Cytotoxic Activity of Two Cembranoid Diterpenes from Nicotiana Sylvestris Against Three Human Cancer Cell Lines

RESEARCH ARTICLE Cytotoxic Activity of Two Cembranoid Diterpenes from Nicotiana Sylvestris Against Three Human Cancer Cell Lines Amir Reza Jassbi, Marzieh Vafapour, Ardeshir Shokrollahi, Omidreza Firuzi, Mehdi Zare, Jima N. Chandran, Bernd Schneider and Ian T. Baldwin Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Department of Phytochemistry, Yasouj university, Yasouj 75914-353, Iran Research group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology, Jena, Germany Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745 Jena, Germany


INTRODUCTION
Nicotiana sylvestris Spegazzini & Comes is a Solanaceae, known by the common names South American tobacco, woodland or flowering tobacco.N. sylvestris is a perennial plant, indigenous to northwestern Argentina.This plant is considered to be one of the ancestors of Nicotiana tabacum [1].
The cembranoids 1 and its C-4 epimer (2) are two major constituents of different tobacco species [3].They are thought to function as insecticides and as inhibitors of plant-growth and fungal-spores germination [4,5].Additionally, they are reported as aldose reductase and prostaglandin inhibitors.They inhibited behavioral sensitization to nicotine in rats and blocked several types of nicotine acetylcholine receptors [6,7].Compounds 1, 2 and their semisynthetic derivatives, which were transformed by catalytic action of terrestrial and marine bacteria or via chemical transformation, have been reported as anticancer agents by different authors in animal and in vitro models against human cancer cells [8 -11].However, to the best of our knowledge, their activity against LS180 (human colon adenocarcinoma), MCF-7 (human breast adenocarcinoma) and MOLT-4 (human lymphoblast leukemia) is discussed here for the first time.

General Experimental Procedures
Optical rotations were measured by a Kruss Optronic polarimeter in chloroform.IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer.The 1 H and 13 C NMR spectra were recorded by Bruker Avance 400 and 500 spectrometer ( 1 H: 400 and 500 MHz, 13 C: 100 and 125 MHz) using CDCl 3 as solvent and TMS as the internal standard.Mass spectra were recorded on an Agilent 5975 C inert GC/MSD.TLC analyses were performed on pre coated silica gel 60 F 254 0.5 mm.The TLC plates were impregnated with 5% AgNO 3 .The silica gel (230-400 mesh) impregnated with silver nitrate was used as the stationary phase for flash column chromatography (FCC) and silica gel (70-230 mesh) for the gravity column chromatography (CC).

Plant Material and Extraction
The seeds of N. sylvestris were obtained from Max Planck Institute for Chemical Ecology (MPICE's) glass house and cultivated in the greenhouse of Medicinal and Natural Products Chemistry Research Center (MNCRC) in December 2012.The cultivation procedure was the same as previously reported [12].Briefly, about 20 seeds after sterilization were germinated on phytagel agar and kept in a growth chamber with 16/8 h day and night, the day and night temperature was set at 30 and 20±1°C respectively.After 10 d, the seedlings were transferred to soil and transferred to glasshouse with the same light and temperature regimes and irrigations once a day.After 34 d, the plants were harvested and their leaves were subjected to extraction.
At room temperature, the leaves of the plants (136.0 g) were placed in DCM (200 mL) for 30 seconds.Then, the surface-extracted leaves were subjected to chloroform extraction for three days.The extracts were concentrated in vacuum at 40°C.The two extracts were subjected to silica gel TLC analyses and after assuring their similarity, were pooled (2.6 g).AgNO 3 (3%)-impregnated silica gel (230-400 mesh, 100 g) was used for purification of the extract's constituents.Elution of the column started with n-hexane, continued with stepwise increasing the EtOAc portion up to 100% to increase the polarity of the eluent mixture, and ended up with 5% MeOH in EtOAc.Fractions 18-21, each 75 mL, were checked with 5% AgNO 3 impregnated silica gel-TLC and found to be of a similar composition.Similar fractions were pooled (437 mg).Then flash chromatography was employed (column dimensions: 202 cm, 15 g silica gel, n-hexane-EtOAc solvent system) to obtain compound 1 (78 mg) as a pure gummy material.Compound 2 (34.0 mg) was obtained from fraction 22 by flash chromatography (silica gel (230-400 mesh, 5 g), and elution with n-hexane-EtOAc (1: 1)..00 (C-20) (Fig. 1F).

