Abstract
Background/Aim: Thanks to its biologically active constituents, Ruta graveolens L. (Rutaceae) is a widely used medicinal plant. In our study, six furanoacridone alkaloids isolated from Ruta graveolens were investigated for their antiproliferative and pro-apoptotic effects on human breast cancer cell lines (MCF-7, MDA-MB-361, MDA-MB-231 and T47D). Materials and Methods: The cell lines were pretreated with alkaloid components (rutacridone, isogravacridone chlorine (IGC), gravacridonediol monomethyl ether, gravacridonediol, gravacridonetriol, a 1:1 mixture of gravacridonetriol and - diol monoglucosides) and their antiproliferative effects were determined by the MTT assay. Results: IGC had the most marked effect on cell proliferation of MDA-MB-231 (half maximal inhibitory concentration (IC50)=2.27 μM). Cell-cycle analysis was applied to quantify the effect of IGC on subpopulations of MDA-MB-231 and MCF-7 cells. It caused a cell-cycle disturbance by decreasing the G2/M and G0/G1 and increasing the S phase and the appearance of the subdiploid (sub-G1) population. Hoechst 33258-propidium iodide staining was used to evaluate the morphological changes in IGC-pretreated MDA-MB-231 and MCF-7 cells, revealing the appearance of apoptotic features. IGC was found to cause a modest activation of caspase-3 and -9, but not caspase-8, indicating the activation of an intrinsic apoptotic pathway in MDA-MB-231 cells. Conclusions: These in vitro findings indicate that furanoacridones are suitable candidates for anticancer drug development.
Thanks to its biologically active constituents, such as photosensitizer furanocoumarins (e.g. psoralen) or the flavonoid rutoside that protects capillary fragility, Ruta graveolens L. (Rutaceae) is a widely used medicinal herb both in traditional and in modern medicine (1, 2). Acridone alkaloids, secondary metabolites of the Rutaceae family, are exclusively characteristic of this plant family and potential anticancer agents due to their structural relationship with the heterocyclic polyaromatic acridines (3, 4). These chemicals are able to intercalate into the double-stranded DNA and, as a result, acridines disturb the vital cell functions during cell division, inhibiting topoisomerase II and telomerase enzymes (5).
Breast cancer is by far the most frequent malignant disease and accountable for most cancer-related deaths among the female population. The data support the urgent need for the development of new antiproliferative agents with higher efficacy and better tolerability profiles. Based on studies by Réthy et al., selected furanoacridone alkaloids isolated from Ruta graveolens L. were investigated for their antiproliferative effects on human breast cancer cell lines (MCF-7, MDA-MB-361, MDA-MB-231 and T47D) (6, 7). Further studies were carried out to determine the effects on the cell cycle and morphology. The studied breast cancer cell lines differ from each other in their receptor profiles. MDA-MB-231 is a triple-negative cell line that does not express estrogen and progesterone receptors or HER2/neu positivity. Patients with triple-negative breast cancer have less favorable prognosis than those with hormone receptor-positive tumors and, therefore, new chemotherapeutic agents acting on these types of tumors would be desirable (8). Patients with triple-negative breast cancer are at a higher risk of recurrence with higher metastasis rates, earlier relapse and a lower benefit of the conventional adjuvant anthracycline/ taxane-based therapy than their hormone receptor-positive counterparts. Since 15% of breast cancers belong in this group, targeted therapy with adjuvant and neoadjuvant therapy supplemented with compounds with new targets is urgently required (9).
Apoptosis initiation is a key factor of potential lead molecules in cancer research and pro-apoptotic features are indispensable in the mechanism of action of antitumor candidates. There is ample evidence of the anticancer effects of pyran ring-containing acridones; however, furanoacridones are much less investigated from this respect (10). Furanoacridones isolated by Szendrei et al. from the roots and herbs of Ruta graveolens L. were previously studied on different cancer cell lines for their cytotoxic and apoptosis-inducing activities. Those findings revealed pronounced cytotoxic activities on murine and human cancer cells, as well as pro-apoptotic characteristics on cellular and mRNA levels (6, 7). In our experiments, the previously investigated furanoacridones were further studied on a breast cancer panel consisting of four different breast cancer cell lines and a non-cancerous cell line, e.g. an immortalized human mammary epithelial cell line and a human lung fibroblast cell line.
