Cytotoxic effects of hyperatomarin, a prenylated phloroglucinol from Hypericum annulatum Moris subsp. annulatum, in a panel of malignant cell lines.

Abstract

The cytotoxic effects of hyperatomarin-a prenylated phloroglucinol isolated from Hypericum annulatum Moris subsp. annulatum were assessed in a broad spectrum of tumor cell lines originating from leukemias, lymphomas and solid malignancies. The tested compound exerted strong concentration-dependent cytotoxic effects (IC50 values ranging 0.14-15.7[micro]M), comparable to and even outclassing in some cell lines those of the established anti-cancer drug daunorubicin. Exposure of different human tumor cell lines to hyperatomarin resulted in strong mono- and oligo-nucleosomal fragmentation of genomic DNA, as evidenced by 'Cell death detection' ELISA kit and by DNA-electrophoresis, which unambiguously indicates that the induction of apoptosis is implicated in the cytotoxic mode of action of the tested compound.

Keywords: Hyperatomarin; Prenylated phloroglucinols; Cytotoxicity; Apoptosis.

Introduction

Exploring the plant chemical diversity appears to be a vital strategy for development of novel anticancer drugs characterized by alternative mode of action or lower toxicity. The intensive screening of plant-derived com-pounds for antineoplastic activity during the last several decades has led to the introduction of several drug classes occupying a significant part of the anticancer drug market (Cragg and Newman, 2000).

Substantial data of evidence suggest that the members the Guttiferae family, e.g. diverse Hypericum, Garcinia, Clusia, Cratoxylum species, appear to be a valuable resource of cytotoxic compounds. Prominent cytotoxic activity has been established for a variety of secondary metabolites bearing complex prenylation patterns, such as phloroglucinols (Winkelmann et al., 2003), xanthones (Chen et al., 2004; Ho et al., 2002; Laphookhieo et al., 2006; Suksamrarn et al., 2006; Thoison et al., 2000), benzophenones (Chaturvedula et al., 2002; Matsumoto et al., 2003), and depsidones (Permanaa et al., 2005; Xu et al., 2000). An important example is hyperforin, a prenylated phloroglucinol from Hypericum perforatum, which in addition to its well-known antidepressant activity has been shown to exhibit potent cytotoxic and proapoptotic effects against tumor cell lines, in low micromolar concentrations, and to inhibit tumor-induced angiogenesis (Schempp et ah, 2002; Quiney et ah, 2006a, b, c).

Hypericum annulatum Moris subsp. annulatum (H. atomarium subsp. degenii) is a herbaceous plant, indigenous for the Balkan Peninsula and Sardinia (Jordanov and Kozucharov, 1970; Robson, 1993). In previous phytochemical studies of this plant the presence of flavonoids, catechins, hypericins, xanthones and benzophenones has been established (Kitanov, 2001; Kitanov and Nedialkov, 2000, 2001; Nedialkov and Kitanov, 2002). The latter were found to exhibit antioxidant and cytoprotective effects in different experimental systems (Mitcheva et al. 2006; Momekov et al., 2006). Recently, hyperatomarin, a prenylated phloroglucinol derivative (Fig. 1), has been isolated from the title plant and found to exert antibacterial activity against some Gram-positive bacteria (Savikin-Fodulovic et al., 2003).

[FIGURE 1 OMITTED]

Taking into account the cytotoxic effects of hyperfor-in and structurally related prenyl-phloroglucinols, isolated from Hypericum species, we sought to determine the antineoplastic activity of hyperatomarin in a panel of malignant cell lines, originating from different neoplastic diseases. A pharmacodynamic evaluation of the ability of hyperatomarin to trigger apoptosis is presented as well.

Materials and methods

Plant material

The aerial parts of Hypericum annulatum Moris subsp. annulatum were collected during the flowering season from wild habitat at the Central Rhodope Mountains in July 2006. A voucher specimen (No. 144296) has been deposited at the Herbarium of Botany Institute of Sofia (SOM).

