Novel Evidence for Curcumin and Boswellic Acid-Induced Chemoprevention through Regulation of miR-34a and miR-27a in Colorectal Cancer

Abstract Colorectal cancer is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Both curcumin and boswellic acids are wellestablished dietary botanicals with potent antitumorigenic properties that have been shown to modulate multiple oncogenic pathways. Recent data suggest that the chemopreventive effects of these botanicals may, in part, be mediated through regulation of key cancer-related microRNAs (miRNA) and their downstream gene targets. Here, we investigated the antitumorigenic effects of curcumin and 3 acetyl-11-keto-b-boswellic acid (AKBA) on modulation of specific cancer-related miRNAs in colorectal cancer cells and validated their protective effects in vivo using a xenograft mouse model. Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cellcycle arrest in colorectal cancer cell lines, and these effects were significantly enhanced with combined treatment. Gene-expression arrays revealed that curcumin and AKBA regulated distinct cancer signaling pathways, including key cell-cycle regulatory genes. Combined bioinformatics and in silico analysis identified apoptosis, proliferation, and cell-cycle regulatory signaling pathways as key modulators of curcumin and AKBA-induced anticancer effects. We discovered that curcumin and AKBA induced upregulation of tumor-suppressive miR-34a and downregulation of miR-27a in colorectal cancer cells. Furthermore, we demonstrated in a mouse xenograft model that both curcumin and AKBA treatments suppressed tumor growth, which corresponded with alterations in the expression of miR-34a and miR-27a, consistent with our in vitro findings. Herein, we provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in colorectal cancer. Cancer Prev Res; 8(5); 431-43. Ó2015 AACR

Differential effects of dietary whey, casein and soya on colonic DNA damage and large bowel SCFA in rats fed diets low and high in resistant starch

Feeding higher levels of dietary animal protein (as casein or red meat) increases colonic DNA damage and thins the colonic mucus barrier in rats. Feeding resistant starch (RS) reverses these changes and increases large bowel SCFA. The present study examined whether high dietary dairy (casein or whey) or plant (soya) proteins had similar adverse effects and whether dietary RS was protective. Adult male rats were fed diets containing 15 or 25 % casein, whey or soya protein with or without 48 % high amylose starch (as a source of RS) for 4 weeks. DNA damage was measured in isolated colonocytes using the comet assay. Higher dietary casein and soya (but not whey) increased colonocyte DNA damage. DNA damage was highest with soya when fed at 15 or 25 % protein without RS. Dietary RS attenuated protein-induced colonocyte DNA damage in all groups but it remained significantly higher in rats fed 25 % soya compared with those fed 15 % protein. Dietary protein level did not affect colonic mucus thickness overall but the barrier was thinner in rats fed high dietary casein. This effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were higher in rats fed RS compared with digestible starch. Caecal and faecal SCFA were unrelated to genetic damage but correlated with mucus thickness. The present data confirm that higher dietary protein affected colonocyte DNA and colonic mucus thickness adversely but that proteins differ in their effects on these indices of colon health. The data show also that these changes were reversed by RS.Shusuke Toden, Anthony R. Bird, David L. Topping and Michael A. Conlo

Resistant Starches Protect against Colonic DNA Damage and Alter Microbiota and Gene Expression in Rats Fed a Western Diet123

Resistant starch (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), opposes dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed Western-type diets moderate in fat (19%) and protein (20%) containing digestible starches [low amylose maize starch (LAMS) or low amylose whole wheat (LAW)] or RS [HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference] for 11 wk (n = 10/group). A control diet included 7% fat, 13% protein, and LAMS. Colonocyte DNA single-strand breaks (SSB) were significantly higher (by 70%) in rats fed the Western diet containing LAMS relative to controls. Dietary HAW, HAMS, and HAMSB opposed this effect while raising digesta levels of SCFA and lowering ammonia and phenol levels. SSB correlated inversely with total large bowel SCFA, including colonic butyrate concentration (R2 = 0.40; P = 0.009), and positively with colonic ammonia concentration (R2 = 0.40; P = 0.014). Analysis of gut microbiota populations using a phylogenetic microarray revealed profiles that fell into 3 distinct groups: control and LAMS; HAMS and HAMSB; and LAW and HAW. The expression of colonic genes associated with the maintenance of genomic integrity (notably Mdm2, Top1, Msh3, Ung, Rere, Cebpa, Gmnn, and Parg) was altered and varied with RS source. HAW is as effective as HAMS and HAMSB in opposing diet-induced colonic DNA damage in rats, but their effects on the large bowel microbiota and colonocyte gene expression differ, possibly due to the presence of other fiber components in HAW

A microRNA-based liquid biopsy signature for the early detection of esophageal squamous cell carcinoma: a retrospective, prospective and multicenter study

Background Currently, there is no clinically relevant non-invasive biomarker for early detection of esophageal squamous cell carcinoma (ESCC). Herein, we established and evaluated a circulating microRNA (miRNA)-based signature for the early detection of ESCC using a systematic genome-wide miRNA expression profiling analysis. Methods We performed miRNA candidate discovery using three ESCC tissue miRNA datasets (n = 108, 238, and 216) and the candidate miRNAs were confirmed in tissue specimens (n = 64) by qRT-PCR. Using a serum training cohort (n = 408), we conducted multivariate logistic regression analysis to develop an ESCC circulating miRNA signature and the signature was subsequently validated in two independent retrospective and two prospective cohorts. Results We identified eighteen initial miRNA candidates from three miRNA expression datasets (n = 108, 238, and 216) and subsequently validated their expression in ESCC tissues. We thereafter confirmed the overexpression of 8 miRNAs (miR-103, miR-106b, miR-151, miR-17, miR-181a, miR-21, miR-25, and miR-93) in serum specimens. Using a serum training cohort, we developed a circulating miRNA signature (AUC:0.83 [95%CI:0.79–0.87]) and the diagnostic performance of the miRNA signature was confirmed in two independent validation cohorts (n = 126, AUC:0.80 [95%CI:0.69–0.91]; and n = 165, AUC:0.89 [95%CI:0.83–0.94]). Finally, we demonstrated the diagnostic performance of the 8-miRNA signature in two prospective cohorts (n = 185, AUC:0.92, [95%CI:0.87–0.96]); and (n = 188, AUC:0.93, [95%CI:0.88–0.97]). Importantly, the 8-miRNA signature was superior to current clinical serological markers in discriminating early stage ESCC patients from healthy controls (p < 0.001). Conclusions We have developed a novel and robust circulating miRNA-based signature for early detection of ESCC, which was successfully validated in multiple retrospective and prospective multinational, multicenter cohorts

The interaction between dietary proteins and resistant starch on large bowel health.