Materials and Methods
Chemicals. The 6 tested acridone alkaloids (A-F, Figure 1) were isolated from the roots and aerial parts of Ruta graveolens L. (Rutaceae) by Reisch and Szendrei et al. (11-14). The purities of the compounds were investigated by proton nuclear magnetic resonance (1H-NMR) spectroscopy and high-performance liquid chromatography (HPLC), using a LiChrospher100 RP-18 (250×4 mm, 5 μm) column and MeOH-H2O (6:4 and 8:2)/0.1% trifluoroacetic acid (TFA) as mobile phase, with detection at 273 nm. The degree of purity of the tested compounds was >96%, with the exception of gravacridonediol for which a purity of 91% was found. Stock solutions of the acridones (10 mM) were prepared in dimethyl sulfoxide (DMSO). Substances were purchased, if not otherwise specified, from Sigma-Aldrich, Budapest, Hungary.
Cell culturing and determination of antiproliferative effects of the tested compounds. Human breast adenocarcinoma cell lines, MCF-7, MDA-MB-361, MDA-MB-231 and T-47D, as well as MRC-5 (non-cancerous fetal lung fibroblast) cells, were cultivated in minimal essential medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids and an antibiotic–antimycotic mixture. All cell lines were purchased from ECACC (Salisbury, UK). For cultivation of the immortalized hTERT-HME1 cell line, mammary epithelial cell growth medium (MEGM) was applied since this cell line requires a serum-free environment. MEGM is supplemented with insulin, human epidermal growth factor (hEGF), hydrocortisone, bovine pituitary extract and an antibiotic-antimycotic mixture. All media and supplements were obtained from PAA Laboratories GmbH, Pasching, Austria. Near-confluent cancer cells were seeded onto a 96-well microplate (5,000/well; 10,000/well for MDA-MB-361) and attached to the bottom of the well overnight. On the second day, 200 μl of new medium containing the tested compound in serial dilutions was added. After incubation for 72 h at 37°C in humidified air with 5% CO2, the living cells were assayed by the addition of 20 μl of 5 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution. MTT was converted by intact mitochondrial reductase and precipitated as blue crystals during a 4-h incubation period. The medium was then removed and the precipitated crystals were dissolved in 100 μl of DMSO during a 60-min period of shaking at 37°C. Finally, the reduced MTT was assayed at 545 nm, using a microplate reader; wells with untreated cells were utilized as controls (15). Sigmoidal dose-response curves were fitted to the measured data in order to determine the half maximal inhibitory concentration (IC50) values by means of GraphPad Prism 4.0 (GraphPad Software; San Diego, CA, USA). All in vitro experiments were carried out on two microplates with at least five parallel wells. Antiproliferative properties of furanoacridones on MCF-7 cells have been determined previously by Réthy et al. (6). Cisplatin was used as positive control. The highest DMSO content of the medium (0.3%) did not have any substantial effect on cell proliferation.
Calculated IC50 values of the tested compounds.
Cell cycle analysis by flow cytometry. Cellular DNA content was determined by means of flow cytometry analysis, using a DNA-specific fluorescent dye, propidium iodide (PI). MDA-MB-231 and MCF-7 cells were seeded in a 6-well plate and cultured overnight. The cultured cells were treated with various concentrations of the tested compound (isogravacridone chlorine (IGC)) for 24 h and 48 h, the medium was removed and the cells were washed with phosphate-buffered saline (PBS) and trypsinized. The harvested cells were suspended in medium and centrifuged at 1,900 rpm for 15 min at 4°C. The supernatant was then removed and the cells were re-suspended in 1 ml of PBS. After a second centrifugation, 1 ml of −20°C 70% EtOH was added dropwise to the cell pellet. The cells were stored at −20°C until DNA staining. On the day of DNA staining, the samples were washed with PBS and suspended in 1 ml of DNA staining buffer containing PI, ribonuclease-A, Triton-X and sodium citrate. After incubation for 1 h at room temperature, protected from light, the samples were analyzed with a Partec CyFlow instrument (Partec GmbH, Münster, Germany). For each experiment, 20,000 events were counted and the percentages of the cells in the different cell-cycle phases (sub-G1, G1, S and G2/M) were determined by means of ModFit LT software (Verity Software House, Topsham, ME, USA) (16).
