechanism by which this compound induced apoptosis cell in prostate cancer remains unknown. Erythrophlesin H induced apoptosis in a dose-dependent manner. Acridine orange and annexin V-FITC/PI double staining confirmed that erythrophlesin H effectively induces apoptosis in PC-3 cells. Ó 2014 Elsevier Ltd. All rights reserved.
Prostate cancer (PCA) is the most commonly diagnosed cancer in men, and it continues to be a major problem throughout the developed world. PCA is important health concern due to the growing worldwide population of men over the age of 50 years, and it is the most common cancer in men and the leading cause of male cancer death after lung cancer.1 Conventional therapy to eradicate tumor cells can prolonged survival and cure some PCA patients.2 However, relapse and metastases occur frequently, and in general, these tumors are unresponsive to conventional therapy. In normal prostate tissue, androgens regulate the growth and differentiation of epithelial cells. In the early stages of prostate cancer, proliferation is increased by androgens, and it can be kept in check using various therapies aimed at either decreasing circulating androgens or blocking the androgen receptor. However, in advanced stages of prostate cancer, growth and development typically become refractory to androgen inhibitors, and tumor cells continue to grow unchecked.3 PC-3 cells are epithelial cells derived from a human prostate adenocarcinoma whose growth is androgen-independent.4 This androgen-insensitive cell line provides an excellent model system for both basic studies and for the evaluation of clinical strategies to treat human prostatic carcinoma. Currently, no therapy has been proven to prolong the survival of PCA patients.1 Therefore, new therapeutic approaches are needed, with the objective of
⇑ Corresponding author. Tel.: +82 53 850 3613; fax: +82 53 850 3602. E-mail address: [email protected] (B.S. Min). http://dx.doi.org/10.1016/j.bmcl.2014.09.025 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
overcoming tumor cell resistance and reducing drug-mediated toxicity. In this regard, many phytochemicals with diversified biological properties have shown promising responses for the prevention and/or intervention of prostate cancer.5,6 During our continued search for anti-cancer agents from natural plants, the methylene chloride fraction of the methanol extract of Erythrophleum fordii Oliver (Leguminosae) demonstrated cytotoxic activity against PC-3 cancer cells. E. fordii is widely distributed in China, Taiwan, and Vietnam, and it is used in Chinese traditional medicine to invigorate and promote blood circulation.7 In prior studies on the genus Erythrophleum, cassaine diterpenoid amines, amides, and oleanane-type triterpene saponins were identified as the main constituents of this genus;8 minor amounts of cassaine acids were also identified.9 Furthermore, cassaine alkaloids produced a digitalis-like action on the heart,10 and it was cytotoxic activity to several tumor cell lines.8 Recently, a phytochemical investigation on the leaves of E. fordii resulted in the isolation of cassaine diterpenoid–diterpenoid amide dimers and cassaine diterpenoid amides with cytotoxic activities against several human cancer cell lines.11 In our previous study on this plant, we reported the isolation and structural elucidation of serial mono cassaine diterpenoid amines and described their anti-angiogenic activity.12 In this study, we provided clear evidence that cassaine diterpenoid– diterpenoid amide dimer derivatives induce apoptosis induction in PC-3 human prostate cancer cells. Repeated column chromatography (silica gel, RP-18) of the CH2Cl2-soluble fraction resulted in the isolation of compounds 1
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O
12 11 15 20 13 1 9 H 14 17 10 8 5 H 7 4 O 6
2
O 16' 12'
15'
11' 13' 20' 1' 9' H 14' 2' 17' 8' 10' 5' H 3' 4' 7' O H 6' O 19' 18'
3
O O
22
N 16
OH
21
O
O
H
O
H O O
H
O
O
H
24
O
OH
H
19 18
1
N
H
HO H
O
O
2
O
21'
Figure 1. Chemical structure of isolated compounds (1–2) from E. fordii.
Table 1 NMR spectral data of compounds 1 and 2 Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 a b c
Compound 1
Compound 2
dHa (in CD3OD, J in Hz)
d Cb
dHa (in CD3OD, J in Hz)
d Cb
1.72(1H, m), 1.21 (1H, m) 2.08 (1H, m), 1.22 (1H, m) 4.63 (1H, dd, 12.0, 4.4)
37.9 25.3 80.3 48.3 52.1 41.1 210.9 52.0 47.9 36.2 28.4 26.1 167.5 40.2 113.1 169.3 12.6 23.6 178.7 15.5 54.1 60.5 41.0 54.5 38.9 25.5 37.7 39.8 54.8 41.0 212.1 52.1 46.7 42.9 28.6 25.3 169.1 41.0 113.2 172.6 12.0 28.4 175.2 15.5 53.0
1.85 (1H, m), 1.34 (1H, m) 2.45 (1H, m), 1.78 (1H, m) 4.64 (1H, dd, 12.0, 4.6)
37.5 26.2 80.4 49.1 53.8 39.9 211.3 53.0 47.9 37.5 28.5 27.2 171.1 39.8 116.7 172.6 12.5 21.5 175.2 15.2 53.8 60.7 38.9 54.5 37.6 25.2 80.4 49.5 54.5 41.0 211.4 53.0 47.9 42.9 28.5 25.4 171.4 41.0 116.9 172.6 12.0 28.2 175.2 15.4 52.0
1.43 (1H, dd, 14.0, 2.5) 2.62 (1H, d, 2.5), 2.86 (1H, d, 14.0) 2.24 (1H, m) 1.80 (1H, m) 2.12 (1H, m), 1.30 (1H, m) 3.74 (1H, m), 2.03 (1H, m) 2.83 (1H, m) 5.76 (1H, s) 1.08 (3H, d, 6.0) 1.26 (3H, s) 0.83 3.78 4.31 3.23 3.64 1.78 1.80 2.09
(3H, (2H, (2H, (3H, (3H, (1H, (1H, (1H,
s) m) t, 4.8) s) s) m), 1.20 (1H, m)c m), 1.65 (1H, m) m), 1.15 (1H, m)
1.57 (1H, dd, 15.0, 4.5) 2.60 (1H, m), 2.98 (1H, m) 1.90 (1H, m) 1.85 (1H, m) 1.95 (1H, m), 1.14 (1H, m) 3.30 (1H, m), 2.04 (1H, m) 2.62 (1H, m) 5.80 (1H, s) 1.08 (3H, d, 6.0) 1.22 (3H, s) 0.89 (3H, s) 3.71 (3H, s)
Recorded at 400 MHz. Recorded at 100 MHz. Overlapped signals. All assignments were confirmed by DEPT-135, HMQC and HMBC experiments.
