Abstract
Chemopreventative phytochemicals having antitumour and antioxidant properties can overcome problems of chemoresistance and nonspecific toxicity towards normal cells that are associated with platinum-based chemotherapy against cancer. These agents exert their effects by bringing into play numerous cellular proteins that in turn affect multiple steps in pathways leading to tumourigenesis. In this study, combinations of two cytotoxic phytochemicals anethole and curcumin were applied in binary combination with platinum drugs cisplatin and oxaliplatin to three epithelial ovarian cancer cell lines: A2780 (parent), A2780cisR (cisplatin-resistant) and A2780ZD0473R (ZD0473-resistant). Cell viability was quantified using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay and the combined drug action was analyzed based on the equations derived by Chou and Talalay (1984). Greatest synergism was observed when the phytochemical was added first followed by the platinum drug 2 h later and additiveness to antagonism in combined drug action was observed when the two compounds were administered as a bolus. If confirmed in vivo, the appropriate sequenced combinations of platinum with the phytochemicals may provide a means of overcoming drug resistance.
- Drug resistance
- drug combination
- anethole
- curcumin
- cisplatin
- oxaliplatin
With more than 10 million new cases diagnosed every year, cancer is one of the most dreaded diseases of our time. The global burden of cancer continues to grow largely because of ageing, increase in world population and increasing adaptation to cancer-causing behaviours (1). It is expected that by 2030 there will be 27 million cases of cancer and 17 million cancer deaths per annum, and 75 million persons will be alive with cancer, being within five years of diagnosis (2). Although ovarian cancer accounts for only 3% of all cancer cases (3), it is the leading cause of death from gynaecological type of cancer in the Western world (4), in spite of a vast amount of clinical and basic research, improved surgical techniques, and an ever-expanding armamentarium of chemotherapeutic agents (5). The poor prognosis of ovarian cancer stems mainly from the high percentage of cases diagnosed at an advanced stage. Although most patients with advanced ovarian cancer respond to first-line chemotherapy, 80% of the patients ultimately succumb to death due to recurrence (6).
Resistance to platinum-based chemotherapy is considered to be a major obstacle in the treatment of human ovarian cancer. Several key proteins associated with cell survival are found to be overexpressed, resulting in drug resistance. Thus agents are needed that would improve the efficacy of platinum drugs on cancer cells and also prevent damage to normal cells and tissues (caused by direct and bystander effects of platinum drugs) but without introducing systemic toxicity themselves. It is thought that chemopreventative phytochemicals that are found in tea, vegetables, herbs, fruits and spices would meet these requirements. Such compounds have been extensively investigated for their anticancer activities due to their safety, low toxicity, and general availability (7). The present study focused on the combination of the platinum drugs cisplatin and oxaliplatin with two such phytochemicals, anethole and curcumin (Figure 1).
Anethole is the chief component of anise oil, fennel oil and camphor (8). It is an aromatic unsaturated ether (1-methoxy-4-(prop-1-enyl) benzene) that is able to cause striking metabolic effects. For example, anethole and its derivative anethole dithiolethione (ADT) have been shown to increase intracellular levels of glutathione and glutathione-S-transferase (8-10). These two compounds and two other derivatives (eugenol and isoeugenol) can also act as antioxidants (11) with the ability to suppress tumour necrosis factor (TNF)-induced lipid peroxidation and generation of reactive oxygen species (ROS), and to reduce oxidative stress by acting as scavengers of hydroxyl radicals (8, 12). Anethole has also been shown to block both inflammation and carcinogenesis. It suppresses activation of the activator protein 1 (AP-1) and nuclear factor-kappa B (NF-κB) (13, 14), TNF-induced activation of c-JUN N-terminal kinase and mitogen-activated protein kinases (MAPK) (15, 16). It is also a potent inhibitor of kappaB-alpha (IκBα) phosphorylation and degradation, and of expression of NF-κB reporter gene. Another phytochemical, curcumin (diferulolyl methane), is a key component of turmeric and has anti-inflammatory, antitumour and antioxidant properties (17). Curcumin also has been found to suppress expression of TNFα and NF-κB, and to sensitize cancer cells towards apoptosis due to cisplatin and taxol (17, 18). Recent studies have demonstrated that diverse activities of curcumin in terms of antioxidant, antiproliferative, and antiangiogenic functions are mediated through multiple signalling pathways (19, 20) and that the molecular targets of curcumin include transcription factors, growth factors, cytokines, enzymes, and other gene products (21, 22). As noted earlier, similar to anethole, curcumin has been found to inhibit NF-κB activation that is known to be induced by TNF, phorbol ester, hydrogen peroxide, or interleukin (IL) 1. The inhibitory effect of curcumin on NF-κB activation is believed to be due to the inhibition of IκB kinase (IKK) activity (23-25). When NF-κB is down-regulated, decreased expression of inflammatory enzymes such as cyclooxygenase (COX2) and inducible nitric oxide synthase (iNOS) (24, 26, 27) will result. Curcumin has also been found to down-regulate the expression of IL1, IL6, TNFα (II), and angiogenic factors such as vascular endothelial growth factor (VEGF) (28, 29).