Double staining with Hoechst 33258 and PI. Cells were seeded into a 96-well plate and incubated with various concentrations of the tested compound for 24 and 48 h. The medium was then removed and 100 μl of medium with 10 μl of staining solution was added to the cells. The final concentrations of Hoechst 33258 and PI were 5 and 3 μg/ml, respectively. After incubation for 60 min at 37°C, the cells were examined on a Nikon Fluorescent Microscope equipped with a Digital Sight Camera System, including appropriate filters for Hoechst 33258 and PI (17).
Chemical structures of studied furanoacridones.
Caspase assays. Caspase-3, -8 and -9 activity assays were performed to assess the pro-apoptotic effects of the tested compounds, determined by colorimetric assays with the substrates Asp-Glu-Val-Asp p-nitroanilide (DEVD-pNA), Ile-Glu-Thr-Asp p-nitroanilide (IETD-pNA) and Leu-Glu-His-Asp p-nitroanilide (LEHD-pNA), respectively. The peptide substrate was cleaved by the caspase, resulting in the release of the chromophore pNA, and the absorbances were measured by a microplate reader at the wavelength of 405 nm. MDA-MB-231 cells were treated with IGC in different concentrations (10 and 30 μM) and incubated for 48 h. Cells were scraped from the surface of cultivating flasks and incubated on ice with cell lysis buffer in proportion with the cell number for 10 min. The lysate was centrifuged for 5 min at 10,000 rpm at 4°C and the supernatant was collected. The protein contents of the lysates were measured by means of Pierce BCA Protein Assay Kit (Thermo Fischer Scientific; Waltham, MA, USA) and samples were diluted so as to have the same protein content in each tube. Each sample was prepared in three parallels and the results were expressed in fold increase of caspase activity as compared with the control results (18).
Statistical analysis. Statistical analysis of the obtained data was performed by analysis of variance (ANOVA) followed by Dunnett's post-test. All analyses were performed with GraphPad Prism 5. (GraphPad Software; San Diego, CA, USA).
Results
The antiproliferative effects of the investigated furanoacridones are summarized in Table I.
Half of the tested compounds exerted antiproliferative effects on the selected breast cancer cell lines in the applied concentration range. IGC (B; Figure 1) had the most pronounced antiproliferative activity, which was comparable to that of the positive control cisplatin. The triple negative breast cancer cell MDA-MB-231 line proved to be the most sensitive of the selected ones; the tested compounds exerted different efficiency on this cell line in the sequence B > C > D > A. Compounds E and F did not show any antiproliferative activity up to a final concentration of 30 μM. IGC was tested on the non-cancerous mammary epithelial cell line too in order to evaluate its differential effects; the selected compound had a substantial antiproliferative effect on hTERT-HME1; however, IC50 was higher for the immortalized cell line and the selectivity of IGC was more favorable, as compared with the cytostatic effect of cisplatin.
Cell-cycle distribution of 10 μM and 30 μM IGC-pretreated MDA-MB-231 triple negative cell line after 24 h and 48 h of incubation; the sub-G1 population refers to apoptotic cells. *, ** and *** denote p<0.05, p<0.01 and p<0.01, respectively, as compared to the control condition.
On the basis of the antiproliferative effects of this study and the previous results of Réthy et al., IGC was selected for testing its possible pro-apoptotic effects and influence on the cell-cycle distribution in the MDA-MB-231 and MCF-7 cell lines, respectively (6). The distribution of the cell cycle phases after 24- and 48-h treatment with IGC (10 and 30 μM) are shown in Figures 2 and 3. In MDA-MB-231 cells, IGC caused a slight, but significant, increase in the subG1 population after a 48-h incubation. There were substantial changes in the G1 and S phases, e.g. the G1 population decreased and the S phase increased after a 48-h treatment with 30 μM IGC. IGC caused a slight cell cycle disturbance in the MCF-7 cells without the appearance of the subG1 apoptotic population.
Cell-cycle distribution of 10 μM and 30 μM IGC-pretreated MCF-7 estrogen receptor-positive cell line after 24 h and 48 h of incubation; sub-G1 population refers to apoptotic cells. *, ** and *** denote p<0.05, p<0.01 and p<0.01, respectively, compared to the control condition.
