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CURCUMIN AND TURMERIC ATTENUATE ARSENIC-INDUCED ANGIOGENESIS IN OVO Panayotis Pantazis, ; Aarthi Varman, ; Cindy Simpson-Durand, ; Jessica Thorpe, ; Satish Ramalingam, ; Dharmalingam
PhD BS BS BS
Subramaniam, PhD; Courtney Houchen, MD; Michael Ihnat, PhD; Shrikant Anant, PhD; Rama P. Ramanujam, PhD
Trivalent arsenic [As(III)] is currently approved by the FDA for the treatment of chronic and acute leukemias. However, As(III) has also demonstrated damaging effects on human health, including development of cardiovascular disease, diabetes, and cancer. Further, As(III) is a potent angiogenic agent. In this context, curcumin, an active ingredient in the dietary agent turmeric, has demonstrated potent antiproliferative, antiinﬂammatory, and antiangiogenic properties. In this report, we have shown that both curcumin and turmeric inhibit expres-
sion of vascular endothelial growth factor in HCT-116 human colon cancer cells exposed to As(III). Further, in the chicken chorioallantoic membrane assay model, treatment with low As(III) concentrations results in extensive increase in blood vessel density, which, however, is reduced in the presence of curcumin or turmeric. Collectively, the ﬁndings reported here strongly suggest that turmeric and curcumin can dramatically attenuate the process of angiogenesis induced by low As(III) concentrations. (Altern Ther Health Med. 2010;16(2):12-14.)
Panayotis Pantazis, PhD, is a senior scientist, Aarthi Varman, BS, is a research assistant, Cindy Simpson-Durand, BS, is a research associate, and Rama P. Ramanujam, PhD, is principal investigator and president at Swaasth, Inc, Oklahoma City, Oklahoma. Jessica Thorpe, BS, is a research assistant and Michael Ihnat, PhD, is an associate professor in the Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City. Satish Ramalingam, PhD, and Dharmalingam Subramaniam, PhD, are postdoctoral fellows in the Department of Medicine, Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center. Courtney Houchen, MD, is an associate professor in the Department of Medicine, Digestive Diseases and Nutrition, and chief of gastroenterology at the OU Cancer Institute, University of Oklahoma Health Sciences Center. Shrikant Anant, PhD, is an associate professor in the Department of Cell Biology, director of research in the Department of Medicine, Digestive Diseases and Nutrition, and program leader at the OU Cancer Institute, University of Oklahoma Health Sciences Center. Corresponding author: Rama P. Ramanujam, PhD E-mail: email@example.com t has been estimated that more than 50% of all patients diagnosed with cancer explored complementary and alternative medicine, especially herbal medicine.1 In this regard, consumption of certain foodstuffs such as spices, fruits, vegetables, and whole grains can reduce the risk of inﬂammation and cancer.2 Speciﬁcally, turmeric (Curcuma longa L), a spice that is used worldwide as a culinary seasoning, has exhibited antiproliferative and antiinflammatory effects in vitro and in animals.3
Curcumin, the active ingredient in turmeric, traditionally has been used in the Indian subcontinent and in Southeast Asia to treat throat ulcers, inﬂammation, skin wounds, and cancer. Further, laboratory studies have demonstrated the antiproliferative, antiinﬂammatory, and antiangiogenic properties of curcumin.3 In addition, current clinical trials of curcumin in humans assess its role in the prevention or treatment of several types of cancer, rheumatoid arthritis, irritable bowel syndrome, and Alzheimer’s disease.4 Arsenic compounds have been used since ancient times to treat medical conditions, but at the same time, these compounds also have been associated with a range of detrimental effects on human health, including increased incidence of ischemic heart disease, arrhythmias, hypertension, and peripheral vascular disease.5 Although the mechanism(s) for these vascular effects remains unknown, it has been demonstrated that some effects are doserelated. Angiogenic effects of arsenic also have been shown to be dose-dependent, with lower doses being angiogenic and higher doses antiangiogenic.6 Angiogenesis, the process of generating new blood vessels, is a critical step in cancer growth and metastasis, and increased tumor angiogenesis has been associated with an increased incidence of distant metastasis.7,8 Therefore, the characterization of natural and synthetic agents that can inhibit angiogenesis is of great clinical and public interest. In the present study, we have demonstrated the ability of the plant-derived products, turmeric and curcumin, to attenuate the production of a major angiogenic factor, vascular endothelial growth factor (VEGF), in trivalent arsenic [As(III)]–treated human colon cancer cells in culture. Further, we have demonstrated that turmeric and curcumin inhibit angiogenesis induced by low As(III) concentrations in the chick chorioallantoic membrane (CAM) model.
