Diterpenoids from the Chinese Liverwort Heteroscyphus tener and

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Diterpenoids from the Chinese Liverwort Heteroscyphus tener and Their Antiproliferative Effects Zhao-Min Lin,† Yan-Xia Guo,‡ Shu-QI Wang,† Xiao-Ning Wang,† Wen-Qiang Chang,† Jin-Chuan Zhou,† Huiqing Yuan,‡ and Hongxiang Lou*,† †

Department of Natural Products Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of China ‡ Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of China S Supporting Information *

ABSTRACT: Four new ent-labdane diterpenoids, heteroscyphins A−D (1−4), and four known diterpenoids (5−8) were isolated from the Chinese liverwort Heteroscyphus tener (Steph.) Schiffn. The absolute configuration of compound 1 was defined by single-crystal X-ray diffraction using Cu Kα radiation. Cytotoxicity tests revealed that compounds 3 and 5 exhibited modest activity against seven cancer cell lines. Compound 5 showed inhibitory effects on prostate cancer (PCa) cell proliferation but with less inhibition on nonneoplastic prostate epithelial cells. Compound 5 markedly caused cell growth arrest at the G0/G1 phase and induced cellular apoptosis through ROS-mediated DNA damage in PCa cells.

V

arious terpenoids and aromatic compounds derived from liverworts have attracted attention because of their diverse biological activities, including cytotoxic, antifungal, antimicrobial, insect antifeedant, and antioxidant properties.1−4 Chemical investigations of the Heteroscyphus species have identified a variety of structurally diverse diterpenoids such as the clerodane,5−7 neoverrucosane,6 halimane,7 and retinane8 types, and sesquiterpenoids such as the ent-aromadendrane,6−10 calamenene,6,11 and cadinane11 types, as well as other aromatic compounds.8,12 However, no chemical studies have been conducted on the liverwort Heteroscyphus tener (Steph.) Schiffn. As part of our ongoing research on bioactive products from Chinese liverworts,13−15 chemical investigation of the liverwort H. tener, collected in the mountainous areas (alt. 1500 m) of the Guangxi Zhuang Autonomous Region, South China, led to the isolation of one known (5) and four new (1−4) ent-labdane diterpenoids, as well as three known fusicoccane-type diterpenoids (6−8). Herein, the isolation, structure determination of all compounds, as well as their cytotoxicity on human cancer cell lines and normal epithelial cell lines are described.

NMR data (Table 1) of 1 displayed resonances for four tertiary methyls [δH 0.91 (s, H3-18), 1.05 (s, H3-19), 1.23 (s, H3-20), and 1.32 (s, H3-16)], two oxygenated methines [δH 4.56 (d, J = 6.6 Hz, H-6) and 5.24 (d, J = 5.4 Hz, H-17)], and three vinylic protons [δH 5.91 (dd, J = 17.4, 10.8 Hz, H-14), 5.18 (d, J = 17.4 Hz, H-15a), and 5.01 (d, J = 10.8 Hz, H-15b)]. The 13C NMR data (Table 2) in conjunction with the HSQC data indicated that 1 contained two vinylic carbons [δC 111.5 (C-15) and 145.7 (C-14)], four methyl, six methylene, four methine, of which two were oxygenated [δC 73.1 (C-6) and 94.4 (C-17)], and four quaternary carbons, of which two were oxygenated [δC 74.3 (C-13) and 81.4 (C-8)]. Three segments, −C-1(H2)−C2(H2)−C-3(H2)−, −C-5(H)−C-6(H)−C-7(H2)−, and −C-

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RESULTS AND DISCUSSION Structure Elucidation. Heteroscyphin A (1) was assigned the molecular formula C20H32O3 on the basis of the 13C NMR data and an [M + Na]+ ion at m/z 343.2245 by HRESIMS (calcd 343.2244), which indicated five indices of hydrogen deficiency. The IR spectrum showed an absorption band at 3490 cm−1, indicating the presence of an OH group. The 1H © 2014 American Chemical Society and American Society of Pharmacognosy

Received: January 18, 2014 Published: June 18, 2014 1336

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Table 1. 1H NMR Data of Compounds 1−4a position

1

1a 1b 2a 2b 3a 3b 5 6 7a 7b 9 11a 11b 12a 12b 14 15a 15b 16 17 18 19 20a 20b 6-OAc 17-OH

1.67 0.95 1.50 1.71 1.44 1.11 1.03 4.56 2.39 1.48 1.50 1.71 1.61 1.61 1.71 5.91 5.01 5.18 1.32 5.24 0.91 1.05 1.23

