Effect of Abietane Diterpenes from Plectranthus grandidentatus on the Growth of Human Cancer Cell Lines

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Five known abietane diterpenes, fatty acid esters of 7α-acyloxy-6β-hydroxyroyleanone (1), grandidone A (2), 7α-acetoxy-6β-hydroxyroyleanone (3), 6β,7α-dihydroxyroyleanone (4), and coleon U (5), isolated from Plectranthus grandidentatus Gürke, were evaluated for their in vitro antiproliferative activity against five human cancer cell lines MCF-7, NCI-H460, SF-268, TK-10, and UACC-62. Coleon U (5) exhibited the strongest effect among all the assayed compounds. The abietane 3 revealed also a strong inhibitory effect while diterpenes 2 and 4 inhibited only slightly the growth of all cancer cell lines.

Several abietane diterpenes of the royleanone and coleon type have been described as antimicrobial [1], [2], [3], antifungal [1], [2], [4], and antitumoral [4], [5] active metabolites. In the past we reported the antimicrobial activity of several royleanones isolated from Plectranthus hereroensis [1] and P. grandidentatus [2]. Further studies on the constituents of P. grandidentatus resulted in the isolation of five known compounds: fatty acid esters of 7α-acyloxy-6β-hydroxyroyleanone (1) [2], grandidone A (2) [6], 7α-acetoxy-6β-hydroxyroyleanone (3) [7], 6β,7α-dihydroxyroyleanone (4) [7], and coleon U (5) [8]. The structures of these abietanes were confirmed by comparing their physical and spectroscopic data with those reported in the literature. Here we describe the in vitro effect of these abietanes on the growth of five human cancer cell lines MCF-7 (breast), NCI-H460 (lung), SF-268 (CNS), TK-10 (renal) and UACC-62 (melanoma). The results are summarised in Table [1].[*]

Diterpenes 2, 3, 4 and 5 inhibited the growth of all cancer cell lines, after 48 h of continuous exposure, in a dose-dependent way. Compound 1 was found to be inactive, even when tested at 50 μg/ml.

While compounds 2 and 4 showed, in general, a poor inhibitory effect (GI₅₀ > 20 μM), compounds 3 and 5 were found to be potent inhibitors of the growth of the five human cancer cell lines (GI₅₀ < 10 μM). Compound 5 exhibited the strongest effect among all the abietane diterpenes tested, showing GI₅₀ values that range from 2.5 μM to 5.5 μM. Although no significant cell type selectivity was observed with the abietanes tested, compound 2 was found to be more active against the MCF-7 cell lines.

The antiproliferative effect showed by the abietane diterpenes studied seems to be due to growth arrest and not to cell death (cytotoxicity). In the SRB assay, the number of cells that remained after the 48 h exposure period was not inferior to the number of cells before the treatment. Studies of viability, performed by trypan blue assay on the well characterised human breast cell line (MCF-7) with the most potent compounds 3 and 5, confirmed this finding. The viability of MCF-7 cells after treatment with 3 and 5 at theirs GI₅₀ concentrations for 48 hwas 80 % and 90 %, respectively.

The comparison of the growth inhibitory effects of monomeric royleanones (1, 3 and 4) and coleon 5 led us to think that lipophilicity may perform a role in the cell membrane penetration. Acetylated abietane 3 shows a stronger antiproliferative activity than abietane 4, which has the hydroxy group at C-7 free. Compound 4 seems to go through the cell membrane less effectively, in the test system used. One explanation for this is that acetylation causes an increase of lipophilicity, and so may enhance its permeability through cellular membranes. However the presence of a fatty acid chain, increasing even more the lipophilicity, had a negative influence in the inhibitory effect of compound 1. The presence of intramolecular hydrogen bondings may increase the lipophilicity of coleon U (5), which contains no ester group and shows the strongest activity. Dimer 2 (a ketal of 6,7-dioxoroyleanone and coleon U 5) has both chromophoric C ring substructure of monomer 3, and chromophoric B/C ring system of monomer 5, however, it shows smaller inhibitory effects. The larger molecular weight, and/or different tridimensional structure can explain this.