A review of the literature revealed that diet plays an important role in serious human noninfectious large bowel diseases including cancer and inflammatory bowel diseases. Dietary protein (especially as red and processed meats) has been implicated as a positive risk factor for colorectal cancer while starch which is not digested in the small intestine (resistant starch, RS) appears to be protective. The series of experiments described in this thesis were aimed to determine the effects of dietary proteins and RS on indices of colon health in an animal model, the laboratory rat. Genetic damage is a prerequisite for carcinogenesis and this was assessed by a specific assay (the comet assay) which gives a measure of DNA strand breaks. Loss of mucus barrier function is thought to contribute to inflammatory bowel disease by permitting bacterial translocation and this was measured optically using a microscope micrometer. Other biomarkers were measured as described below. There were four major experiments. 1. Effects of dietary red meat and casein on colonic DNA damage and interaction with resistant starch Previous studies had shown that higher dietary protein (as casein) induced genetic damage in rat colonocytes and that RS (fed as a high amylose maize starch) was protective. This study was aimed at establishing whether a high protein diet fed as cooked red meat had similar effects and whether RS was protective. Rats were fed diets containing either 15 % or 25% casein or 25% barbecued lean red beef, each with or without 48% high amylose maize starch (as a source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic single-strand DNA breaks compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% more DNA damage than the high casein diet but reduced mucus thickness to a similar degree as casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. It is thought that RS promotes large bowel health through the SCFA produced by the large bowel bacteria. One acid in particular (butyrate) has been associated particularly with promotion of normal large bowel function and protection against disease. In keeping with this hypothesis, caecal and faecal short chain fatty acid pools (including those of butyrate) were increased by inclusion of RS in the diet. DNA damage is an early step in the initiation of cancer and these findings agree with the population data which suggest that total dietary protein and red meat promote risk of colorectal cancer. However, inclusion of resistant starch in the diet could significantly reduce that risk. 2. Differential effects of dietary whey, soy and casein on colonic DNA damage and interaction with resistant starch The preceding experiments showed that high levels of animal-derived proteins increased colonocyte genetic damage and loss of the mucus barrier in rats. This second experiment was designed to determine whether diets high in different types of dairy protein (casein or whey) or a plant protein isolate (soy) had similar adverse effects on colonic DNA and mucus barrier function and whether inclusion of RS in the diet was protective. Adult male Sprague Dawley rats were fed a diet containing 15 % or 25 % casein, whey or soy protein, each with or without 48 % high amylose maize starch for 4 weeks. In confirmation of the earlier studies, higher levels of dietary casein increased colonocyte DNA damage significantly. However, whey did not increase genetic damage. Colonic DNA damage was highest for soy when fed at both 15% and 25% protein in the absence of RS. Inclusion of RS in the diet attenuated colonocyte DNA damage due to higher dietary protein in all three groups. The colonic mucus barrier was thinner in rats fed higher dietary protein but the effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were low in rats fed the digestible starch and were higher in rats fed RS. However, there was no relationship between caecal or faecal SCFA and genetic damage or mucus thickness. Caecal and colonic tissue weight and colon length were higher in rats fed RS, consistent with greater SCFA supply. These data confirm that higher dietary protein of animal (casein) or plant (soy) origin increases genetic damage and loss of the mucus barrier indicating that this is an effect of protein and not its source. These findings accord with the epidemiological data which link dietary protein to greater risk of colorectal cancer and inflammatory bowel disease. However, the data show also that dietary proteins differ in their specific actions on genetic damage and mucus thickness. Further, the data from the feeding of whey suggest that not all proteins are equivalent in their capacity to provoke adverse changes in colonic integrity. While the data show that RS raised large bowel and faecal SCFA, they indicate their levels were not related directly to these biomarkers. 3. Dose response effects of resistant starch on protein induced colonic DNA damage The accumulated data linking greater protein intakes to adverse changes in the colon were obtained at dietary levels which were not unreasonable in terms of animal or human consumption. However, the dietary level of RS which were fed were relatively high (48% by weight) so this study was conducted to determine its effectiveness at lower levels of dietary inclusion. It was also important to ascertain whether there was a dose-response relationship between RS intake and the observed effects. One of the mechanisms proposed for the induction of colorectal cancer by high dietary protein intakes is oxidative damage to DNA. In this experiment, this was done by assaying with endonuclease III. Adult male rats were fed a diet containing 25% casein with 0%, 10%, 20%, 30% or 40% high amylose maize starch for 4 weeks. As in the preceding studies comet tail moment was greatest and the mucus barrier thinnest in rats fed 0% RS. DNA damage was reduced and the mucus barrier thickened in a logarithmic dose-dependent manner by RS. There was no significant difference between dietary groups associated with oxidative DNA damage as measured by endonuclease III. Caecal and faecal short chain fatty acid (SCFA) pools rose with the increased level of dietary RS. DNA damage of colonocytes correlated negatively with caecal SCFA but the strongest correlation was with caecal butyrate, which is consistent with the proposed role of this SCFA in promoting a normal cell phenotype. The data show that RS prevents protein induced colonic DNA damage in a dose-dependent manner. Inclusion of 10% high amylose maize starch was found to be sufficient to oppose colonocyte DNA damage, and to increase caecal and faecal SCFA pools. Intakes of this order are not unreasonable in terms of human consumption of RS. 4. Dose response effects of red and white meat on colonic DNA damage and interaction with resistant starch The accumulated evidence from large prospective human studies links diet to colorectal cancer risk strongly. The evidence from the animal studies described in this thesis that dietary protein induces colonocyte genetic damage supports a role for high protein intakes in increasing risk. Recently, several large epidemiological studies and a meta-analysis of prospective studies have found that consumption of dietary red or processed meats, but not white (poultry) meat, is associated with increased risk of colorectal cancer. This is consistent with the data from the preceding studies that specific proteins affected colonic integrity differentially. A large prospective European study (European Prospective Investigation into Cancer and Nutrition) has reported that dietary fibre was protective. The findings reported in this thesis that RS opposes the effects of high dietary protein accord with that conclusion. This study aimed to compare the effects of cooked red (beef) or white (chicken) meat on DNA damage and mucus barrier thickness in rats. The study was designed to determine whether the relationship between the intakes of these meats was dose-dependent. Double-strand DNA breaks are thought to relate more closely to carcinogenesis than single-strand breaks so both were measured. Adult male Sprague-Dawley rats were fed a diet containing 15%, 25% or 35% cooked beef or cooked chicken each with or without 20% high amylose maize starch for four weeks. Both red and white meat increased colonic DNA damage dose-dependently. However, both single and double strand breaks were significantly greater when the rats were fed the red meat diets compared to those fed the white meat. Colonocyte DNA damage was reduced by the consumption of RS while large bowel SCFA were increased. The findings of this study are consistent with the epidemiological data which show that red meat consumption is associated with greater risk of colorectal cancer but that white meat is not. Summary The data reported in this thesis support the findings of prospective population studies that high dietary protein, red meat in particular, appears to be harmful to the health of the large bowel. However, the data demonstrate also that different protein types have differential effects on the integrity of the colonocyte DNA. Furthermore, the addition of RS to the diet protects against protein-induced colonic DNA damage and maintenance of the colonic mucus barrier, apparently through increased SCFA production by colonic fermentation. The results of these experiments indicate a strong potential for RS to be effective in maintenance of large bowel integrity in the face of high dietary protein.Thesis(Ph.D.)-- School of Molecular and Biomedical Science, 2007