1.38 (1H, dd, 14.0, 2.5) 2.65 (1H, d, 2.5), 2.89 (1H, d, 14.0) 2.48 (1H, dd, 13.0, 3.5) 1.82 (1H, m) 1.88 (1H, m), 1.30 (1H, m) 3.21 (1H, m), 2.14 (1H, m) 2.87 (1H, m) 5.91 (1H, s) 1.30 (3H, d, 6.4) 1.40 (3H, s) 1.14 3.50 3.67 3.36 3.71 1.78 2.10 4.60
(3H, (2H, (2H, (3H, (3H, (1H, (1H, (1H,
s) m) t, 5.6) s) s) m), 1.31 (1H, m) m), 1.72 (1H, m) dd, 12.0, 4.0)
1.68 (1H, dd, 15.0, 4.5) 2.62 (1H, m), 2.80 (1H, m) 1.91 (1H, m) 1.80 (1H, m) 2.10 (1H, m), 1.31 (1H, m) 3.04 (1H, m), 1.96 (1H, m) 2.54 (1H, m) 6.00 (1H, s) 1.06 (3H, d, 6.0) 1.23 (3H, s) 0.95 (3H, s) 3.72 (3H, s)
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T. M. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4989–4994
O
O
O O
N
O
OH
O
O
O O O
HO O
O O
O
O
N
H
O
O COSY
O
1
O
H
H
H
2
HO OH
O
HMBC
O
O
H H
H
H
H H
H
H HO H
O 1
OH
O
O
O
N
right unit
NOE
O
2
left unit
Figure 2. Selected COSY, HMBC and NOEs correlations for 1 and 2.
Figure 3. Cytotoxic activity of compounds 1 and 2.
and 213a (Fig. 1). Compound 1 was isolated as a white powder. A quasi-molecular ion at m/z 780.5514 (calc. 780.5525) [M+H]+ in the positive HR-ESI-MS indicated a molecular formula of C45H65NO10. Its IR spectrum showed absorption bands at 3375 and 1720 cm1, indicative of the presence of hydroxyl and carbonyl groups in the molecule.13b The 1H NMR spectrum of 1 revealed signals for six methyl groups, two methoxy groups (dH 3.64, 3H, s, H-24 and dH 3.71, 3H, s, H-210 ), two exocyclic olefinic protons (dH 5.76, 1H, s, H-15 and dH 5.80, 3H, s, H-150 ), one oxymethine (dH 4.63, 1H, dd, 12.0, 4.4 Hz, H-3), and a methyl amide N–CH3 (dH 2.67, 3H, s, H-23) (Table 1). The 13C NMR spectrum of 1 displayed resonances for two ketones at dC 210.9 (C-7) and 212.1 (C-70 ), four ester carbonyl groups
at dC 178.7 (C-19), 175.2 (C-190 ), 169.3 (C-16), and 172.6 (C-160 ), two sp2 methines at dC 113.1 (C-15) and 113.2 (C-150 ), two quaternary sp2 carbons at dC 167.5 (C-13) and 169.1 (C-130 ), one oxymethine at dC 80.3 (C-3), and two methoxy groups at dC 54.5 (C-24) and 53.0 (C-210 ) (Table 1). All signals in the above spectra were further supported by HMQC spectroscopic analyses. Since most of the 1H and 13C NMR signals of 1 were observed as pairs, it was inferred that 1 had a dimeric structure consisting of two tricyclic diterpenoids in Erythrophleum sp.11,14 Detailed analysis of 1H–1H COSY and HMBC correlation data indicated that compound 1 was constructed from two cassaine diterpenoids connected through an ester linkage in the presence of a 7-keton group for both monomer units (left and
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Con
3 M
10 M
30 M
G0/G1 S G2/M
0 66.6 14.3 19.1
3 59.9 15.1 24.9
10 49.9 10.9 39.2
30 43.5 10.1 46.4
Figure 4. DNA content analysis in erythrophlesin H treated PC-3 cells. Erythrophlesin H was treated as described above and sub-G1 cell populations stained with propidium iodide (PI) were determined by flow cytometry (FACS). Fluorescence intensities are shown on a log scale. Results show a typical experiment which has been repeated three times.
right units). The 1H–1H COSY spectrum showed proton connectivity for H-5/H-6 and H-50 /H-60 , confirming the location of the ketone group at C-7 for both the left and right units (Fig. 2). A conjugate ester moiety is located at the C-13/C-15/C-16 position of both units, as revealed from the HMBC correlations for H-14/C-13, H-14/C-15, H2-12/C-15, H-15/C-12, and H-15/C-16 observed for each unit. The C-16 ester carbonyl of the right unit at dC 169.3 was connected to an N-methyl amide group that was assigned by dH 2.67 (H-23), 3.87 (H-21), and 4.31 (H-22) and dC 54.1 (C-21), 60.5 (C-22), and 40.6 (N-Me, C-23) in 1H and 13C NMR spectra. The HMBC spectrum confirmed correlations between protons H-23, H-21 and C-16; H-23 and C-21; and H-21 and C-22 (Fig. 2). The adjacent C-4 position of the both of left and right units bore a methoxycarbonyl group and a tertiary methyl group due to the diagnostic HMBC correlation observation between H-18, C-4, and C-19, as well as between H-24 and C-19. In addition, the C-160 ester carbonyl of the left unit at dC 172.6 displayed a HMBC correlation signal with an oxymethine at dH 4.46 (H-3) on the right unit (Fig. 2), indicating the connection of the two monomer units.11,14 A comparison of the 1H and 13C NMR spectroscopic data from 1 with those of erythrophlesin D suggested they share the same skeleton, except for the location of 6,60 -methylene and 7,70 -keto groups in 1.11 This difference was supported by cross-peaks from H-5/H-50 to H-6/60 in the COSY spectrum (Fig. 2). According to the biogenetic isoprene rule of the cassaine skeleton, the two methyl groups attached to the rings were in the 10b and 14a orientations in both of the units.11,14 The J value of H-3 (dd, J = 12.0, 4.0 Hz) suggested an b-orientation for the ether linkage. The aforementioned considerations were further substantiated by significant NOE correlations between H-3, H-5 and H-18, H-5 and H-9, and H-9 and H-17 (a-orientation) (Fig. 2). The NOE correlations between H-8 and H-14, H-8 and H-20, and H-20 and H-24 indicated
that these protons were b-orientated (Fig. 2). Furthermore, in both monomer units, the NOE correlation between H-14 and H-15 indicated that the double bond at C-13/C-15 had the E-conformation.11,14 From these results, we concluded that the structure of erythrophlesin H was 1. Erythrophlesin I (2) had a molecular formula C45H65NO11, as established by HR-ESI-MS data (m/z 796.5269, calcd. for C45H66NO11 [M+H]+, 796.5273), which was 16 mass units greater than the co-occurring erythrophlesin H compound 1.13 Inspection of the 1D (1H and 13C) and 2D (HMQC, COSY and HMBC) NMR data of 2 established the presence of nearly identical structural features to those of 1 (Table 1). Comparison of the 1H and 13C NMR spectra of 2 with 1 revealed that these compounds were similar cassaine diterpenoid–diterpenoid amide dimmers, except resonances for the hydroxyl group at position three of the left unit of 2 was present at dH 4.60 (1H, dd, J = 12.0, 4.4 Hz, H-30 ) and dC 80.3 (C-30 ) (Table 1). This was confirmed by HMBC correlations from the H-50 to dC 80.4 (C-30 ) and dH 4.65 (H-30 ) to dC 48.0 (C-40 ) (Fig. 2). An HMBC correlation of H-3 to the ester carbonyl at dC 172.6 (C-160 ) confirmed the left unit attachment to C-3 of the right unit (Fig. 2). Moreover, in addition to similar correlation signals with 1, the oxygenated methine at dH 4.60 (H-30 ) was present as a double doublet (J = 12.0, 4.4 Hz), and NOE correlations between H-30 , H-50 , and H-180 in the left unit of 2 indicated that these protons were in the same a-orientation; therefore, the hydroxyl group at C-30 was b-orientated (Fig. 2).14 From these results, we concluded that the structure of erythrophlesin I was 2. The cytotoxic activity of compounds 1 and 2 were evaluated against a panel of human cancer cell lines (A549, MCF-7, PC-3 and HepG2). Cell growth was measured using MTT assays.15 Compound 1, erythrophlesin H, displayed potently cytotoxic activity
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flow cytometry (BD FACSCalibur flow).15 Annexin V-FLUOS/PI double-staining analysis demonstrated that erythrophlesin H decreased the number of PC-3 cells in a dose-dependent manner. Fig. 4 presents histograms of the DNA content from PC-3 cells treated with erythrophlesin H for 24 h. Sub-G1 DNA increased up to 48 h in response to 3, 10 and 30 lM erythrophlesin H, reached approximately 46.4%. Moreover, the number of cells in the early and late stages of apoptosis increased in a dose-dependent manner (Fig. 5A). Erythrophlesin H induced apoptosis in PC-3 cells, especially late stage apoptosis. The percentage of apoptotic cells, including early and late stage of apoptosis, increased from 0% (control) to appropriate 10% in response to erythrophlesin H treatment (Fig. 5B). Thus, erythrophlesin H showed a dramatically induced apoptosis in PC-3 cells in a dose-dependent manner. The development of anti-cancer drugs that inhibit abnormal cancer cell proliferation and induce cell death through apoptosis is a fundamental objective of cancer research. Here, we demonstrate that erythrophlesin H, a compound present in the bark of E. fordii, induces apoptosis in PC-3 cancer cells. We are currently investigating the feasibility of further developing this chemical structure as a chemo-preventive agent. Acknowledgments This research was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (KRF-2012R1A2003547) and BK-21 Plus (22A20130000073). We are grateful to the Korean Basic Science Institute (KBSI) for supplying the MASS spectra. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09. 025. References and notes Figure 5. Flow cytometric analysis of erythrophlesin H-induced apoptosis in PC-3 cell using annexin-V-FITC/PI. Cells (1 106 cells) were incubated with indicated concentration of erythrophlesin H for 48 h and stained with annexin-V-FITC/PI to analysis apoptosis and necrotic cell populations. Cells in the lower right quadrant represented apoptosis and in the upper right quadrant. Data are representative of one of three similar experiments.
against all the cell lines in a dose-dependent manner with IC50 values of 24.5, 17.2, 12.5 and 25.2 lM, respectively (Fig. 3). Camptothecin, which was effectively used as a positive control, exhibited the cytotoxic activity with IC50 values ranging from 0.54 to 1.02 lM against all kind of cancer cell lines. The structure of 1 and 2 are similar, except for the substitution at C-3 of left unit in the cassaine diterpenoid–diterpenoid amide dimmer skeleton. Compound 2, erythrophlesin I with a hydroxyl group at C-30 , exhibited weak cytotoxicity against all cancer cell lines. These data indicate that the substitution at C-30 might influence its cytotoxic activity. Because erythrophlesin H (1) exhibited the most potent cytotoxic activity against PC-3 cells (IC50 12.5 lM), we further evaluated its inhibition of PC-3 cell proliferation.15 Cells were treated with various concentrations of erythrophlesin H (3–30 lM), incubated for 7 days, and counted at 1-day intervals using the trypan blue exclusion method. The growth of cells treated with erythrophlesin H was significantly inhibited after 2 days compared to control cells (data not shown). To quantify the modes of erythrophlesin H (1)-induced cell death, PC-3 cell were treated with erythrophlesin H (3–30 lM) for 48 h, stained with annexin V-FLUOS and PI, and analyzed by
1. Jemal, A.; Tiwari, R. C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer, E. J.; Thun, M. J. CA Cancer J. Clin. 2004, 54, 8. 2. Brawley, O. W.; Barnes, S.; Parnes, H. Ann. N.Y. Acad. Sci. 2001, 952, 145. 3. Feldman, B. J.; Feldman, D. Nat. Rev. Cancer 2001, 1, 34. 4. Kaighn, M. E.; Narayan, K. S.; Ohnuki, Y.; Lechner, I. F.; Jones, L. W. Invest. Urol. 1979, 17, 16. 5. Surh, Y. J. Nat. Rev. Cancer 2003, 3, 768. 6. Barnes, S. Epidemiol. Rev. 2001, 23, 102. 7. Chen, J. S.; Zhen, S. Chinese Virose Plant, 1st ed.; Science Press: Beijing, 1987. p. 321. 8. (a) Culvenor, C. C. J.; Loder, J. W.; Nearn, R. Phytochemistry 1971, 10, 2793; (b) Cronlund, A.; Sandberg, F. Acta Pharm. Suec. 1976, 13, 35; (c) Qu, J.; Hu, Y. C.; Yu, S. S.; Chen, X. G.; Li, Y. Planta Med. 2006, 72, 442; (d) Du, D.; Fang, L.; Qu, J.; Yu, S.; Ma, S.; Lv, H.; Liu, J.; Liu, Y.; Wang, J.; Wang, X. Planta Med. 2011, 77, 1631. 9. (a) Li, N.; Yu, F.; Yu, S. S. Acta Bot. Sin. 2004, 46, 371; (b) Yu, F.; Li, N.; Yu, S. S. J. Asian Nat. Prod. Res. 2005, 7, 19; (c) Qu, J.; Wang, Y. H.; Li, J. B.; Yu, S. S.; Li, Y.; Liu, Y. B. Rapid Commun. Mass Spectrom. 2007, 21, 2109; (d) Tsao, C. C.; Shen, Y. C.; Su, C. R.; Li, C. Y.; Liou, M. J.; Dung, N. X.; Wu, T. S. Bioorg. Med. Chem. 2008, 16, 9867. 10. Verotta, L.; Aburjai, T.; Rogers, C. B.; Dorigo, P.; Maragno, I.; Fraccarollo, D.; Giovanni, S.; Gaion, R. M.; Floreani, M.; Carpenedo, F. Planta Med. 1995, 61, 271. 11. Du, D.; Qu, J.; Wang, J. M.; Yu, S. S.; Chen, X. G.; Xu, S.; Ma, S. G.; Li, Y.; Ding, G. Z.; Fang, L. Phytochemistry 2010, 71, 1749. 12. Hung, T. M.; Cuong, T. D.; Kim, J. A.; Tae, N.; Lee, J. H.; Min, B. S. Bioorg. Med. Chem. Let. 2013, 24, 168. 13. E. fordii Oliver (Leguminosae) bark was collected from Quang Nam province, Vietnam, in May 2012. Professor Tran Cong Luan at Hochiminh City University of Medicine and Pharmacy performed the botanical identification, and a voucher specimen (CUD-3177) was deposited at Herbarium of the College of Pharmacy, Catholic University of Daegu, Korea. The bark (2.0 kg) was extracted with MeOH three times (3 h 3 L) under reflux. After solvent removal under reduced pressure, the residue (426.0 g) was suspended in H2O and partitioned sequentially versus CH2Cl2 and EtOAc. The CH2Cl2 soluble fraction (40.0 g) was separated on a silica gel column (50 10 cm) using a stepwise gradient of CHCl3/MeOH (40:1 ? 1:1, v/v) to yield 27 fractions (A1–A27), according to their TLC profiles. Fraction A27 (3.7 g) was subjected to silica gel column
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chromatography (60 5 cm) using CHCl3–MeOH plus 0.1% acetic acid (7:1) as an eluent to yield seven sub-fractions (A27-1–A27.7). Fraction A27-2 (106.0 mg) was chromatographed on a silica gel column (50 2.5 cm) using a CHCl3–MeOH gradient (7:1) to produce compound 1 (5.0 mg). Fraction A27.5 (146.0 mg) was further chromatographed on a silica gel column (50 2.5 cm) using a CHCl3–MeOH (6:1) gradient to yield 2 (4.0 mg); (a): Erythrophlesin H (1): White amorphous powder; [a]25 D 39.0 (c 0.05, MeOH); UV (MeOH) kmax (log e): 223 (3.20) nm; IR mmax (KBr): 3380, 2945, 2850, 1720, 1654, 1630, 1 1 1549 cm ; H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanold4) spectroscopic data, see Table 1; HR-ESI-MS m/z 780.5514 [M+H]+ (calcd for 780.5525) [M+H]+, C45H65NO10); (b) Erythrophlesin I (2): White amorphous powder; [a]25 D 58.5 (c 0.10, MeOH); UV (MeOH) kmax (log e): 225 (4.16) nm; IR mmax (KBr): 3415, 2942, 1721, 2864, 1765, 1640, 1610, 1435 cm1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) spectroscopic data, see Table 1; HR-ESI-MS m/z 796.5269 [M+H]+ (calcd for 796.5273 [M+H]+, C45H66NO11). 14. Miyagawa, T.; Ohtsuki, T.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Tetrahedron Lett. 2009, 50, 4658. 15. The human prostate cancer cell line, PC-3, was obtained from the American Type Tissue Collection (ATCC, Rockville, MD). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum were obtained from GIBCO (GIBCO BRL Co., New York, USA). EDTA (ethylenediaminetetraacaetic acid, disodium salt dihydrate, –Na2 EDTA2H2O), FBS (fetal bovine serum), penicillin, streptomycin, l-glutamine, trypsin, and MTT [3-(4,5 dimethylthiazol-2-yl)2,5,-diphenyl, tetrazolium bromide] solution were purchased from Sigma Chemical Co. (Ontario, Canada). All other reagents were of analytical grade. (a) Cell culture. Based on the manufacturer’s instruction, PC-3 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 50 U/mL penicillin, and
50 lL/mL streptomycin in 75 cm2 T-Flasks. The cells were grown at 37 °C, 85% humidity and 5% CO2 atmosphere. PC-3 cells were digested with 0.25% trypsin and 0.02% EDTA solution, and they were propagated when the cell density reached approximately 80%. (b) Cell proliferation assays. MTT cell proliferation assays were used to measure the proliferation of PC-3 cells. PC-3 cells in the logarithmic growing phase were plated at a density of 1 105 cells/well in 96well flat bottomed cell culture plates and incubated at 37 °C. Twenty-four hours after incubation, different concentrations of compound were added to each well (except for control wells) in 100-lL volume. After 48-h incubation, 10-lL MTT solution was added to each well to form formazan salt crystals, and the plates were incubated for 4 h. One hundred microliters of solubilization solution (10% SDS in 0.01 M HCl) was added, and the plates were incubated overnight at 37 °C. The amount of formazan produced was proportional to the number of viable cells. After incubation, the MTT-formazan was solubilized in 2-propanol, and absorbance was measured at 570 nm using a multiwell plate reader (Model 680, Bio-Rad, Richmond, CA, USA). (c) Cell-cycle distribution analysis using fluorescence-activated cell sorting. Briefly, PC-3 cells were trypsinized, washed twice with cold PBS, and centrifuged. The pellet was fixed in 70% (vol/vol) ethanol for 1 h at 4 °C, washed once with PBS, and resuspended in cold propidium iodide (PI) solution (50 lg/mL) containing RNase A (0.1 mg/mL) in PBS (pH 7.4) for 30 min in the dark. Flow cytometric analyses were performed using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Forward light scatter characteristics were used to exclude cell debris from the analysis, and CellQuest software was used to analyze the data (Becton-Dickinson). (d) Annexin-V and PI double staining assay. Dual propidium iodide (PI) and annexin-V labeling for cell death was carried out using an Annexin-V-FLUOS staining kit, according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA).