2

m (α-H) td (12.6, 4.2) (β-H) mb (α-H) m (β-H) m (α-H) td (13.2, 4.2) (β-H) s (β-H) d (6.6) (β-H) dd (10.2, 6.6) (α-H) d (10.2) (β-H) mb (β-H) mb (α-H) mb (β-H) mb (α-H) mb (β-H) dd (17.4, 10.8) d (10.8) d (17.4) s (3H) d (5.4) (α-H) s (3H) s (3H) s (3H)

1.88 1.14 1.38 1.50 1.47 1.15 1.17 4.40 2.39 1.56 1.42 1.87 1.63 1.48 1.65 5.91 5.00 5.18 1.38 5.19 0.94 1.14 3.51 4.49

3

m (α-H) m (β-H) m (α-H) m (β-H) m (α-H) m (β-H) s (β-H) d (6.0) (β-H) dd (10.8, 6.0) (α-H) m (β-H) m (β-H) m (α-H) m (β-H) m (α-H) m (β-H) dd (17.4, 10.8) dd (10.8, 1.2) dd (17.4, 1.2) s (3H) s (α-H) s (3H) s (3H) d (10.8) d (10.8)

1.72 1.02 1.50 1.72 1.50 1.16 1.29 4.84 2.69 1.84 1.57 1.68 1.59 1.59 1.89 5.89 4.99 5.17 1.55

mb (α-H) m (β-H) mb (α-H) mb (β-H) mb (α-H) td (13.8, 4.8) (β-H) s (β-H) d (6.0) (β-H) dd (10.8, 6.0) (α-H) d (10.8) (β-H) m (β-H) m (α-H) mb (β-H) mb (α-H) m (β-H) dd (17.4, 10.8) d (10.8) d (17.4) s (3H)

0.96 s (3H) 1.05 s (3H) 1.13 s (3H)

4 1.70 0.89 1.48 1.70 1.38 1.16 1.11 5.51 2.88 1.60 1.60 2.20 1.64 1.76 1.92 5.87 4.96 5.14 1.24

mb (α-H) td (13.8, 4.8) (β-H) m (α-H) mb (β-H) br d (13.2) (α-H) td (13.2, 3.6) (β-H) s (β-H) br s (β-H) dd (14.4, 3.0) (α-H) mb (β-H) mb (β-H) m (α-H) m (β-H) m (α-H) m (β-H) dd (17.4, 10.8) d (10.8) d (17.4) s (3H)

0.95 s (3H) 0.95 s (3H) 1.21 s (3H) 1.96 s

3.20 br s

Recorded at 600 MHz in CDCl3, δ in ppm, and J in Hz. Assignments were made on the basis of HSQC, 1H−1H COSY, and NOESY data. bSignals overlapped.

a

Table 2. 13C NMR Data of Compounds 1−4a

a

position

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 6-OAc

41.6b, CH2 19.1, CH2 41.6b, CH2 33.7, C 59.5, CH 73.1, CH 46.0, CH2 81.4, C 53.8, CH 38.7, C 17.1, CH2 36.1, CH2 74.3, C 145.7, CH 111.5, CH2 24.4, CH3 94.4, CH 33.4, CH3 22.9, CH3 16.1, CH3

37.1, CH2 18.7, CH2 41.8, CH2 33.3, C 58.7, CH 74.2, CH 49.7, CH2 79.4, C 48.3, CH 39.1, C 16.4, CH2 34.5, CH2 73.7, C 146.5, CH 111.2, CH2 22.6, CH3 105.2, CH 34.0, CH3 22.1, CH3 65.3, CH2

42.1, CH2 18.8, CH2 41.5, CH2 33.4, C 57.6, CH 74.3, CH 50.0, CH2 78.2, C 53.4, CH 38.4, C 16.3, CH2 35.0, CH2 75.2, C 146.2, CH 111.2, CH2 26.8, CH3 177.7, C 33.7, CH3 22.9, CH3 17.0, CH3

41.4, CH2 18.7, CH2 43.7, CH2 33.8, C 56.8, CH 69.3, CH 43.2, CH2 74.9, C 53.9, CH 38.3, C 15.2, CH2 34.3, CH2 76.1, C 146.5, CH 111.2, CH2 26.5, CH3 178.0, C 33.3, CH3 23.0, CH3 17.1, CH3 170.1, C 21.4, CH3

Figure 1. Selected 1H−1H COSY (bold lines), HMBC (H → C), and NOESY (H↔H) correlations for compound 1.