Isolation and identification of hyperatomarin

The air-dried and powdered herb (700 g) was extracted with n-hexane (21 x 6) at room temperature. The combined extracts were evaporated in vacuo to give dark green residue (48 g) which was then subjected to column chromatography on silica gel 60 (Merck; 0.063-0.2 mm; 7.5 x 40 cm) and was eluted with CH[Cl.sub.3] and mixtures of CH[Cl.sub.3]-MeOH (99:1 [right arrow] 94:6). Six pooled fractions (A, B, C, D, E and F) were obtained. Fraction B (18 g) was mixed with Celite [R] 545 (Merck; 0.01-0.2mm; 100 g) and was washed with MeOH 500 (ml). Evaporation of methanol solution resulted in orange semi-transparent oil (13 g). A portion (6g) of this oil was subjected to vacuum liquid chromatography on silica gel 60 (Merck; 0.04-0.063mm; 7.5 x 20cm) and was eluted with mixtures of iz-hexane-EtOAc (95:5 [right arrow] 87:13). Ten pooled fractions ([B.sub.1]-[B.sub.10]) were obtained. A [N.sub.2] driven (ca. 1 atm) CC on LiChroprep [R] RP-18 (Merck; 40-63 [micro]m; 4x 16 cm) of a portion of [B.sub.3] (300 mg) eluted with a mixture of [CH.sub.3]CN-[H.sub.2]O (9:1) gave hyperatomarin (96% purity, HPLC). The structure was confirmed by means of spectral methods (UV, IR, [.sup.1]H--and [.sup.13]C-NMR, EI-MS).

Cell lines and culture conditions

The panel of cell lines used in this study consisted of LAMA-84, K-562 (chronic myeloid leukemias), SKW-3 (a KE-37 derivative; T-cell leukemia), U-266 (multiple myeloma), DOHH-2 (non-Hodgkin lymphoma), HD-MY-Z (Hodgkin's lymhoma), EJ (urinary bladder carcinoma), MCF-7 (breast cancer), SAOS-2 (osteosarcoma) and Neuro-2A (murine neuroblastoma). EJ cells were obtained from the American Type Cell Cultures while the other cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were maintained in controlled environment--cell culture flasks at 37 [degrees]C in a 'Heraeus' incubator with 5% [CO.sub.2] humidified atmosphere. MCF-7 were cultured in 90% RPMI1640 medium supplemented with 10% FBS, MEM nonessential amino acids, 1 mM sodium pyruvate and 10 [micro]g/ ml human insulin. Neuro-2A cells were maintained in growth medium consisting of 90% Dulbecco's MEM, 10% FBS, supplemented with non-essential amino acids. For all of the remaining cell lines the growth medium was 90% RPMI-1640, supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine.

Cytotoxicity determination

Stock solutions of the tested compounds were prepared in purified water (daunorubicin) or DMSO (hyperatomarin) and were consequently diluted with RPMI-1640 medium to yield the desired final concerntrations. At the final dilutions obtained, cells were never exposed to DMSO concentrations exceeding 0.5%.

For the cytoxicity determination cells were seeded into 96-well plates (100 [micro]l/well at a density of 1 x [10.sup.5] cells/ml) and exposed to various concentrations of the tested compounds for 72h. Cell survival was determined with the MMT dye-reduction assay as previously described (Mosmann, 1983), with some modifications (Konstantinov et al., 1999). Briefly, after the incubation with the test-compound, MTT solution (10 mg/ml in PBS) was added (10 [micro]/well). Plates were further incubated for 4h at 37[degrees]C and the formazan crystals obtained were dissolved by adding 100[micro]/well 5% formic acid in 2-propanol. Absorption was measured on a microprocessor-controlled microplate reader (Labexim LMRI[R]) at 540 nm. For each concentration at least eight wells were used. As a bank solution 100[micro]l RPMI 1640 medium with 10[micro]l MTT stock and 100 [micro]l 5% formic acid in 2-propanol was used.

