Study on the Cytotoxic Activity of Drimane Sesquiterpenes and Nordrimane Compounds against Cancer Cell Lines

Twelve drimanes, including polygodial (1), isopolygodial (2), drimenol (3), confertifolin (4), and isodrimenin (5), were obtained from natural sources. Semi-synthetic derivatives 6–12 were obtained from 1 and 2, and cytotoxic activity was evaluated in vitro against cancer cell lines (HT-29, MDA-MB231, DHF, MCF-7, PC-3, DU-145, and CoN). IC50 values were determined at concentrations of 12.5–100 µM of each compound for 72 h. In addition, it was found that polygodial (1), 8, and 12 induced changes in mitochondrial membrane permeability in CoN, MCF-7, and PC-3 cells.


Introduction
The third leading cause of cancer-related deaths in women, breast cancer incidence has increased worldwide, although this may be in part due to improvements in early detection. Just as for breast cancer in women, prostate cancer is the most common non-skin malignancy in men. In the United States, for example, there were an estimated 244,000 new cases and 40,400 deaths from this cause in 1995 alone. In Chile, the risk of death from this disease has tripled in the last 40 years; the figures for 1998 indicate 1,218 deaths were recorded from prostate cancer in the country, determining a mortality rate of 16.6 per 100,000 men [1]. In Chile, prostate cancer is the second leading cause of cancer death in men, similar to developed countries. Despite the high prevalence and mortality, there are no established screening programs for early detection [2]. Due to this need, several drugs with anticancer effects have been extracted from plants in the last 20 years, many of which are particularly effective against breast cancer cells [3].
As part of a general attempt to study structure-activity relationships for unsaturated dialdehydes from natural sources, several compounds have been investigated using the Salmonella-microsome assay (strains TA 98, TA 2637, and TA 100). Polygodial and isopolygodial showed no mutagenic activity at their highest non-toxic concentration [9]. Furthermore, unsaturated sesquiterpene dialdehydes were tested for antimicrobial, cytotoxic, and mutagenic activity. Where Polygodial exhibited antibacterial and cytotoxic activity, isopolygodial was slightly less active [10].
Although the claims that 1 possesses cytotoxic activity were consistent with previous reports [11][12][13][14], contradictory reports were found in the literature regarding the cytotoxic activities of its natural analogue 2, which was variously reported to be either active or inactive [15].
In the course of our search for bioactive compounds from natural sources, we recently reported the antifungal and antifeedant activity of naturally occurring drimanes and their semi-synthetic derivatives, including polygodial, isopolygodial, drimenol, and confertifoline [15,16]. In order to clarify the main features required for drimanes to display cytotoxic activities, we tested herein a series of 12 compounds 1-12 (most of them not previously assayed for cytotoxic activities) for antitumoral properties against a unique panel of five cancer cell lines.

Compounds Obtained from 3
Scheme 2 shows the modifications performed on compound 3. In this respect, we recently reported the synthesis of epoxinordrimane 9 [17]. In order to allow the synthesis of isomer 12, we proceeded to treat 3 with LDA in refluxing benzene, which allowed us to obtain 11-nordrimanic acetate 10. Scheme 2. Modifications performed on 3 to obtain compounds 9-12. CH  The subsequent epoxidation of 10 with m-chloroperbenzoic acid (MCPBA) produced epoxide 11. Subsequent hydrolysis of 11 under alkaline conditions and posterior oxidation with PCC in dichloromethane lead to epoxinordrimane 12. The identities of 6, 7, and 8 were established through comparisons with previously reported data [16]. Compounds 11 and 12, which were only previously reported once, are described in detail in this work.
We recently reported a derivative series from drimenol and polygodial [15]. The assignment of the configuration in C-8 and C-9 for compound 12 was proposed on the basis of NOE-spectra observations. From a 1 H-1D -NOESY experiment, when CH3-12 was selectively irradiated, only long-range interactions with H-9 and CH3-15 were observed. In addition, when H-9 was selectively irradiated, a long-range interaction with CH3-15 was observed. NMR experiments thus confirm the α spatial orientation of the epoxide ring and the stereochemistry of C-8 and C-9, as shown in Figure 2.

Biological Results
Previous programs designed to discover more active compounds from D. winteri and its derivatives, had yielded results suggesting that these compounds showed cytotoxic activity. Based on that information, and knowing that this tree has high activity potential metabolites, a growth regulatory study on cancer cell lines has been carried out.