IGC-pretreated MCF-7 and MDA-MB-231 cells were studied by Hoechst33258/PI double staining in order to reveal the possible morphological changes in the cell nuclei (Figure 4). IGC treatment resulted in the appearance of apoptotic events in the MDA-MB231 cell line with typical nucleic morphological changes, including bright-blue chromatin condensation, granulation, membrane blebbing and cellular shrinkage. It is worth noting that signs of necrosis dominated in the MCF-7 cells, which displayed red fluorescence due to the membrane disintegration. The apoptotic and necrotic features demonstrated a concentration- and time-dependent behavior.
In confirmation of previous results, the enzymatic background of apoptosis was elicited after the IGC treatment of MDA-MB-231 cells (Figure 5). The caspase-3-, -8- and -9-inducing activity was tested by colorimetric assays after a 48-h incubation. The enzyme-inducing activity was modest but significant when the cells were treated with the higher concentration, while IGC elevated the caspase-3 and -9 levels in the pretreated cells at 30 μM. This effect was less pronounced in the case of caspase-3.
Fluorescence microscopy images of IGC-treated MDA-MB-231 and MCF-7 cells after 24 h and 48 h of incubation visualized by Hoechst33258 and PI.
Discussion
Plant-derived compounds and their semisynthetic congeners may serve as sources of potential new cytotoxic drugs that might be used in the treatment of malignant diseases. Acridone alkaloids are secondary metabolites of plants belonging in the Rutaceae family (19). Two types of acridone alkaloids can be distinguished: tricyclic and tetracyclic acridones. Compounds of tetracyclic acridones can carry a pyran or a furan ring in an angular position attached to the acridine nucleus. Pyranoacridones have been proven by in vitro and in vivo studies to possess antiparasitic and antitumor effects (20). Acronycine, a pyranoacridone originally isolated from Acronychia baueri, exerts anticancer properties on solid tumor models (21, 22). Its antitumor potency is modest and the compound displays poor solubility in a water-based environment, which hinders in vivo studies. Clinical trials with oral administration of acronycine were discontinued due to its gastrointestinal side-effects and weak therapeutic potency (23). Consequently, rational drug design-based modifications of the original structure were highly desirable in order to develop potent structural analogs of acronycine that exert antitumor effects and have better solubility in biocompatible solvents (24). The systematic synthesis of acronycine derivatives revealed important structural requirements: a 1,2-double bond is indispensable for antitumor activities and a methoxy electron-donating group at C-6 is also necessary (25). Further investigations clarified that acronycine epoxide is an active metabolite of acronycine in vivo and the metabolite should be responsible for the alkylation of nucleophilic targets in the cancer cells (26). The most promising benzo[b]acronycine derivative is the diacetate S23906-1, which was selected for preclinical development (27). This compound forms an adduct with the exocyclic amino group of the guanine residues in the minor groove of the DNA helix destabilizing base pairing to cause helix opening. It induces checkpoint kinase 1 and cyclines resulting in a mitotic catastrophe of the treated cells (28). Flow cytometry analysis of S23906-1-pretreated HT-29 colon cancer cells indicated that this acronycine derivative caused reversible accumulation of the G2/M population and irreversible arrest of the cell cycle in the S phase (29). The anticancer effects of S23906-1 were tested in murine orthotopic cancer models as well. The tested compound inhibited tumor growth, caused marked tumor regression in the case of C38 colon adenocarcinoma and showed better antitumor properties and tolerability profile than acronycine itself (27, 30, 31). Abundant information is available on the antitumor effects of pyaranoacridones; however, the pharmacological activities of furan-condensed acridones have been less well-studied. In the present experiments, one of the tested furanoacridones, IGC, showed a noteworthy antiproliferative effect on the selected breast cancer cell lines as compared with the control cytotoxic agent, cisplatin. It has been previously reported that the lipoid characters of acridone alkaloids determine the cytotoxic mechanism and the crossing of the cellular membranes (6). IGC, as the most potent furanoacridone, was selected for further investigations in order to assess its in vitro tumor selectivity. The IC50 value of IGC was determined on an hTERT-HME1-immortalized human mammary epithelial cell line and MRC-5 fetal lung fibroblasts so as to establish if there is an antiproliferative effect on non-cancerous cells. Tumor selectivity is preferable to avoid toxic adverse effects. The IC50 of IGC on non-cancerous mammary epithelial cells has the same order of magnitude as its cancerous counterparts, although the antiproliferative effects of IGC on MRC-5 are relatively high.