From this, it is logical to suppose that appropriate combinations of the platinum drugs cisplatin and oxaliplatin with the phytochemicals anethole and curcumin may cause enhanced cell kill. Such combination treatments may also reduce systemic toxicity caused by chemotherapies and radiotherapies. The present research constitutes a part of continued study on combinations of platinum drugs and phytochemicals in models of human ovarian tumour (30-33).
Materials and Methods
Drugs. Cisplatin was prepared according to the Dhara method (34) and powdered oxaliplatin was obtained from Sigma-Aldrich, Sydney, Australia. They were initially dissolved in dimethyl formamide (DMF) followed by the addition of milli-Q water (at a ratio of 1:5) to give 1 mM stock solutions. Both of the phytochemicals were also obtained from Sigma-Aldrich, anethole in liquid form and curcumin in powder form. Anethole and curcumin were dissolved in dimethyl sulfoxide (DMSO) and ethanol, respectively, for making stock solutions (1 M for anethole and 1 mM for curcumin). The solutions were filtered using a DISMIC-25cs ADVANTEC filter (cellulose acetate, 0.20 μm hydrophilic, pressure limitation: 0.51 MPa; Millipore Ireland ltd.Tullagreen, Carrigtwohill, Co Cork, IRL) to sterilize. The stock solutions were serially diluted with freshly prepared RPMI-1640 medium to produce a range of final concentrations from 0.0005 to 100 μM for cisplatin, oxaliplatin and curcumin, and 0.0005 to 100 mM for anethole.
Cell lines. Human ovarian cancer cell lines A2780 (parent), A2780cisR (cisplatin-resistant) and A2780ZD0473R (ZD0473-resistant) were obtained from Dr. Mei Zhang from the Royal Prince Alfred Hospital, University of Sydney, Australia. The A2780 cell line was originally established from tumour tissue of an untreated patient, the A2780cisR cell line was developed by chronic exposure of A2780 cells to increasing concentration of cisplatin and the A2780ZD0473R cell line was developed by in vitro exposure of A2780 cells to increasing concentration of the drug ZD0473 from 0.5 to 12.5 μM for a period of seven months (35). The cell lines were subcultured in RPMI-1640 medium that was prepared in 10% fetal calf serum (FCS), 1 mM Hepes, 5.6% sodium bicarbonate and 200 mM glutamine.
Cytotoxicity assays. Cytotoxic activities of both platinum drugs and phytochemicals towards A2780, A2780cisR and A2780ZD0473R cancer cell lines were determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay (36). In short, between 4000 to 5000 cells per well were seeded into a flat-bottomed 96-well culture plate in 10% FCS/RPMI-1640 culture medium and allowed to attach overnight. For single treatments, drugs were added at a range of at least three to five different concentrations to triplicate wells and left in the incubator (37°C, 5% carbon dioxide in air, pH 7.4) for 72 h. After preparation of serial fivefold dilutions of the drugs in 10% FCS/RPMI-1640 medium (oxaliplatin: 0.064-8 μM, cisplatin: 0.32-40 μM, anethole: 0.4-50 mM and curcumin: 0.64-80 μM), 100 μl of drugs were added to equal volumes of cell culture in triplicate wells; cells were then left to incubate under normal growth conditions for 72 h at 37°C in a humidified atmosphere. For combination studies, cells were treated with increasing concentrations of compounds at constant ratios of their half maximal inhibitory concentration (IC50) values using the sequences: 0/0, 0/2 and 2/0, where 0/0 mean that both the drugs were added simultaneously, 0/2 that cisplatin or oxaliplatin was added first followed by the anethole or curcumin 2 h later and 2/0 the converse. The concentration ranges were: cisplatin: 0.26-4.09 μM, 1.66-26.52 μM and 0.57-9.11 μM; oxaliplatin: 0.16-2.62 μM, 0.59-9.41 μM and 0.42-6.75 μM; anethole: 0.97-15.49 mM, 0.69-11.01 mM and 0.54-8.70 mM; curcumin: 2.73-43.73 μM, 3.86-61.78 μM and 3.31-52.89 μM for A2780 (parent), A2780cisR (cisplatin-resistant) and A2780ZD0473R (ZD0473-resistant) cell lines respectively. At the completion of the 72 h period of incubation, the MTT assay was performed as in previous experiments (36).