Viability Assay
The cytotoxicity of compounds 1-12 was evaluated in vitro against different cancer cell lines: MCF-7 breast cancer, DU-145 and PC-3 human prostate cancer, and one non-tumor cell line, human colon epithelial cells CCD 841 CoN (CoN). A colorimetric assay was set up to estimate the IC50 values, which represent the concentration of a drug that is required for 50% inhibition in vitro after 72 h of continuous exposure to the test compounds. Four serial dilutions (from 12.5 to 100 μM) for each sample were evaluated in triplicate. The IC50 obtained from these assays are shown in Table 1.   The highest cytotoxicity values were observed for polygodial (1) and compound 8 in all cell lines tested and were more active than those of the rest of compounds. The cytotoxicity of compounds in colon epithelial cells (CoN) is lower than in the cancer cell lines under study, indicating that the compounds are some selectivity to cancer cells.
Since compounds 1, 8 and 12 had inhibitory effects on the growth of the cancer cell types tested, the effect of these compounds required study in greater detail. Moreover, the compounds 1 and 8 associated with α,β-unsaturated systems and dialdehyde presence was included to probe the relevance of these groups in their biological activity on cancer cells.
First, the appearance of morphological changes in the cells treated with 50 μM compound for 24 h was analyzed. Direct observation, using a phase contrast microscope, revealed that the morphologies of PC-3, MCF-7 and CoN cells were severely distorted and cells became rounded after treatment with compounds 1, 8, and 12. Moreover, the cells showed a reduction in number, indicating an increasing progression toward cell death. The control-treated cells (1% ethanol) displayed normal and healthy shapes ( Figure 3).  Figure 4). After exposure to the compounds for 24 h, cells treated with compounds 1 and 8 significantly showed chromatin condensation and karyopyknosis compared to control cells (1% ethanol) and similar to control positive (5-fluorouracil, 5-FU) which are typical characteristics of apoptotic process [18]. Mitochondria play a crucial role in the apoptotic cascade by serving as a convergent center of apoptotic signals originating from both the extrinsic and intrinsic pathways [19]. Changes induced in the mitochondrial membrane potential (MMP) have been reported previously to represent a determinant in the execution of cell death [20]. As polygodial has been described as an inhibitor of mitochondrial ATPase and also as nonionic surfactant [21][22][23]. This effect may be determinant in cell death, we has analyzed the effect of compounds 1, 8 and 12 on the mitochondrial membrane potential using flow cytometry with rhodamine 123 stain [24]. As shown in Table 2, the percentage of rhodamine 123 stained-cells were 111.6% ± 8.9%, 77.0% ± 6.5% and 49.1% ± 6.6% in the CoN, MCF-7 and PC-3, respectively, after treatment with compound 1 (50 μM); and 96.0% ± 7.6%, 93.3% ± 7.1% and 116.2 ± 18.8 respectively, after treatment with compound 8, as compared to 90%-95% in the control cells (1% ethanol). Similar differences were observed after treatments with compounds 1 and 8 at 100 μM. Table 2 shows that compound 1 increased mitochondrial membrane permeability in cancer cells with a greater effect than compounds 8 and 12. Moreover the compounds have a weaker effect on mitochondrial membrane potential for the epithelial colon cells, CoN, than in cancer cell lines. Thus, compounds 1 and 8 induced loss of mitochondrial membrane potential, correlated with increased cell death (see Table 2). However, compound 8 has a weaker effect than compound 1, indicating that the aldehyde group may be important in the loss of mitochondrial membrane potential. Moreover, compound 12 did not change the mitochondrial membrane potential when compared to control cells.    Depletion of mitochondrial membrane potential leads to the release of apoptogenic factors, such as cytochrome c in the apoptotic cascade, and activation of caspases [25,26]. Then we investigated the effects of our compounds on caspase activity. We focused on caspase-3, which is a main executor of apoptosis playing a central role in its biological processing. We analyzed the effect of treatment with the compound 1, 8 and 12 on caspase-3 activation in normal and cancer cells. As shown in Figure 5, the activation of caspase-3 in cells exposed to compounds 1 and 8 is increased versus control-treated cells (1% ethanol). Compound 1 increased the activity of caspase-3 by 3.4 ± 0.1, 1.4 ± 0.1 and 2.8 ± 0.1 times versus control cells in MCF-7 (black), CoN (white) and PC-3 (gray) cells, respectively. Compound 8 increased the activity of caspase 3 by 2.4 ± 0.1, 1.3 ± 0.2 and 2.7 ± 0.1 times versus control cells in MCF-7 (black), CoN (white) and PC-3 (gray) cells, respectively. Finally, compound 12 did not change the activity of caspase-3 when compared with control cells.

Plant Material
The stem bark of D. winteri adult trees was collected from the Malleco Province (Chile), in February 2010. A voucher specimen (N° Dw-10113) was deposited at the Herbarium of the Natural Products Laboratory, "Dr. Herbert Appel A.", Department of Chemistry, Universidad Técnica Federico Santa María, Valparaíso, Chile. Fresh bark was carefully washed with abundant distilled water to remove any residue. Afterwards, it was dried in an oven at 35 °C to a constant weight. Once dried, it was stored in hermetically sealed plastic containers at 4 °C.