Cell Lines and Culture
The following human cancer cell lines were purchased from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran: LS180 (human adenocarcinoma), MCF-7 (human breast adenocarcinoma) and MOLT-4 (human lymphoblastic leukemia) cells.The cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum (10% v/v), penicillin (100 units/mL) and streptomycin (100 µg/ml).MOLT-4 cells were grown in suspension, while LS180 and MCF-7 cells were grown in monolayer cultures in humidified air containing 5% CO 2 at 37°C.

Cytotoxicity Assay
The inhibitory effect of 1 and 2 against cancer cell growth was evaluated by the MTT reduction assay.This colorimetric assay is based on the conversion of the yellow 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to the purple formazan by the action of mitochondrial enzyme succinate dehydrogenase in viable cells.Compounds 1 and 2 were dissolved in dimethylsulfoxide (DMSO) and diluted at least 400 times in growth medium before being incubated with cells.LS180, MCF-7 and MOLT-4 cells were seeded in 96-well plates at the densities of 50,000, 30,000 and 50,000 cells/mL in 100 µL, respectively and incubated for 24 h.Then, 50 mL of medium was replaced with fresh medium containing 3-4 different concentrations of the compounds.After 72 h of incubation, the medium of each well was replaced by fresh RPMI without phenol red containing 0.5 mg/ml MTT and incubated at 37 °C for 4 h.DMSO was used to solubilize the formed formazan crystals.The absorbance of different wells was measured at 570 nm, with background correction at 655 nm using a microplate reader.The potency of cell growth inhibition for each compound was expressed as IC 50 value, defined as the concentration that caused 50% of maximum inhibition of cell viability.IC 50 values were calculated with software CurveExpert, version 1.3 for Windows.

RESULTS AND DISCUSSION
The structures of compounds 1 and 2 were elucidated using spectroscopic analysis, including 1 H NMR and APT 13 C NMR together with EIMS spectra and comparing them with those of reference compounds (Fig. 1A-F) [11,13].The stereochemistry of compound 1 was determined by X-ray crystallography (4), and later the absolute configuration of compound 2 was established by its ozonolysis followed by X-ray crystallography of the resulting degradation product [14] and at C-6, with chemical transformation to a related cembratriene-4,6,11-triol [15].The carbon signals in the 13 C NMR spectra of both compounds 1 and 2 had identical or very near chemical shifts to those reported for the authentic samples in the literature [13].The 1 H NMR spectra of the compounds were compared to those reported for compounds 1 and 2 and their related cembranoids [11,13].
Inverting the stereochemistry at C-4 affected the chemical shifts of C-4 (δ 71.48 to 72.45), and had about 2 ppm changes in the chemical shifts at gamma positions: C-2 (δ 130.40 to 127.79) and C-6 (δ 64.45 to 66.34) in compound 1 compared to those recorded for 2, respectively.The epimerization at C-4 also affected even more drastically the The cytotoxic potentials of compounds 1 and 2 were tested against three human cancer cell lines, LS180, MCF-7 and MOLT-4, and were presented as IC 50 s in Table (1) in comparison to the standard anticancer agent cisplatin.The inhibitory effect of compounds 1 and 2 against tumor promotion has been previously reported.It has been shown that E F these compounds are able to inhibit the skin tumor promoting effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice [8].The tumorigenisis inhibition of cembranoid diterepenoids against prostate cancer cell lines has also been reported for different derivatives of cembranoids (1S,2E,4S,6E,8S,11E)-2,6,11-cembratriene-8-O-methyl-4,8-diol and the known (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4-O-methyl-4,6-diol isolated from N. tabacum and their biotransformed, mostly oxidized products, by the action of two fungal strains: Cunninghamella NRRL 5695 and Mucor ramannianus ATCC 9628 [9].Compound 1 was converted into its C-6 hydroxyl esters with different carbamate functionalities and also to different hydroxylation reaction biocatalyzed by symbiotic Bacillus species isolated from a Red sea marine sponge [11].Compound 1 and its derivatives were tested on highly invasive prostate cancer cell lines and a SAR conclusion indicated their high potential against cancer cell lines due to the polar substitution on C-6 hydroxy group [11].
The semisynthetic products and a secocembranoid diterpenoid resulted from the reaction of compound 1 with different halogenated carbamic acids and also reactions catalyzed in the presence of marine Bacillus species and M. ramannianus ATCC 9628 and C. elegans showed antiproliferative activity against highly malignant +SA mammary epithelial cells with an IC 50 range of 15-30 μM [10].
The relatively good cytotoxic activity of compounds 1 and 2 (Table 1) in comparison to the standard anticancer agent cisplatin against the tested cancer cell lines in the present study is compatible with the previous reports.

Table 1 . Cytotoxic activity of compounds 1 and 2 against human cancer lines determined by MTT reduction assay.
The results are presented as mean ± S.E.M. of 3-4 experiments.