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In vitro apoptotic effect of cassaine-type diterpene amides from Erythrophleum fordii on PC-3 prostate cancer cells Tran Manh Hung a, To Dao Cuong a, Jeong Ah Kim b, Jeong Hyung Lee c, Mi Hee Woo a, Byung Sun Min a,⇑ a
College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Republic of Korea College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea c College of Natural Science, Kangwon National University, Kangwon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 6 June 2014 Revised 26 August 2014 Accepted 9 September 2014 Available online 16 September 2014 Keywords: Erythrophleum fordii Leguminosae Cassaine diterpenoid amide Erythrophlesin H PC-3 apoptosis
a b s t r a c t Cytotoxic activity-guided fractionation of Erythrophleum fordii led to the isolation of two new cassaine diterpenoid–diterpenoid amide dimers, erythrophlesins H–I (1, 2). Spectral data indicated that they consist of asymmetrical dimeric structure via an ester bond between two cassaine diterpenoids. MTT assay confirmed that compound 1, erythrophlesin H, had the strongest cytotoxic effect toward the human prostate cancer cell line, PC-3. The molecular mechanism by which this compound induced apoptosis cell in prostate cancer remains unknown. Erythrophlesin H induced apoptosis in a dose-dependent manner. Acridine orange and annexin V-FITC/PI double staining confirmed that erythrophlesin H effectively induces apoptosis in PC-3 cells. Ó 2014 Elsevier Ltd. All rights reserved.
Prostate cancer (PCA) is the most commonly diagnosed cancer in men, and it continues to be a major problem throughout the developed world. PCA is important health concern due to the growing worldwide population of men over the age of 50 years, and it is the most common cancer in men and the leading cause of male cancer death after lung cancer.1 Conventional therapy to eradicate tumor cells can prolonged survival and cure some PCA patients.2 However, relapse and metastases occur frequently, and in general, these tumors are unresponsive to conventional therapy. In normal prostate tissue, androgens regulate the growth and differentiation of epithelial cells. In the early stages of prostate cancer, proliferation is increased by androgens, and it can be kept in check using various therapies aimed at either decreasing circulating androgens or blocking the androgen receptor. However, in advanced stages of prostate cancer, growth and development typically become refractory to androgen inhibitors, and tumor cells continue to grow unchecked.3 PC-3 cells are epithelial cells derived from a human prostate adenocarcinoma whose growth is androgen-independent.4 This androgen-insensitive cell line provides an excellent model system for both basic studies and for the evaluation of clinical strategies to treat human prostatic carcinoma. Currently, no therapy has been proven to prolong the survival of PCA patients.1 Therefore, new therapeutic approaches are needed, with the objective of
⇑ Corresponding author. Tel.: +82 53 850 3613; fax: +82 53 850 3602. E-mail address: [email protected] (B.S. Min). http://dx.doi.org/10.1016/j.bmcl.2014.09.025 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
overcoming tumor cell resistance and reducing drug-mediated toxicity. In this regard, many phytochemicals with diversified biological properties have shown promising responses for the prevention and/or intervention of prostate cancer.5,6 During our continued search for anti-cancer agents from natural plants, the methylene chloride fraction of the methanol extract of Erythrophleum fordii Oliver (Leguminosae) demonstrated cytotoxic activity against PC-3 cancer cells. E. fordii is widely distributed in China, Taiwan, and Vietnam, and it is used in Chinese traditional medicine to invigorate and promote blood circulation.7 In prior studies on the genus Erythrophleum, cassaine diterpenoid amines, amides, and oleanane-type triterpene saponins were identified as the main constituents of this genus;8 minor amounts of cassaine acids were also identified.9 Furthermore, cassaine alkaloids produced a digitalis-like action on the heart,10 and it was cytotoxic activity to several tumor cell lines.8 Recently, a phytochemical investigation on the leaves of E. fordii resulted in the isolation of cassaine diterpenoid–diterpenoid amide dimers and cassaine diterpenoid amides with cytotoxic activities against several human cancer cell lines.11 In our previous study on this plant, we reported the isolation and structural elucidation of serial mono cassaine diterpenoid amines and described their anti-angiogenic activity.12 In this study, we provided clear evidence that cassaine diterpenoid– diterpenoid amide dimer derivatives induce apoptosis induction in PC-3 human prostate cancer cells. Repeated column chromatography (silica gel, RP-18) of the CH2Cl2-soluble fraction resulted in the isolation of compounds 1
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O
12 11 15 20 13 1 9 H 14 17 10 8 5 H 7 4 O 6
2
O 16' 12'
15'
11' 13' 20' 1' 9' H 14' 2' 17' 8' 10' 5' H 3' 4' 7' O H 6' O 19' 18'
3
O O
22
N 16
OH
21
O
O
H
O
H O O
H
O
O
H
24
O
OH
H
19 18
1
N
H
HO H
O
O
2
O
21'
Figure 1. Chemical structure of isolated compounds (1–2) from E. fordii.
Table 1 NMR spectral data of compounds 1 and 2 Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 a b c
Compound 1
Compound 2
dHa (in CD3OD, J in Hz)
d Cb
dHa (in CD3OD, J in Hz)
d Cb
1.72(1H, m), 1.21 (1H, m) 2.08 (1H, m), 1.22 (1H, m) 4.63 (1H, dd, 12.0, 4.4)
37.9 25.3 80.3 48.3 52.1 41.1 210.9 52.0 47.9 36.2 28.4 26.1 167.5 40.2 113.1 169.3 12.6 23.6 178.7 15.5 54.1 60.5 41.0 54.5 38.9 25.5 37.7 39.8 54.8 41.0 212.1 52.1 46.7 42.9 28.6 25.3 169.1 41.0 113.2 172.6 12.0 28.4 175.2 15.5 53.0
1.85 (1H, m), 1.34 (1H, m) 2.45 (1H, m), 1.78 (1H, m) 4.64 (1H, dd, 12.0, 4.6)
37.5 26.2 80.4 49.1 53.8 39.9 211.3 53.0 47.9 37.5 28.5 27.2 171.1 39.8 116.7 172.6 12.5 21.5 175.2 15.2 53.8 60.7 38.9 54.5 37.6 25.2 80.4 49.5 54.5 41.0 211.4 53.0 47.9 42.9 28.5 25.4 171.4 41.0 116.9 172.6 12.0 28.2 175.2 15.4 52.0
1.43 (1H, dd, 14.0, 2.5) 2.62 (1H, d, 2.5), 2.86 (1H, d, 14.0) 2.24 (1H, m) 1.80 (1H, m) 2.12 (1H, m), 1.30 (1H, m) 3.74 (1H, m), 2.03 (1H, m) 2.83 (1H, m) 5.76 (1H, s) 1.08 (3H, d, 6.0) 1.26 (3H, s) 0.83 3.78 4.31 3.23 3.64 1.78 1.80 2.09
(3H, (2H, (2H, (3H, (3H, (1H, (1H, (1H,
s) m) t, 4.8) s) s) m), 1.20 (1H, m)c m), 1.65 (1H, m) m), 1.15 (1H, m)
1.57 (1H, dd, 15.0, 4.5) 2.60 (1H, m), 2.98 (1H, m) 1.90 (1H, m) 1.85 (1H, m) 1.95 (1H, m), 1.14 (1H, m) 3.30 (1H, m), 2.04 (1H, m) 2.62 (1H, m) 5.80 (1H, s) 1.08 (3H, d, 6.0) 1.22 (3H, s) 0.89 (3H, s) 3.71 (3H, s)
Recorded at 400 MHz. Recorded at 100 MHz. Overlapped signals. All assignments were confirmed by DEPT-135, HMQC and HMBC experiments.
