Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis. In colon cancer, RXRα, an important dimerization partner with other nuclear transcription factors, is silenced through this mechanism. We previously found that colon tumors in Apc Min/+ mice had diminished levels of RXRα protein and expression levels of this gene were restored by treatment with a green tea intervention, due to reduced promoter methylation of RXRα. We hypothesized that CIMP+ cell lines, which epigenetically silence key regulatory genes would also evidence silencing of RXRα and EGCG treatment would restore its expression. We indeed found EGCG to restore RXRα activity levels in the human cell lines, in a dose dependent manner and reduced RXRα promoter methylation. EGCG induced methylation changes in several other colon cancer related genes but did not cause a decrease in global methylation. Numerous epidemiological reports have shown the benefits of green tea consumption in reducing colon cancer risk but to date no studies have shown that the risk reduction may be related to the epigenetic restoration by tea polyphenols. Our results show that EGCG modulates the reversal of gene silencing involved in colon carcinogenesis providing a possible avenue for colon cancer prevention and treatment.
The anti-inflammatory actions of Vitamin D have long been recognized and its importance in modulating colon cancer and colitis development is becoming apparent. The Vitamin D receptor (VDR) is downregulated in human Ulcerative Colitis (UC) and colitis-associated cancer (CAC); however, its status in murine models of colitis has yet to be explored. Snail and Snail2, zinc-finger transcription factors regulated by inflammatory pathways and able to transcriptionally silence VDR, are upregulated in human UC and are associated with localized VDR silencing. To signal, VDR must heterodimerize with Retinoid X Receptor alpha (RXRα). If either VDR or RXRα are compromised, Vitamin D cannot regulate inflammatory pathways. RXRα is downregulated in human colorectal cancer (CRC), yet its expression in human and murine colitis has yet to be investigated. To explore the importance of Vitamin D and VDR in murine colitis, we utilized acute and chronic Azoxymethane/Dextran Sulfate Sodium (AOM/DSS) models of murine colitis. VDR was downregulated early in the onset of colitis whereas RXRα downregulation only occurred as colitis became chronic and developed into CAC. Receptor downregulation was associated with an early increase in the expression of the inflammatory markers, Snail and Snail2. The acute colitis model induced in combination with a Vitamin D deficient diet resulted in increased morbidity, receptor downregulation, inflammatory marker expression and Snail and Snail2 upregulation. These experiments demonstrate the importance of Vitamin D and VDR in modulating murine colitis development.
Background In order for vitamin D to signal and regulate inflammatory pathways, it must bind to its receptor (VDR) which must heterodimerize with the retinoid X receptor alpha (RXRα). Although the role that vitamin D signaling plays in the development and progression of colitis, a disease characterized by excessive inflammation, has been suggested, little research has been done on determining the role that RXRα plays in acute colitis development. Aims This study sought to determine the effects that reduced availability of RXRα would have on the development of acute murine colitis. Expression of inflammatory markers, VDR and RXRα were investigated to determine if the reduction in expression of RXRα in RXRα+/− mice would result in increased inflammatory signaling and receptor downregulation as compared to their wild-type littermates. Methods An acute murine model of colitis, the axozymethane (AOM) and dextran sulfate sodium (DSS) model was utilized in wild-type and RXRα+/− mice. Gross manifestations of colitis measured included weight loss and colitis score. Immunblots and real-time PCR were performed for inflammatory markers and receptor expression. Results RXRα+/− mice induced with AOM/DSS colitis demonstrated increased gene expression of Snail and Snail2, transcription factors downstream of inflammatory mediators, as compared to their wild-type littermates. Conclusions This demonstrates the importance of RXRα in regulating inflammation in acute colitis and also identifies RXRα expression as a new consideration when developing successful interventions for acute colitis due to the requirement of numerous receptors for RXRα.
The development of colon cancer, the third most diagnosed cancer and third leading cause of cancer deaths in the United States, can be influenced by genetic predispositions and environmental exposures. As 80% of colon cancer cases are sporadic in nature, much interest lies in determining risk factors that may foster its development, as well as identifying compounds that could inhibit colon cancer development or halt progression. A major risk factor for sporadic colon cancer is a high fat, Western diet which has been linked to a cancer-prone, pro-inflammatory state. Cultures which place an emphasis on fresh fruits and vegetables demonstrate lower colon cancer incidences. Diet not only has the potential to encourage colon cancer development, but recent evidence demonstrates that certain dietary natural products can halt colon cancer development and progression via epigenetic regulation. Epigenetic dysregulation may contribute to inflammation-driven diseases, such as cancer, and can lead to the inappropriate silencing of genes necessary to inhibit cancer development. Natural compounds have shown the ability to reverse epigenetic dysregulation in in vitro and in vivo models. As current allopathic medicines aimed at reversing epigenetic silencing are accompanied with the risk of toxicity and side effects, much interest lies in being able to harness the disease preventing properties in natural products. Here, we discuss the epidemiology of colon cancer, describe the need for natural approaches to inhibit disease development and highlight natural products which have been shown to inhibit gastrointestinal cancer initiation and progression in vitro or in vivo through epigenetic modulation.
Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis. In colon cancer, RXRα, an important dimerization partner with other nuclear transcription factors, is silenced through this mechanism. Previous studies from our laboratory have shown that ApcMin/+ mice treated with the carcinogen azoxymethane (AOM) will develop colonic tumors but when treated with green tea a reduction in tumor numbers was found. We found that colon tumors had lower expression of RXR protein but expression levels were restored by treatment with green tea. We also found that the degree of promoter methylation of the RXR gene was reduced. To translate these findings to human colon cancer we treated CpG Island Methylator Phenotype sensitive and insensitive (CIMP+ & CIMP-) human colon cancer cell lines with epigallocatechin gallate (EGCG - major polyphenolic compound in green tea). We hypothesized that CIMP+ cell lines which epigenetically silence key regulatory genes would demonstrate silencing of RXRα and that EGCG would restore its expression. To test this we used the following human colon cancer cell lines: HCT116, SW48, HCT15 - CIMP+; HT29, SW480, SW620 - CIMP- and IEC-6 - normal control. We found EGCG to restore RXR activity levels in the CIMP+ lines, in a dose dependent manner (0, 50, 100 & 150 μM EGCG was tested for 48 and 72 hour durations). EGCG also reduced RXRα promoter methylation in one CpG island from a CIMP+ cell line compared to a CIMP-line. We found that EGCG produced methylation changes in several other colon cancer related genes but did not cause a decrease in global methylation. Numerous epidemiological reports have shown the benefits of green tea consumption in reducing colon cancer risk but to date no studies have shown that some of the risk reduction may be related to the epigenetic regulation of key genes by constituent tea polyphenols. Our results here show that EGCG, a common tea polyphenolic compound, can modulate the reversal of gene-silencing involved colon carcinogenesis and maybe a possible avenue for colon cancer treatment. Supported by USPHS grant CA96694 from the National Cancer Institute. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A39.
This book chapter discusses the overall trends of the Westernization of the diet and dietary acculturation, and provides country-specific evidence (Okinawa, Japan; Inuit and remote Alaskan populations, USA; Grenada, Caribbean; China; Japan; and the Mediterranean) for the adoption of a Western diet. Examples are presented of some traditional diets and their disease-fighting constituents that are slowly being forgotten. The chapter then reviews the depletion of phytochemicals and the resulting risk for cancer and other chronic diseases. Finally, the chapter presents some phytochemicals that are associated with the prevention of chronic diseases such as cancer.
Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis of numerous cancers. We have previously shown that Retinoid X Receptor alpha (RXRα) is silenced in tumors of the colon cancer AOM-APC/min+ mouse model. Upon treatment with green tea, RXRα expression was restored and intestinal tumorigenesis was inhibited. We examined human colonic tumor microarrays by immunohistochemcial staining and found a marked decrease in RXR expression in tumor tissue compared to normal matched tissues, inversely β-Catenin levels increased. To determine how RXRα loss is reversed in human cancers by green tea, we treated HT-29 (methylation insensitive) and HCT116 (methylation sensitive) human colon cancer cell lines with different concentrations of Epigallocatechin gallate (EGCG - major polyphenolic compound in green tea). We found by western blotting nuclear protein fractions that EGCG can restore RXRα levels in HCT116 cells in a dose dependant manner. Using these same lysates, we probed for presence of the most common DNA Methyltransferases (DNMTs) and Histone Deacetylases (HDACs). We found that expression of DNMTs as well as HDACs was inhibited in a dose dependent manner following treatment with EGCG. Additionally we examined by real time PCR, the message levels of DNMTs in cells treated with EGCG to determine if they correlate with the observed downregulation in protein expression. We can conclude that the silencing of RXRα may be due in part to by repressing effects of EGCG on DNMT as well as HDAC activity. Our results here show that EGCG, a common dietary compound, can modulate the reversal of gene-silencing involved colon carcinogenesis and maybe a possible avenue for colon cancer therapy and demonstrates be one potential mechanism of cancer chemoprevention in green tea that outlines a new epigenetic pathway. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A40.
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