Cell Lines
The experimental cell cultures were obtained from the American Type Culture Collection (Rockville, MD, USA). MCF-7 cells (breast cancer cell line), PC-3 and DU-145 (prostate cancer cell lines), and human colon epithelial cells CCD 841 CoN (non-tumoral cell line) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mM glutamine. Cells were seeded into 96 well microtiter plates in 100 μL at a plating density of 5 × 10 3 cells/well. After 24 h incubation at 37 °C under a humidified 5% CO2 atmosphere to allow cell attachment, the cells were treated with different concentrations of drugs and incubated for 72 h under the same conditions. Stock solutions of compounds were prepared in ethanol and the final concentration of this solvent was kept constant at 1%. Control cultures received 1% ethanol alone.

Cell Viability
The sulforhodamine B assay was used according to the method of Skehan et al. [32]. Briefly, the cells were set up at 3 × 10 3 cells per well of a 96 200 μL well, flat-bottomed, microplate. Cells were incubated at 37 °C in a humidified 5% CO2/95% air mixture and treated with the compounds at different concentrations for 72 h. At the end of drug exposure, cells were fixed with 50% trichloroacetic acid at 4 °C. After washing with water, cells were stained with 0.1% sulforhodamine B (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 1% acetic acid (50 μL/well) for 30 min, and subsequently washed with 1% acetic acid to remove unbound stain. Protein-bound stain was solubilized with 100 μL of 10 mM unbuffered Tris base, and the cell density was determined using a fluorescence plate reader (wavelength 540 nm). Values shown are the mean ± SD of three independent experiments in triplicate. The software used to calculate the IC50 values was GraphPad (GraphPad Software, San Diego, CA, USA).

Morphological Assessment of Cell Apoptosis
Morphological changes in the nuclear chromatin of cells undergoing apoptosis were revealed by a nuclear fluorescent dye, Hoechst 33342. Briefly, on 24-well chamber slides, 1 × 10 4 cells/mL were cultured and exposed to compounds for 24 h. The control group was also exposed to 1% ethanol. The cells were washed twice with phosphate buffer solution, fixed with 3.7% formaldehyde, and washed again with phosphate buffer solution. Following the addition of 1 μM Hoechst 33342 (Sigma-Aldrich, Santiago, Chile), they were reacted in a dark room at room temperature for 30 min. After washing, they were examined under an immunofluorescence microscope (IX 81 model inverted microscope, Olympus, Santiago, Chile).

Analysis of Mitochondrial Membrane Permeability
Rhodamine 123, a cationic, voltage-sensitive probe that reversibly accumulates in mitochondria, was used to detect changes in transmembrane mitochondrial membrane potentials. Exponentially growing cells were incubated with the compound as indicated in the figure legends. Cells were labelled with 1 μM rhodamine 123 at 37 °C in cell medium for 60 min before terminating the experiment. Cells were detached from the plate, after washing with ice cold PBS, and the samples were analyzed by flow cytometry. Data are expressed in percentage of cells with rhodamine 123 [24].

Caspase 3 Activity Assay
Caspase activity was measured using a colorimetric assay [33]. Briefly, the cells exposed to compounds were collected by centrifugation at 1000 rpm and lysed with lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, 1 mM PMSF, 1 g/mL aprotinin, 1 mg/mL leupeptin). Thereafter, the lysates were transferred to wells in a 96-well microplate and were incubated with DEVD-pNA (final concentration 200 μM) specific for caspase-3/7, at 37 °C for 1 h. The intensity of the developed color was read at 405 nm in a microplate reader (SpectraMax, Winooski, VT, USA). The results are expressed as percentages of the control level.

Statistical Analysis
Data shown in tables are average results obtained by means of three or 10 replicates and are presented as average ± standard errors of the mean (SEM). Data were subjected to analysis of variance (ANOVA) with significant differences between means identified by GLM procedures. Results are given in the text as probability values, with p < 0.05 adopted as the criterion of significance. Differences between treatment means were established with a Student-Newman-Keuls (SNK) test. The EC50 values for each activity were calculated by PROBIT analysis based on percentage of mortality obtained at each concentration of the samples. EC50 is half maximal effective concentration. Complete statistical analysis was performed by means of the MicroCal Origin 6.0 statistics and graphs PC program.

Conclusions
The results show that only polygodial (1), isodrimenin (5), aldehyde 8 and compounds 9 and 12 had IC50 values lower than 200 µM against a panel of cancer cell lines. We found that a higher concentration of polygodial (1) decreases the cell viability of DU-145 and PC-3, MCF-7. Drimenol (3) and confertifolin (4) have an effect on the cell lines at higher concentrations, but only in PC-3 did they show activity against tested cells, DU-145, at lower concentrations. Compounds 1 and 8 showed a good apoptotic response against prostate cancer cell cultures. Moreover, compounds 1 and 8 also depleted the mitochondrial membrane potential and increased caspase-3 activity. Compound 12 neither changed the mitochondrial membrane permeability nor the activity of caspase-3, suggesting that the presence of a dialdehyde had relevance for the apoptotic activity in the cell lines studied.