For centuries, the concept of synergy between two or more plant constituents to maximize the desired therapeutic effect has been applied successfully in the Indian Ayurvedic and traditional Chinese systems of medicine [1–3]. However, recognition of merits of multiple drug therapy is relatively recent in modern medical practice. With the advent of sophisticated and sensitive technologies to study combination therapies and an understanding of its underlying principles through theoretical models [4–6], synergy research with anticancer plant products and conventional drugs has now emerged as an active area in the search for new cancer chemotherapy [7–15]. The benefits of synergism include increasing the efficacy of therapeutic effect, decreasing dosage without compromising efficacy, mitigating or obliterating toxic side-effects and minimizing or delaying the induction of resistance to drugs [16–21].
Doxorubicin (Adriamycin) is the current, first drug of choice for the chemotherapy of a wide variety of carcinomas, sarcomas and cancers of hematological origin . However, there are serious side-effects associated with the use of this potent anticancer drug . Nausea, vomiting, neutropenia, alopecia and heart arrhythmias are common at the beginning of the therapy. The potential for developing cardiotoxicity leading to cardiomyopathy and congestive heart failure increases dramatically when the cumulative drug dose reaches >550 mg/m2 upon continued use. Further, at high doses, doxorubicin loses its efficacy against multidrug resistant tumors, such as breast cancer. Recent studies found that several plant constituents such as p-coumaric acid  and other common polyphenols (apigenin, baicalenin, kaempherol, luteolin and quercetin)  and curcumin from Curcuma longa L. (turmeric) ; essential oil terpenes, linalool found in several aromatic plants , thymoquinone from Nigella sativa (black cumin seed)  and sesame oil (from Sesamum indicum) ); extracts of Allium sativum (garlic)  and Ginkgo biloba (ginkgo leaves)  and antioxidants from Morus alba L. (white mulberry), Phyllanthus emblica L. (Indian gooseberry) and Piper rostratum Roxb (Thai herb)  and Spinacea oleracea (spinach)  mitigated adverse side-effects associated with doxorubicin chemotherapy including cardiotoxicity.
Additionally, the following anticancer plant constituents were found to act synergistically with doxorubicin in in vitro and/or in vivo animal model studies: polyphenols from Camellia sinensis (green tea; catechins) , Mangifera indica (mango; mangiferin) , Petroselinum hortense Hoffm. 1814 (parsley root juice; apiin and luteolin) , Semecarpus anacardium (nut kernel; catechol) , Silybum marianum (milk thistle; silymarin extract  and silibinin ), Toona sinensis (gallic acid) ; and curcumin , gambogic acid (gamboge resin constituent)  from several Garcinia species and quercetin ; alkaloids, bisbenzylisoquinoline (tetrandrine) from Stephania tetrandra (fen fangi roots)  and stemona alkaloids (stemocurtisine and oxystemokerrine) from the roots of Stemona aphylla and S. burkilli (Thai herbs) ; anthraquinone (aloe emodin) from Rheum palmatum (rhubarb stalks) ; benzoquinone (thymoquinone) ; saponins from Panax ginseng (ginseng; ginsenosides)  and Pulsatilla chinensis (bunge roots; betulinic acid derivative)  and extracts of the fruits of Phyllanthus emblica L. (Indian gooseberry) and Terminalia bellerica (beleric)  and Morinda citrifolia (Noni)  and seeds of Vitis vinifera (grape) .