The intracellular mode of action how these royleanones and coleon inhibit the mechanisms of cell proliferation may be suggested. Compounds 1 - 5 probably exhibit a multiple mechanism of action in tumour cells, in a similar way as the antibiotics containing quinone that can produce free radical species [9]. Besides, the intercalation into DNA as well as chelation of transition metal ions, such as iron or copper, should also be taken into account. This is supported by the fact that compound 5, with a larger coplanar area and being a stronger chelating agent, shows better activity than compound 3.

Materials and Methods

The acetonic extract was obtained from the whole plant P. grandidentatus Gürke. This specimen collected in 1994, was obtained from cultivated plants, as previously described by us [2], and a voucher specimen was deposited as C. Marques S/N° (LISC) (Centro de Botânica, Instituto de Investigação Científica e Tropical, MCT, Portugal).

Repeated column chromatography of the acetone extract of the plant (silica gel Merck 9385, hexane-EtOAc) as previously described [2], allowed the isolation of the following compounds: fatty acid esters of 7α-acyloxy-6β-hydroxyroyleanone (1, 10 mg, yellowish thick oil, [α]_D + 11.7°), grandidone A (2, 7.5 mg, mp 280 - 282 °C, [Θ]₂₆₈: + 32, [Θ]₃₀₈: -2.6), 7α-acetoxy-6β-hydroxyroyleanone (3, 200 mg, mp 227 - 228 °C, [α]_D ²¹ 0°; see also [7]) 6β,7α-dihydroxyroyleanone (4, 10.6 mg, mp 200 - 201 °C, [α]₅₇₈ ²¹: -76°), (hexane-EtOAc 8 : 2, ordered by increasing chromatographic polarity), and coleon U (5, 20 mg, mp 171 - 173 °C, [Θ]₂₈₁: + 10.8, [Θ]₃₀₄: -2.6), (hexane-EtOAc 3 : 7). All the pure isolated compounds were identified by their physical (mp, [α] or [Θ]) and spectroscopic (¹H-NMR) data.

Stock solutions of compounds were prepared in DMSO and then diluted to the desired final concentrations in culture medium. All compounds were completely soluble in the experimental conditions. Final concentrations of DMSO showed no interference with the biological activity tested.

Five human tumour cell lines were used, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), SF-268 (CNS cancer), TK-10 (renal cancer) and UACC-62 (melanoma). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 5 % heat-inactivated foetal bovine serum, 2 mM glutamine and 50 μg/ml gentamicin, at 37 °C, in an humidified air incubator containing 5 % CO₂. They were maintained at concentrations below 1 × 10⁶ cells/ml to ensure exponential growth. Cell cultures were periodically tested for mycoplasma infection with the DNA fluorochrome DAPI.

The effects of compounds on the growth of human cancer cell lines were evaluated according to the procedure adopted in the NCI’s in vitro anticancer compound screening that use the SRB assay to assess growth inhibition [10]. Growth inhibition of 50 % (GI₅₀) as well as cytoxicity were calculated as described elsewhere [11]. The viability of MCF-7 cells was determined by trypan blue exclusion assay after exponential growing cells had been exposed for 48 h to GI₅₀ concentration of compounds. Attached cells were combined with non-adherent ones and counted in a haemocytometer using the trypan blue (0.1 %) exclusion dye. Percentage of cell viability was calculated by comparing the number of viable exposed cells with those of the unexposed control.

Acknowledgements

The authors thank FCT (I&D N°8/94 and N°226/94), POCTI (QCA III) and FEDER for financial support. PhD grants to Cristina Gaspar Marques (Praxis XXI/BD/18 046/98) and to Madalena Pedro (SFRH/BD/1456/2000) are acknowledged to FCT. We are grateful to the investigator Eurico S. Martins (IICT-MCT, Portugal) for plant identification, and NCI (USA) for the generous provision of the tumour cell lines.