from the olefinic protons H2-15 to C-13 and C-14 indicated that C-12, C-16, and the terminal vinyl group were connected to the oxygenated carbon C-13. In light of the above data, this compound was determined to be a manoyloxide-type labdane diterpenoid.16−18 The HMBC correlations from H-6 to C-4 (δC 33.7), C-10 (δC 38.7), C-7 (δC 46.0), C-8, and C-17 and from H-17 to C-6, C-7, and C-8 suggested the presence of an oxygen atom between C-6 and C-17, which fulfilled the total indices for hydrogen deficiency. The relative configuration of 1 was determined from the NOESY spectrum (Figure 1). Correlations of H-6/H-5/H-9 and H3-16/H-17/H3-20 suggested that these respective protons were cofacial. To determine the absolute configuration, compound 1 was subjected to singlecrystal X-ray diffraction analysis with Cu Kα radiation, which unambiguously confirmed the structure and the depicted absolute configuration to be that of an ent-labdane diterpenoid (Figure 2). Accordingly, the structure of compound 1 was defined as 6α,17:8β,13β-diepoxy-ent-labd-14-en-17α-ol. The molecular formula of heteroscyphin B (2) was determined to be C20H30O3 from 13C NMR data and

Recorded at 150 MHz in CDCl3, and δ in ppm. bInterchangeable.

9(H)−C-11(H2)−C-12(H2)− (shown as bold lines in Figure 1), were established on the basis of the 1H−1H COSY spectrum. In the HMBC spectrum (Figure 1), the long-range correlations from H3-16 to C-12 (δC 36.1), C-13, and C-14 and 1337

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3. Therefore, the structure of compound 4 was defined as 6αacetoxy-8β,13β-epoxy-ent-labd-14-en-17-oic acid. The known compounds isomanool (5),19 anadensin (6),20 fusicoauritone (7),21 and 4α-hydroxyfusicocca-3(7)-en-6-one (8)22 were identified by comparison of their NMR and MS data with reported data. In Vitro Antiproliferation Activity. All compounds were evaluated for their inhibitory effect against a panel of cancer cell lines (PC3, DU145, K562, A549, NCI-H292, NCI-H446, and NCI-H1299) and two human nontumorigenic cell lines, human bronchial epithelial cell HBE, and human prostate epithelial cell RWPE-1, using a published method.23 Cisplatin was used as the positive control. As shown in Table 3, compounds 3 and 5 both exhibited modest antiproliferation activity against cancer cell lines with IC50 values ranging from 10.7 to more than 40.0 μM. However, compounds 1, 2, 4, 7, and 8 did not show cytotoxicity against any of the tested cell lines (IC50 > 40.0 μM, data not shown). Because the yield of compound 5 (24.0 mg) was higher than that of compound 3 (3.6 mg) and because compound 5 appeared to have a better therapeutic ratio in prostate cancer versus normal prostate cells (IC50 in cancer cell lines 26.6 and 28.5 μM, in nontumorigenic RWPE-1 81.0 μM), we chose compound 5 for further investigations. Isomanool (5) Arrests the Cell Cycle at the G0/G1 Phrase in PCa Cells. As shown in Figure 3A, 5 markedly inhibited the proliferation of prostate cancer cells. Because regulation of the cell cycle is critical for cell growth, we investigated the effect of 5 on cell cycle progression using flow cytometry. As shown in Figure 3B, 5 arrested the cell cycle at the G0/G1 phase in both PC3 and DU145 cells in a concentration-dependent manner, which induced a corresponding decrease in the number of cells in the S-phase. In PC3 cells, treatment with 5 increased the percentage of cells in the G0/ G1 phase to 73 and 82% at concentrations of 10 and 20 μM, respectively, compared to 66% in the control group. Similar results were observed in DU145 cellsthe percentage of cells in the G0/G1 phase increased up to 68 and 75% with increasing concentrations of 5, while the cells in the G0/G1 phase was 63% in untreated control. The cell cycle distribution is summarized in Figure 3C. We next assessed the effect of 5 on key modulators associated with the G0/G1 phase by Western blotting. The results revealed that cyclin D1 and E were both reduced after treatment with 5 in a time-dependent manner in PC3 and DU145 cells (Figure 3D). Activation of cyclin-dependent kinase 4 (Cdk4) is essential for facilitation of the cell cycle via association with cyclin D1.24−26 Treatment with 5 also led to decreased Cdk4 in cells, as shown in Figure 3D. Meanwhile, p21CIP1, an important cyclin-dependent kinase inhibitor, was up-regulated in both cells. Taken together, the down-regulation

Figure 2. ORTEP drawing of compound 1.