While the above findings of synergistic activity of anticancer plant products with cancer drug doxorubicin are significant, their clinical application is hampered by safety concerns of the new combinations. This is because such safety data in humans are generally not available, and pharmaceutical companies are required to conduct extensive, new safety studies on all new drug combinations as mandated by Food and Drug Administration (FDA) in the US, European Medicines Agency (EMA) in the European Union and other drug regulatory agencies around the world . In this regard, it is noteworthy that our recent comparative cytotoxic investigation with red beetroot (Beta vulgaris L.) extract and doxorubicin in several human cancer cell lines indicated their potential for synergistic activity [49, 50]. Moreover, we reported earlier that red beetroot extract consistently reduced multi-organ tumor formations in various animal models when administered in drinking water [51–53]. This led us to postulate that a long-term daily exposure to small quantities of beetroot extract through diet might be safe and sufficient to produce cancer chemopreventive effect in humans [51, 53]. Since red beetroot extract is approved by FDA and EMA for use in humans as red food color E162 and considered safe with no known toxicity , it seemed logical to evaluate its synergistic cytotoxicity with doxorubicin. Accordingly, we performed the present study of their synergistic growth inhibition potential in pancreatic (PaCa), breast (MCF-7) and prostate (PC-3) cancer cell lines of human origin. The combination index (CI) method of Chou and Talalay [5, 54] based on the median-effect principle was used to evaluate synergy between beetroot extract and doxorubicin in their cytotoxicity in the above three cancer cell lines.
Materials and methods
Red beetroot (B. vulgaris L.) extract was purchased from Kasei Tokyo Chemical Industry Co., Ltd, Tokyo, Japan marketed under the trade name “Betanin” (Catalog Number B0397). It is described as red beetroot extract diluted with Dextrin, rendered acidic with citric acid, pH 5.4 and stabilized with ascorbic acid, Batch Number GA01 with specification: λmax 530.0–536.0 nm with 1.6 min absorbance. Doxorubicin (Adriamycin) was procured from Sigma Chemical Co., St. Louis, MO, USA.
Human pancreatic cancer cells (PaCa) were purchased from D.S. Pharma Biomedical Co., Ltd., Osaka, Japan. The estrogen receptor-positive human breast cancer cells (MCF-7) and androgen-independent human prostate cancer cells (PC-3) were procured from Dainippon Pharmaceutical Co., Osaka, Japan. The cells were cultured in suspension at 37°C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and maintained in a humidified atmosphere containing 5% CO2 in air. Nunclon 3.5 cm cell culture dishes from Nunc Co., Denmark were used in all cell culture studies.
Cytotoxicity evaluation of red beetroot extract and doxorubicin, individually and in combination
Cytotoxic studies were conducted per experimental procedures described in detail earlier [55, 56]. The cancer cells, PaCa, MCF-7 and PC-3 in exponential growth phase, were seeded and separately incubated with different concentrations (0.29–290 μg/ml) of red beetroot extract (B) and doxorubicin (D), individually and in three different combinations (B:D ratios:1:1, 5:1 and 1:5). For cytotoxicity evaluation, the cancer cells were incubated with various concentrations, individually and in various combinations of the test substances, and the viability of cells was assessed after 3 days by the trypan-blue staining method . At the end of each experiment, 0.1 ml of treated cells was suspended in phosphate-buffered saline solution and stained with 0.1 ml of 0.25% trypan-blue solution. The dying cells were stained blue and the viable, non-blue cells were counted.
All results are expressed as growth inhibition or percent viability of cells, and each data point represents the mean of three independent experiments (n = 3) with ±SD values ranging from ±0.3 to ±8.4. Statistical significance between groups was determined by the analysis of variance and p values ≤0.05 were considered significant. The 50% growth inhibitory concentrations (IC50) of beetroot extract and doxorubicin were calculated from dose–response curves.
Evaluation of synergistic cytotoxicity of red beetroot extract and doxorubicin when tested in combination
Synergism was evaluated by the CI method of Chou and Talalay which is based on the median-effect principle [5, 54]. The CIs were calculated by the Chou–Talalay equations for multiple drug effects which take into account both potency (IC50) and shape (slope, m) of dose–effect curve. The general equation used for the classic isobologram (CI1) is: CI(D)1/(Dx)1+(D)2/(Dx)2, where (Dx)1 and (Dx)2 in the denominators are the concentrations of D1 (e.g. beetroot extract) and D2 (e.g. doxorubicin) alone that gives x% inhibition, whereas (D)1 and (D)2 in the numerators are the concentrations of D1 and D2 in combination that also inhibits x% (i.e. isoeffective).
The CI values obtained from the above classic (mutually exclusive) isobologram equation were used to quantitate synergistic cytotoxicity of red beetroot extract when combined with doxorubicin in PaCa, MCF-7 and PC-3 cancer cell lines. The CIs and their plots versus the fraction affected (Fa) were obtained from the Chou–Talalay median-effect analysis computer program (http://www.compusyn.com). This software provides automated simulation of synergism (defined as more-than-expected additive effect) and antagonism (defined as less-than-expected additive effect) at all dose or effect levels. It displays the dose–effect curve, median-effect plot, CI plot, isobologram, polygonogram and dose-reduction index (DRI) plot . Also, the median-effective dose (Dm) that produces 50% cell death; m, the slope of the median-effect plot and r, the conformity parameter (the linear correlation coefficient) for goodness of fit can be calculated from the median-effect plot using the above program. The value of CI<1 indicates synergism; CI>1 indicates antagonism and CI=1 indicates additive effect.
DRI is a measure of how many folds the dose of beetroot extract or doxorubicin in a synergistic combination may be reduced at a given effect level compared with their doses when used alone and is defined as follows: (DRI)1=(Dx)1/(D)1 and (DRI)2=(Dx)2/(D)2. The relationship between CI and DRI is given by the equation: CI=(Dx)1/(D)1+(Dx)2/(D)2=1/(DRI)1+1/(DRI)2. Both potency (Dm) of a dose (D) and shape (slope, m) of dose–effect curve are taken into account in the median-effect plots using the equation: log (Fa/Fu)=m log(D)–m log(Dm), where Fa=fraction affected and Fu=fraction unaffected.
Cytotoxicity of red beetroot extract and doxorubicin in human cancer cell lines
The growth inhibitory effects of red beetroot extract and doxorubicin alone or in various combinations (B:D ratios: 1:1, 5:1 and 1:5) in human pancreatic (PaCa), breast (MCF-7) and prostate (PC-3) cancer cells are shown in Figure 1. A dose-dependent growth inhibition was observed with B, D and their three combinations in all three cancer cell lines tested. The B:D combination ratio of 1:5 exhibited the highest growth inhibition in all three tested cancer cell lines.
The IC50 values for red beetroot extract ranged from 310.0 ± 3.2 µg/ml to 328.0 ± 2.2 µg/ml in PaCa, PC-3 and MCF-7 cancer cells while those for doxorubicin ranged from 2.2 ± 4.3 µg/ml to 32.2 ± 42 µg/ml (Table 1). All IC50 values were significantly lower for doxorubicin when compared to those for beetroot extract in all three cancer cell lines evaluated (p < 0.001). Similarly, IC50 values for B:D combination ratios of 1:1 and 5:1 were lower than those for beetroot extract. The B:D (1:5) ratio exhibited significantly lower IC50 values when compared to those for doxorubicin in all three cancer cell lines tested (p < 0.001). These dose-reduction levels were different and specific for each beetroot extract–drug combinations and the three cancer cell lines tested.
Combination effects of red beetroot extract with doxorubicin in human cancer cell lines
The combination effects of red beetroot extract with doxorubicin in PaCa, PC-3 and MCF-7 cancer cell lines are summarized in Table 2. The dose–effect relationships were calculated from cytotoxicity data using the Chou–Talalay median-effect analysis computer program. They are represented by the median-effect dose (Dm), the slope of the median-effect plot (m), the linear correlation coefficient (r), the CI, DRI and the dose–effect levels of cancer cell growth inhibition (IC50, IC75 and IC90). The median-effect and log CI plots of various B:D combinations for PaCa, PC3 and MCF-7 cell lines are given in Figures 2 and 3, respectively. Different B:D ratios responded differently but were specific to each combination and cancer cell line tested. The median-effect plots showed that the 1:5 ratio of B:D was better than other ratios, especially at higher doses, because the plots were lower and close to intersection (Figure 2). However, in PaCa cell line, the B:D ratio of 1:5 was better at all dose levels. In contrast, the lower dose levels of the 1:5 ratio were inferior to other combinations of B and D in PC-3 and MCF-7 cell lines.