Data analysis. The combined action of the drugs was studied using median–effect analysis. The combination index (CI) was calculated based on the pooled data from five individual experiments each comprising at least three data points for each drug alone and for the drug combinations. The CI for two drugs in combination can be calculated using the following equation (37, 38).
Equation 1 where D1 and D2 represent the concentrations of compounds 1 and 2 in combination required to achieve x% inhibition whereas D1x and D2x represent concentrations of compounds 1 and 2 required to achieve x% inhibition when present alone. Dx can be readily calculated from equation 2. In the equation, Dx denotes the dose of drug, Dm is the median–effect dose or IC50, fa is the fraction of cells affected (killed) by the dose, fu is the fraction of cells remaining unaffected so that fu=1-fa, and m is the exponent defining the shape of the dose–effect curve.
CI of <1, =1 and >1 indicates synergism, additiveness and antagonism respectively of the combined drug action. The CI, Dm and r values were calculated using Calcusyn software (V2) (Biosoft, UK). Dm reflects the IC50 value. The linear correlation coefficient, r, indicates the goodness of fit for the pooled data (where r=1 is a perfect fit).The linear correlation coefficient, r, of the median–effect plot for the cell culture system should be greater than 0.95 (r>0.95) (37, 38).
A new term called the ‘enhancement factor’ (EF) has been defined to give a direct relationship between drugs in combination (30). The EF at the median–effect level (EFm) is defined by the equation: Equation 3 Where X1 and X2 are the mole-fractions of drugs 1 and 2 in the mixture, Dm is the concentration of the drugs required to cause the median effect, IC501 and IC502 are the concentrations of drugs 1 and 2 respectively required for 50% cell kill when acting alone.
Results
Cytotoxic effect of single drugs. Table I gives the IC50 values and resistance factors (RF) of cisplatin, oxaliplatin, curcumin and anethole as applied to human ovarian cancer cell lines A2780, A2780cisR and A2780ZD0473R. RF is defined as the ratio of the concentration of the drug required for 50% cell kill in the resistant cell line to that in the parent cell line. Among the four compounds, oxaliplatin was found to be most active and anethole had the lowest activity. However, anethole had a greater activity in the resistant cell lines than did the rest of the compounds.
Cytotoxic effects of drugs in combination. As stated earlier, the main objective of the present study was to investigate synergism in activity of the combinations of the selected platinum compounds with the phytochemicals anethole and curcumin in three human ovarian cancer cell lines. Both dose–response curves and CIs were used as measures of combined drug action. Whereas dose–response curves give a visual representation of the combined drug action that is qualitative in nature, CIs give a quantitative measure. Table II gives dose–effect parameters in terms of median–effect dose, shape (sigmoidicity), conformity (linear correlation coefficient), represented as Dm, m and r respectively. Figures 2 and 3 give dose–response curves for platinum drugs and the phytochemicals administered alone and in combination using 0/0 h, 0/2 h and 2/0 h sequences of administration for the human ovarian A2780, A2780cisR and A2780ZD0473R cancer cell lines.
Dose-response curves for anethole (alone) could not be included in Figure 2 as the compound has a much lower activity than the rest of the compounds (IC50 at mM level for anethole and μM level for cisplatin, oxaliplatin and curcumin).
Both the CIs and dose–response curves show that administration of anethole or curcumin and platinum drug with a 2 h time gap produces much greater cell kill than the bolus administration and that the combined drug action is greater when phytochemicals are added first rather than the converse.
Discussion
Among all the four compounds, anethole was found to have the lowest cytoxicity. However, it had greater activity against the resistant cell lines A2780cisR and A2780ZD0473R than the parent cell line A2780 so that it has lower (<1) resistant factor. The results indicate that anethole is apparently better able to induce programmed cell death i.e. apoptosis of the resistant cell lines than of the parent cell line. More importantly, 2/0 combinations of anethole with both cisplatin and oxaliplatin were found to be highly synergistic, indicating that pretreatment of ovarian cancer cells with the phytochemical serves to sensitize them to platinum (cisplatin and oxaliplatin) action. Likewise, curcumin was also found to sensitize ovarian cancer cells to platinum action.
