Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells.

Advanced colon cancer is extremely difficult to cure, underscoring the need to develop novel therapeutic agents. Prenylated curcumins that are semisynthetic curcumin derivatives with significant anti-cancer potential have been studied herein to assess their therapeutic potential for colon cancer and tested to this aim in vitro for their growth inhibitory properties against 5-fluorouracil + oxaliplatin resistant human colon cancer CR-HT29 and HCT-116 cells. The resulting most active product, gercumin (mono-O-geranylcurcumin), has been further tested for its synergistic effects with FOLFOX (a combination of 5-fluorouracil and oxaliplatin) on the same cell lines. Activity of this combination on colonosphere formation was also investigated. Gercumin was able to suppress the growth of cancer cells with a potency similar to that of curcumin. A synergistic effect of this compound and FOLFOX was also observed. doses tested for synergy in the colonosphere assays did not show greater suppression of colonosphere formation than independent treatment with either reagent alone. Only one of the combinations was shown to be more effective at suppressing colonosphere formation [gercumin 5  µM + FOLFOX (2x)]. Thus, the growth inhibitory effects of curcumin against human cancer cells can be modulated and enhanced by the introduction of hydrophobic chains, normally found in several natural compounds, like the geranyl one. Such compounds are also able to synergize with known chemotherapeutics.

Blockade of CCR5-mediated myeloid derived suppressor cell accumulation enhances anti-PD1 efficacy in gastric cancer.

PURPOSE: Myeloid derived suppressor cells (MDSC) play an important role in tumor immune evasion and its level significantly increased in patients with gastric cancer. Studies confirmed the associations between MDSC and various cytokines in the peripheral blood. However, little is known about the mechanism drawing MDSC into tumor parenchyma. This study was to analyze the correlation between MDSC subsets and CCR5 level in gastric cancer. MATERIALS AND METHODS: G-MDSC and M-MDSC from the peripheral blood and tumor parenchyma were analyzed by flow cytometry. CCR5 ligand CCL5 was detected by ELISA. CCR5 was detected by real-time PCR, western blot and flow cytometry. Furthermore, the therapeutic effects of CCR5 blockade was assessed by the tumor model. RESULTS: CCR5 ligand, gene and protein expression of CCR5, and surface expression of CCR5 significantly increased in blood and tumor of tumor-bearing mice, suggesting MDSC may be attracted into the parenchyma by CCL5/CCR5. Anti-CCR5 treatment decreased G-MDSC and M-MDSC in the periphery and tumor. In addition, combination treatment enhanced CD4+ and CD8+ T cell infiltration and decreased the tumor burden of tumor-bearing mice. CONCLUSIONS: This study elucidated a possible association between MDSC subsets and CCR5, in addition to provide a new potential target to enhance the efficacy of immunotherapy in patients with gastric cancer.

A novel mechanism of lncRNA and miRNA interaction: CCAT2 regulates miR-145 expression by suppressing its maturation process in colon cancer cells.

BACKGROUND: Although both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. CCAT2 (Colon cancer-associated transcript-2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in CRC and is found to promote tumor growth. miRNAs, a class of naturally occurring short RNAs negatively control the expression of target genes by cleaving mRNA or through translation repression. Recently, we reported that miR-145 and miR-21 cooperate to regulate colon cancer stem cell (CSC) proliferation and differentiation. Considering that CCAT2 is mainly located in the nucleus and miRNA maturation process begins in the nucleus, we hypothesize that CCAT2 selectively blocks miR-145 maturation process, resulting in decreased mature miR-145 affecting colon CSC proliferation and differentiation. METHODS: The levels of CCAT2 were manipulated by transfection of CCAT2 expression plasmid or knockdown by siRNA or by CRISPR/Cas9. Quantitative RT-PCR was performed to examine the expression of CCAT2 and pri-, pre- and mature miR-145/21. Fluorescence in situ hybridization (FISH) was used to visualize CCAT2 in the cells. In vitro processing of pri-miRNA-145 was performed using T7 RNA polymerase and recombinant human Dicer. RESULTS: We have observed that modulated expression of CCAT2 regulates the expression of miR-145 in colon cancer HCT-116 and HT-29 cells. Knockout of CCAT2 increases miR-145 and negatively regulates miR-21 in HCT-116 cells, impairs proliferation and differentiation. In contrast, stable up-regulation of CCAT2 decreases mature miR-145 and increases the expression of several CSC markers in colon cancer cells. We have also observed that CCAT2 is enriched in the nucleus and correlates with the expression of pre-miR-145 but not pre-miR-21 in HCT-116 cells. These results indicate CCAT2 selectively blocks miR-145 maturation by inhibiting pre-miR-145 export to cytoplasm. Further, we revealed that CCAT2 blocks cleavage of pre-miR-145 by Dicer in vitro. CONCLUSIONS: Our results identify CCAT2 as a negative regulator of miRNA-145 biogenesis, and expose a novel mechanism of lncRNA-miRNA crosstalk.

miR-21 and miR-145 cooperation in regulation of colon cancer stem cells.