Comparison of the Dm values showed a reduction by 36.5% for doxorubicin when combined with red beetroot extract in the B:D ratio of 1:5 in PaCa cell line (Table 2). Such reduction in Dm values for red beetroot extract was also observed in all combinations with doxorubicin (B:D ratios: 1:1, 5:1 and 1:5) in PaCa and with B:D ratios 1:1 and 1:5 in PC-3 and MCF-7 cell lines. The CI values indicated that 1:5 combination of beet root extract and doxorubicin exhibited synergistic effects at IC50, IC75 and IC90 dose levels in PaCa cells, because the values were <1. Similarly, the CI value was <1 at IC90 dose level in MCF-7 cells treated with beet root extract and doxorubicin at 1:5 ratio, indicating synergism.
The DRIs at IC50, IC7 5 and IC90 dose levels were positive and low (0.01–0.68) for all three combination ratios of B:D and in all three cancer cell lines except in high value (DRI>1) situations where CI values were <1, as in the cases of B:D ratio of 1:5 in PaCa and MCF-7 cells (Table 2). Overall, positive reduction in drug concentration was achieved by doxorubicin when combined with red beetroot extract in various ratios in all three cancer cell lines tested. The synergistic activity was best when B:D ratio of 1:5 was used in PaCa cancer cells at IC50, IC75 and IC90 dose levels (CI values 0.36–0.53). Similarly, synergistic cytotoxicity was also observed with B:D ratio of 1:5 in MCF-7 cancer cells at IC90 dose level but to a lesser extent (CI value 0.89). The B:D combination ratios of 1:1 and 5:1 were antagonistic in all three cancer cell lines, while B:D (1:5) ratio was also antagonistic in PC-3 and MCF-7 cells except at IC90 dose level in the latter.
The inadequacy of standard dose–effect plots in establishing whether a drug combination is synergistic, additive or antagonistic is now well established . Defining synergism in drug combinations has been an arduous task and only recently its theoretical basis, experimental design and computerized simulation have been accomplished [4–6, 54]. According to the generally accepted definition, synergism is a combined effect that is more than additive effects of two or more drugs. However, a combined effect greater than each drug alone does not necessarily indicate synergism. Also, the additive effect of two or more drugs is not the simple “arithmetic sum” of effects of individual drugs. The merging of the mass-action law principle with mathematical induction–deduction is proven to be a unique and effective scientific method for developing theoretical basis of synergism. Computer software based on the median-effect principle and its mass-action law is now available to evaluate whether a single compound or entity can be used beneficially in combination therapies with other drugs or modalities [4, 5].
In the present study, we have evaluated synergistic cytotoxicity of red beetroot extract with doxorubicin in several human cancer cells in vitro utilizing the CI method of Chou and Talalay [4, 5, 24] which is based on the median-effect principle. With the aid of a versatile computer program (http://www.compusyn.com), the log of CI was plotted against Fa (fraction affected) to obtain the following dose–effect data: the median effective dose (Dm) that produces 50% cancer cell death, the slope of the median-effect plot (m), the linear correlation coefficient (r), CIs at IC50–IC90 dose levels and the DRI, a measure of how many folds the dose of each drug in a synergistic combination may be reduced at a given effect level when compared with the doses of each drug alone. The median-effect analysis was used to analyze the type of interaction between beetroot extract and doxorubicin when tested in combination (by quantitative determination of drug interactions) where CI<1, =1 and >1 indicate synergism, additive effect and antagonism, respectively.