The enhanced cell kill resulting from 2/0 combinations of platinum drugs and the phytochemicals cannot be simply attributed to the antioxidant role played by the phytochemicals because when the phytochemicals act as antioxidants, they would serve to reduce oxidative stress, resulting in sparing of cellular antioxidants such as glutathione. Indeed, anethole and its derivative, ADT, and curcumin have been shown to increase the intracellular levels of glutathione and glutathione-S-transferase (8-10). Increased glutathione concentration in turn would cause increased deactivation of platinum drugs before their binding with DNA (32). The reduced level of platinum–DNA binding would be expected to cause reduced cell kill if binding with the DNA was an essential (although not sufficient) requirement to induce apoptosis. However, the above argument is simplistic in that it fails to take into account the fact that cisplatin and oxaliplatin induce apoptosis by both extrinsic and intrinsic pathways (39-41). Besides acting as antioxidants, the two phytochemicals are known to be involved in various pathways associated with apoptosis and inflammation (6, 8, 12). As stated earlier, platinum resistance is associated with increased expression of transcription factor NF-κB and up-regulation of serine/threonine-specific protein kinase (AKT) and COX2 pathways (resulting in cell survival), whereas curcumin and anethole are known to do the converse (6, 12). It is thus suggested that the enhanced cell kill resulting from treatment of cancer cells with the phytochemicals (2/0 sequence of administration) is rather related to the reduced expression of NF-κB and down-regulation of AKT and COX2 pathways. It is possible that the duality in behaviour of the phytochemicals (antioxidant role and pro-apoptotic function) may lead to one role being more dominant at different concentrations, the antioxidant role may be more significant at a lower concentration and the pro-apoptotic function (due to involvement in multiple pathways) becomes more dominant at a higher concentration. In support of this idea, it may be stated that anethole promotes cell survival at a lower concentration (0.2 mM) and promote cell death at higher concentrations (25 mM).
Based on the current knowledge, about 15% of all solid tumours are driven by NF-κB as a player, whereas most cancer preventative agents are believed to be NF-κB inhibitors (42). Aberrant activation of NF-κB can provide protection against apoptosis and stimulate proliferation of malignant cells, and its overexpression is causally linked to phenotypic changes that are characteristic of neoplastic transformation. Activation of NF-κB occurs in response to a wide variety of stimuli, such as cytokines, growth factors, physiological, physical and oxidative stress, and certain pharmacological drugs and chemicals (43). The convergent step in signal-induced activation of NF-κB is the phosphorylation of IκBα which is carried out by IKK, leading to its ubiquitination and degradation. Once IκBα is degraded, the active NF-κB is translocated into the nucleus. Anethole is known to inhibit this crucial step (12). The inhibitory effect of curcumin on NF-κB activation is also believed to be due to the inhibition of IKK activity (23, 24).
As to the question of why the bolus addition of platinum drugs and the selected phytochemicals is found to be from additive to antagonistic in action, it is suggested that concurrent administration of the two compounds in some cases failed to sensitize the ovarian cancer cells to platinum action, perhaps due to less significant reduction in the expression of NF-κB (32). Thus, the prior incubation of ovarian cancer cells with anethole and curcumin appears to be the critical determinant in lowering the expression of NF-κB. Curcumin has also been shown to suppress angiogenic factors such as VEGF, which is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis (6). As solid tumours cannot grow beyond a limited size without an adequate blood supply, it is thought that combination treatment with curcumin may also inhibit cancer progression and development.
In a previous study, it was reported that both the bolus administration and sequenced combinations of cisplatin with curcumin using 4 h time gap (0/4 h and 4/0 h) produced synergistic outcomes in A2780, A2780cisR and A2780ZD0473R cell lines, whereas 24/0 h combination was found to be antagonistic (31). The results indicate that the duration of treatment with the phytochemical is also a critical determinant of cytotoxicity. Proteomic studies designed to characterize key proteins that are differentially expressed in resistant cell lines as compared to the parent cell line and whose functions are restored to normalcy due to treatment with successful drug combinations may shed further light on this matter. If confirmed in vivo, the results of the present study may have profound implications in cancer treatment.
Acknowledgements
Meher Un Nessa is grateful to the Australian Department of Education, Employment and Workplace Relations (DEEWR) for the Endeavour Postgraduate Award and to the Sydney Medical School, University of Sydney, for Part-fee scholarship which have enabled her to conduct this research work. Meher Un Nessa is also grateful to Khulna University, Bangladesh for providing study leave to carry out this study at the University of Sydney, Australia. Special thanks are expressed to the Biomedical Science Research Initiative Grant and Biomedical Science Cancer Research Donation Fund for their part support in this project.
Footnotes
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Conflicts of Interest
Meher Un Nessa, Philip Beale, Charles Chan, Jun Qing Yu and Fazlul Huq declare that they have no financial or personal relationships with other people or organizations that could inappropriately influence their work.
- Received July 17, 2012.
- Revision received September 25, 2012.
- Accepted September 27, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved