Ferutinin, an Apoptosis Inducing Terpenoid from Ferula ovina

A current hurdle in cancer management is the intrinsic or acquired resistance of cancer cells to chemical agents that restricts the efficacy of therapeutic strategies. Accordingly, there is an increasing desire to discover new natural compounds with selective toxicity to combat malignancies. In present study, the cytotoxic and apoptosis-inducing activities of ferutinin, a terpenoid derivative from Ferula ovina , were investigated on human breast (MCF7) and bladder (TCC) cancer cells as well as normal fibroblasts (HFF3).The toxicity and DNA damage inducing effects of ferutinin were studied by MTT and comet assays, DAPI and PI staining and DNA laddering. The IC 50 values of ferutinin were identified and compared with routine prescribed drugs, doxorubicin and vincristine, by MTT test. Alkaline comet assay and DAPI staining revealed DNA damage due to ferutinin, which was significantly (p<0.001) higher in MCF7 and TCC than HFF3 cells. Apoptosis induction was evidenced by PI staining and DNA laddering. Our results suggest that ferutinin could be considered as an effective anticancer agent for future in vivo and clinical experiments.

In present study, ferutinin was isolated from the roots of F. ovina and evaluated for its cytotoxic and apoptosisinducing effects in vitro. The investigations were carried out on human breast (MCF7) and bladder (TCC) cancerous cells, and also human foreskin fibroblasts (HFF3). To study the activity of ferutinin, MTT assay, alkaline single cell gel electrophoresis approach (comet assay), 4', 6diamidino-2-phenylindole (DAPI) and propodium iodide (PI) stainings and DNA laddering were used. Furthermore, to better compare the toxic effects of ferutinin, vincristine and doxorubicin, routine anticancer drugs prescribed for bladder and breast malignancies (Gurib-Fakim, 2006;Ibrahim et al., 2000), were also used as positive controls.

Materials and Methods
Extraction and isolation of ferutinin F. ovina was collected from Binaloud Mountain, north of Iran, in May 2007, and voucher specimens were deposited in the herbarium of the School of Pharmacy, Mashhad University of Medical Sciences under accession No. 1011. The concentrated dichloromethane extract of F. ovina roots was subjected to normal phase column (60×5cm) chromatography; the elution of column was done by petroleum ether and subsequently ethyl acetate. The fractions were compared by TLC (Silica gel using petroleum ether-acetone as solvent), and those giving similar spots were combined. Three fractions were finally obtained; fraction 3 was assigned as ferutinin (a sesquiterpene) and its structure was confirmed by 1Dand 2D-nuclear magnetic resonance (NMR) spectra (Figure 1), which was in agreement with those previously described in the literature (Abd El-Razek et al., 2003).

Culture of cancerous and normal cells
MCF7 and TCC cells were purchased from Pasteur Institute (Tehran, Iran), while HFF3 cells were kindly provided by Royan Institute (Tehran, Iran). All cells were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco, Scotland) supplemented with 10% fetal bovine serum (Gibco, Scotland) and incubated at 37ºC in a humidified atmosphere of 5% CO 2 in air. To subculture the cells, 0.25% trypsin and 1 mM EDTA were used for 3-5 min. For morphological alterations, treated MCF7, TCC and HFF3 cells with various concentrations of ferutinin were observed under invert microscope 24, 48 and 72h after treatments.

MTT cytotoxicity assay
To determine the half maximal inhibitory concentration (IC 50 ) of all reagents used in present study, MTT assay was used as previously described (Rassouli et al., 2011a). Briefly, 24h after cells were seeded at a density of 8×10 3 cells/well in 96-well plates, they were treated with increasing concentrations of ferutinin, vincristine or doxorubicin for 24, 48 and 72h. Then, 5 mg/ml MTT solution (Sigma, Germany) was added to each well, plates were incubated at 37°C for 4h, and finally insoluble produced formazan was dissolved in DMSO. The optic density of each well was measured spectrophotometrically at 570 nm; all tests were performed in triplicate. The viability of cells in each treatment was calculated by dividing the absorbance of treated cells in each concentration to the mean absorbance of control cells.