Photometric determination of apoptosis

The characteristic for apoptosis mono-and oligonucleosomal fragmentation of genomic DNA was detected using a commercially available 'Cell Death Detection' ELISA kit (Roche Diagnostics, Germany). Exponentially growing HD-MY-Z, DOHH-2, SKW-3, and EJ cells were plated in sterile petri dishes and exposed to equipotent concentrations ([IC.sub.50]) of hyperatomarin for 24h. Cytosolic fractions of 1 x [10.sup.4] cells per group (treated or untreated) served as an antigen source in a sandwich ELISA, utilizing primary anti-histone anti-body-coated microplate and a secondary peroxidaseconjugated anti-DNA antibody. The photometric immunoasay for histone-associated DNA fragments was executed according to the manufacturer's instructions at 405 nm, using a microplate reader (Labexim LMR-1). The results were calculated as the oligonucleosome enrichment factor (representing a ratio between the absorption in the treated versus the untreated control samples) and expressed as percentage (untreated control = 100%).

DNA isolation and gel electrophoresis

Oligonucleosomal DNA fragments were isolated from the cytosolic fraction of hyperatomarin or daunorubicin treated SKW-3 cells after 24h continuous exposure, and analysed by gel eletrophoresis as described elsewhere (Konstantinov et al., 1999). Briefly, exponentially growing SKW-3 cells were exposed to equitoxic concentrations of the tested compounds and after the treatment period the cells were pelleted and washed thrice in PBS. Thereafter the cell pellets were resuspended in 0.25 ml PBS and lysed through addition of 0.5 ml lysis buffer (0.5% Triton X-100, 20 mM Tris HCl, 1mM EDTA, pH = 7.4). After centrifugation (13,000 rpm for 20 min) the supernatants were transferred into Eppendrof safe lock tubes. DNA was precipitated via addition of 0.187 ml 6M NaCl solution and 0.937 ml 2-propanol. After mixing the samples were incubated at --20[degrees]C overnight and thereafter were spun at 13,000 rpm for 20 min. DNA precipitates were then washed with 1 ml 70% ethanol. The DNA pellets were air dried and dissolved in 20[micro]l distilled water. The samples were analysed by 0.8% agarose gel electrophoresis and visualized by ethidium bromide staining and UV transillumination. The DNA laddering was documented using an UV-transilluminator with CCD camera (UVBioDoc-It [TM] System, al-Biotech GmbH, Martinried, Germany).

Data processing and statistics

The cytoxicity assays were carried out in eight separate experiments, whereas the apoptosis induction evaluation was conducted in quadruplicate. The MTT data were fitted to sigmoidal concentration-response curves and the corresponding [IC.sub.50] values were calculated using non-linear regression analysis (GraphPad Prizm package). Statistical processing exploited Student's t-test with p[less than or equal to] 0.05 set as significance level.

Results and discussion

The antineoplastic activity of hyperatomarin was evaluated in vitro in a panel of malignant cell lines after 72h continuous exposure. The cell viability was assessed using the MTT-dye reduction assay and the corresponding [IC.sub.50] values were calculated as the concentrations of tested compounds causing 50% decrease of cell survival (Table 1). The clinically utilized antineoplastic drug daunorubicin (Daunoblastin[R], Pharmacia) was ezploited as a cytotoxic reference compound.

The prenylated phloroglucinol derivative exerted prominent cytotoxic activity, whereby most of the cell lines were found to be highly sensitive with very low micromolar [IC.sub.50] values, generally comparable to those of the referent drug daunorubicin. The most distinguished cytoxicity was encountered with U-266, DOHH-2 and MCF-7 cells, whereby hyperatomarin significantly outclassed the reference compound in terms of relative potency. The CML-derived cell lines LAMA-84 and K-562, the urinary bladder carcinoma EJ, as well as the murine neuroblastoma Neuro-2A proved to be less responsive with [IC.sub.50] values approximately an order of magnitude higher relative to those obtained with the other cell lines.

Table 1. Antineoplastic activity of hyperatomarin and daunorubicin
in a panel of tumor cell lines after 72h exposure (MTT-dyereduction
assay)

                              [IC.sub.50] ([micro]M)

Cell line   Cell type      Hyperatomarin   Daunocubicin

LAMA-84    Chronic        12.65 [+ or -]  1.27 [+ or -]
           myeloid        2.07            0.09
           leukemia
           (human)

K-562      Chronic        15.71 [+ or -]  1.51 [+ or -]
           myeloid        1.11            0.05
           leukemia
           (human)

SK W-3     T-cell          3.04 [+ or -]  0.55 [+ or -]
(KH-37)    leukemia       0.03            0.01
           (human)

U-266      Malignant       0.49 [+ or -]  1.21 [+ or -]
           myeloma        0.02            0.02
           (human)

DOHH-2     Non-Hodgkin     0.14 [+ or -]  1.07 [+ or -]
           lymphoma       0.03            0.07
           (human)

HD-MY-Z    Hodgkin         4.97 [+ or -]  2.07 [+ or -]
           lymphoma       0.39            0.11
           (human)

EJ         Urinary         8.75 [+ or -]  2.41 [+ or -]
           bladder        1.34            0.04
           carcinoma
           (human)

MCF-7      Breast          0.79 [+ or -]  1.22 [+ or -]
           carcinoma      0.04            0.04
           (human)

SAOS-2     Osteogenic      1.18 [+ or -]  0.91 [+ or -]
           sarcoma        0.05            0.03
           (human)

Neuro-2A   Neuroblastoma   9.35 [+ or -]  4.21 [+ or -]
           (murine)       2.18            0.19

These results well correlate with the established prominent cytotoxic effects of the structurally related phloroglucinol hyperforin, which displayed similar potency against a panel of human and murine malignant cell lines. Interestingly, the juxtaposition of the cytoxicity indicates that both compounds are practically equipotent against the human breast cancer derived cell line MCF-7 with [IC.sub.50] values of approximately 0.8[micro]M (Schempp et al., 2002).

In order to assess the ability of hyperatomarin to induce apoptosis, we evaluated the formation of mono-and oligonucleosomal DNA fragments in treated cells using a commercially available ELISA kit. As evident from the results depicted in Fig. 2 the 24 h exposure of tested cell lines to hyperatomarin resulted in a significant level of DNA fragmentation, which indicates the recruitment of the apoptotic cell signaling pathways. The established proapoptotic effects were corroborated further by agarose electrophoresis of DNA fragments isolated from treated SKW-3 cells. Evident from the electrophoregram in Fig. 3 treatment with hyperatomarin or with the reference antineoplastic drug daunor-ubicin evoked prominent DNA-laddering, indicative for apoptotic mono-and oligonucleosomal fragmentation of genomic DNA.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

While the intimate mechanisms of the pro-apoptotic effect of hyperatomarin and the recruited cell signaling pathways need further clarification the results obtained by two complimentary read-out systems unambiguously indicate that the induction of nroprammed cell death plays crucial role for the anticancer cytoxicity of this phloroglucinol. These data are in concordance with previous studies, pointing out that the induction of apoptosis mediates the cytotoxicity of other prenylated compounds, extracted from the Guttiferae family (Matsumoto et al. 2003; Quiney et al. 2006a; Schempp et al. 2002).

This is the first account to address the antineoplastic potential of hyperatomarin.

Our data indicate that the compound is highly effective in a broad-spectrum of human tumor cell lines, representative for some important types of neoplastic disease.

Additional beneficial features of hyperatomarin are its abundance in relatively high concentrations ca. 3% in the plant source and its superior stability vs. hyperforin (Savikin-Fodulovic et al. 2003). These together with the established prominent cytotoxicity of hyperatomarin and its ability to recruit the apoptotic signaling pathways in sensitive cells give us reason to consider this phloroglucinol derivative as a promising natural anticancer compound, which deserves further more detailed pharmacological evaluation.

Acknowledgements

This study was supported financially by the Medical Science Council at the Medical University-Sofia through Grant 12/2007.

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* Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (G. Momekov).

0944-7113/$--see front matter [C] 2008 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2008.04.008

G. Momekov (a), *, D. Ferdinandov (a), (b), D. Zheleva-Dimitrova (c), P. Nedialkov (c), U. Girreser (d), G. Kitanov (c)

(a) Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University-Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria

(b) Department of Neurosurgery, "St. Ivan Rilski" University Hospital, Medical University-Sofia, Bulgaria

(c) Department of Pharmacognosy, Faculty of Pharmacy, Medical University-Sofia, Bulgaria

(d) Institute of Pharmacy, Christian-Albrechts-University, Kiel, Germany