HRESIMS, suggesting one more index of hydrogen deficiency than in 1. Comparison of the 1H and 13C NMR data of 1 and 2 (Tables 1 and 2) revealed that they possessed a similar entlabdane skeleton, except for the presence of an oxygenated methylene group [δH 3.51 (d, J = 10.8 Hz, H-20a), 4.49 (d, J = 10.8 Hz, H-20b); δC 65.3 (C-20)] in 2 instead of a methyl group [δH 1.23 (s, H3-20); δC 16.1 (C-20)] in 1 and the downfield shift of 10.8 ppm for C-17 (δC 105.2) in 2. HMBC correlations from the oxygenated H2-20 to C-17 and from H-17 [δH 5.19 (s, 1H)] to C-20 (δC 65.3) suggested the presence of an oxygen atom between C-17 and C-20, which explained the molecular formula and the deshielding of C-17 relative to that of 1. The relative configuration of 2, obtained from the NOESY spectrum, resembled that of 1. Consequently, the structure of compound 2 was defined as 6α,17:8β,13β:17,20-triepoxy-entlabd-14-ene. Heteroscyphin C (3) was analyzed for C20H30O3 by 13C NMR data and HRESIMS. The 1H and 13C NMR data (Tables 1 and 2) were similar to those of 1. The only difference was the presence of a carbonyl carbon [δC 177.7 (C-17)] in 3 and the absence of the acetalic methine [δH 5.24; δC 94.4 (C-17)] in 1. The HMBC correlation from H-6 [δH 4.84 (d, J = 6.0 Hz)] to C-17, together with the six indices of hydrogen deficiency, confirmed the presence of a lactone moiety between C-6 and C-17. Consequently, the structure of 3 was defined as 8β,13βepoxy-ent-labd-14-en-17,6β-olide. The 13C NMR data and HRESIMS of heteroscyphin D (4) revealed a molecular formula of C22H34O5. The 1H and 13C NMR data of 4 (Tables 1 and 2) closely resembled those of 3, except for the presence of an acetyl group [δH 1.96 (s, 3H); δC 21.4 and 170.1]. The acetoxy group at C-6 was confirmed by the HMBC correlations of H-6 with the acetyl carbonyl (δC 170.1). On the basis of similar NOE relationships, the configuration of 4 was determined to be the same as that of Table 3. Effect of Compounds 3, 5, and 6 on Cell Proliferationa compd

PC3

DU145

K562

A549

3 5 6 cisplatinb

20.0 ± 0.1 26.6 ± 1.0 >40 19.9 ± 0.4

18.9 ± 1.1 28.5 ± 1.1 >40 29.2 ± 0.6

34.0 ± 1.9 15.8 ± 0.5 >40 20.2 ± 0.8

21.5 ± 1.1 >40 >40 25.2 ± 1.5

NCI-H292 10.7 17.1 39.9 18.7

± ± ± ±

0.4 0.1 0.4 0.5

NCI-H1299

NCI-H446

RWPE-1

HBE

± ± ± ±

16.7 ± 0.7 39.5 ± 2.1 >40 31.4 ± 1.9

26.4 ± 0.7 81.0 ± 1.5 >40 33.8 ± 2.0

53.2 ± 2.5 53.9 ± 3.1 >40 27.2 ± 0.2

21.9 35.1 27.0 21.9

0.9 0.4 0.6 0.9

Results are expressed as the mean values of IC50 ± SD in μM. bCisplatin was used as the positive control. PC3 and DU145, human hormoneindependent prostate carcinoma cell lines; K562, human myelogenous leukemia cell line; A549, NCI-H292, and NCI-H1299, human non-small-cell lung cancer cell lines; NCI-H446, human small-cell lung cancer cell line; RWPE-1, human prostate epithelial cell; HBE, human normal bronchial epithelial cell. The experiments were performed three times. a

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Figure 3. Inhibitory effect and cell cycle progression of 5 in PCa Cells. (A) Cytotoxic effects of 5 in PC3 and DU145 cells over 24 h as measured by MTT assay. Data are presented as the means ± SD of three independent experiments, **P < 0.01, ***P < 0.001 compared to the control group. (B) Cells were treated with 5 (0, 10, 20 μM) for 24 h. For the cell cycle analysis, cells were harvested by trypsinization, fixed, stained with PI, and analyzed by flow cytometry. Data are given as the percentage of cells in each cell cycle phase. (C) Quantitative analysis of percent gated cells at the sub-G1, G0/G1, S, and G2/M phases were shown. All values are expressed as the means ± SD of three independent experiments *P < 0.05, **P < 0.01 compared to the untreated control group. (D) The levels of cyclin D1, cyclin E, Cdk4, and p21 in PC3 and DU145 cells were measured by Western blotting with GAPDH as the loading control.

