Antiproliferative Activity of Abietane Diterpenoids against Human

Article pubs.acs.org/jnp

Antiproliferative Activity of Abietane Diterpenoids against Human Tumor Cells Olga Burmistrova,† M. Fátima Simões,‡ Patrícia Rijo,‡,§ José Quintana,† Jaime Bermejo,⊥ and Francisco Estévez*,† †

Departamento de Bioquímica y Biología Molecular, Unidad Asociada al Consejo Superior de Investigaciones Científicas (CSIC), Instituto Canario de Investigación del Cáncer, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain ‡ Faculdade de Farmácia da Universidade de Lisboa, iMed.UL, Avenida Prof. Gama Pinto, 1649-003 Lisboa, Portugal § CBIOS, Universidade Lusófona, Campo Grande 376, 1749-024 Lisboa, Portugal ⊥ Instituto de Productos Naturales y Agrobiología, CSIC, Avenida Astrofísico Francisco Sánchez 3, 38206 La Laguna, Tenerife, Spain ABSTRACT: In the present study, the cytotoxicity of 30 diterpenoids with an abietane or a halimane skeleton was determined against five human tumor cell lines (HL-60, U937, Molt-3, SK-MEL-1, and MCF-7). Diterpenoids containing an abietane skeleton including taxodone (1) and taxodione (2), as well as the semisynthetic derivatives 12, 14, 15, 17, and 22, were the most cytotoxic compounds for human leukemia cells. Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-xL did not confer resistance to abietane diterpene-induced cytotoxicity. Studies performed on HL-60 cells indicated that growth inhibition triggered by compounds 1, 12, 14, and 15 was caused by induction of apoptosis. This was prevented by the nonspecific caspase inhibitor Z-VAD-FMK and, in the case of compounds 14 and 15, reduced by the selective caspase-8 inhibitor Z-IETD-FMK. Cell death induced by these abietane diterpenes was found to be associated with the release of mitochondrial proteins, including cytochrome c, Smac/DIABLO, and AIF (apoptosis-inducing factor), accompanied by dissipation of the mitochondrial membrane potential (ΔΨ), and modulated by inhibition of extracellular signal-regulated kinases signaling and the p38 mitogen-activated protein kinase pathway.

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that was isolated previously from the aerial parts of Salvia clevelandii.6 This diterpenoid contains two double bonds conjugated with a carbonyl group. Taxodone (1) has been isolated also from the aerial parts of Salvia phlomoides,7 but so far the potential significance of this abietane diterpene in antitumor therapy is largely unexplored. The main aim of this work was to investigate the cytotoxic effects of 30 diterpenoids, including taxodone (1) and related abietane diterpenes as well a series of diterpenoids based on a halimane skeleton, against five human tumor cell lines and also to determine the mechanism of action of selected compounds of these structural types. The HL-60 tumor cell line was used as a cellular model to elucidate whether caspase activation and the MAPK (mitogen-activated protein kinase) cascade are involved in the mechanism of cell death triggered by these compounds.

any antitumor compounds have been shown to cause death in sensitive cells through the induction of apoptosis, a type of cell death defined by characteristic changes in nuclear morphology. In the area of cancer, over approximately the last 70 years, 49% of small-molecule anticancer agents are either natural products or directly derived from these.1 It is generally accepted that natural products and/ or natural product structures will continue to play a highly significant role in the drug discovery and development process. Abietane diterpenes display an array of biological activities including cytotoxic and antiproliferative activities against human tumor cells.2,3 Tanshinone IIA, a 20-norditerpene with an abietane-type skeleton containing a quinone moiety in the C-ring, has been proposed as a promising cytotoxic compound for human leukemia cells.4 Carnosol, an orthodiphenolic diterpene with an abietane skeleton, targets multiple deregulated pathways associated with inflammation and cancer, and it has selective toxicity toward cancer cells versus nontumorigenic cells.5 Abietane diterpenes such as 7α-acetoxyroyleanone and royleanone have been demonstrated to possess alkylating properties, but no clear correlation between the alkylating properties and cytotoxicity has been observed.3 Taxodone (1) is a naturally occurring diterpene based on an abietane skeleton © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Abietane Diterpenes Inhibit the Cell Viability of Human Tumor Cell Lines. In the present study, the potential cytotoxic properties of taxodone (1) as well as a series of Received: February 28, 2013

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dx.doi.org/10.1021/np400172k | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Chemical structures of the diterpenoids evaluated biologically.

