Effects and synergy of feed ingredients on canine neoplastic cell proliferation
© The Author(s). 2016
Received: 13 February 2016
Accepted: 16 July 2016
Published: 2 August 2016
Adjunctive use of nutraceuticals in human cancer has shown promise, but little work has been done in canine neoplasia. Previous human research has shown that polyphenols and carotenoids can target multiple pathways in vitro and in vivo. These compounds could synergize or antagonize with currently used chemotherapies, either increasing or decreasing the effectiveness of these drugs. Considering the routine and well controlled feeding practices of most dogs, the use of nutraceuticals incorporated into pet food is attractive, pending proof that the extracts are able to improve remission rates. The aim of this study was to examine five feed ingredients for antiproliferative effects, as well as the interaction with toceranib phosphate and doxorubicin hydrochloride, when treating canine neoplastic cell lines in vitro.
Screening using MTT proliferation assays showed that green tea, turmeric, and rosemary extracts were the most effective. Turmeric extract (TE) was the most potent and exhibited synergy with a rosemary extract (RE) at concentrations from 1 to 25 μg mL−1. This combination had an additive or synergistic effect with chemotherapeutic agents at selected concentrations within each cell line. No significant effects on cell viability were observed when the combination therapy was used with normal primary cells.
The use of turmeric and rosemary extracts in combination may be worthwhile to investigate in the pre-clinical and clinical neoplastic considering there are no negative effects on traditional chemotherapy treatment. Further studies into the pharmacokinetics and mechanisms of action of these extracts should be investigated.
Neoplasia is the cause of death in 20–30 percent of dogs in the United States, and the incidence and mortality increases to 50 % in dogs over the age of ten [1–3]. Risk factors for developing cancer vary based on age, breed, sex, medical history, and nutrition . The incidence and, in some cases, tumor origin are different between dogs and humans, with lymphoma, mast cell disease, osteosarcoma, and mammary neoplasia being particularly prevalent in dogs . Current treatments rely on the use of chemotherapeutics, radiation therapy, and, when practical, surgical interventions. The efficacy of such treatments varies greatly and relies heavily on the stage of cancer progression when treatment is provided, genetic predispositions, and tolerance to the chemotherapy and/or radiation treatments.
Most chemotherapeutic treatments are limited and target DNA replication (cell cycle), cellular metabolism or a single regulatory pathway. In the case of high grade and refractory tumors, multiple drug combinations are often used in order to achieve remission. As the number of treatment modalities increase there can be compounding side effects that are not always well tolerated by patients .
The use of natural compounds, or nutraceuticals, in combination with traditional chemotherapy might provide value, and 40 % of pet owners admit to using nutritional supplements as a complementary therapy . Natural products have been used in the initial design of chemotherapies, with well over half of all compounds synthesized or screened being more potent derivations of natural products . The effects of these compounds, whether natural or synthetically made, act through a variety of mechanisms that may contribute to the anti-cancer properties [9, 10]. In fact, a single compound might be able to induce apoptosis through several different pathways . Much of the natural product research performed has been on human or rodent cell lines, particularly of epithelial origin, with little research being done on canine cell lines . More research is necessary to determine the specific effects of these natural products in canine cells and in combination with chemotherapies commonly used in veterinary medicine, as there is a paucity of information in veterinary medicine [13–15].
Plant extracts consist of various polyphenols, terpines, stilbenoids and carotenoids . Several epidemiological studies in humans have found benefits to the inclusion of polyphenol and carotenoid rich fruits and vegetables in the diet [17, 18]. A case–control study has been completed in dogs, and the results indicated an inverse relationship between the consumption of vegetables and the risk of developing transitional cell carcinoma of the urinary bladder in Scottish Terriers . The potential use of certain polyphenols and carotenoids as a treatment option is ongoing in pre-clinical trials with treatment of human epithelial carcinomas on the horizon [20–22]. However; it is necessary to investigate the potential effectiveness in canine cancers which represent other cellular lineages with round, mesenchymal and epithelial neoplasias all being prevalent. In veterinary patients, there have been few intervention studies to examine nutraceutical dietary additions, with two studies examining possible treatment of lymphoma in dogs [23, 24]. Considering that the typical daily feeding patterns of companion dogs are consistent (once or twice per day), the potential for incorporation of natural anti-carcinogenic ingredients is feasible, so long as the added herbal, fruit, or vegetable extract is considered generally reliable and safe by the American Association of Feed Control Officials .
The objective of this study was to examine the antiproliferative effects of five commonly used natural food extracts chosen from an initial cytotoxic screening, which could be utilized in commercial dog food, on canine mastocytoma, mammary carcinoma, and osteosarcoma cell lines. After defining efficacy of the leading extracts we set forth to determine if combinations of the most potent extract, turmeric extract [85 % curcuminoids] (TE) could work synergistically with the other extract, and whether these extracts had an additive effect when used in conjunction with the chemotherapeutic drugs most often utilized in the treatment of each neoplastic disease represented in the cell culture systems.
Characteristics of natural extracts
Compound of interest
Green tea extract
Pomegranate extract [40 % punicosides]
Rosemary extract INOLENS70
Three canine neoplastic primary cell lines were used, representing round, mesenchymal and epithelial tumor types: mastocytoma C2 (University of California, San Fransisco, USA), mammary gland carcinoma CMT-12 (Auburn University, Alabama, USA), and osteosarcoma D17 (#CCL-183; ATCC, Manassas, VA, USA). The cell lines were grown on tissue culture-treated plates (Laboratory Product Sales [LPS], Rochester, NY, USA) with appropriate medium containing 10 % heat inactivated fetal bovine serum (HI-FBS; Invitrogen, Carlsbad, CA, USA) and 1 % antibiotic-antimycotic (Invitrogen). Cell lines were grown at 37 °C and 5 % CO2 for all experiments and passage of cells, unless otherwise noted. Canine primary dermal fibroblasts (Applied Biological Materials [ABM], Richmond, BC, Canada) were used to investigate effects on normal cells and were propagated and maintained on PriCoat T25 flasks (ABM) in Prigrow II medium (ABM) containing 10 HI-FBS (Invitrogen) and 1 % penicillin/streptomycin (Invitrogen).
MTT proliferation assay
Cells were plated at a density of 4 × 103 cells per well on 96-well tissue culture-treated flat bottom plates (LPS) and incubated overnight in complete medium. Cells were treated the following day with DMSO vehicle control or extract using a twofold serial dilution for eight final concentrations ranging from 0.4 to 100 μg mL−1 for 48 h representing physiological and supra-physiological concentrations to assess all extracts for potential effectiveness at reducing cellular proliferation. A twofold serial dilution of chemotherapy was also tested ranging from 1.7 to 100 nM toceranib phosphate for the C2 cell line, and 0.03 to 2 μM doxorubicin hydrochloride for the CMT-12 and D17 cell lines. To quantify cellular proliferation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT dye; Alfa Aesar, Ward Hill, MA, USA) assays were performed by adding 30 μL of MTT dye (5 mg mL−1 in phosphate-buffered saline solution) to each well and incubating at 37 °C for 1 h. Media were then aspirated and the cells were solubilized in 200 μL of isopropanol. The optical density of each well was analyzed on a spectrophotometric plate reader (Epoch; Biotek, Winooski, VT, USA) at a wavelength of 570 nm as previously described . Single extract experiments were assayed in duplicate in four independent experiments.
Synergy between extracts was examined using combinations of two extracts at six concentrations: 0.8, 1.7, 3.1, 6.3, 12.5, or 25 μg mL−1 as a representation of potentially high end physiological concentrations. All pairwise combinations of extract concentrations were tested. The percent proliferating cells of control for each treatment was pooled from all experiments and is reported as mean ± standard error of the mean.
Interaction with toceranib phosphate/doxorubicin hydrochloride using proliferation assay
Cells were plated at a density of 4 × 103 cells per well on 96-well tissue culture-treated plates (LPS) and incubated overnight in complete medium. Cells were treated the following day with 50 μL of a combination of TE (0.8, 1.7, 3.1, or 6.3 μg mL−1), RE (0.8, 1.7, 3.1, or 6.3 μg mL−1), and chemotherapeutic drug. The C2 cell line was treated with toceranib phosphate (3.1, 6.3, 12.5, 25, 50, 100 nM) and the CMT-12 and D17 cell lines were treated with doxorubicin hydrochloride (0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 μM). DMSO was used as a vehicle control for all treatments. Cells were treated for 48 h before performing MTT assays. Wells treated with DMSO were considered to represent 100 % proliferating cells. All combinations were tested in duplicate in two independent experiments.
Trypan blue exclusion assay of cell viability
The trypan blue exclusion assay was performed on canine primary dermal fibroblasts (CDF) due to the slow rate of proliferation and low metabolic activity of these normal canine cells, precluding productive MTT assays. The effects of extract treatments were compared to the results obtained on the C2, CMT-12, and D17 cell lines. For the CDF cells, Applied Cell Extracellular Matrix (ABM) was applied overnight to 24-well tissue culture-treated plates (LPS). For all cell lines, cells were plated at a density of 5 ×103 cells per well and incubated until 60 % confluent before treatment with DMSO vehicle control, 6.3 μg mL−1 TE, 6.3 μg mL−1 RE, or a combination of 3.1 μg mL−1 each of TE and RE. After 48 h of treatment, cells were collected and centrifuged at 1,900 g for 10 min. With the exception of the C2 cell line, cells were detached with 0.05 % Trypsin/EDTA. The cell pellet was resuspended in 0.1 % trypan blue (Sigma) in phosphate-buffered saline solution and 1 % FBS, loaded on a hemocytometer, and visualized on an inverted microscope. Cells which stained blue were considered non-viable. All treatments were performed in triplicate and the percent of viable cells were averaged. All values were standardized to the vehicle control treatment which was considered to represent 100 % viable cells.
Data management and calculations
Raw data from proliferation assays (optical density of each well) were normalized to the vehicle alone treatment for individual assays, considered to represent 100 % proliferating cells (single or combined treatment). The percent proliferating cells was then averaged across each replicate. The IC50 for each extract was then calculated across experiments by Probit analysis.
Where (D)1 and (D)2 are the doses of both compounds in combination and (Dx)1 and (Dx)2 are the doses of each compound alone at x percent of inhibition. CI values ≤0.9 indicate synergism, a CI value >0.9 and <1.1 indicates an additive effect, and CI values ≥1.1 indicate antagonism.
All statistical analysis on the outcome of percent proliferating cells as measured by MTT assay was performed using JMP Pro (v. 11.2.1; SAS Institute Inc., Cary, NC, USA). The residuals of the statistical model were evaluated for normality and found to be not normally distributed. Therefore, non-parametric Kruskal-Wallis test was used to compare differences in percent proliferating cells for every treatment concentration used within each cell line across experiments. Comparisons between each treatment group and vehicle control group were carried out using the Steel method adjusting alpha risk for multiple comparisons.
For the outcome of percent viability determined by the trypan blue exclusion assay, residuals of the statistical model were found to be normally distributed, and therefore analyzed using analysis of variance with Tukey’s method for comparison between all treatments, controlling for multiple comparisons. Differences were considered statistically significant at p < 0.05.
Single treatment versus dual combination treatment on three types of cancer cell lines
IC50 of the extracts and chemotherapy determined by MTT assays (single treatment)
Green tea extract
Pomegranate extract [40 % punicosides]
Rosemary extract [70 % Carnosic Acid]
Black pepper extract
12.5 nM (6.2 ng mL−1)
0.3 μM (163.1 ng mL−1)
0.5 μM (271.8 ng mL−1)
Turmeric extract (TE) and rosemary extract [70 % carnosic acid] (RE) combination
Natural extracts and chemotherapy interaction on growth inhibition of cancer cell lines
Cytotoxic activity of TE and RE against cancer cell lines without affecting normal cells
The use of bioactive polyphenol and/or carotenoids from feed ingredients is well studied in human cancer cells, and there may be efficacy in the utilization of many of these bioactive components, particularly in combination [28, 29]. In the current study we surveyed five extracts which were selected based an initial screen of feed ingredients most abundant anti-proliferative compounds that could hinder cell proliferation at 25 μg mL−1, which would represent the high end of physiologically achievable concentration of approximately 10–20 μM for the presumed main component in the extracts examined [14, 15, 30]. Our results indicate that TE was the most potent at inhibiting proliferation at low microgram quantities. To further confirm the single extracts and potential interaction of TE and RE observed in the MTT assay, trypan blue exclusion assays were also performed to assess viable versus non-viable cells. The modest differences in terms of inhibitory effects when comparing the trypan blue and MTT assays may be a reflection of membrane permeability and cytotoxicity being measured with the trypan blue assay versus metabolic cellular activity being measured with the MTT assay. This is particularly evident in the D17 cell line which was less sensitive than the C2 or CMT-12 cell lines, showing diminished activity on MTT proliferative assays and no increase in cell death in the trypan blue assay, suggesting a diminished cell proliferation response rather than cell death response. The response of C2 and CMT-12 cell lines showed that the combination effect of TE with RE was equal, if not superior, in diminishing cell growth as well as increasing the number of non-viable cells than treatment with TE alone, suggesting synergy in this treatment strategy.
Of the two plant extracts that were most effective, TE, which contains 87 % curcuminoids, showed the most potent anti-proliferative effects. These effects could be seen at a concentration below 15 μg mL−1 in all cell lines examined. The high potency of curcumin may be related to its binding affinity for at least 30 different proteins . The anti-proliferative effects have been examined in cell culture models of nearly every type of neoplastic condition including leukemia, breast, prostate, bladder, melanocyte, skin, ovarian, hepatic and uterine cancer cells . In most cell-based models, low micromolar concentrations of curcumin thought to be physiological, affect various intracellular pathways ranging from transcriptional activation, to induction of apoptosis, to halting of the cell cycle . These include: transcriptional activators AP-1, Nf-kB, β-catenin, STAT-3, hypoxia induced factor-1, and notch-1; receptor signaling cascades EGF, HER2, VEGF, PDGF, IGF, and FGF; all three major MAP Kinases ERK1/2, p38, and JNK and protein kinase C; intrinsic mitochondrial apoptosis induction by changes in mitochondrial membrane permeability via Bcl-2 and Bax family member proteins are also documented . The wide array of molecular targets has led to over 100 clinical trials in humans to study the use of curcumin to treat various pathologies from obesity related diseases to neurological diseases to various models of neoplastic conditions . Although curcumin and turmeric extracts are effective in vitro, the bioavailability and absorption of curcuminoids is somewhat limited. Typically, less than 10 % of curcumin is absorbed, even with aggressive treatment regimens, leading to high nanomolar serum concentrations in rodents and humans [36, 37]. The curcumin that is absorbed is quickly glycosylated, sulfated, or hydroxylated, and it is unclear if these metabolites are as effective as the unconjugated curcumin [38, 39]. Several approaches to increase bioavailability are being examined including the use of curcumin analogs , liposomal curcumin , curcumin nanoparticles , and adjuvant therapy  with bioavailability enhancers such as piperine from black pepper extract . These modifications have led to increased blood curcumin concentrations and a half-life of nearly 10 h. Recently, there have been several studies looking into using whole turmeric extract mixtures instead of pure curcumin alone. Specifically, extract formulations including turmerones has led to increased solubility, absorption, and bioavailability . Generally, treatment with curcumin has exhibited no adverse side effects even at high doses and because of this, a maximum tolerated dose has not been established . Only two canine studies have been completed, with low oral dosing regimens of 4 mg kg−1 given orally twice a day showing no side effects after 2 months of treatment [46, 47]. These studies did not measure specifically measure bioavailability, but one study was able to measure transcriptional changes after 20 days of curcumin supplementation .
Rosemary extract rich in carnosic acid (RE) also generated interesting results considering the IC50 for cellular proliferation is likely within physiological ranges, but more specifically because of its synergistic effects with TE. Rosmarinus officinalis contains several phenolic compounds including carnosic acid, carnosol, and rosmarinic acid . In our study, as well as others, carnosic acid and carnosol were more potent in decreasing cellular proliferation than rosmarinic acid in various types of cancer cell lines at concentrations below 20 μM [49, 50]. Carnosic acid and carnosol have been shown to have several mechanisms of action including cell cycle arrest, induction of apoptosis, free radical scavenging, inhibition of metastatic markers, and inhibition of P-glycoprotein mediated drug efflux [51–53]. Intracellular pathways affected include inhibition of PI3-Kinase/AKT/Nf-kB signaling , down-regulation of cyclins A and B , induction of apoptosis by decreases in Bcl-2 , and inhibition of all three major MAP Kinases ERK1/2, p38, and JNK . In rodent studies, the use of a topical  or oral  rosemary extract has been well tolerated and effective. Toxicity studies in rats have shown that up to 3 g kg−1 of rosemary oil is acceptable [60, 61] and biologically relevant levels of around 10 μM can be reached through dietary administration , however canine studies are lacking.
We found synergy between TE and RE, which agrees with previous in vitro studies using the same combination [63, 64]. While RE alone was only effective at concentrations above 6.3 μg mL−1 in all three cancer cell lines, its use with TE significantly decreased the concentrations needed to reduce cell proliferation. In all three tumor cell lines, these extracts worked synergistically at concentrations between 1 – 10 μg mL−1 of each extract. When used in combination, extrapolation of our data accounting for the percentage of the compound of interest (curcumin and carnosic acid) suggest that the IC50 is 6.8 μM curcumin and 7.6 μM carnosic acid for C2, 12 μM curcumin and 13 μM carnosic acid for CMT-12, and 18 μM curcumin and 20 μM carnosic acid for D17. Neither of the extracts, when used alone or in combination, showed effects on cell viability in the normal canine dermal fibroblasts, suggesting the effects on normal cell death or proliferation is minimal. Other control cells were considered, including the canine fibroblast A-72 tumor cell line and Madin-Darby Canine Kidney (MDCK) epithelial cells, but due to the highly proliferative and potentially tumorigenic nature of these cell lines they were not used. CDF cells were chosen due to their seemingly normal phenotype, ease of maintenance, and commercial availability. Further studies could examine the effects on primary lymphocytes or epithelial cells, but these cell types were not available at the time this study was completed.
When the C2 cell line was incubated with the TE/RE combination in the presence of toceranib phosphate, a synergistic or additive effect was seen when either extract was used at 6.3 μg mL−1, or when TE was used at 3.1 μg mL−1 or higher. When the CMT-12 cell line was treated with the TE/RE combination in the presence of doxorubicin hydrochloride, there was a modest antagonistic effect at lower concentrations of both extracts when used alone (below 3.1 μg mL−1 of each), but a synergistic or additive effect could be seen with a higher concentration of 6.3 μg mL−1 of both extracts individually. The D17 cell line showed considerable additive and synergistic effects with all extract combinations at the IC25 and IC75 of doxorubicin hydrochloride in general. Mild antagonism was seen when extracts were used at 3.1 μg mL−1 or lower, but this was diminished or absent when either extract reached a concentration of 6.3 μg mL−1. Considering these findings, further testing of TE and RE with other chemotherapies to ensure similar synergy, additive, or antagonistic effects is warranted. Furthermore, considering the lack of basic pharmacokinetics with oral TE and RE canine studies are needed to examine whether there feed ingredients would have any utility. The synergistic effect of these compounds with chemotherapies is necessary, not only due to the potential to decrease the administered dose for treatment, but also to examine alterations in chemotherapy metabolism.
Other extracts examined in the MTT assay were VetPerine® (piperine), pomegranate extracts and green tea extracts. Effective IC50 for these extracts across the cell lines were typically above 25 μg mL−1 which would be considered supraphysiological. This takes into account that most animals cannot reach concentrations greater than approximately 10–20 μM of any specific bioactive component from these extracts when used at relatively large doses for any significant period of time. That said, these compounds were also tested for synergy, antagonism, or additive effects and were not observed to increase TE or RE effectiveness (data not shown) and were discounted for further examination with commonly used chemotherapeutics.
This study of commonly used feed ingredients showed that a combination of TE and RE diminished the growth of cancer cells. This synergistic effect was observed at 10 μg mL−1 and below indicating a potential for physiological effects, however in vivo pharmacokinetic and efficacy studies are needed. Although we are unsure of the bioactive molecules inducing these effects, the high concentrations of curcuminoids and carnosic acid are likely involved. The anti-proliferative effects of chosen chemotherapeutics were not hindered when these extracts were used in combination at concentrations of 3.1 and 6.3 μg mL−1 across a range of chemotherapeutic concentrations. Further testing of other chemotherapies with these specific extracts is warranted to ensure no distinct antagonism is evident. In addition, further examination of the potential apoptotic effects and cellular pathways affected by these extracts individually and in combination may be fruitful in determining the similarities and differences of their effects between cell lines.
CDF, canine dermal fibroblasts; CI, combination index; DMSO, dimethyl sulfoxide; IC50, 50 % inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RE, rosemary extract [70 % carnosic acid]; TE, Turmeric extract
We are grateful to the University of California San Francisco, especially Dr. Warren Gold and Dr. George Caughey, for supplying the C2 canine mastocytoma cell line; and to Auburn University, especially Dr. R. Curtis Bird, for supplying CMT-12 canine mammary gland carcinoma cell line.
The aforementioned study was funded by Royal Canin. Royal Canin (JB and VB) collaborated on the study design and interpretation of data collected.
Availability of data and materials
Additional data regarding combinations of extracts will be made available upon request to corresponding author.
CBL carried out the technical experimentation, performed statistical analysis, and drafted the manuscript. JB and VB conceived the study and participated in its design and coordination as well as manuscript editing. JJW helped to conceive the study, supervised the study, and helped to draft the manuscript. All authors have read and approved the final manuscript.
JB and VB are employed by Royal Canin. JJW receives honoraria from and is on the advisory council for Nestle Purina, Annamaet Petfoods, and Mars Inc.
Consent for publication
Ethics approval and consent to participate
Not applicable. Cell culture study institutional biosafety committee # 15902.
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