BACKGROUND: Acquired drug resistance is one of the major reasons for failing cancer therapies. Although the reasons are not fully understood, they may be related to the presence of cancer stem cells (CSCs). We have reported that chemo-resistant (CR) colon cancer cells, highly enriched in CSCs, exhibit a marked up-regulation of miR-21 and that down-regulation of this miR renders the CR cells more susceptible to therapeutic regimens. However, the underlying molecular mechanism is poorly understood. The aim of this investigation is to unravel this mechanism. METHODS: The levels of miR-145 and miR-21 were manipulated by transfection of mature, antago-miRs or pCMV/miR-145 expression plasmid. Quantitative RT-PCR or/and Western blots were performed to examine the expression of CD44, ß-catenin, Sox-2, PDCD4, CK-20 and k-Ras. Colonosphere formation and SCID mice xenograft studies were performed to evaluate the tumorigenic properties of CSC-enriched colon CR cells. RESULTS: We investigated the role that microRNAs (miRs), specifically miR-21 and miR-145 play in regulating colon CSCs. We found the expression of miR-21 to be greatly increased and miR-145 decreased in CR colon cancer cells that are highly enriched in CSC, indicating a role for these miRNAs in regulating CSCs. In support of this, we found that whereas forced expression of miR-145 in colon cancer cells greatly inhibits CSCs and tumor growth, up-regulation of miR-21 causes an opposite phenomenon. In addition, administration of mature miR-145 or antagomir-21 (anti-sense miR-21) greatly suppresses the growth of colon cancer cell xenografts in SCID mice. This was associated with decreased expression of CD44, ß-catenin, Sox-2 and induction of CK-20 indicating that administration of miR-145 or antagomir-21 decreases CSC proliferation and induces differentiation. In vitro studies further demonstrate that miR-21 negatively regulates miR-145 and vice versa. k-Ras appears to play critical role in regulation of this process, as evidenced by the fact that the absence of k-Ras in CR colon cancer cells increases miR-145 expression, suppresses miR-21, and interrupts the negative cooperation between miR-21 and miR-145. CONCLUSIONS: Our current observations suggest that miR-21, miR-145, and their networks play critical roles in regulating CSCs growth and/or differentiation in the colon cancer and progression of chemo-resistance.

Auraptene and its effects on the re-emergence of colon cancer stem cells.

Recent studies indicate that auraptene (7-geranyloxycoumarin, AUR), a geranyloxycoumarin extracted from fruits of edible plants belonging to the Rutaceae family, may represent a novel lead compound for dietary colon cancer chemoprevention in rodents. As a continuation of studies aimed to better depict the pharmacological effects and mechanism of action of the title natural compound, the current investigation was undertaken to determine whether AUR would be able to prevent the growth and sphere (surrogate tumors) formation of FOLFOX-resistant colon cancer cells that are highly enriched in cancer stem cells (CSCs). Our results demonstrate that AUR at a concentration of 10 µM was able to inhibit the growth and formation of colonospheres of FOLFOX-resistant colon cancer HT-29 cells in vitro. The corresponding parental cells were also similarly affected by AUR at the same concentration level. The reduction in the growth and colonospheres formation in FOLFOX-resistant HT-29 was also associated with a concomitant decrease in phospho-epidermal growth factor receptor. These findings suggest that AUR could prevent the re-emergence of CSCs.

MicroRNA-21 induces stemness by downregulating transforming growth factor beta receptor 2 (TGFßR2) in colon cancer cells.