Caspase-inducing activity of IGC on MDA-MB-231 breast carcinoma cells after a 48-h treatment. *, ** and *** denote p<0.05, p<0.01 and p<0.01, respectively, compared to the control condition.
The pro-apoptotic characteristic of antitumor agents is determinative and favorable in their mechanism of action in contrast with necrosis; during the development of anticancer drugs, the demonstration of apoptosis induction is a major factor. In our series of experiments, the pro-apoptotic features of IGC were investigated. Since the triple negative breast cancers had the least favorable outcome during treatment, further investigations were carried out on the MDA-MB-231 cell line. This cell line was more susceptible to IGC than to cisplatin. Experiments were also performed on the estrogen receptor-positive MCF-7 cell line. Dual staining with Hoechst 33258/PI disclosed the key qualitative features of apoptosis, e.g. chromatin condensation, cellular shrinkage, appearance of apoptotic bodies and enhanced membrane permeability after a 48-h treatment with IGC. The quantitative evaluation of apoptosis was investigated by flow cytometry analysis of pre-treated cells by determining the cellular DNA content. A significant elevation of subdiploid (sub-G1) population was observed in the IGC-pretreated MDA-MB-231 cell line after 48 h of incubation. The cell cycle analysis demonstrated a marked increase of the S phase with a consequent alteration of the G1 and G2/M populations. A similar behavior of IGC was previously observed in HeLa (human cervix adenocarcinoma) cells without any effect on the G2 phase but with a consequent increase in the S/G2 ratio (6). In the case of MCF-7, a slight disturbance of the cell cycle was noticeable with an increase in the G1 population at 10 μM IGC and a decrease at 30 μM after 24 h and 48 h of incubation. IGC treatment altered the S phase in the MCF-7 cells only after 24 h of incubation. Acridone alkaloids gave rise to a substantial increase in the late apoptotic cell population of murine L5178 MDR lymphoma cell line after a 24-h treatment in the Annexin-V assay (7, 32). IGC also proved to cause a concentration-dependent increase in the ratio Bax/Bcl-2 at the RNA expression level, indicating the pro-apoptotic effect on HeLa cells (6).
Since IGC displayed pro-apoptotic characteristics on the RNA level, it was also assumed that this effect might also be exhibited on the apoptotic enzyme activity. Caspases are cystein proteases that are present in all cells in their zymogen forms and activated by apoptotic signals resulting in a proteolytic processing that converts the inactive precursors to active enzymes. In our studies, caspase-3, -8 and -9-inducing activities were studied. Caspase-3 is regarded as an effector caspase, while caspase-8 and -9 are initiators. Caspase-8 is the key initiator enzyme of death receptor-mediated apoptosis (extrinsic pathway) and caspase-9 is regarded as the crucial enzyme of activation of the intrinsic mitochondrial pathway (4). By activating one of the two latter enzymes, it can be distinguished which of the main apoptotic pathways is activated. In our studies, a significant activation of caspase-9 could be detected and, therefore, it can be concluded that the mitochondrial pathway was initiated by IGC. This finding was in concert with the elevated level of activated caspase-3 and serves as a further evidence of the pro-apoptotic effects of the selected furanoacridone alkaloid. The mitochondrial pathway of apoptosis is the most commonly deregulated type of cell death in cancer cells and the intrinsic pathway of programmed cell death would, therefore, be a suitable target in cancer therapy. Mitochondrial membrane permeabilization is a defining event during the intrinsic pathway, with a consequential release of cytochrome c whereupon it forms apoptosomes with the apoptotic protease activating factor 1 (APAF-1) adaptor molecule. The apoptosome recruits and activates pro-caspase-9, which consecutively triggers the activation of executioner caspases, e.g. caspase-3 and caspase-7 (33).
The kingdom of plants has always played a substantial role in the history of cancer treatment and serves as a source for clinically relevant lead structures of natural origin for the development of semisynthetic cytostatic drugs (34). Furanoacridones have been only sparsely investigated in this respect so far; however, our findings, on their antiproliferative and pro-apoptotic activities, support the application of naturally occurring acridone alkaloids as lead molecules.
Acknowledgements
The Authors are grateful to the Hungarian Scientific Research Fund for financial support (OTKA K109293).
- Received March 11, 2016.
- Revision received April 18, 2016.
- Accepted April 19, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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