1.38 (1H, dd, 14.0, 2.5) 2.65 (1H, d, 2.5), 2.89 (1H, d, 14.0) 2.48 (1H, dd, 13.0, 3.5) 1.82 (1H, m) 1.88 (1H, m), 1.30 (1H, m) 3.21 (1H, m), 2.14 (1H, m) 2.87 (1H, m) 5.91 (1H, s) 1.30 (3H, d, 6.4) 1.40 (3H, s) 1.14 3.50 3.67 3.36 3.71 1.78 2.10 4.60
(3H, (2H, (2H, (3H, (3H, (1H, (1H, (1H,
s) m) t, 5.6) s) s) m), 1.31 (1H, m) m), 1.72 (1H, m) dd, 12.0, 4.0)
1.68 (1H, dd, 15.0, 4.5) 2.62 (1H, m), 2.80 (1H, m) 1.91 (1H, m) 1.80 (1H, m) 2.10 (1H, m), 1.31 (1H, m) 3.04 (1H, m), 1.96 (1H, m) 2.54 (1H, m) 6.00 (1H, s) 1.06 (3H, d, 6.0) 1.23 (3H, s) 0.95 (3H, s) 3.72 (3H, s)
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O
O
O O
N
O
OH
O
O
O O O
HO O
O O
O
O
N
H
O
O COSY
O
1
O
H
H
H
2
HO OH
O
HMBC
O
O
H H
H
H
H H
H
H HO H
O 1
OH
O
O
O
N
right unit
NOE
O
2
left unit
Figure 2. Selected COSY, HMBC and NOEs correlations for 1 and 2.
Figure 3. Cytotoxic activity of compounds 1 and 2.
and 213a (Fig. 1). Compound 1 was isolated as a white powder. A quasi-molecular ion at m/z 780.5514 (calc. 780.5525) [M+H]+ in the positive HR-ESI-MS indicated a molecular formula of C45H65NO10. Its IR spectrum showed absorption bands at 3375 and 1720 cm1, indicative of the presence of hydroxyl and carbonyl groups in the molecule.13b The 1H NMR spectrum of 1 revealed signals for six methyl groups, two methoxy groups (dH 3.64, 3H, s, H-24 and dH 3.71, 3H, s, H-210 ), two exocyclic olefinic protons (dH 5.76, 1H, s, H-15 and dH 5.80, 3H, s, H-150 ), one oxymethine (dH 4.63, 1H, dd, 12.0, 4.4 Hz, H-3), and a methyl amide N–CH3 (dH 2.67, 3H, s, H-23) (Table 1). The 13C NMR spectrum of 1 displayed resonances for two ketones at dC 210.9 (C-7) and 212.1 (C-70 ), four ester carbonyl groups
at dC 178.7 (C-19), 175.2 (C-190 ), 169.3 (C-16), and 172.6 (C-160 ), two sp2 methines at dC 113.1 (C-15) and 113.2 (C-150 ), two quaternary sp2 carbons at dC 167.5 (C-13) and 169.1 (C-130 ), one oxymethine at dC 80.3 (C-3), and two methoxy groups at dC 54.5 (C-24) and 53.0 (C-210 ) (Table 1). All signals in the above spectra were further supported by HMQC spectroscopic analyses. Since most of the 1H and 13C NMR signals of 1 were observed as pairs, it was inferred that 1 had a dimeric structure consisting of two tricyclic diterpenoids in Erythrophleum sp.11,14 Detailed analysis of 1H–1H COSY and HMBC correlation data indicated that compound 1 was constructed from two cassaine diterpenoids connected through an ester linkage in the presence of a 7-keton group for both monomer units (left and
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Con
3 M
10 M
30 M
G0/G1 S G2/M
0 66.6 14.3 19.1
3 59.9 15.1 24.9
10 49.9 10.9 39.2
30 43.5 10.1 46.4
Figure 4. DNA content analysis in erythrophlesin H treated PC-3 cells. Erythrophlesin H was treated as described above and sub-G1 cell populations stained with propidium iodide (PI) were determined by flow cytometry (FACS). Fluorescence intensities are shown on a log scale. Results show a typical experiment which has been repeated three times.
right units). The 1H–1H COSY spectrum showed proton connectivity for H-5/H-6 and H-50 /H-60 , confirming the location of the ketone group at C-7 for both the left and right units (Fig. 2). A conjugate ester moiety is located at the C-13/C-15/C-16 position of both units, as revealed from the HMBC correlations for H-14/C-13, H-14/C-15, H2-12/C-15, H-15/C-12, and H-15/C-16 observed for each unit. The C-16 ester carbonyl of the right unit at dC 169.3 was connected to an N-methyl amide group that was assigned by dH 2.67 (H-23), 3.87 (H-21), and 4.31 (H-22) and dC 54.1 (C-21), 60.5 (C-22), and 40.6 (N-Me, C-23) in 1H and 13C NMR spectra. The HMBC spectrum confirmed correlations between protons H-23, H-21 and C-16; H-23 and C-21; and H-21 and C-22 (Fig. 2). The adjacent C-4 position of the both of left and right units bore a methoxycarbonyl group and a tertiary methyl group due to the diagnostic HMBC correlation observation between H-18, C-4, and C-19, as well as between H-24 and C-19. In addition, the C-160 ester carbonyl of the left unit at dC 172.6 displayed a HMBC correlation signal with an oxymethine at dH 4.46 (H-3) on the right unit (Fig. 2), indicating the connection of the two monomer units.11,14 A comparison of the 1H and 13C NMR spectroscopic data from 1 with those of erythrophlesin D suggested they share the same skeleton, except for the location of 6,60 -methylene and 7,70 -keto groups in 1.11 This difference was supported by cross-peaks from H-5/H-50 to H-6/60 in the COSY spectrum (Fig. 2). According to the biogenetic isoprene rule of the cassaine skeleton, the two methyl groups attached to the rings were in the 10b and 14a orientations in both of the units.11,14 The J value of H-3 (dd, J = 12.0, 4.0 Hz) suggested an b-orientation for the ether linkage. The aforementioned considerations were further substantiated by significant NOE correlations between H-3, H-5 and H-18, H-5 and H-9, and H-9 and H-17 (a-orientation) (Fig. 2). The NOE correlations between H-8 and H-14, H-8 and H-20, and H-20 and H-24 indicated