Although microRNA-21 (miR-21) is emerging as an oncogene and has been shown to target several tumor suppressor genes, including programmed cell death 4 (PDCD4), its precise mechanism of action on cancer stem cells (CSCs) is unclear. Herein, we report that FOLFOX-resistant HCT-116 and HT-29 cells that are enriched in CSCs show a 3- to 7-fold upregulation of pre- and mature miR-21 and downregulation of PDCD4. Likewise, overexpression of miR-21 in HCT-116 cells, achieved through stable transfection, led to the downregulation of PDCD4 and transforming growth factor beta receptor 2 (TGFßR2). In contrast, the levels of ß-catenin, TCF/LEF activity and the expression of c-Myc, Cyclin-D, which are increased in CSCs, are also augmented in miR-21 overexpressing colon cancer cells, accompanied by an increased sphere forming ability in vitro and tumor formation in SCID mice. Downregulation of TGFßR2 could be attributed to decreased expression of the receptor as evidenced by reduction in the activity of the luciferase gene construct comprising TGFßR2-3' untranslated region (UTR) sequence that binds to miR-21. Moreover, we observed that downregulation of miR-21 enhances luciferase-TGFßR2-3' UTR activity suggesting TGFßR2 as being one of the direct targets of miR-21. Further support is provided by the observation that transfection of TGFßR2 in HCT-116 cells attenuates TCF/LEF luciferase activity, accompanied by decreased expression of ß-catenin, c-Myc and Cyclin-D1. Our current data suggest that miR-21 plays an important role in regulating stemness by modulating TGFßR2 signaling in colon cancer cells.

EGFR regulation of colon cancer stem-like cells during aging and in response to the colonic carcinogen dimethylhydrazine.

One of the most consistent pathological conditions in the gastrointestinal tract with advancing age is malignancy, particularly gastrointestinal cancers, the incidence of which increases sharply with aging. Although the reasons for the age-related rise in colorectal cancer are not fully understood, we hypothesize that aging increases susceptibility of the colon to carcinogen(s)/toxicant(s), leading to an increase in cancer stem-like cells (CSLCs) that express cancer stem cell markers, in the colonic mucosa. The current study demonstrates that aging is associated with increased expression of several colon CSLC markers [CD44, CD166, and aldehyde dehydrogenase 1 (ALDH-1)] and a higher proportion of cells expressing these markers. Aging is also accompanied by increased expression of miR-21 in colon. These increases are further increased in response to the colonic carcinogen dimethylhydrazine (DMH). Aging is also associated with increased tyrosine-phosphorylated epidermal growth factor receptor (EGFR). Inhibition of EGFR using the EGFR inhibitor cetuximab abrogated the age-related increase in CD166 and ALDH-1 as well as miRNA (miR)-21. Our results provide new evidence that aging and DMH are associated with increases in CSLC biomarkers and miR21, each of which have been linked to colorectal cancer. EGFR inhibition attenuates these changes, indicating a role for EGFR in age- and mutagen-associated changes in CSLCs.

Associations between markers of colorectal cancer stem cells and adenomas among ethnic groups.

BACKGROUND AND PURPOSES: Most colorectal tumors develop from adenomatous polyps, which are detected by colonoscopy. African Americans (AAs) have higher incidence of colorectal cancer (CRC) and greater mortality from this disease than Caucasian Americans (CAs). We investigated whether differences in predisposition to CRC and its surrogate (colonic adenomas) between these ethnic groups were related to numbers of cancer stem or stem-like cells (CSCs) in colonocytes. METHODS: We analyzed colonic effluent from 11 AA and 14 CA patients who underwent scheduled colonoscopy examinations at the John D. Dingell Veterans Affairs Medical Center. We determined proportions of cells that expressed the CSC markers CD44 and CD166 by flow cytometry. RESULTS: The proportion of colonocytes that were CD44(+)CD166(-) in effluent from patients with adenomas was significantly greater than from patients without adenomas (P = 0.01); the proportion of CD44(+)CD166(+) colonocytes was also greater (P = 0.07). Effluent from AAs with adenomas had 60 % more CD44(+)166(-) colonocytes than from CAs with adenomas. Using cutoff values of 8 % for AAs and 3 % for CAs, the proportion of CD44(+)166(-) colonocytes that had positive predictive value for detection of adenomas was 100 % for AAs and CAs, determined by receiver operator characteristic curve analysis. CONCLUSION: The proportion of CD44(+)166(-) colonocytes in colonic effluent can be used to identify patients with adenoma. AAs with adenomas have a higher proportion of CD44(+)166(-) colonocytes than CA. The increased proportion of CSCs in colonic tissue from AA might be associated with the increased incidence of CRC in this population.

Curcumin enhances dasatinib-induced inhibition of growth and transformation of colon cancer cells.

Colorectal cancer is the third most common form of malignancy, behind prostate and lung cancers. Despite recent advances in medicine, mortality from colorectal cancer remains high, highlighting the need for improved therapies. Numerous studies have demonstrated increased activation of EGFR and its family members (EGFRs), IGF-1R as well as c-Src in colorectal cancer. The current study was undertaken to examine the effectiveness of combination therapy of dasatinib (BMS-354825; Bristol-Myers Squibb), a highly specific inhibitor of Src family kinases (SFK) and a nontoxic dietary agent; curcumin (diferuloylmethane), in colorectal cancer in in vitro and in vivo experimental models. For the latter, we utilized C57BL/6 APC(Min+/-) mice. Initial in vitro studies revealed synergistic interactions between the two agents. Additionally, we have observed that combination treatment causes a much greater inhibition of the following metastatic processes than either agent alone: (i) colony formation, (ii) invasion through extracellular matrix and (iii) tubule formation by endothelial cells. Dasatinib affects the cell adhesion phenotype of colon cancer HCT-116 cells whereas the combination therapy enhances this effect to a greater extent. Preclinical investigation revealed that the combination therapy to be highly effective causing an over 95% regression of intestinal adenomas in Apc(Min+/-) mice, which could be attributed to decreased proliferation and increased apoptosis. In conclusion, our data suggest that combination treatment of dasatinib and curcumin could be a potential therapeutic strategy for colorectal cancer.

Do nutraceutics play a role in the prevention and treatment of colorectal cancer?

Colorectal cancer is the third most common cancer worldwide with a 5-year survival of 50%. Current chemotherapeutic regimens used for advanced colorectal cancer provide an average survival of approximately 20 months. Non-toxic agents such as nutraceutics and supplements have been shown to aid in the prevention and adjuvant treatment of colorectal cancer. This article will discuss the epidemiology, progression, prevention, treatment, and recurrence of colorectal cancer and the role of nutraceutics and supplements in the treatment process.

Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells.

We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.

Difluorinated-curcumin (CDF): a novel curcumin analog is a potent inhibitor of colon cancer stem-like cells.

PURPOSE: Recurrence of colon cancer, which affects nearly 50% of patients treated by conventional therapeutics, is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs). Therefore, development of therapeutic strategies for targeted elimination of CSCs would be a novel strategy. The current study examines whether difluorinated-curcumin (CDF), a novel analog of the dietary ingredient of curcumin, in combination with 5-fluorouracil and oxaliplatin (5-FU + Ox), the mainstay of colon cancer chemotherapeutic, would be effective in eliminating colon CSCs. METHODS: Multiple methodologies that include real-time RT-PCR, Western blot, MTT assay, caspase-3 activity, colonosphere formation, Hoechst-33342 dye exclusion and NF-κB-ELISA were used. RESULTS: We observed that CDF together with 5-FU + Ox were more potent than curcumin in reducing CD44 and CD166 in chemo-resistant colon cancer cells, accompanied by inhibition of growth, induction of apoptosis and disintegration of colonospheres. These changes were associated with down-regulation of the membrane transporter ABCG2 and attenuation of EGFR, IGF-1R, and NF-κB signaling consistent with inactivation of ß-catenin, COX-2, c-Myc and Bcl-xL and activation of the pro-apoptotic Bax. CONCLUSIONS: Our results suggest that CDF together with the conventional chemotherapeutics could be an effective treatment strategy for preventing the emergence of chemo-resistant colon cancer cells by eliminating CSCs.

The Wnt/beta-catenin pathway regulates growth and maintenance of colonospheres.

BACKGROUND: Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. The current investigation was undertaken to examine the role of Wnt/beta-catenin pathway in regulating the growth and maintenance of colonospheres. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant) were used. RESULTS: Colonospheres formed in vitro exhibited higher expression of colon CSCs markers LGR5, CD44, CD166 and Musashi-1 along with putative CSC marker EpCAM, compared to the corresponding parental cancer cells and also exhibit the ability to form spheroids under extreme limiting dilution, indicating the predominance of CSCs in colonospheres. Colonospheres formed by HCT-116 cells show over 80% of the cells to be CD44 positive, compared to

Schlafen 3 induction by cyclic strain regulates intestinal epithelial differentiation.