References

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Dr. Mª. Fátima A. Simões

Faculdade de Farmácia

Universidade de Lisboa - CECF

Av. das Forças Armadas

1649-019 Lisboa, Portugal

Email: fasimoes@ff.ul.pt

Fax: 351-217946470

References

• 1 Batista O, Duarte A, Nascimento J, Simões F, de la Torre M C, Rodríguez B. Structure and antimicrobial activity of diterpenes from the roots of Plectranthus hereroensis .  J Nat Prod. 1994 ;  57 858-61
  CrossrefPubMedSearch in Google Scholar
• 2 Teixeira A P, Batista O, Simões M F, Nascimento J, Duarte A, de la Torre M C, Rodríguez B. Abietane diterpenoids from Plectranthus grandidentatus .  Phytochemistry. 1997;  44 325-7
  CrossrefPubMedSearch in Google Scholar
• 3 Yang Z, Kitano Y, Chiba K, Shibata N, Kurokawa H, Doi Y, Arakawa Y, Tada M. Synthesis of variously oxidised abietane diterpenes and their antibacterial activities against MRSA and VRE.  Bioorg Med Chem. 2001;  9 347-56
  CrossrefPubMedSearch in Google Scholar
• 4 Ulubelen A, Öksüz S, Kolak U, Tan N, Bozok-Johansson C, Çelik C, Kohlbau H -J, Voelter W. Diterpenoids from the roots of Salvia bracteata .  Phytochemistry. 1999;  52 1455-9
  CrossrefPubMedSearch in Google Scholar
• 5 Engler T A, Umashanker S, Sriram N, Vander V D, Fusao T. A new general synthetic approach to diterpenes: application to syntheses of (±)-taxodione and (±)-royleanone.  J Org Chem. 1989;  54 5712-27
  CrossrefPubMedSearch in Google Scholar
• 6 Uchida M, Miyase T, Yoshizaki F, Bieri J H, Rüedi P, Eugster C H. 14-Hydroxytaxodion als Hauptditerpen in Plectranthus grandidentatus Guerke; Isolierung von sieben neuen dimeren Diterpenen aus P. grandidentatus, P. myrianthus Briq. und Coleus carnosus Hassk: Strukturen der Grandidone A, 7-epi-A, B, 7-epi-B, C, D und 7-epi-D.  Helv Chim Acta. 1981;  64 2227-50
  CrossrefPubMedSearch in Google Scholar
• 7 Hensch M, Rüedi P, Eugster C H. Horminon, Taxochinon und weitere Royleanone aus 2 abyssinischen Plectranthus - Spezies (Labiatae).  Helv Chim Acta. 1975;  58 1921-34
  CrossrefPubMedSearch in Google Scholar
• 8 Miyase T, Rüedi P, Eugster C. Diterpenoid Drüsenfarbstoffe aus Labiatien: Coleone U, V, W und 14-O-Formyl-coleon-V sowie 2 Royleanone aus Plectranthus myrianthus Briq; cis- und trans-A/B-6, 7-Dioxoroyleanon.  Helv Chim Acta. 1977;  60 2770-9
  CrossrefPubMedSearch in Google Scholar
• 9 Foye W O. Cancer Chemotherapeutic agents, ACS Professional Reference Book. Washington; American Chemical Society 1995: p. 211, 273
  Search in Google Scholar
• 10 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren J, Bokesch H, Kenney S, Boyd M J. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 11 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A, Gray-Goodrich M, Campbell H, Mayo J, Boyd M. New colorimetric cytotoxicity assay for anticancer-drug screening.  J Natl Cancer Inst. 1991;  83 757-66
  CrossrefPubMedSearch in Google Scholar

Dr. Mª. Fátima A. Simões

Faculdade de Farmácia

Universidade de Lisboa - CECF

Av. das Forças Armadas

1649-019 Lisboa, Portugal

Email: fasimoes@ff.ul.pt

Fax: 351-217946470