70.25% and 4.22 to 23.75% in PC3 and DU145 cells, respectively (Figure 4A,B). Caspases are a family of cysteine proteases that play important roles in apoptosis, of which caspase-3 is considered to be a critical effector that can be cleaved and activated during apoptosis.30 As shown in Figure 4C, incubation of both cells with 5 resulted in a timedependent increase in caspase-3 activity. Activation of caspase-3 was further confirmed by Western blotting. In both cell lines, 5induced activation of caspase-3 was evidenced by appearance of

of cyclin D1, E, and Cdk4 resulted in a decrease in the cyclin/ Cdk complex by 5, which contributed to the induction of the G0/G1 phase arrest. Isomanool (5) Induces Apoptosis in PCa Cells. We next examined whether 5 caused cell cycle arrest is associated with induction of apoptosis, because many antitumor agents exert their therapeutic effect through the induction of apoptosis.27−29 A flow cytometry assay showed that the apoptotic cells accumulated in a time-dependent manner from 8.02 to 1339

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Figure 4. Analysis of apoptosis in 5-treated PCa Cells. (A) PC3 and DU145 cells were treated with 20 μM 5 for the indicated times. Cells were collected and stained with FITC-conjugated annexin V and PI and immediately subjected to flow cytometry. (B) Mean percentage of annexin V/(PI + plus PI−) cells obtained from three independent experiments in the indicated cell lines, *P < 0.05, **P < 0.01 compared to the untreated control group. (C) Activation of caspase-3 in PC3 and DU145 cells induced by 5. Cells were treated with 20 μM 5 for the indicated times. Caspase-3 activity was measured with whole cell extracts by a fluorometric method. *P < 0.05, **P < 0.01 compared to the untreated control group. (D) 5-induced activation of caspase and cleavage of PARP. The expression of cleaved caspase-3, caspase-9, and PARP were assayed by Western blotting in cells treated with 5 at 20 μM for the indicated times. GAPDH served as the loading control.

Isomanool (5) Triggers ROS in PCa Cells. The activation of caspase-9 and caspase-3 suggested that 5-induced apoptosis may be mediated by the mitochondrial pathway.32 Thus, the mitochondrial production of ROS that can trigger cellular apoptosis was assessed using the fluorometric probe DCFHDA. As shown in Figure 5A,B, a significant increase in ROS was observed in PC3 cells after 30 min of treatment and maintained up to 4 h, which was followed by a slight decrease until 6 h after

cleaved caspase-3 after 12 h of treatment (Figure 4D), which was consistent with the results of caspase-3 activity analysis. PARP (poly ADP-ribose polymerase) cleavage, a downstream of caspase-3 in the apoptosis pathways,31 was found to increase in both cell lines (Figure 4D). Simultaneously, caspase-9 was also activated with increased cleavage of caspase-9 in a timedependent manner. The results indicated that 5-induced cell death is, at least partially, due to the arrest of the cell cycle and induction of apoptosis in PCa cells. 1340

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Figure 5. Effect of 5 on the generation of ROS. (A) PC3 and DU145 cells exposed to 20 μM 5 for the indicated times were collected, stained with DCFH-DA, and detected by flow cytometry. (B) Statistical analysis of the relative ratio of ROS formation, *P < 0.05, **P < 0.01 compared to the untreated control group. (C) Effect of the antioxidant NAC on 5-induced ROS generation and cell viability. PC3 and DU145 cells were pretreated with 5 mM NAC or vehicle for 1 h and then incubated with 5 in the presence or absence of NAC for 24 h. ROS production and cell viability were then assessed. Data are expressed as the means ± S.D., **P < 0.01 compared to the NAC-untreated control groups.

treatment. However, in DU145 cells, the increase in the ROS production was sustained up to 6 h during treatment. To determine the role of ROS in 5-mediated cell death, we tested whether abolishment of ROS could reverse the effect of 5. As shown in Figure 5C, addition of an antioxidant, Nacetylcysteine (NAC), dramatically diminished 5-induced ROS level and significantly attenuated the inhibitory effect of 5 on cell viability in both PC3 and DU145 cells. Therefore, our data demonstrated that 5-triggered ROS production, at least in part, played an important role in 5-mediated cell death. Isomanool-Induced ROS Are Involved in DNA Damage. Excessive ROS disturb redox-signaling pathways, which in turn trigger oxidative stress, resulting in DNA damage, cell cycle arrest, loss of cell function, and apoptosis.33 We therefore examined whether DNA damage occurred in cells with high ROS levels induced by 5. As shown in Figure 6, 5 induced DNA damage in PC3 and DU145 cells in a time-dependent manner, as demonstrated by the accumulation of phosphorH2AX at Ser139 (γH2AX), a typical marker of DNA damage,34 without increase of the total H2AX protein levels. The DNA damage response network is largely regulated by the ATM/Chk2 and ATR/Chk1 signaling pathways.35,36 We also observed that the activation of ATR/Chk1 was clear, as evidenced by the accumulation of phosphor-Chk1 (Ser296) after 12 h of treatment in both PC3 and DU145 cells (Figure 6). It was noted that the total Chk1 proteins were reduced by treatment with 5 in both cells. However, the mechanism of this decline was unknown, although previous studies showed that phosphorylation of Chk1 at Ser345 resulted in the degradation

Figure 6. Western blotting analysis of DNA damage and ATRsignaling-related proteins. PC3 and DU145 cells were treated with 20 μM 5 for the indicated times. GAPDH served as the loading control.

of Chk1.37,38 Because inactivation of Chk1/2 may reduce DNA repair and increase genomic instability to a level that is not compatible with cellular survival,39 we were prompted to analyze the changes of DNA repair protein BRCA1 in response to 5-induced DNA damage. As expected, in PC3 cells, the activation of BRCA1 (phosphorylation at Ser1524) declined after 8 h of treatment, which was not due to the decrease of total BRCA1 protein level. A similar result was found in DU145 cells (Figure 6). The down-regulation of phospho-BRCA1 may lead to repair failure and apoptosis. 1341

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which was purified using semipreparative HPLC (MeCN/H2O, 70:30) to yield 1 (36.0 mg, tR = 25.3 min) and 4 (10.2 mg, tR = 29.6 min). Furthermore, separation of Fr. 2.5 (73.0 mg) by semipreparative HPLC (MeOH/H2O, 90:10) yielded 5 (24.0 mg, tR = 18.5 min). Fraction 3 (0.4 g) was subjected to a Sephadex LH-20 (MeOH) and separated using reversed-phase C18 (MeOH/H2O, 7:3 to 9:1) to yield four subfractions (Fr. 3.1−3.4). Fr. 3.3 (58.0 mg) was purified by semipreparative HPLC (MeCN/H2O, 70:30) to yield 3 (3.6 mg, tR = 22.6 min) and 2 (0.9 mg, tR = 24.5 min). Heteroscyphin A (1): colorless crystals (MeOH); mp 154− 156 °C; [α]D25 −8 (c 0.1, MeOH); IR νmax 3490, 2936, 1123, 1083 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 343.2245 [M + Na]+ (calcd for C20H32O3Na, 343.2244). Heteroscyphin B (2): colorless crystals (MeOH); mp 153− 155 °C; [α]D25 −11 (c 0.1, MeOH); IR νmax 2925, 1452, 1369, 1026 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 336.2536 [M + NH4]+ (calcd for C20H34O3N, 336.2533). Heteroscyphin C (3): colorless crystals (MeOH); mp 163− 164 °C; [α]D25 +21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (2.11) nm; ECD (MeOH) λmax (Δε) 228 (+1.53) nm; IR νmax 2925, 1773, 1681, 1372, 1126 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 336.2535 [M + NH4]+ (calcd for C20H34O3N, 336.2533). Heteroscyphin D (4): colorless crystals (MeOH); mp 192− 193 °C; [α]D25 −35 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 225 (2.17) nm; ECD (MeOH) λmax (Δε) 235 (+0.27), 207 (−0.60) nm; IR νmax 3415, 2928, 1728, 1715, 1269, 1031 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS (positive mode) m/z 396.2752 [M + NH4]+ (calcd for C22H38O5N, 396.2745). X-ray Crystal Structure Analysis. Colorless crystals of heteroscyphin A (1) were obtained from MeOH. Intensity data were collected on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97.40 Refinements were performed with SHELXL-97 using full-matrix least-squares with anisotropic displacement parameters for all the non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with SHELXS-97. Crystallographic data (excluding structure factor tables) for the structure of heteroscyphin A (1) have been deposited with the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 972083 for 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, U.K. [fax: int. +44(0) (1223) 336 033; e-mail: [email protected]]. Crystal Data for Heteroscyphin A (1). C20H32O3: M = 320.46, monoclinic, a = 23.9804(18) Å, b = 6.4997(5) Å, c = 11.1603(9) Å, α = 90.00°, β = 92.105(3)°, γ = 90.00°, V = 1738.3(2) Å3, T = 100(2) K, space group C2, Z = 4, μ(Cu Kα) = 0.628 mm−1, 6780 reflections measured, 2664 independent reflections (Rint = 0.0389). The final R1 values were 0.0430 [I > 2σ(I)]. The final wR(F2) values were 0.1248 [I > 2σ(I)]. The final R1 values were 0.0431 (all data). The final wR(F2) values were 0.1250 (all data). The goodness of fit on F2 was 1.056. Flack parameter = 0.1(2). The Hooft parameter is 0.16(7) for 1057 Bijvoet pairs.