The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. In vitro, cyclic strain promotes intestinal epithelial proliferation and induces an absorptive phenotype characterized by increased dipeptidyl dipeptidase (DPPIV) expression. Schlafen 3 is a novel gene recently associated with cellular differentiation. We sought to evaluate whether Schlafen 3 mediates the effects of strain on the differentiation of intestinal epithelial cell (IEC)-6 in the absence or presence of cyclic strain. Strain increased Schlafen 3 mRNA and protein. In cells transfected with a control-nontargeting siRNA, strain increased DPPIV-specific activity. However, Schlafen 3 reduction by siRNA decreased basal DPPIV and prevented any stimulation of DPPIV activity by strain. Schlafen 3 reduction also prevented DPPIV induction by sodium butyrate (1 mM) or transforming growth factor (TGF)-beta (0.1 ng/ml), two unrelated differentiating stimuli. However, Schlafen-3 reduction by siRNA did not prevent the mitogenic effect of strain or that of EGF. Blocking Src and phosphatidyl inositol (PI3)-kinase prevented strain induction of Schlafen 3, but Schlafen 3 induction required activation of p38 but not ERK. These results suggest that cyclic strain induces an absorptive phenotype characterized by increased DPPIV activity via Src-, p38-, and PI3-kinase-dependent induction of Schlafen 3 in rat IEC-6 cells on collagen, whereas Schlafen 3 may also be a key factor in the induction of intestinal epithelial differentiation by other stimuli such as sodium butyrate or TGF-beta. The induction of Schlafen 3 or its human homologs may modulate intestinal epithelial differentiation and preserve the gut mucosa during normal gut function.

Colorectal cancer: chemopreventive role of curcumin and resveratrol.

Colorectal cancer (CRC) is a second leading cause of cancer deaths in the Western world. Currently there is no effective treatment except resection at a very early stage with or without chemotherapy. Of various epithelial cancers, CRC in particular has a potential for prevention, since most cancers follow the adenoma-carcinoma sequence, and the interval between detection of an adenoma and its progression to carcinoma is usually about a decade. However no effective chemopreventive agent except COX-2 inhibitors, limited in their scope due to cardiovascular side effects, have shown promise in reducing adenoma recurrence. To this end, natural agents that can target important carcinogenic pathways without demonstrating discernible adverse effects would serve as ideal chemoprevention agents. In this review, we discuss merits of two such naturally occurring dietary agents-curcumin and resveratrol-for chemoprevention of CRC.

Natural agents inhibit colon cancer cell proliferation and alter microbial diversity in mice.

The current study was undertaken to investigate the effect of differentially formulated polyphenolic compound Essential Turmeric Oil-Curcumin (ETO-Cur), and Tocotrienol-rich fraction (TRF) of vitamin E isomers on colorectal cancer (CRC) cells that produce aggressive tumors. Combinations of ETO-Cur and TRF were used to determine the combinatorial effects of ETO-Cur and TRF-mediated inhibition of growth of CRC cells in vitro and HCT-116 cells xenograft in SCID mice. 16S rRNA gene sequence profiling was performed to determine the outcome of gut microbial communities in mice feces between control and ETO-Cur-TRF groups. Bacterial identifications were validated by performing SYBR-based Real Time (RT) PCR. For metagenomics analysis to characterize the microbial communities, multiple software/tools were used, including Quantitative Insights into Microbial Ecology (QIIME) processing tool. We found ETO-Cur and TRF to synergize and that the combination of ETO-Cur-TRF significantly inhibited growth of HCT-116 xenografts in SCID mice. This was associated with a marked alteration in microbial communities and increased microbial OTU (operation taxonomic unit) number. The relative abundance of taxa was increased and the level of microbial diversity after 34 days of combinatorial treatment was found to be 44% higher over the control. Shifting of microbial family composition was observed in ETO-Cur-TRF treated mice as evidenced by marked reductions in Bacteroidaceae, Ruminococcaceae, Clostridiales, Firmicutes and Parabacteroids families, compared to controls. Interestingly, during the inhibition of tumor growth in ETO-Cur treated mice, probiotic Lactobacillaceae and Bifidobacteriaceae were increased by 20-fold and 6-fold, respectively. The relative abundance of anti-inflammatory Clostridium XIVa was also increased in ETO-Cur-TRF treated mice when compared with the control. Our data suggest that ETO-Cur-TRF show synergistic effects in inhibiting colorectal cancer cell proliferation in vitro and in mouse xenografts in vivo, and might induce changes in microbial diversity in mice.

Combination of aging and dimethylhydrazine treatment causes an increase in cancer-stem cell population of rat colonic crypts.