that these protons were b-orientated (Fig. 2). Furthermore, in both monomer units, the NOE correlation between H-14 and H-15 indicated that the double bond at C-13/C-15 had the E-conformation.11,14 From these results, we concluded that the structure of erythrophlesin H was 1. Erythrophlesin I (2) had a molecular formula C45H65NO11, as established by HR-ESI-MS data (m/z 796.5269, calcd. for C45H66NO11 [M+H]+, 796.5273), which was 16 mass units greater than the co-occurring erythrophlesin H compound 1.13 Inspection of the 1D (1H and 13C) and 2D (HMQC, COSY and HMBC) NMR data of 2 established the presence of nearly identical structural features to those of 1 (Table 1). Comparison of the 1H and 13C NMR spectra of 2 with 1 revealed that these compounds were similar cassaine diterpenoid–diterpenoid amide dimmers, except resonances for the hydroxyl group at position three of the left unit of 2 was present at dH 4.60 (1H, dd, J = 12.0, 4.4 Hz, H-30 ) and dC 80.3 (C-30 ) (Table 1). This was confirmed by HMBC correlations from the H-50 to dC 80.4 (C-30 ) and dH 4.65 (H-30 ) to dC 48.0 (C-40 ) (Fig. 2). An HMBC correlation of H-3 to the ester carbonyl at dC 172.6 (C-160 ) confirmed the left unit attachment to C-3 of the right unit (Fig. 2). Moreover, in addition to similar correlation signals with 1, the oxygenated methine at dH 4.60 (H-30 ) was present as a double doublet (J = 12.0, 4.4 Hz), and NOE correlations between H-30 , H-50 , and H-180 in the left unit of 2 indicated that these protons were in the same a-orientation; therefore, the hydroxyl group at C-30 was b-orientated (Fig. 2).14 From these results, we concluded that the structure of erythrophlesin I was 2. The cytotoxic activity of compounds 1 and 2 were evaluated against a panel of human cancer cell lines (A549, MCF-7, PC-3 and HepG2). Cell growth was measured using MTT assays.15 Compound 1, erythrophlesin H, displayed potently cytotoxic activity
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flow cytometry (BD FACSCalibur flow).15 Annexin V-FLUOS/PI double-staining analysis demonstrated that erythrophlesin H decreased the number of PC-3 cells in a dose-dependent manner. Fig. 4 presents histograms of the DNA content from PC-3 cells treated with erythrophlesin H for 24 h. Sub-G1 DNA increased up to 48 h in response to 3, 10 and 30 lM erythrophlesin H, reached approximately 46.4%. Moreover, the number of cells in the early and late stages of apoptosis increased in a dose-dependent manner (Fig. 5A). Erythrophlesin H induced apoptosis in PC-3 cells, especially late stage apoptosis. The percentage of apoptotic cells, including early and late stage of apoptosis, increased from 0% (control) to appropriate 10% in response to erythrophlesin H treatment (Fig. 5B). Thus, erythrophlesin H showed a dramatically induced apoptosis in PC-3 cells in a dose-dependent manner. The development of anti-cancer drugs that inhibit abnormal cancer cell proliferation and induce cell death through apoptosis is a fundamental objective of cancer research. Here, we demonstrate that erythrophlesin H, a compound present in the bark of E. fordii, induces apoptosis in PC-3 cancer cells. We are currently investigating the feasibility of further developing this chemical structure as a chemo-preventive agent. Acknowledgments This research was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (KRF-2012R1A2003547) and BK-21 Plus (22A20130000073). We are grateful to the Korean Basic Science Institute (KBSI) for supplying the MASS spectra. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09. 025. References and notes Figure 5. Flow cytometric analysis of erythrophlesin H-induced apoptosis in PC-3 cell using annexin-V-FITC/PI. Cells (1 106 cells) were incubated with indicated concentration of erythrophlesin H for 48 h and stained with annexin-V-FITC/PI to analysis apoptosis and necrotic cell populations. Cells in the lower right quadrant represented apoptosis and in the upper right quadrant. Data are representative of one of three similar experiments.
against all the cell lines in a dose-dependent manner with IC50 values of 24.5, 17.2, 12.5 and 25.2 lM, respectively (Fig. 3). Camptothecin, which was effectively used as a positive control, exhibited the cytotoxic activity with IC50 values ranging from 0.54 to 1.02 lM against all kind of cancer cell lines. The structure of 1 and 2 are similar, except for the substitution at C-3 of left unit in the cassaine diterpenoid–diterpenoid amide dimmer skeleton. Compound 2, erythrophlesin I with a hydroxyl group at C-30 , exhibited weak cytotoxicity against all cancer cell lines. These data indicate that the substitution at C-30 might influence its cytotoxic activity. Because erythrophlesin H (1) exhibited the most potent cytotoxic activity against PC-3 cells (IC50 12.5 lM), we further evaluated its inhibition of PC-3 cell proliferation.15 Cells were treated with various concentrations of erythrophlesin H (3–30 lM), incubated for 7 days, and counted at 1-day intervals using the trypan blue exclusion method. The growth of cells treated with erythrophlesin H was significantly inhibited after 2 days compared to control cells (data not shown). To quantify the modes of erythrophlesin H (1)-induced cell death, PC-3 cell were treated with erythrophlesin H (3–30 lM) for 48 h, stained with annexin V-FLUOS and PI, and analyzed by
1. Jemal, A.; Tiwari, R. C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer, E. J.; Thun, M. J. CA Cancer J. Clin. 2004, 54, 8. 2. Brawley, O. W.; Barnes, S.; Parnes, H. Ann. N.Y. Acad. Sci. 2001, 952, 145. 3. Feldman, B. J.; Feldman, D. Nat. Rev. Cancer 2001, 1, 34. 4. Kaighn, M. E.; Narayan, K. S.; Ohnuki, Y.; Lechner, I. F.; Jones, L. W. Invest. Urol. 1979, 17, 16. 5. Surh, Y. J. Nat. Rev. Cancer 2003, 3, 768. 6. Barnes, S. Epidemiol. Rev. 2001, 23, 102. 7. Chen, J. S.; Zhen, S. Chinese Virose Plant, 1st ed.; Science Press: Beijing, 1987. p. 321. 8. (a) Culvenor, C. C. J.; Loder, J. W.; Nearn, R. Phytochemistry 1971, 10, 2793; (b) Cronlund, A.; Sandberg, F. Acta Pharm. Suec. 1976, 13, 35; (c) Qu, J.; Hu, Y. C.; Yu, S. S.; Chen, X. G.; Li, Y. Planta Med. 2006, 72, 442; (d) Du, D.; Fang, L.; Qu, J.; Yu, S.; Ma, S.; Lv, H.; Liu, J.; Liu, Y.; Wang, J.; Wang, X. Planta Med. 2011, 77, 1631. 