cells (4.3 ± 1.3 μM). Moreover, SK-MEL-1 human melanoma cells were especially sensitive to coleon U (6) (IC50 10 ± 1.8 μM). Compound 6 exhibited IC50 values that were lower than 5, except in MCF-7 cells. Although the difference cannot be attributed exclusively to the methylation of the hydroxy group at C-12, previous studies on carnosic acid derivatives have shown that their cytotoxicity against P388 murine leukemia cells tends to decrease when the phenolic hydroxy groups are acetylated and methylated.9 The rearranged abietane diterpenoids 2-oxocandesalvone A (7) and teuvincenone A (8) displayed distinct cytotoxic effects. While 2-oxocandesalvone A (7) was not found to be cytotoxic for any of the five cell lines assayed, teuvincenone A (8) exhibited cytotoxic activity against leukemia cells. The synthetic derivatives 11,12-di-O-acetyl-14-deoxycoleon U (9) and 6,11,12-tri-O-acetyl-14-deoxycoleon U (10) displayed similar cytotoxic effects in all cell lines assayed, except for MCF-7 cells (IC50 > 30 μM). However, a triacetate derivative (10) showed IC50 values that were slightly lower than for the diacetate (9) in the four cell lines assayed. Differences in lipophilicity could explain the increase in cytotoxicity. The triacetyl derivative (10) might pass through biological membranes more easily than the diacetyl derivative (9) and reach a higher intracellular concentration. Although 7α-acetoxy-6β-hydroxyroyleanone (11) did not display potent cytotoxic properties for the human leukemia and the SK-MEL-1 human melanoma cell line used, it displayed antiproliferative activity against the MCF-7 human breast cancer cell line (IC50 6.8 ± 1.3 μM). Thus, compound 11 affected cell proliferation in a cell-type-specific manner. Since compound 11 has two hydroxy groups, derivatives were obtained with different radicals. Greater activity against human leukemia cell lines was shown by the corresponding

compounds including derivatives of 1 and a series of halimane diterpenoids were evaluated using several tumor cell lines (Figure 1, Table 1). Human myeloid leukemia cell lines (HL-60 and U937), including the lymphoid Molt-3 cell line, were, in general, the most sensitive to abietane compound-induced cytotoxicity, while the human breast cancer MCF-7 and human melanoma SK-MEL-1 cell lines were more resistant. Taxodone (1) and taxodione (2) showed similar IC50 (concentrations inducing a 50% inhibition of cell growth) values (ca. 1−4 μM) in all leukemia cell lines in which they were evaluated. These compounds exhibited also IC50 values of approximately 10 μM for the SK-MEL-1 and MCF-7 solid tumor cell lines, except for taxodione (2) against MCF-7, for which the IC50 value was >30 μM. These results suggest that the presence of a hydroxy group at C-6 is important in conferring cytotoxicity against MCF-7 cells. The IC50 values were approximately 5−10 μM for compounds 1 and 2 against the melanoma cell line (Table 1). Royleanone (3) and its 7α-ethoxy derivative (4) displayed IC50 values of >30 μM for all cell lines assayed. The only exception was the effect of 3 in Molt-3 cells, showing an IC50 value of 13.6 μM. The low cytotoxicity of royleanone (3) has been also observed in the MIA PaCa-2 human pancreatic cell line.3 Although 7α-ethoxyroyleanone (4) is not cytotoxic against all cell lines assayed in this study, its analogue, 7αacetoxyroyleanone, has been reported to display cytotoxicity against melanoma cells but not against leukemia cells.8 Inuroyleanol (5) displayed cytotoxic properties against U937 cells, but it was less active against HL-60 cells, while the SKMEL-1, MCF-7, and Molt-3 cell lines were highly resistant. Coleon U (6) was cytotoxic against both myeloid cell lines used, namely, HL-60 and U937. The IC50 value against HL-60 cells was 4.1 ± 1.8 μM, similar to the value obtained for U937 B



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Table 1. Effects of Diterpenoids on the Growth of Human Tumor Cell Linesa IC50 (μM) compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

HL-60 1.8 4.8 − − 20.8 4.1 − 3.5 11.5 4.1 25.4 3.6 5.7 2.7 2.4 8.4 3.8 9.0 4.7 15.9 11.8 5.6 − − − − − − − −

± 1.3 ± 0.6

± 1.9 ± 1.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 1.7 0.6 2.8 1.4 0.9 0.4 1.1 1.2 1.7 1.9 1.0 4.7 2.4 2.7

U937 1.5 3.0 − − 11.1 4.3 − 1.5 8.4 4.6 13.7 1.8 5.6 1.4 1.1 1.1 1.4 6.4 3.9 12.1 7.0 3.4 − 24.7 − − − − − −

Molt-3

± 0.6 ± 0.7

1.4 1.8 13.6 − − 8.8 − 2.7 13.4 4.6 12.7 6.9 − 1.5 1.4 10.0 1.5 3.0 10.8 5.4 6.3 3.5 − − − − − − − −

± 3.5 ± 1.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 2.5 1.6 4.0 0.6 2.6 0.2 0.1 0.1 0.2 1.4 0.2 1.6 0.6 0.1

± 0.9

± 0.2 ± 0.3 ± 12.7

± 5.2 ± ± ± ± ±

0.2 3.6 1.8 0.3 4.0

± ± ± ± ± ± ± ± ±

0.2 0.1 1.0 0.8 1.5 4.9 2.1 2.1 0.3

MCF-7 9.6 −b − − − 27.5 − − − − 6.8 25.5 − 3.1 2.9 11.4 9.1 5.9 28.3 23.3 17.1 6.5 − − − − − − − −