In conclusion, the current study describes the isolation and identification of five ent-labdane diterpenoids, four of which were new (1−4), and three known fusicoccane-type diterpenoids (6−8) from the liverwort H. tener. A sufficient amount of compound 5 was available to perform pharmacological analysis in vitro. Our results indicated that 5 enforced cell cycle arrest at the G0/G1 phase in both DU145 and PC3 cells, accompanied by induction of apoptosis. Further study demonstrated that 5 induced cellular apoptosis associated with ROS-induced DNA damage in PCa cells. Depletion of ROS by NAC, a ROS scavenger, blocked cell death induced by 5, which indicated that ROS might be a major inducer of cell death. Like many other agents, the DNA damage response to 5 was involved in the activation of the ATR/Chk1 signaling pathway.

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EXPERIMENTAL SECTION General Experimental Procedures. Melting points were determined on an X-6 melting-point apparatus (Beijing TECH Instrument Co. Ltd.) and were uncorrected. Optical rotations were measured on a PerkinElmer 241 MC polarimeter. UV spectra were obtained with an Agilent 8453E UV−vis spectroscopy system. ECD spectra were obtained on a Chirascan spectropolarimeter. IR spectra were recorded on a Nicolet iN 10 Micro FTIR spectrometer. NMR spectra were obtained using a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz with TMS as an internal standard. HRESIMS was carried out on an LTQOrbitrap XL. HPLC was performed on an Agilent 1100 system equipped with a G1310A isopump, a G1322A degasser, and a G1314 UV detector and a ZORBAX SB-C18 column (9.4 mm × 250 mm, 5 μm). All solvents used were of analytical grade. MCI-gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.), C18 reversed-phase silica gel (150−200 mesh, Merck), and Sephadex LH-20 (25−100 μm; Pharmacia Biotek, Denmark) were used for column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.) were used for TLC. Spots were visualized with UV light or by spraying with H2SO4−EtOH (1:9, v/v) followed by heating. Plant Material. The liverwort H. tener was collected from Maoer Mountain (1500 m), Guangxi Zhuang Autonomous Region, P. R. China, in April 2011 and was identified by Prof. Yuan-Xin Xiong, College of Life Sciences, Guizhou University, P. R. China. A voucher specimen (no. 20110416-16) was deposited at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, P. R. China. Extraction and Isolation. The air-dried powder of the whole plant material of H. tener (80.0 g) was extracted with 95% EtOH at room temperature (3 × 1.5 L, each for 1 week). Evaporation of the solvent in vacuo provided a dark residue (9.0 g), which was suspended in H2O (150 mL) and partitioned with Et2O (3 × 150 mL). The Et2O extract (5.5 g) was separated by MCI gel column chromatography (MeOH/H2O, 3:7 to 9:1) to yield four fractions (Fr. 1−4). Fraction 2 (0.8 g) was chromatographed on a Sephadex LH-20 (MeOH) and then separated using reversed-phase C 18 (MeOH/H2O, 6:4 to 9:1) to yield five subfractions (Fr. 2.1− 2.5). Fr. 2.3 (18.0 mg) was purified by semipreparative HPLC (MeOH/H2O, 75:25) to yield 7 (2.8 mg, tR = 15.6 min) and 8 (1.2 mg, tR = 18.2 min). Fr. 2.4 (203.5 mg) was separated using semipreparative HPLC (MeOH/H2O, 80:20) to obtain 6 (2.0 mg, tR = 8.6 min) and fraction a (58.0 mg, tR = 27.4 min), 1342

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Cell Culture and Treatments. The human hormoneindependent prostate carcinoma cell lines PC3 and DU145, myelogenous leukemia cell line K562, non-small-cell lung cancer cell lines A549, NCI-H292, and NCI-H1299, small-cell lung cancer cell line NCI-H446, and human normal bronchial epithelial cell HBE were cultured in RPMI 1640 medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin. The human prostate epithelial cell line RWPE1 was maintained in Keratinocyte1 medium (K-SFM) supplemented with 50 mg/L bovine pituitary extract and 5 μg/L epidermal growth factor (Gibco, Grand Island, NY). The cells were maintained in 5% CO2 at 37 °C until they reached approximately 50−70% confluence, and the cells were then treated with various concentrations of compounds. DMSO was used as the control vehicle. Cell Proliferation Assay. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide, Sigma, St. Louis, MO] assay was used to measure the proliferation of cells treated with different compounds in 96-well plates. After treatment with vehicle, the compounds alone, or cisplatin as a positive control for 24 h, the cells were incubated with 10 μL of MTT (5 mg/mL) for 4 h. Formazan product was then solubilized with 100 μL of DMSO, and the absorbance was measured at 570 nM by a plate reader (Bio-Rad, U.S.A.). Viable cells were represented as the percentage cell viability compared to control cells as per the equation below. The IC50 value was calculated on the basis of % cell viability using GraphPad Prism 5.0 (La Jolla, CA). All experiments were conducted at least three times.