Alkaline comet assay and DAPI staining
To semi-quantitatively study the DNA damaging effect of ferutinin, alkaline comet assay and DAPI staining were performed as previously described (Rassouli et al., 2011a). Briefly, untreated MCF7, TCC and HFF3 cells, and cells treated with IC 50 values of ferutinin, as well as their DMSO controls, were trypsinized and washed in phosphate buffered saline (PBS).
For comet assay, cell pellets were suspended in 0.75% (w/v) low melting point agarose (LMA, Fermentas, Germany), dispensed onto slides precoated with 1% (w/v) normal melting agarose (NMA, Helicon, Russia), solidified on ice, and covered with 0.75% (w/v) LMA. After immersing slides in lysing buffer (pH 10) and 4h incubation at 4°C, they were kept in alkaline electrophoresis buffer (pH 13) for 30 min. Then, electrophoresis was conducted for 20 min at 4°C, in an electric field of 300 mA and 25 V. Afterwards, slides were washed with neutralizing buffer (pH 7.5), dried and stained with ethidium bromide (EtBr), and finally analyzed using a fluorescent microscope (Olympus, Japan). The percentage of DNA in tail, an indicator for DNA fragmentation, was determined using TriTek Cometscore version 1.5 software.
For DAPI staining, cell pellets were fixed in 4% paraformaldehyde (Sigma, Germany), permeabilized with 0.1% Triton X-100 and stained with 2 μg/ml DAPI (Merck, Germany) for 10 min. Stained cells were then counted under fluorescent microscope, while chromatin condensation and nuclear fragmentation were the criteria used to demonstrate apoptosis.

Statistical analysis
Significant level was ascertained by one way ANOVA, followed by Tukey multiple comparison test, using SPSS software. Values are expressed as mean±SD. A p-value of <0.001 was considered as significant.

Results
To investigate the cytotoxicity of ferutinin by MTT test, MCF7, TCC and HFF3 cells were treated with increasing concentrations of this sesquiterpene, as well as equivalent DMSO dilutions as controls, and also vincristine and doxorubicin, for 24, 48 and 72h. As summarized in Table  1, ferutinin induced its toxic effects in a dose and time dependent manner; 72h after treatment, the IC 50 values of ferutinin on MCF7, TCC and HFF3 cells were 29, 24 and 36 µg/ml, respectively. Interestingly, in comparison with this terpenoid, vincristine and doxorubicin induced their toxic effects in higher concentrations, as the IC 50 values of doxorubicin and vincristine were 64 µg/ml on MCF7 cells and 50 µg/ml on TCC cells, respectively.
Examining morphological alterations revealed that in comparison with untreated and control cultures, treatment with IC 50 value of ferutinin reduced the number of cells and their attachment, and also induced cytoplasmic granulation in all cell lines (Figure 2).
To determine the mechanisms involved in ferutinin activity, DNA damage was analyzed by alkaline version of comet assay. Based on MTT results and morphological alterations, comet assay was carried out on untreated MCF7 cells, cells incubated with DMSO control, and cells treated with IC 50 values of ferutinin for 24h ( Figure 3A-C). As it is shown, 37 µg/ml ferutinin induced approximately 42% DNA damage, significantly (p<0.001) higher than that induced by its relevant controls ( Figure 3G).
Due to its great activity, ferutinin effects on nuclear morphology was investigated by DAPI staining on MCF7 cells ( Figure 3D-F). Demonstrating apoptotic morphology revealed that 71% of MCF7 cells treated with 37 μg/ml ferutinin presented condensed chromatins and fragmented nuclei, significantly (p<0.001) higher than its relevant controls ( Figure 3G).
As it is demonstrated in Figure 4A-F, flow cytometry analysis after PI staining revealed that in comparison with untreated MCF7 cells and cells treated with 0.18% and 0.36% DMSO, about 10% and 45% of cells treated with 37 and 74 μg/ml ferutinin were detected in sub-G1 peak, respectively, which is comparable to doxorubicin effects (60%).
To further analyze ferutinin effects on DNA structure, DNA laddering was applied on MCF7 cells treated with 74 and 148 μg/ml ferutinin and their relevant DMSO and untreated controls. As it is presented in Figure 4G, only in cells treated with 74 and 148 μg/ml ferutinin severe DNA fragmentations were observed.