± 3.8

± 9.4

± 1.3 ± 2.1 ± ± ± ± ± ± ± ± ±

0.4 0.7 1.0 2.9 1.7 2.4 8.5 3.8 2.1

SK-MEL-1 7.2 9.6 − − − 10 − − 24.1 9.9 29.1 3.1 − 7.2 4.0 5.7 6.7 8.1 27.4 16.2 9.0 3.6 − − − − − − − −

± 2.8 ± 1.5

± 1.8

± ± ± ±

9.1 0.5 3.6 1.1

± ± ± ± ± ± ± ± ±

2.2 1.1 0.5 1.4 2.1 2.2 4.1 4.1 0.7

Cells were cultured for 72 h, and the IC50 values were calculated as described in the Experimental Section. The data shown represent the means ± SEM of 3−5 independent experiments with three determinations in each. b− not active, IC50 values >30 μM.

a

Members of a series of halimane diterpenoids (compounds 23−30) were investigated for their cytotoxicity against the HL60, U937, Molt-3, SK-MEL-1, and MCF-7 cell lines. However, none of the halimane diterpenoid derivatives displayed any cytotoxic properties (IC50 < 10 μM) for any of the five cell lines. The antitumor agent etoposide was used as a positive control for both HL-60 (IC50 0.5 ± 0.1 μM) and U937 cells (IC50 1.4 ± 0.2 μM). It was concluded that taxodone (1) and the semisynthetic derivatives 12, 14, 15, 17, and 22 display cytotoxic properties for human leukemia cells (HL-60, U937, and Molt-3), for the human melanoma cell line SK-MEL-1, and for the human breast cancer cell line MCF-7. Using SK-MEL-1 cells, the IC50 values were between 3.1 and 7.2 μM for compounds 1, 12, 14, 15, 17, and 22. These results are interesting given that melanoma frequently resists chemotherapy. Further studies are needed to determine the mechanisms involved in SK-MEL-1 cell death. In contrast, compound 2 was cytotoxic against leukemia cells but not against MCF-7 cells. Among the 22 abietane diterpenoids presented in Table 1, 14 compounds (abietane diterpenoids 3, 4, and 11−22) contain a p-benzoquinone moiety. The presence of this moiety does not play a crucial role for cytotoxicity, since compound 4 was not cytotoxic against all cell lines assayed and compound 3 exhibited cytotoxicity only against Molt-3 cells. Although no straightforward structural prerequisites for potent cytotoxic

derivatives 12−22, containing a monoester or diester functional group. The only exception was compound 13, with two introduced p-chlorobenzoyl radicals, which exhibited a lower potency than compound 11 against Molt-3, SK-MEL-1, and MCF-7 cells. Among the different monobenzoyl derivatives (at the quinone ring) there were no differences between an electron-donating substituent (−OCH3, compound 14) and an electron-withdrawing substituent (−Cl, compound 15) at the benzene moiety. However, the introduction of the electronwithdrawing substituent nitro group (compound 16) at the benzene moiety reduced the resultant cytotoxic activity. The pnitrobenzoyl derivative 17 was at least 6−10-fold more potent than 11 for all cell lines except for MCF-7. The diacetyl derivative 18 was more cytotoxic than the monoacetyl derivative (compound 20) in all cell lines assayed. A similar trend was observed with the dipropionyloxy derivative of compound 11 (compound 19) for the HL-60 and U937 cell lines. However, the monoacetyl derivative (compound 20) was more cytotoxic than the dipropionyloxy derivative 19 when evaluated against Molt-3, MCF-7, and SK-MEL-1 cells. The influence of the length of the hydrocarbon chain in compounds 20−22 was also explored. An increase in the number of carbons from two to four led to an increase in the cytotoxicity of all cell lines assayed, except for Molt-3. In this cell line the IC50 values for compounds 20−22 were similar. C



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Figure 2. Effects of abietane diterpenes 1, 12, 14, and 15 on human leukemia cells. (A) Differential effect of abietane diterpenes on proliferation of normal peripheral blood mononuclear cells (PBMC) vs HL-60 cells. PBMC and HL-60 cells were cultured in the presence of the concentrations indicated of each abietane diterpene for 24 h, and thereafter cell viability was determined by the MTT assay (*p < 0.05, significantly different from untreated control). (B) HL-60 cells were treated with 3 μM of the specified abietane diterpene for 24 h, and apoptosis was evaluated by flow cytometry. Values represent means ± SE of three independent experiments each performed in triplicate (*p < 0.05, significantly different from untreated control). (C) Cells were incubated as above, and the cell cycle phase distribution was determined by flow cytometry after propidium iodide staining. (D) Photomicrographs of representative fields of cells stained with Hoechst 33258 to evaluate nuclear chromatin condensation (i.e., apoptosis) after treatment with abietane diterpenes. (E) Flow cytometry analysis of annexin V-FITC and propidium iodide-stained HL-60 cells after treatment with 3 μM abietane diterpenes for 24 h. Cells appearing in the lower right quadrant show positive annexin V-FITC staining, which indicates phosphatidylserine translocation to the cell surface, and negative propidium iodide (PI) staining, which demonstrates intact cell membranes, both features of early apoptosis. Cells in the top right quadrant are double positive for annexin V-FITC and PI and are undergoing necrosis. Data are representative of three separate experiments. (F) Transmission electron micrographs of HL-60 cells after 24 h incubation with 3 μM taxodone 1 and compound 12.

activity can be described for the 22 abietane diterpenoids, it can be concluded that the introduction of a hydroxy group at C-6 and an acetoxy at C-7 in royleanone (3) increased the cytotoxicity (compound 3 vs compound 11) against all cell lines assayed. Also, in general, the esterification of the hydroxy groups at C-6, C-12, or both in compound 11 increased the resultant cytotoxicity. The only exception was compound 13,

which was less cytotoxic than 11 against Molt-3, MCF-7, and SK-MEL-1. Since the abietane compounds 1, 12, 14, and 15 showed similar levels of cytotoxicity against human leukemia cells, further experiments were performed with these compounds. It was evaluated as to whether they display cytotoxic properties in Bcl-2- and Bcl-xL-overexpressing human leukemia cells. The D



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Figure 3. Involvement of caspases in the induction of apoptosis triggered by abietane diterpenes on human leukemia cells. (A) Immunoblotting for the cleavage of caspases and PARP in HL-60 cells after 24 h incubation with 3 μM of the indicated compounds. (B) Activation of caspases in response to the selected abietane diterpenes. Cells were treated as above, and cell lysates were assayed for caspase-9, -8, and -3/7 activities. Results are expressed as n-fold increases in caspase activity compared with control. Values represent the means ± SEs of three independent experiments each performed in triplicate. (C) Effects of pancaspase inhibitor on the percentage of hypodiploid cells. Cells were pretreated with Z-VAD-FMK (100 μM) before the addition of the indicated abietane diterpene (3 μM), and apoptotic cells were analyzed by flow cytometry. Bars represent the mean ± SE of three independent experiments each performed in triplicate. (D) Effects on apoptosis of caspase inhibitors. Cells were pretreated with the corresponding inhibitor for 1 h and then incubated with abietane diterpenes for 24 h (*p < 0.05, significantly different from untreated control, #p < 0.05, significantly different from abietane diterpene treatment alone).

that bypasses the mitochondria or that they are able to inactivate the protection conferred by these proteins. Therefore, these compounds could be useful in the treatment of human cancers, because the increased expression of Bcl-2 and/ or Bcl-xL has been associated with chemoresistance particularly in the case of hematologic malignancies,12 and there is also a poor prognostic outcome with increased Bcl-xL expression.13 Normal quiescent lymphocytes (PBMC) showed no appreciable toxicity up to 3 μM of compounds 12, 14, and 15 for 24 h (Figure 2A). Although compound 1 (3 μM) was slightly toxic against PBMC (∼80% cell viability), it was more cytotoxic against HL-60 cells (36% cell viability). Abietane Diterpenes Induce Apoptosis on Human Myeloid Leukemia Cells. To elucidate the mechanism responsible for inhibition of cell proliferation, compounds 1, 12, 14, and 15 were examined for the induction of apoptosis in

results obtained indicated that human leukemia cell lines overexpressing Bcl-2 (U937/Bcl-2) and Bcl-xL proteins (HL60/Bcl-xL) were also sensitive to these compounds (1, 12, 14, and 15). The IC50 values in U937/Bcl-2 cells were 2.6 ± 0.4, 0.6 ± 0.1, 2.1 ± 0.1, and 1.1 ± 0.3 μM for compounds 1, 12, 14, and 15, respectively, and were similar to the values obtained for U937 cells. The IC50 values in HL-60/Bcl-xL cells were 2.7 ± 0.5, 1.1 ± 0.1, 2.9 ± 0.6, and 2.2 ± 0.5 μM for compounds 1, 12, 14, and 15, respectively, similar to the values obtained for HL-60/neo cells. The Bcl-2 family members confer resistance against apoptosis by inhibiting the mitochondrial permeability transition, the cytosolic accumulation of cytochrome c, and the activation of the executioner caspases of apoptosis.10,11 The fact that the mitochondria-protecting proteins Bcl-2 and Bcl-xL were unable to prevent the cytotoxicity of the abietane diterpenoids suggests that these compounds trigger an alternative pathway E



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In the intrinsic pathway, diverse proapoptotic signals provoke the translocation of cytochrome c from mitochondria to the cytosol and caspase-9 activation, which cleaves and activates downstream caspases. Cytochrome c release from the mitochondria is regulated tightly by B-cell lymphoma 2 (Bcl2) family proteins that control the permeabilization of the outer mitochondria membrane.16 To determine whether caspases are involved in the cell responses to abietane diterpenoids, these compounds were examined for the production of proteolytic processing of caspases and PARP [poly(ADP-ribose) polymerase] cleavage. To this end, HL-60 cells were treated with 3 μM of compounds for 24 h, and initiator caspases (caspases-8 and -9) were determined by Western blots using antibodies that bind both the proenzyme (caspase precursors) and the cleaved caspases (Figure 3A). The results indicated that compounds 1, 12, 14, and 15 stimulate the cleavage of inactive pro-caspase-9 to the active 35−37 kDa fragment. Also evaluated was the potential contribution of the extrinsic apoptotic pathway. As shown in Figure 3A, compounds 1, 12, 14, and 15 induced a significant pro-caspase-8 processing. Compound 15 induced the most potent hydrolysis of this zymogen. These compounds also stimulate the proteolytic processing of executioner caspase-3. Compounds 1 and 12 induced a more potent hydrolysis of procaspase-7 than pro-caspase-6. However, compounds 14 and 15 did not induce a clear processing of pro-caspase-6. Selected abietane diterpenes were assessed to determine if they could induce cleavage of pro-caspase-4, which is involved in endoplasmic reticulum stress, and observed a reduction (compound 1) and processing (compounds 12, 14, and 15) of this zymogen after 24 h of treatment. Poly(ADP-ribose)polymerase (PARP) protein, which is normally involved in DNA repair and a known substrate for caspase-3, was effectively hydrolyzed to the 85 kDa fragment by compounds 1, 12, 14, and 15. Although compound 1 induced the lowest activation of caspase-3, as determined by Western blot, it induced the highest PARP cleavage. The reason is that besides caspase-3, caspase-7 also cleaves PARP,17 and, as shown in Figure 3A, taxodone (1) also induced a processing/activation of caspase-7. Pro-caspase processing does not always correlate with activity, and so the enzymatic activities of caspase-3-like proteases (caspase-3/7) and of caspase-8 and -9 were also investigated in extracts of control or HL-60 cells treated with selected abietane diterpenoids. Cell lysates were assayed for cleavage of the tetrapeptides DEVD-pNA, IETD-pNA, and LEHD-pNA as specific substrates for caspase-3/7, caspase-8, and caspase-9, respectively. Induction of caspase-9, caspase-8, and caspase-3 activities was detectable significantly after 24 h of treatment with compounds 1, 12, and 14 (Figure 3B), although there was not a clear caspase-9 and -8 activation after treatment with compound 15. Caspase-3/7 activity increased 4.6-fold in taxodone (1)-treated cells, while caspases-9 and -8 activities increased 2.1-fold and 1.5-fold over the control, respectively. A 2-fold increase in caspase-3/7 activity was observed in compound 12-treated cells, and similar increases were observed in cells treated with compounds 14 and 15. To investigate whether taxodone (1) and the abietane diterpenes 12, 14, and 15 require the activation of caspases to induce apoptosis, HL-60 cells were pretreated with the broadspectrum caspase inhibitor Z-VAD-FMK. The results indicated that apoptosis was either reduced significantly (compound 1) or almost completely suppressed (compounds 12, 14, and 15) (Figure 3C), indicating that the abietane diterpenes selectively

HL-60 cells. Evaluation of the number of hypodiploid cells by flow cytometry showed that the percentage of apoptotic cells increased approximately 5-fold (20.3 ± 1.9% vs 4.4 ± 0.8%), 11-fold (48.0 ± 15.9% vs 4.4 ± 0.8%), 8-fold (34.5 ± 4.8% vs 4.4 ± 0.8%), and 9-fold (40.4 ± 22% vs 4.4 ± 0.8%) after 24 h exposure to compounds 1, 12, 14, and 15, at a concentration of 3 μM, respectively (Figure 2B). A significant number of apoptotic cells was already detected at 12 h of treatment and increased in a time-dependent manner (Figure 2C). Although the abietane diterpenoids 17 and 22 showed IC50 values similar to compounds 1, 12, 14, and 15 in all human tumor cell lines assayed, the flow cytometry experiments revealed that 17 and 22 were less potent apoptotic inducers (results not shown). It is possible that other mechanisms that induce cell growth inhibition may be triggered by these diterpenoids in HL-60 cells. A similar observation has been described for the abietane diterpenoid cryptotanshinone. This diterpene strongly inhibited the cell growth of HepG2 cells but did not cause apoptosis or significant changes to the cell cycle.14 It was determined also whether these compounds (1, 12, 14, and 15) induce morphological changes characteristic of apoptotic cell death by fluorescent microscopy. Whereas untreated cells exhibit a typically nonadherent, approximately round morphology, cells exposed to the abietane diterpenes selected display condensation and fragmentation of chromatin, which is detectable after only 6 h of treatment (Figure 2D). These compounds also induced DNA fragmentation, considered the end point of the apoptotic pathway (results not shown), as well as externalization of phosphatidylserine in HL60 cells (Figure 2E). Transmission electronic microscopy was used to visualize the apoptosis-associated ultrastructural changes (Figure 2F). Cells with fragmented and/or condensed nuclei characteristic of apoptotic cells were clearly visible. Cells treated with compounds 1 and 12 showed a dense perinuclear condensation and also abundant vacuoles. To study the effect of abietane diterpenes on the cell cycle, cells were cultured in the presence of selected abietane diterpenes and analyzed by flow cytometry. When the subdiploid (sub-G1) portions of the histograms were excluded from the analyses, taxodone (1) caused an arrest of ∼57% cells in the G0−G1 phase of the cell cycle, which was accompanied by a concomitant decrease in cells in the S phase of the cell cycle. The percentage of control-treated cells in the S phase was ∼33%, which decreased to ∼16% after treatment with taxodone (1) for 24 h. Diterpenoids 14 and 15 caused a significant G1 arrest (∼56−59% vs ∼44% in control cells) and also a decrease in the percentage of cells in the G2−M phase (∼12% vs ∼23% in control cells). Lower concentrations than 3 μM of abietane diterpenoids 1, 12, 14, and 15 were unable to induce apoptosis. Since a significant increase in the percentage of cells in sub-G1 (i.e., hypodiploid) cells was observed only at 3 μM of these compounds, this concentration was used in the following experiments. Effects of Abietane Compounds 1, 12, 14, and 15 on Caspase and PARP Processing. Apoptosis can occur with or without the activation of caspases, a family of conserved cysteine aspartate-specific proteases that are expressed constitutively as zymogens.15 In general, two major pathways of caspase activation during apoptosis have been described. The extrinsic pathway involves death receptors, such as tumor necrosis factor or Fas receptors, and is dependent on the initiator caspase-8, which activates the downstream effector caspases (caspase-3, -6, and -7), inducing a cascade of caspases. F



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Figure 4. (A) Effects of abietane diterpenes (1, 12, 14, and 15) on mitochondrial protein release and on Bcl-2 family members. Cells were incubated in the presence of the abietane diterpene indicated, and whole cell lysates or cytosolic fractions (in the case of mitochondrial proteins) were probed with antibodies raised against the indicated proteins. β-Actin was used as a loading control. (B) Selected abietane diterpenes reduce the mitochondrial membrane potential (ΔΨm). HL-60 cells were treated with 3 μM abietane diterpenes and harvested at 24 h, and ΔΨm analyzed with JC-1. The intensity of JC-1 fluorescence was analyzed by flow cytometry as described in the Experimental Section. Similar results were obtained in two separate experiments with each performed in triplicate.

abietanes 14 and 15, as demonstrated by pro-caspase-8 processing (Figure 3A). It seems that different caspases are important in cell death signaling triggered by abietane diterpenoids 1, 12, 14, and 15 in HL-60 cells. On comparing the structures of these abietane diterpenoids, these results indicate that the presence of a p-chlorobenzoyl substituent and to a lesser extent a p-methoxybenzoyl unit at position C-12 of 7α-hydroxy-6β-hydroxyroyleanone (11) is important in conferring activation of the death receptor pathway of cell death. To determine whether the apoptosis induced by these abietane diterpenoids involves the release of mitochondrial proteins, cytosolic preparations were analyzed by immunoblotting. Consistent with effects on apoptosis, treatment with abietane diterpenes resulted in a substantial release of the proapoptotic mitochondrial proteins cytochrome c, Smac/ DIABLO, and AIF (apoptosis-inducing factor) into the cytosolic fraction (Figure 4A). Moreover, Bcl-2 levels were clearly decreased after treatment with abietane diterpenes 1 and 15, while there was a slight decrease in cytosolic Bax (Figure 4A). Taxodone (1) also induced a decrease in Bid levels, in accordance with caspase-8 activation.

induced cell death by a caspase-dependent mechanism. To identify the caspases that are important, the effects of the cellpermeable caspase inhibitors Z-LEHD-FMK (a caspase-9 inhibitor) and Z-IETD-FMK (a caspase-8 inhibitor) were examined. As shown (Figure 3D) the caspase-9 inhibitor did not block apoptosis stimulated by the abietane diterpenes selected. These results are consistent with those observed for compounds 14 and 15, since they displayed a minimal effect on pro-caspase-9 processing (Figure 3A) and/or caspase-9 activity (Figure 3B). The lack of a significant effect of the caspase-9 inhibitor on apoptosis induced by 1 and 12 was surprising, given the presence of proteolytic processing of pro-caspase-9 (Figure 3A) and also the increase in caspase-9 activity (Figure 3B). Compensatory mechanisms such as increases in the activation of other caspases, such as caspase-8, when a particular caspase activity is suppressed (i.e., caspase-9) might be involved, as previously described.18 In contrast, the caspase-8 inhibitor reduced the percentage of hypodiploid cells induced by 14 and 15, but it was unable to suppress cell death induced by 1 and 12 (Figure 3D). These results suggest a main role of the extrinsic apoptotic pathway in cell death induced by G



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Figure 5. (A) Effects of mitogen-activated protein kinase inhibitors on abietane diterpene-induced apoptosis. HL-60 cells were preincubated with PD98059 (PD, 10 μM), U0126 (U0, 10 μM), SP600125 (SP, 10 μM), and SB203580 (SB, 2 μM) for 1 h and then treated with abietane diterpenes for 24 h. Apoptosis was quantified by flow cytometry, and bars represent the means ± SE of three independent experiments each performed in triplicate (*p < 0.05, significantly different from untreated control; #p < 0.05, significantly different from the corresponding abietane diterpene treatment alone). (B) Representative Western blots showing the time-dependent phosphorylation of MAPK by the abietane diterpenes indicated. (C) Abietane diterpene-induced ROS generation. HL-60 cells were incubated with 3 μM of abietane diterpenes for the time points indicated, and the fluorescence of oxidized H2DCF was determined by flow cytometry. Similar results were obtained from three independent experiments.

To examine whether a disruption of the mitochondrial membrane potential (ΔΨm) is required for the release of mitochondrial proteins, cells were stained with JC-1 (5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and analyzed by flow cytometry. It was found that the ΔΨm dropped only after 24 h of treatment (Figure 4B), suggesting that the dissipation of ΔΨm is not an early event in abietane diterpene-induced apoptosis. In accordance with these results, neither of the permeability transition pore inhibitors cyclosporin A (25 μM, 24 h) and bongkrekic acid (25−50 μM, 24 h) had any effect on abietane diterpene-induced apoptosis (results not shown). Abietane Diterpenes Activate Mitogen-Activated Protein Kinases. MAPK pathways can mediate signals that either promote or suppress the growth of malignant hematopoietic cells.19 To determine whether the phosphorylation of MAPKs plays a key role in abietane diterpeneinduced apoptosis, the effects of specific inhibitors were examined initially on taxodone (1)-induced cell death. Treatment of cells with PD98059 and/or U0126, inhibitors

of mitogen-activated extracellular kinases 1/2 (MEK1/2), and with the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) inhibitor SP600125 did not alter the rate of taxodone (1)-mediated apoptosis (Figure 5A), suggesting that activation of ERK1/2 (extracellular signal-regulated kinases 1/ 2) or JNK/SAPK is not required for cell death. However, inhibition of the p38 mitogen-activated protein kinases (p38MAPK), using SB 203580, enhanced taxodone (1)-induced cell death. The percentage of apoptotic cells increased from 15.1 ± 3.8% in taxodone (1)-treated cells to 54.3 ± 7.6% with SB203580. In HL-60 cells, taxodone (1) and SB203580 combined caused almost four times the rate of cell death when compared with taxodone (1) alone (Figure 5A) and 15 times more cell death than SB203580 alone (results not shown). The 4-fold (or 15-fold) increase in apoptosis was calculated by comparing the sub-G1 readings of treated samples with the value of the untreated control as 1.0. These results have possible implications for the use of taxodone (1) in combination with p38MAPK inhibitors as potential therapeutic agents. The effects of selective MAPK inhibitors on cell death H



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the full potential of taxodone (1) and the abietane diterpenoids as chemopreventive or chemotherapeutic agents.

induced by 12 were different from those triggered with taxodone (1). Neither the selective JNK/SAPK inhibitor SP600125 nor the p38MAPK or specific MEK1/2 inhibitors attenuated or influenced cell death (Figure 5A), suggesting that activation of these MAPKs is not required for apoptosis. In contrast, differing effects were observed for the combination of compounds 14 and 15 and MAPK signaling inhibitors. Both the MEK1/2 inhibitors and SP600125 enhanced the apoptosis induced by compound 14. However, inhibition of p38MAPK was found to almost completely suppress cell death induced by compound 14 in the combination group (compound 14 + SB203580). In contrast, the p38 MAPK inhibitor did not display any significant effect on apoptosis triggered by the abietane diterpene 15. These data suggest that activation of p38 MAPK is involved in apoptosis induced by compound 14. Incubation of HL-60 cells with taxodone (1) leads to phosphorylation of p38MAPK, which was detected 1 h after addition of 1 and remained elevated for at least 6 h. However, the activation peak of JNK/SAPK was observed transiently after 4 h, and the phosphorylation of ERK1/2 (extracellular signalregulated kinases 1/2) was not detected under the same experimental conditions (Figure 5B). These results indicated that abietane diterpene 1 treatment leads to activation of p38MAPK and JNK/SAPK following different kinetics. In contrast to the results observed with taxodone (1), there was not any clear phosphorylation of MAPK for at least 6 h in compound 14-treated cells, but a decrease in the phosphorylation of phosphor-p38MAPK and also a faint decrease in total p38MAPK were evident. Reactive Oxygen Species (ROS) Were Not Required for Abietane Diterpene-Induced Cell Death. Since increased ROS production in leukemic cells may lead to the activation of MAPKs and cell death,20 it was decided to investigate whether ROS are involved in abietane diterpeneinduced apoptosis. To this end, abietane diterpene-treated cells were loaded with the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCF-DA) and then analyzed by flow cytometry. ROS formation was detected at 30 min, 1 h, and 4 h after treatment with abietane diterpenes 15, 12, and 1, respectively (Figure 5C). However, no intracellular ROS generation occurred after exposure to compound 14. To determine whether the generation of ROS is involved in cell death induced by 1, 12, and 15, the effects of the antioxidants N-acetyl-L-cysteine (NAC, 10 mM), α-tocopherol (vitamin E, 25 μM), and trolox (2 mM) were investigated. NAC was the only antioxidant that blocked cell death, which suggests that ROS generation is crucial for abietane diterpene-induced cell death (results not shown). However, the protective role of NAC appears to be a consequence of a direct binding to the abietane diterpenes, since cell death was not observed when abietane diterpenes and NAC were preincubated for 2 h and then added to the cells (results not shown). These findings suggest that abietane diterpene-induced apoptosis is independent of ROS generation. In conclusion, the abietane diterpenes evaluated in the present study are cytotoxic against several human tumor cell lines, and in the case of human leukemia cells their cytotoxicity involves activation of both the extrinsic and intrinsic pathways of apoptosis. The abietane diterpene taxodone (1) also enhances the mitogen-activated protein kinase pathway, and the combined treatment of 1 and the p38MAPK inhibitor leads to enhanced cell death. Further studies are needed to understand

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EXPERIMENTAL SECTION

Diterpenoids Evaluated. The compounds assayed were either natural diterpenoids isolated from plant species belonging to the Lamiaceae family or semisynthetic derivatives obtained from naturally occurring compounds. All compounds have been described previously, and their isolation procedures or chemical preparation is reported in the references cited. Abietane diterpenoids taxodone (1), taxodione (2), and royleanone (3) were isolated from Salvia phlomoides,7 7αethoxyroyleanone (4) and inuroyleanol (5) were isolated from Salvia lavandulaefolia,21 and coleon U (6) was isolated from Plectranthus grandidentatus.22 The rearranged abietane diterpenoids 2-oxocandesalvone A (7) and teuvincenone A (8) were isolated as constituents of S. palaestina23 and Teucrium polium subsp. vincentinum,24 respectively. The semisynthetic derivatives 11,12-di-O-acetyl-14-deoxycoleon U (9) and 6,11,12-tri-O-acetyl-14-deoxycoleon U (10) were prepared starting from 14-deoxycoleon U, as described previously.7 Large amounts of 7α-acetoxy-6β-hydroxyroyleanone (11) were obtained from P. grandidentatus,25 and the semisynthetic derivatives 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (12), 7α-acetoxy6β-(4-chloro)benzoyloxy-12-O-(4-chloro)benzoylroyleanone (13), 7α-acetoxy-6β-hydroxy-12-O-(4-methoxy)benzoylroyleanone (14), 7α-acetoxy-6β-hydroxy-12-O-(4-chloro)benzoylroyleanone (15), 7αacetoxy-6β-hydroxy-12-O-(4-nitro)benzoylroyleanone (16), 7α-acetoxy-6β-(4-nitro)benzoyloxyroyleanone (17), 6β,7α-diacetoxy-12-Oacetylroyleanone (18), 7α-acetoxy-6β-propionyloxy-12-O-propionylroyleanone (19), 6β,7α-diacetoxyroyleanone (20), 7α-acetoxy-6βpropionyloxyroyleanone (21), and 7α-acetoxy-6β-butyryloxyroyleanone (22) were prepared from 11, as already described.26 The synthetic derivatives (11R*,13E)-11-acetoxyhalima-5,13-dien15-ol (23), (11R*,13E)-15-propionyloxyhalima-5,13-dien-11-ol (24), (11R*,13E)-15-butyryloxyhalima-5,13-dien-11-ol (25), (11R*,13E)15-benzoyloxyhalima-5,13-dien-11-ol (26), (11R*,13E)-15-(4methoxy)benzoyloxyhalima-5,13-dien-11-ol (27), (11R*,13E)-halima5,13-diene-11,15-diol (28), and (11R*,13E)-11-acetoxyhalima-5,13dien-15-oic acid methyl ester (29) were obtained as described previously,27 starting from (11R*,13E)-11-acetoxyhalima-5,13-dien15-oic acid (30), a 5-halimene diterpenoid isolated from P. ornatus.22 Cell Culture. The HL-60, U937, and Molt-3 human leukemia and SK-MEL-1 human melanoma cells were grown in RPMI 1640 containing 2 mM L-glutamine supplemented with 10% (v/v) heatinactivated fetal bovine serum. These cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The MCF-7 human breast cancer cell line was cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. HL-60 cells transfected with the pSFFV-neo plasmid (HL-60/neo) and/or pSFFV-bcl-xL plasmid (HL-60/Bcl-xL) were established by Dr. Kapil N. Bhalla (Medical College of Georgia Cancer Center, USA), donated by Dr. Angelika Vollmar (University of Munich, Germany), and cultured as described.28 The U937 cell line overexpressing human Bcl-2 (kindly provided by Dr. Jacqueline Bréard, INSERM U749, Faculté de Pharmacie Paris-Sud, France) was cultured as described.29 Human peripheral blood mononuclear cells were isolated from heparinanticoagulated blood of healthy volunteers by centrifugation with Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Cytotoxicity Assays on Human Cancer Cell Lines. The cytotoxicity of compounds was evaluated by using a colorimetric 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, 1 × 104 exponentially growing cells were seeded in 96-well microculture plates with various compound concentrations. After the addition of MTT (0.5 mg/mL), cells were incubated at 37 °C for 4 h. Sodium dodecyl sulfate (10% w/v) in 0.05 M HCl was added to the wells and then incubated at room temperature overnight under dark conditions. The absorbance was measured at 570 nm. Concentrations inducing a 50% inhibition of cell I



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Statistical Analysis. Statistical significance of differences between means of control and treated samples was calculated using Student’s ttest, with p values of

Antiproliferative Activity of Abietane Diterpenoids against Human

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