ALTERNATIVE THERAPIES, mar/apr 2010, VOL. 16, NO. 2
Curcumin and Turmeric Attenuate Arsenic-induced Angiogenesis in Ovo
Difference was considered signiﬁcant at P<.0 software (Graphpad Software. VOL. and As(III)+30 μM curcumin for 72 hours. Compounds for treatment were applied to 6-mm diameter paper discs and placed on the chorioallantoic membrane. puriﬁed growth factors are added locally to the highly vascularized CAM to induce angiogenesis. P<. San Diego. as compared to addition of As(III) alone to levels approaching those of untreated CAMs. The CAM assay used in these studies was performed according to the modiﬁed version of Brooks. HCT-116 human colon cancer cells (American Type Culture Collection. **P<. As(III)+50 μg/mL turmeric. 2 13 . respectively. RESULTS Curcumin Inhibits As(III)-induced VEGF Expression We initially examined whether addition of curcumin or turmeric extract to As(III)-treated cells would affect expression of VEGF. All reagents used in routine molecular biology experimentation were purchased from Sigma-Aldrich (St Louis. The results shown were derived from four to six samples from two independent experiments.9 In this assay. as compared to As(III) exposure alone. Virginia) were grown in Dulbecco’s modiﬁed Eagle medium. Missouri). the blood vessel density/branching of the treated area of the CAM is determined. VEGF Enzyme-linked Immunosorbent Assay HCT-116 cells were exposed to 0. the eggs were candled. Winooski.6 addition of either 200 ng/mL VEGF or 0.1 μM As(III) alone.05. Unpaired two-tailed Student’s t-test was used to compare treated groups to one another. It was found that the addition of curcumin to As(III) cells treated for 72 hours resulted in a signiﬁcant decrease in soluble VEGF amounts. The plate was read on a BioTek Synergy HT luminometer (BioTek Instruments.1 μM As(III) in absence or presence of 30 μM curcumin or 50 mg/mL turmeric for 72 hours with serum-free media used for the last 24 hours of the experiment.10 Brieﬂy. and a small opening was made at the top of the live eggs. presence of curcumin extensively inhibited angiogenesis induced by the presence of VEGF in CAM (Figure 2). 16. Manassas.01 by one-way ANOVA to compare all treatments vs untreated samples. a primary angiogenic factor in colon cancer cells. New Jersey).001 300 ** 200 VEGF Levels (% control) 150 100 50 0 As Curcumin Turmeric + + + + + * FIGURE 1 Turmeric and Curcumin Attenuate As(III)-induced Angiogenesis in the Chick CAM Model HCT-116 cells were treated with 0. addition of turmeric resulted in a lesser decrease in VEGF amounts in As(III)-treated cells.10 On day 10.1 μM As(III) for 48 hours resulted in a signiﬁcant stimulation of angiogenesis in the CAM assay as demonstrated by an extensive increase in blood vessel density (Figure 2). Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) to compare the means of all groups. The addition of either curcumin or turmeric along with As(III) resulted in a signiﬁcant decrease in blood vessel density. Inc (St Paul. and digitized images were recorded using a dissecting microscope (Amscope. to levels approaching those in untreated cells (Figure 1). Minnesota) and Sabinsa Corporation (Piscataway. The cells were serum-starved for the ﬁnal 24 hours. The Dunnett’s multiple comparison test was used to compare the treatments vs the control groups and unpaired two-tailed Student’s t-test was used to compare treatment groups to one another. Inhibitors are then added to the same localized area of the membrane and. California). Supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) analysis using the QuantiGlo Chemiluminescent ELISA Human VEGF kit from R&D Systems (Minneapolis. *P<. However. after a desired incubation period. mar/apr 2010.05. All analyses were performed with the aid of Prism 5. Results were quantiﬁed in relative light units after considering the control value as 100%. Chick CAM Assay of Angiogenesis The chick CAM assay is a standard assay for testing antiangiogenic agents. subsequently the CAMs were ﬁxed. Texas) were incubated for 10 days and turned at regular intervals to achieve nearly complete vasculogenesis and blood vessel development progressing mostly through angiogenesis. DISCUSSION The preliminary experimental ﬁndings we have presented in this report demonstrate that turmeric and curcumin attenuate or completely prevent low As(III) concentrations from Curcumin and Turmeric Attenuate Arsenic-induced Angiogenesis in Ovo ALTERNATIVE THERAPIES. Vermont). Minnesota) according to instructions provided by the manufacturer.6 The eggs were incubated for 48 hours. model MD600). Cameron. supplemented with 10% heat-inactivated fetal bovine serum and standard antibiotics (Sigma-Aldrich) in a 37°C–humidiﬁed incubator containing 5% CO2. and supernatants were subjected to ELISA to detect VEGF protein expression. The results are shown upon direct visualization of microscopy images and measuring the relative angiogenic index (Figure 2). fertile leghorn chicken eggs (Ideal Poultry Breeding Farms. Turmeric and Curcumin Inhibit As(III)-induced Angiogenesis As previously shown. NO. Further.MATERIALS AND METHODS Reagents and Cells Curcumin powder and turmeric powder were purchased from LKT Laboratories.
Rao CV. Soucy NV.105(15):1804-1809. and metastasis of carcinomas.75(4):787-809. 2008.01 P<. Betz JM. Anant S. Ihnat MA. therapeutic resistance during tumor development. Data represent the average values of nine samples from two independent experiments. Kamat CD. 1999. 6. turmeric and related spices are actively promoted for their suggested antiinﬂammatory and anticancer activities and are available in powder or tablet form from a number of suppliers worldwide.01 300 Mean vessel branch point (% untreated) * ** In conclusion. Approaches towards evaluation of medicinal plants prior to clinical trials. 2003.gov/ct2/results?term=curcumin. The quantitative results (angiogenesis index) for each experimental variable are summarized below.Control VEGF Arsenic Arsenic and Curcumin Arsenic and Turmeric P<. List results: Found 37 studies with search of: curcumin.5(12):2485-2501. 2. Use of the 10-day-old chick embryo model for studying angiogenesis. Lead. Nat Rev Clin Oncol. Tufan AC. Biochem Pharmacol. invasion and development of anti-angiogenic agents.5(3-4):137-153. 2002. VOL. In addition to being used in cooking. J Environ Pathol Toxicol Oncol. 3.21(2):155-164. as we detected using high-performance liquid chromatographyhydride generation-inductively coupled plasma mass spectrometry (HPLC-HG-ICPMS) analysis (unpublished data) in agreement with a recent report that some commercial preparations of herbal medicines contain detectable toxic metals such as arsenic. We also have determined that low concentrations of As(III) can protect IκB-modulated TNF-α induction of apoptosis in HCT-116 cells in vitro in agreement with a published report11 and a manuscript in preparation. Brooks PC. Kunnumakkara AB. as well as to identify contaminants that may be health hazards. Proc Found Med Res Mumbai-India. et al. Phillips RS. Houchen CW. Novel imaging system for determining anti-cancer activity. angiogenesis. Heath VL. 2009. JAMA. 14 ALTERNATIVE THERAPIES. Sleeman BD.76(2):271-279. Acknowledgment This work was funded in part by grant 5R44AT004118-03 from the National Institutes of Health.01 by Dunnett’s t-test. 2008. However. NF-kappa B: a new player in angiostatic therapy. IκB is a regulatory protein that inhibits degradation of the nuclear factor-κB (NF-κB) that has been implicated in the proliferation of various tumor cells. Griffioen AW. 4. Cheresh DA. Biological gradient between long-term arsenic exposure and carotid atherosclerosis. Novel approaches for colon cancer prevention by cyclooxygenase-2 inhibitors. Sehgal A. 8.11(1):101-106. Subramaniam D RR. Toxicol Sci. 7. Methods Mol Biol. 2004.5(4):249-266. Yip PK. progression. 13. Wong J. J Theor Med. Saper RB. NO. Jeng JS.12 It should be noted that in the experiments described in this report. 2003. No authors listed. 5. 100 0 VEGF As Curcumin Turmeric - + - + - + + - + + FIGURE 2 Effect of Turmeric and Curcumin on As(III)-induced VEGF Expression in HCT-116 Cells CAMs were treated with the reagents and concentrations indicated in the ﬁgure for 48 hours as described in the Materials and Methods section. mercury. 11. Boon H.6(7):395-404. we caution that these commercially available products may contain traces or low levels of arsenic.05. inducing VEGF production in HCT-116 cells and angiogenesis in the CAM model. *P<. Anticancer strategies involving the vasculature. 10. 2009. mar/apr 2010. Angiogenesis. Arsenic stimulates angiogenesis and tumorigenesis in vivo. Expert Opinion Pharmacother. Visual results from a representative experiment are shown above. et al. Plank MJ. 2002. The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis. Available at: http://clinicaltrials. 16. Tabruyn SP. 2005. **P<. 200 REFERENCES 1. et al. Aggarwal BB. and arsenic in US. Murali PM. Tumor-induced angiogenesis: a review. Accessed October 26.300(8):915-923. Montgomery AM.and Indianmanufactured Ayurvedic medicines sold via the Internet. the experimental ﬁndings described in this report demonstrate that turmeric and curcumin attenuate or completely prevent low As(III) concentrations from inducing angiogenesis in the CAM model and that curcumin reduces VEGF production in HCT-116 cells. the HCT-116 cells were treated with As(III) concentrations several-fold lower than the cytotoxic As(III) concentrations of near 100 μM (manuscript in preparation). Goel A. Satiroglu-Tufan NL. Botanical medicine and cancer: a review of the safety and efﬁcacy. 2 Curcumin and Turmeric Attenuate Arsenic-induced Angiogenesis in Ovo . Circulation. Curr Cancer Drug Targets. 9.13 Further studies are needed to investigate the functional activities of different turmeric and related spice preparations. 12. Reddy BS. Curcumin as “Curecumin”: from kitchen to clinic. Wang CH. 2008.129:257-269. Bicknell R. 2006:44-57.
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