Discussion
The intrinsic or acquired resistance of cancer cells to chemical reagents and ionizing radiations is the most important factor that has been introduced for the low efficacy of current therapeutic strategies against malignancies including breast and bladder cancers. This drawback has prompted a good deal of investigations in search for new biosynthetical compounds with antitumor properties.
For years, plant-derived natural products have been used for the development of cancer therapeutic agents in modern medicine. There are several ongoing clinical trials with natural compounds, including green tea, genistein and n-3 poly unsaturated fatty acids that show promising results in the prevention and/or therapy of breast and bladder cancers (Amin et al., 2009). Interestingly, dietary terpenoids have shown antitumor activity on a wide variety of experimental tumors by preventing the formation of neoplastic cancers or regressing the existence of malignant tumors (Crowell et al., 1996;Bardon et al., 1998;Shi et al., 2002;Jagetia et al., 2005).
Ferula species are reputed in traditional medicine for their therapeutic applications against a range of disorders, and terpenoid compounds are the most abundant constituents of Ferula essential oils. A number of medicinal properties including antibacterial, antifungal, antioxidant, anti-inflammatory, anticonvulsant and hypotensive activities have been reported for Ferula species in several studies (Sahebkar and Iranshahi, 2011). Due to these great therapeutic potentials, we selected ferutinin, extracted from F. ovina, to study its probable antitumor and apoptosis inducing activities.
Present results revealed that the IC 50 value of ferutinin on MCF7 cells were less than those of doxorubicine, a currently prescribed drug for breast carcinoma. Moreover, the toxicity of this terpenoid on TCC cells was also higher in comparison with vincristine, a routine anticancer drug used for bladder cancer treatment. Studying the activity of ferutinin during 72h revealed that this compound act in a dose and time dependent manner and show less toxic effects on normal human fibroblasts.
Apoptosis is a highly complex process that is involved in normal cell turnover, and its misregulation may account for tumor development. Nuclear morphological changes in the form of fragmentation and condensation, and also DNA lesions detected as laddering patterns are known as common and important characteristics of apoptosis. Using alkaline version of comet assay revealed that in comparison with control cultures, ferutinin significantly increased DNA lesions in treated cells. Furthermore, we detected a significant higher number of condensed chromatins in cells treated with ferutinin by DAPI staining, which was in agreement with MTT results and morphological observations, and was also confirmed by PI staining and DNA laddering results.
For many years, ferutinin, an aromatic ester of a daucane alcohol, was known as a potent phytoestrogen that has agonist activities over the α subunit of human estrogen receptor (ER), while acts as agonist/antagonist for ERβ (Ignatkov et al., 1990;Ikeda et al., 2002). Recent studies indicated that ferutinin has acetylcholinesterase inhibitory activity and shows anti-inflammatory and antimicrobial effects (Dall'Acqua et al., 2010;Geroushi et al., 2011;Abourashed et al., 2011). This sesquiterpene promotes the redistribution of intracellular Ca 2+ -pools throughout different cellular compartments including the release of Ca 2+ from endoplasmic reticulum into the cytosol and also the accumulation of Ca 2+ in the mitochondria (Zamaraeva et al., 1997;Abramov et al., 2001;Abramov and Duchen, 2003). Furthermore, ferutinin exhibits antiproliferative effects on breast cancer cells (Lhuillier et al., 2005) and induces apoptosis in leukemia cells through a loss in mitochondrial transmembrane potential and an increase in intracellular reactive oxygen species (Macho et al., 2004). Interestingly, it has been shown that ferutinin increases apoptosis in uterine luminal and glandular epithelia, and as a result, has protective function against uterine carcinoma
It has been shown that terpenoid derivatives with antitumor effects act through diverse mechanisms including mitochondrial permeability and depolarization (Chen et al., 2007), activation of enzymes such as c-Jun N-terminal kinase, Fas, caspase-8 and caspase-3 (Choi and Lee, 2009;Mansoor et al., 2009;Choi et al., 2012), microtubule polymerization and lipid peroxidation (Bocca et al., 2004;Jagetia et al., 2005), to clarify ferutinin exact mechanism of action, further and precise studies are needed.
In conclusion, F ovina is an edible plant that has been widely used in Iranian traditional medicine for its therapeutic effects. Present results indicate that ferutinin, sesquiterpene from the roots of F. ovina, has selective cytotoxic effects, and induce apoptosis in vitro. Since the binding of ferutinin to albumin protein guarantees the high stability and well distribution of this compound in plasma (Greige-Gergesa et al., 2008), ferutinin could be considered as a suitable antitumor agent for further preclinical studies.