Aging is associated with increased incidence of colon cancers. It is also becoming evident that cancer stem cells (CSC) play a vital role in the pathogenesis and prognosis of colon cancer. Recently, we reported the presence of colon cancer stem-like cells in macroscopically normal mucosa in patients with adenomatous polyps and that they increase with aging, suggesting that aging may predispose the colon to carcinogenesis. In the current study we have examined the combined effects of aging and carcinogen exposure on the status of colon CSCs in an experimental model. We used young (4-6 months) and aged (22-24 months) rats and exposed them to the carcinogen, dimethylhydroxide (DMH). We investigated the expression of colon cancer stem cell markers, CD44, CD166, EpCam, and ALDH1 as well as EGFR expression in normal colonic crypt epithelium following carcinogen treatment. Our results demonstrate that aging per se or carcinogen treatment alone causes an increase in the number of colon cancer stems cells, as evidenced by increased immunoreactive-CSC-markers positive cells in the colonic mucosa. In aged rats, carcinogen exposure results in a more pronounced increase in colon cancer stem cells. Our study shows that in aging colon the effects of carcinogens are more pronounced, and an increase in colon CSCs is one of the earliest changes preceding tumor development. Moreover, the current investigation of the use of a panel of immunohistochemical markers of colon CSC can potentially serve as a prognostic marker during screening for colon cancer.

Age-related increase in colorectal cancer stem cells in macroscopically normal mucosa of patients with adenomas: a risk factor for colon cancer.

It is becoming increasingly evident that cancer stem cells play a vital role in development and progression of cancers and relapse following chemotherapy. The present study examines the presence of cancer stem-like cells (CSC) in adenomatous polyps and in normal appearing colonic mucosa in humans during aging. The number of polyps was found to increase linearly with advancing age (r(2)=0.92, p<0.02). Immunohistochemical analysis revealed co-localization of CSC markers CD44 and CD166 in colonic polyps. Real-time RT-PCR analysis of normal appearing mucosa from subjects with adenomatous polyps showed an age-related rise in CSC as evidenced by the increased expression of CD44, CD166 and ESA. A similar phenomenon was also observed for EGFR. In addition, the expression each CSC marker was found to be about 2-fold higher in subjects with 3-4 polyps than those with 1-2 polyps. In conclusion, our results show that colon cancer stem-like cells are present in the premalignant adenomatous polyps as well in normal appearing colonic mucosa. Moreover, our observation of the age-related rise in CSC in macroscopically normal colonic mucosa suggests a predisposition of the organ to developing colorectal cancer.

Schlafen-3: a novel regulator of intestinal differentiation.

Schlafen-3 (Slfn-3), a novel gene, has been shown to be a negative regulator of proliferation. The current investigation was undertaken to determine whether Slfn-3 might play a role in regulating cellular differentiation. Butyric acid, a short chain fatty acid, which induced differentiation of intestinal cells as evidenced by increased alkaline phosphatase (ALP) activity in the rat small intestinal IEC-6 cells, also produced a marked increase in Slfn-3 expression. Furthermore, overexpression of Slfn-3 caused stimulation of ALP activity in IEC-6 cells, which was exacerbated by butyrate. On the other hand, downregulation of Slfn-3 by slfn-3-si-RNA greatly attenuated the butyrate-mediated induction of differentiation of IEC-6 cells. Additionally, we observed that increased expression of Slfn-3 in colon cancer HCT-116 cells stimulated TGF-beta expression and modulated expression of its downstream effectors as evidenced by increased expression of p27kip1 and downregulation of CDK-2. In addition, Slfn-3 increases E-cadherin expression but downregulates beta-catenin. In conclusion, our data show that Slfn-3 plays a critical role in regulating intestinal mucosal differentiation. Furthermore our data also show that TGF-beta signaling pathway plays an important role in mediating slfn-3 induced differentiation.

Synergistic role of curcumin with current therapeutics in colorectal cancer: minireview.

Despite the use of surgical resection and aggressive chemotherapy, nearly 50% of patients with colorectal carcinoma develop recurrent disease, highlighting the need for improved therapies. Curcumin (diferuloylmethane), the major active ingredient of turmeric (curcuma longa) with no discernable toxicity, has been shown to inhibit the growth of transformed cells and colon carcinogenesis at the initiation, promotion, and progression stages in carcinogen-induced rodent models. In a Phase I clinical trial, curcumin has been found to be extremely well tolerated and effective. In this review, we summarized the current status of our knowledge about the effectiveness of curcumin when given in combination with current chemotherapeutics such as 5-fluorouracil, oxaliplatin, and gemcitabine in treatment of gastrointestinal cancers with particular reference to colorectal cancer. Existing data suggest that curcumin in combination with chemotherapy is a superior strategy for treatment of gastrointestinal cancer.