9. (a) Li, N.; Yu, F.; Yu, S. S. Acta Bot. Sin. 2004, 46, 371; (b) Yu, F.; Li, N.; Yu, S. S. J. Asian Nat. Prod. Res. 2005, 7, 19; (c) Qu, J.; Wang, Y. H.; Li, J. B.; Yu, S. S.; Li, Y.; Liu, Y. B. Rapid Commun. Mass Spectrom. 2007, 21, 2109; (d) Tsao, C. C.; Shen, Y. C.; Su, C. R.; Li, C. Y.; Liou, M. J.; Dung, N. X.; Wu, T. S. Bioorg. Med. Chem. 2008, 16, 9867. 10. Verotta, L.; Aburjai, T.; Rogers, C. B.; Dorigo, P.; Maragno, I.; Fraccarollo, D.; Giovanni, S.; Gaion, R. M.; Floreani, M.; Carpenedo, F. Planta Med. 1995, 61, 271. 11. Du, D.; Qu, J.; Wang, J. M.; Yu, S. S.; Chen, X. G.; Xu, S.; Ma, S. G.; Li, Y.; Ding, G. Z.; Fang, L. Phytochemistry 2010, 71, 1749. 12. Hung, T. M.; Cuong, T. D.; Kim, J. A.; Tae, N.; Lee, J. H.; Min, B. S. Bioorg. Med. Chem. Let. 2013, 24, 168. 13. E. fordii Oliver (Leguminosae) bark was collected from Quang Nam province, Vietnam, in May 2012. Professor Tran Cong Luan at Hochiminh City University of Medicine and Pharmacy performed the botanical identification, and a voucher specimen (CUD-3177) was deposited at Herbarium of the College of Pharmacy, Catholic University of Daegu, Korea. The bark (2.0 kg) was extracted with MeOH three times (3 h 3 L) under reflux. After solvent removal under reduced pressure, the residue (426.0 g) was suspended in H2O and partitioned sequentially versus CH2Cl2 and EtOAc. The CH2Cl2 soluble fraction (40.0 g) was separated on a silica gel column (50 10 cm) using a stepwise gradient of CHCl3/MeOH (40:1 ? 1:1, v/v) to yield 27 fractions (A1–A27), according to their TLC profiles. Fraction A27 (3.7 g) was subjected to silica gel column
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chromatography (60 5 cm) using CHCl3–MeOH plus 0.1% acetic acid (7:1) as an eluent to yield seven sub-fractions (A27-1–A27.7). Fraction A27-2 (106.0 mg) was chromatographed on a silica gel column (50 2.5 cm) using a CHCl3–MeOH gradient (7:1) to produce compound 1 (5.0 mg). Fraction A27.5 (146.0 mg) was further chromatographed on a silica gel column (50 2.5 cm) using a CHCl3–MeOH (6:1) gradient to yield 2 (4.0 mg); (a): Erythrophlesin H (1): White amorphous powder; [a]25 D 39.0 (c 0.05, MeOH); UV (MeOH) kmax (log e): 223 (3.20) nm; IR mmax (KBr): 3380, 2945, 2850, 1720, 1654, 1630, 1 1 1549 cm ; H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanold4) spectroscopic data, see Table 1; HR-ESI-MS m/z 780.5514 [M+H]+ (calcd for 780.5525) [M+H]+, C45H65NO10); (b) Erythrophlesin I (2): White amorphous powder; [a]25 D 58.5 (c 0.10, MeOH); UV (MeOH) kmax (log e): 225 (4.16) nm; IR mmax (KBr): 3415, 2942, 1721, 2864, 1765, 1640, 1610, 1435 cm1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) spectroscopic data, see Table 1; HR-ESI-MS m/z 796.5269 [M+H]+ (calcd for 796.5273 [M+H]+, C45H66NO11). 14. Miyagawa, T.; Ohtsuki, T.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Tetrahedron Lett. 2009, 50, 4658. 15. The human prostate cancer cell line, PC-3, was obtained from the American Type Tissue Collection (ATCC, Rockville, MD). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum were obtained from GIBCO (GIBCO BRL Co., New York, USA). EDTA (ethylenediaminetetraacaetic acid, disodium salt dihydrate, –Na2 EDTA2H2O), FBS (fetal bovine serum), penicillin, streptomycin, l-glutamine, trypsin, and MTT [3-(4,5 dimethylthiazol-2-yl)2,5,-diphenyl, tetrazolium bromide] solution were purchased from Sigma Chemical Co. (Ontario, Canada). All other reagents were of analytical grade. (a) Cell culture. Based on the manufacturer’s instruction, PC-3 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 50 U/mL penicillin, and
50 lL/mL streptomycin in 75 cm2 T-Flasks. The cells were grown at 37 °C, 85% humidity and 5% CO2 atmosphere. PC-3 cells were digested with 0.25% trypsin and 0.02% EDTA solution, and they were propagated when the cell density reached approximately 80%. (b) Cell proliferation assays. MTT cell proliferation assays were used to measure the proliferation of PC-3 cells. PC-3 cells in the logarithmic growing phase were plated at a density of 1 105 cells/well in 96well flat bottomed cell culture plates and incubated at 37 °C. Twenty-four hours after incubation, different concentrations of compound were added to each well (except for control wells) in 100-lL volume. After 48-h incubation, 10-lL MTT solution was added to each well to form formazan salt crystals, and the plates were incubated for 4 h. One hundred microliters of solubilization solution (10% SDS in 0.01 M HCl) was added, and the plates were incubated overnight at 37 °C. The amount of formazan produced was proportional to the number of viable cells. After incubation, the MTT-formazan was solubilized in 2-propanol, and absorbance was measured at 570 nm using a multiwell plate reader (Model 680, Bio-Rad, Richmond, CA, USA). (c) Cell-cycle distribution analysis using fluorescence-activated cell sorting. Briefly, PC-3 cells were trypsinized, washed twice with cold PBS, and centrifuged. The pellet was fixed in 70% (vol/vol) ethanol for 1 h at 4 °C, washed once with PBS, and resuspended in cold propidium iodide (PI) solution (50 lg/mL) containing RNase A (0.1 mg/mL) in PBS (pH 7.4) for 30 min in the dark. Flow cytometric analyses were performed using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Forward light scatter characteristics were used to exclude cell debris from the analysis, and CellQuest software was used to analyze the data (Becton-Dickinson). (d) Annexin-V and PI double staining assay. Dual propidium iodide (PI) and annexin-V labeling for cell death was carried out using an Annexin-V-FLUOS staining kit, according to the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA).