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Our study investigated the effects of dietary supplementation with curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] on spontaneous metastasis of Lewis lung carcinoma (LLC) in C57BL/6 mice. Mice were fed with the AIN93G control diet or with the diet supplemented with 2 or 4% curcumin for 5 weeks at which time they were injected subcutaneously with 2.5 × 105 viable LLC cells. The subcutaneous primary tumor was surgically removed when it reached ∼ 8 mm in diameter, and the experiment was terminated 10 days after the surgery. There was no difference in pulmonary metastatic yield among the groups. Curcumin supplementation at either dietary level did not significantly increase the size of metastatic tumors; however, the combined data from both curcumin groups showed that curcumin treatment increased metastatic tumor cross-sectional area by 46% (p < 0.05) and volume by 70% (p < 0.05) compared to the controls. Curcumin supplementation increased plasma concentrations of angiogenic factors angiogenin (p < 0.05), basic fibroblast growth factor (p < 0.05) and vascular endothelial growth factor (p < 0.05), as well as inflammatory cytokines interleukin-1β (p < 0.05) and monocyte chemotactic protein-1 (p < 0.05), compared to the controls. These results demonstrate that curcumin does not prevent metastasis and indicate that it can enhance metastatic growth of LLC in mice, perhaps through upregulation of angiogenesis and inflammation.
Curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a phenolic compound derived from the Curcuma longa plant, commonly known as turmeric. Curcumin has been used traditionally in Ayurvedic medicine as it has therapeutic properties including being anti-inflammatory,1 antioxidant2 and antimicrobial.3 In recent years, curcumin has been found to possess anticancer activities. Laboratory studies in rodent models showed that curcumin reduces experimentally induced primary tumorigenesis in different organ sites, for example, mammary glands4 and gastrointestinal tract5; moreover, Phase I clinical trials of curcumin have been conducted with patients with cancer.6, 7
Metastasis, the spread of malignant cells from a primary tumor to different sites of the same organ or to distant organs, is the most devastating aspect of cancer, and its occurrence directly affects the prognosis and survival of patients with cancer. To metastasize, malignant cells must disseminate from primary tumors, intravasate into blood circulation system, arrest in a distant vascular bed, extravasate into the interstitium of a target organ and then proliferate. Angiogenesis plays an important role in malignant spread, particularly during the metastatic formation and growth in a metastatic site, as it provides the metabolic needs for the rapidly proliferating malignant cells. Curcumin has been shown to regulate proangiogenic growth factors.8–10 For example, it has been shown to inhibit basic fibroblast growth factor (bFGF)-stimulated angiogenic responses in mouse and rabbit corneal angiogenesis assays,8 to reduce serum vascular endothelial growth factor (VEGF) levels in mice bearing hepatocellular carcinoma9 and to have antiangiogenic effects on xenograft tumor growth in animals.10 Furthermore, curcumin has been shown to inhibit the production of inflammatory cytokines that are involved in tumorigenesis, including interleukin (IL)-1β and monocyte chemotactic protein-1 (MCP-1).11 These studies suggest that curcumin may reduce malignant spread, as both angiogenesis and inflammation are known to contribute to metastatic formation and growth.
The purpose of our study was to determine whether dietary supplementation with curcumin reduced spontaneous metastasis in a well-characterized model, Lewis lung carcinoma (LLC) in the C57BL/6 mouse.
Material and Methods
Our study was approved by the Animal Care and Use Committee of the U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center. The procedures followed the National Institutes of Health guidelines for the care and use of laboratory animals.12
Animals and diets
Three-week-old female C57BL/6 mice (Harlan, Madison, WI) were housed 3–4 per box, in wire-topped plastic boxes, in a pathogen-free room on a 12:12-hr light–dark cycle at 22°C ± 1°C. Three diets were compared; the AIN93G diet13 as a control diet and that diet supplemented with 2 or 4% curcumin (purity 95%; PureBulk, Roseburg, OR). The gross energy of curcumin and each experimental diet, as determined by oxygen bomb calorimetry (Model 6200; Oxygen Bomb Calorimeter, Parr Instrument, Moline, IL), showed that curcumin contained 6.77 ± 0.01 kcal/g (n = 3), which was sufficient not to affect the energy densities of the curcumin-supplemented diets (control diet: 4.33 ± 0.01 kcal/g; 2% curcumin diet: 4.42 ± 0.01 kcal/g; 4% curcumin diet: 4.41 ± 0.01 kcal/g; n = 3 for each diet). In contrast, density with respect to other nutrients was reduced by the magnitude of curcumin addition (Table 1). All diets were presented in powdered form. Mice had free access to their diets and deionized water; they were weighed weekly.
Table 1. Composition of the experimental diets1
LLC cells (provided by Dr. Pnina Brodt, McGill University, Montreal, QC, Canada) were cultured with RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum. Cells were maintained in a humidified atmosphere of 5% CO2 in air at 37°C, and they were periodically monitored for phenotype (microscopically examining the cell morphology), proliferation properties and metastatic capacity by injecting these cells to mice. All assessments of cell identity and behavior were similar to those of original stocks from the institution providing the cell line.
Mice were maintained on the AIN93G diet for 3 weeks before they were randomly assigned into three groups (n = 17–20 mice per group) for experimental feeding. After 5 weeks on experimental diets, mice were injected subcutaneously with 2.5 × 105 viable LLC cells/50 μl into the lower dorsal region. The subcutaneous tumor was surgically removed 10 days later when it reached ∼8 mm in diameter, and then they were maintained on their respective diets for an additional 10 days. Mice fed with the control diet but receiving no LLC cells were used as the normal controls for quantifying plasma angiogenic factors and inflammatory cytokines. One week before the subcutaneous injection, body composition analysis of fat and lean body mass and total body water of conscious, immobilized mice was performed using quantitative magnetic resonance (Echo whole-body composition analyzer, Model 100; Echo Medical System, Houston, TX). At the end of the experiment, mice were intraperitoneally injected with a mixture of ketamine and xylazine, and their lungs were harvested and fixed with formalin-buffered Bouin's solution. The number of pulmonary metastases was quantified using a dissecting microscope, and the cross-sectional area (a two-dimensional measurement of the total area of the orthographic projection of a tumor) and the average diameter of metastatic tumors were measured using ImagePro-Plus software (Media Cybernetics, Silver Spring, MD) and camera-equipped microscope. The volume of metastatic tumors was calculated using the average diameter measured with the assumption that tumors were spherical.14
Quantification of plasma angiogenic factors and inflammatory cytokines
Plasma concentrations of angiogenin (MyBioSource, San Diego, CA), bFGF (Raybiotech, Norcross, GA), VEGF, platelet-derived growth factor-BB (PDGF-BB), and IL-1β and MCP-1 (R&D System, Minneapolis, MN) were quantified by sandwich ELISA following manufacturers' protocols. Samples were read within the linear range of the assay, and the accuracy of the analysis was confirmed by the controls provided in each ELISA kit.
One-way ANOVA and Tukey contrasts were used to compare differences among the groups. For metastatic tumor cross-sectional area and volume, group differences were tested by a mixed model ANOVA with mouse as the random effect. An a priori contrast was included to test for an overall effect of curcumin compared to the controls. Pearson's correlations were used to test for relationships between metastatic tumor size (cross-sectional area and volume) and plasma concentrations of angiogenic and inflammatory biomarkers. All data are presented as means ± SEM. Differences with a p value of 0.05 or less are considered significant. All statistical analyses were performed using SAS software (version 9.3; SAS Institute, Cary, NC).
Dietary supplementation with 2 or 4% curcumin neither affected animal growth compared to the AIN93G control diet for the duration of the experiment (Fig. 1) nor changed body composition among the groups. The average percent body fat mass, lean mass and total body water were 9.72% ± 0.31%, 79.95% ± 0.33% and 66.29% ± 0.29%, respectively.
Subcutaneous injection of LLC cells resulted in the formation of a primary tumor at the injection site in all of the mice. Curcumin affected neither the primary tumor weight at surgery nor pulmonary metastatic tumor yield at the end of the experiment, with 0.25 ± 0.01 (n = 20), 0.24 ± 0.01 (n = 16) and 0.25 ± 0.01 g/tumor (n = 18) for primary tumor weight and 20 ± 3 (n = 20), 17 ± 3 (n = 17) and 23 ± 3 metastatic tumors per mouse (n = 18) for the control, 2 and 4% curcumin-supplemented groups, respectively. Supplementation with curcumin at 2 and 4% dietary levels resulted in dose-dependent increases in both metastatic tumor cross-sectional area and volume compared to the controls; the differences among the groups were not significant (Figs. 2a and 2c). However, the overall effect of combining data from both curcumin groups showed significant increases in metastatic tumor cross-sectional area by 46% (p < 0.05) and volume by 70% (p < 0.05) compared to the controls (Figs. 2b and 2d).
The presence of LLC resulted in significant increases in plasma concentrations of angiogenin (twofold, p < 0.05; Fig. 3a), bFGF (ninefold, p < 0.05; Fig. 3b), VEGF (sixfold, p < 0.05; Fig. 3c) and PDGF-BB (threefold, p < 0.05; Fig. 3d) compared to the normal controls that received no LLC injection. Compared to the LLC controls, curcumin at 2 and 4% dietary levels resulted in further dose-dependent increase in plasma angiogenin and VEGF, and the differences among the groups were significant at p < 0.05 (Figs. 3a and 3c). Curcumin, at both dietary levels, increased plasma bFGF by ∼60% compared to the LLC controls (p < 0.05; Fig. 3b). Supplementation with curcumin at both levels resulted in ∼25% increase in plasma PDGF-BB compared to the LLC controls; however, the differences were not statistically significant (Fig. 3d). Plasma concentrations of bFGF (p < 0.01) and PDGF-BB (p < 0.05) were positively correlated with metastatic tumor cross-sectional area (Figs. 4b and 4d) and volume (Figs. 4h and 4j).
The presence of LLC resulted in an approximately sixfold increase in plasma concentrations of IL-1β compared to the normal controls that received no LLC injection (p < 0.05; Fig. 3e). Curcumin at 2 and 4% dietary levels resulted in further dose-dependent increase in plasma IL-1β compared to the LLC controls, and the differences among the groups were significant at p < 0.05 (Fig. 3e). The presence of LLC increased plasma MCP-1 levels by 20% compared to the normal controls; mice supplemented with 2 and 4% curcumin showed that plasma MCP-1 levels were ∼ 65% and twofold higher than the LLC controls, respectively (p < 0.05; Fig. 3f). Plasma concentration of IL-1β was marginally correlated with metastatic tumor cross-sectional area (p < 0.07; Fig. 4e).
The results of our study demonstrated that dietary supplementation with 2 or 4% curcumin did not affect pulmonary metastatic yield but increased the cross-sectional area and volume of metastatic tumors. These results indicate that curcumin enhances metastatic growth of LLC in mice.
Quantification of metastatic tumor yield in a distant metastatic site is the primary and the most important measurement of malignant spread. A previous study showed that feeding mice with 1% curcumin diet after an intracardiac injection of mammary carcinoma cells inhibited lung metastasis.15 Lee et al.16 reported that dietary supplementation with 5% curcumin does not affect experimental metastasis of LLC to the lungs using an intravenous injection model. Another study showed that feeding mice with 2% curcumin diet 5 days after surgical removal of the primary mammary tumor decreased the incidence of metastasis to the lungs.17 The experimental metastasis by injection of cancer cells into blood circulation theoretically assesses metastasis from extravasation to the formation of malignant tumors in a target organ. Previous quantitative analyses showed the rapid appearance of radioactively labeled cancer cells in the lungs right after an intravenous injection; however, only 1% of those cells survived to form secondary tumors.18 Such results indicate that methods that use a direct injection of cancer cells into the bloodstream likely assess the survival, formation and growth of malignant tumors in a distant organ, but not malignant spread. Initiation of curcumin feeding after surgical removal of the primary tumor17 made it unlikely that the curcumin treatment affected metastasis, which had already occurred prior to the surgery; instead, it indicates metastatic growth in the target organ. Our study was aimed at determining whether curcumin prevented spontaneous metastasis, for which reason the curcumin-supplemented diets were provided to mice prior to the inoculation of LLC cells. The results showed no differences in pulmonary metastatic yield among the groups, indicating that curcumin does not prevent the metastasis of LLC in this model.
The measurement of tumor size is an assessment of tumor growth. This was assessed by directly measuring the metastatic tumor cross-sectional area and calculating the tumor volume based on the average diameter measured, because pulmonary metastases were too small to be trimmed off and weighed with acceptable accuracy. These results showed that curcumin supplementation significantly increased both metastatic tumor cross-sectional area and volume compared to the controls, indicating that curcumin enhances metastatic growth of LLC in the lungs.
The LLC is a murine-transplantable malignancy that has been widely used for metastasis and angiogenesis studies in immune-competent C57BL/6 strain,19, 20 which is an advantage over the immune-compromised mouse models15, 17 in which immune deficiency may enhance malignant development and affect treatment effects. Significant increases in blood concentrations of angiogenic and inflammatory biomarkers have been reported with metastatic development in this immune-competent model.21–23 The LLC cells used in our study are from a highly metastatic variant with organ specificity to the lungs21; they form pulmonary metastases when subcutaneously injected into mice.21, 22 This model responds to dietary modifications, for example, dietary fat enhances24 and selenium inhibits22 the malignant spread with corresponding increases24 and reductions22 in plasma concentrations of angiogenic and inflammatory biomarkers. These studies demonstrate the usefulness of this LLC mouse model for the study of dietary effects on metastasis.
Curcumin supplementation significantly increased plasma concentrations of angiogenic factors angiogenin, bFGF and VEGF. Angiogenin is an angiogenic ribonuclease, and its expression is upregulated in many types of human cancers.25 FGF and VEGF are prominent regulators of angiogenesis, acting in concert to enhance metastasis. For example, bFGF and VEGF synergistically stimulate vascularization,26 and the simultaneous expression of bFGF and VEGF results in fast-growing tumor xenografts in mice with high vessel density.27 The inhibition of bFGF and VEGF receptor binding and activation markedly inhibits LLC metastasis.28 Furthermore, PDGF-BB is synergistic with VEGF in inducing neovascularization29 and with bFGF in promoting tumor angiogenesis and pulmonary metastasis.30 Overexpression of angiogenic factors, including angiogenin,31 FGF,32 VEGF33 and PDGF,34 is associated with advanced tumor stages and unfavorable prognosis in patients with cancer. Thus, curcumin feeding produced significant increases in plasma concentrations of these angiogenic factors suggests the possibility that curcumin enhances metastatic growth by upregulating angiogenesis in LLC-bearing mice.
Curcumin may enhance metastatic growth through proinflammatory mechanisms. Curcumin feeding in LLC-bearing mice significantly increased plasma concentrations of IL-1β and MCP-1, both of which are important inflammatory cytokines and the expressions of which correlate with increased invasiveness and poor prognosis in patients with cancer.35, 36 Invasive human breast carcinoma expresses IL-1β,37 and elevated serum levels of IL-1β correlates with a high rate of recurrence in patients with breast cancer.38 MCP-1 knockdown23 or blockade by neutralizing antibodies39 reduces pulmonary metastasis in mice. Furthermore, inflammatory cytokines participate in angiogenesis. IL-1β upregulates VEGF expression,40, 41 and through which it promotes the growth of LLC in mice.42 In in vitro assays, VEGF increases MCP-1 mRNA expression, and inhibition of MCP-1 attenuates VEGF-induced angiogenic activity.43
In our study, curcumin was supplemented to the diet at 2 and 4% levels. These levels are within the range from 1 to 5% dietary levels that have been used in studies of curcumin and cancer prevention.5, 16, 44 Consistent with the previous reports,5, 16, 44 curcumin at 2 and 4% levels did not affect body weight or change body composition of the mice. This showed that the addition of curcumin to the diet, which did not affect the energy content, did not affect the rate of food intake. Although the addition of curcumin resulted in corresponding reductions in the contents of other nutrients, such decreases did not affect growth and were unlikely to have been of physiological significance. This also suggests that the observed enhancement of metastatic growth was not due to the effects on the overall health of the mice by the levels of curcumin supplemented.
Curcumin has been shown to inhibit chemically induced primary carcinogenesis of the forestomach, duodenum and colon,5 virus-induced lung cancer progression45 and the formation of prostate adenocarcinoma44 in laboratory animals. In contrast, our results showed that dietary supplementation with curcumin did not inhibit spontaneous metastasis, but enhanced metastatic growth in this model. This enhancement was associated with significant increases in plasma concentrations of angiogenin, bFGF, VEGF, IL-1β and MCP-1, indicating that curcumin may stimulate metastatic growth by upregulating angiogenesis and inflammation. Angiogenin,25 bFGF44, 46, 47 and VEGF33, 46, 48 are expressed widely in many types of malignancies in human patients. Therefore, it is possible that curcumin may play a role in the development and growth of malignancies with rapid progression, including metastasis. However, this hypothesis warrants further investigation.
The author gratefully acknowledges the assistance of the following staff of the Grand Forks Human Nutrition Research Center: Lana DeMars for technical support; James Lindlauf for preparing experimental diets; LuAnn Johnson for statistical analysis and vivarium staff for providing high-quality animal care. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of trade names or commercial products in this article is solely for providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.