While dose–effect plots indicated enhancement of growth inhibitory effect of red beetroot extract and doxorubicin when used in combination in PaCa, PC-3 and MCF-7 cancer cell lines (Figure 1), they failed to identify whether such enhancement was due to additive effect or synergism. In contrast, analysis of cytotoxicity data by the CI method of Chow and Talalay showed synergism between the extract and drug which was not only dose-specific but also depended on their combination ratio when tested together (Figures 2 and 3 and Table 2). Both drug and extract exhibited different potency (the Dm values) and dose–effect curves with different shapes or slopes (the m values). The r values in general were >0.95 indicating the versatility of the median-effect equation and plot used . The extract–drug ratio of 1:5 was found to be the best for their synergistic cytotoxicity at IC50, IC75 and IC90 dose levels in PaCa cancer cells and at IC90 dose level in MCF-7 cancer cells. In contrast, the B:D combination ratios of 1:1 and 5:1 were antagonistic in all three cancer cell lines tested. Also, B:D (1:5) ratio was antagonistic in PC-3 and MCF-7 cells except synergism was observed at IC90 dose level in the latter case.
The mechanism of synergistic cytotoxicity exhibited by red beetroot extract and doxorubicin remains to be delineated. Because of the similarity between the chemical structure and configuration of betanin, the major betalain constituent responsible for red color of beetroot extract and doxorubicin, their intercalation with DNA in cancer cells has been indicated as a possible common mechanism of their antiproliferative activity [49, 53]. Based on cell biology studies and biochemical events at intra- and extra-cellular levels, several additional mechanisms of anticancer activity attributable to various beetroot extract constituents have been proposed [53, 58, 59]. These include involvement of processes such as anti-inflammatory, antiangiogenesis and pro-apoptosis in tumor cells and enzymes such as DNA topomerase I and II, LDL, MPO, DMNT, phase-II and COX-1 and -2. A common feature in the above-proposed anticancer mechanisms is the powerful antioxidant, free radical scavenging property of polyphenolic compounds (doxorubicin, betanin and possibly other betalain constituents of red beetroot extract) with the potential to modulate reactive oxygen species involved in apoptosis process through activation of caspases and other oxidative processes leading to mutation and cancer.
Whether any of the above-proposed mechanisms play a role in the observed synergy between red beetroot extract and doxorubicin cytotoxicity is not known at the present time. Additional studies, both in vitro and in animal models, are needed to further ascertain synergy and mechanism involved as well as clinical validation of doxorubicin combination chemotherapy with red beetroot extract in treating pancreatic, breast and other cancers in humans. The dosage reduction achievable with such combination therapy has the potential to mitigate serious side-effects (cardiotoxicity, immune deficiency, etc.) and development of drug resistance commonly associated with current doxorubicin chemotherapy.
Analysis of cytotoxicity data by the computerized CI method of Chow–Talalay established synergism between beetroot extract and cancer drug, doxorubicin, in several human cancer cell lines. Such synergism was dose-specific as well as dependent on the extract–drug combination ratio and cancer cell line tested with the ratio of 1:5 exhibiting the best synergistic cytotoxicity against pancreatic cancer cells at doses IC50, IC75 and IC90 and breast cancer cells at IC90 dose level.
The present study supports the postulation that dietary constituents with known anticancer activity (e.g. red beetroot extract) when used in right combination and dose could enhance the therapeutic efficacy of potent chemotherapeutic drugs (like doxorubicin), reduce their toxic side-effects through dosage reduction and impede development of cancer cell resistance to drug treatment [6, 11, 16, 18, 53]. The multi-organ cancer treatment potential of red beetroot extract–doxorubicin combination is noteworthy. Favorable clinical trials in humans could lead to expedited review process by regulatory agencies and quick approval of doxorubicin–red beetroot extract combination chemotherapy, since the latter is already approved for use in humans as a safe food colorant (designated as food color E162).
This study was supported in part by Howard University Funds for Academic Excellence and Grant-in-Aid from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan.
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