CHAPS, 10% Glycerol, 2 mM EDTA, 5 mM DTT) were added. Plates were incubated at 37 °C for 2 h, and caspase activity was determined by fluorescence intensity with the excitation wavelength at 380 nm and emission wavelengths at 440 nm. ROS Measurement. Intracellular ROS accumulation was monitored using the fluorescent probe DCFH-DA. After treatment with 5 for the indicated times, cells were incubated with 10 μM DCFH-DA at 37 °C for 30 min. Cells were washed, collected, resuspended in PBS and analyzed immediately using flow cytometry (Becton Dickinson, U.S.A.). In some experiments, cells were pretreated with 5 mM NAC for 1 h prior to exposure with 5 and analyzed for ROS generation. Western Blotting. Cells were collected and lysed with RIPA buffer containing a fresh protease inhibitor mixture (50 μg/mL aprotinin, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 10 mM NaF, and 10 mM glycerol phosphate). Protein concentrations were quantified using BCA (bicinchoninic acid) assay. Equal amounts of proteins were separated by SDS-PAGE (12%) and electro-transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST buffer (20 mM Tris-buffered saline and 0.5% Tween 20) for 1 h at room temperature followed by incubation with the corresponding primary antibodies overnight at 4 °C. After washing with TBST buffer, secondary antibodies were used at 1:2000 dilutions for 45 min at room temperature. Immunoblot proteins were visualized using an enhanced chemiluminescence detection system (Millipore, Germany) followed by exposure to X-ray film. The primary antibodies used were as follows: p21CIP1, cyclin D1, cyclin E, Cdk4, poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cleaved caspase-9, p-Chk1, Chk1, γH2AX, H2AX, p-BRCA1, BRCA1, and glyceraldehyde3-phosphate dehydrogenase (GAPDH). Statistical Analysis. Data are presented as the means ± SD for triplicate experiments. The statistical significance of the differences between treated groups and controls was calculated using Student’s t test. P < 0.05 was considered statistically significant.

%cell viability OD570of treated cells − OD570of blank = × 100% OD570 of untreated control cells − OD570of blank

In some experiments, cells were exposed to 5 mM of the antioxidant N-acetylcysteine (Sigma-Aldrich, U.S.A.) for 1 h prior to treatment with 5. The cell proliferations in response to the compounds were analyzed using the MTT assay. Cell Cycle Analysis. Control and 5-treated cells were collected, washed with PBS, and fixed in iced 70% EtOH overnight at 4 °C. After incubation for 30 min in PBS containing 100 μg/mL RNase (Invitrogen) followed by incubation with 50 μg/mL PI (Sigma-Aldrich, U.S.A.) at 4 °C for 30 min in the dark, the cells were analyzed by flow cytometry, performed on a FACScan cytometry (FACSCalibur, Becton Dickinson, U.S.A.). The data were analyzed using MODFIT and CELLQUEST software (Verity Software House, Topsham, Maine, U.S.A.). Cell Apoptosis Assay. Cells were seeded in 6 well-plates for 24 h. After treatment with 5 for the indicated times, the cells were trypsinized and washed with PBS, then resuspended in the annexin V-FITC/PI staining solution according to the manufacturer’s instruction (Becton Dickinson, U.S.A.). Apoptotic cells were analyzed by flow cytometry (Becton Dickinson, U.S.A.). Determination of Caspase-3 Activity. Treated cells were harvested and collected as cell pellets, which were suspended in cell lysis buffer and incubated on ice for 15 min. After centrifugation (14 000g, 15 min at 4 °C), supernatants were collected and immediately measured for protein concentration and caspase-3 activity. Briefly, cell lysates were placed in 96-well plates, and then specific caspase-3 substrates (Ac-DEVD-AMC) and HEPES reaction buffer (50 mM Hepes pH 7.4, 0.1%

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ASSOCIATED CONTENT

S Supporting Information *

1D NMR, 2D NMR, HRESIMS, and IR spectra of compounds 1−4 and X-ray data of compound 1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-531-8838-2019. Tel.: +86-531-8838-2012. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 30925038 and 30730109).We acknowledge Mrs. J. Ren and Mr. B. Ma for NMR measurements and Mrs. Y.-H. Gao for HRESIMS determination. We are grateful to Dr. X.-N. Li (Kunming Institute of Botany, Chinese Academy of Sciences) for the single crystal X-ray diffraction analyses. 1343

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