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.

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.

Role of Microbiome in Carcinogenesis Process and Epigenetic Regulation of Colorectal Cancer.

Epigenetic changes during the development of colorectal cancer (CRC) play a significant role. Along with factors such as diet, lifestyle, and genetics, oncogenic infection, bacteria alone or whole microbiome, has been associated with this tumor type. How gut microbiome contributes to CRC pathogenesis in the host is not fully understood. Most of the epigenetic studies in CRC have been conducted in populations infected with Helicobacter pylori. In the current review, we summarize how the gut microbiota contributes in colon carcinogenesis and the potential role of epigenetic mechanism in gene regulation. We discuss microbiota-mediated initiation and progression of colon tumorigenesis and have also touched upon the role of microbial metabolites as an initiator or an inhibitor for procarcinogenic or antioncogenic activities. The hypothesis of gut microbiota associated CRC revealed the dynamic and complexity of microbial interaction in initiating the development of CRC. In the multifaceted processes of colonic carcinogenesis, gradual alteration of microbiota along with their microenvironment and the potential oncopathogenic microbes mediated modulation of cancer therapy and other factors involved in microbiome dysbiosis leading to the CRC have also been discussed. This review provides a comprehensive summary of the mechanisms of CRC development, the role of microbiome or single bacterial infection in regulating the processes of carcinogenesis, and the intervention by novel therapeutics. Epigenetic mechanism involved in CRC is also discussed.

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.

Bile acid: a potential inducer of colon cancer stem cells.

BACKGROUND: Although the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer. METHODS: Normal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of ß-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA. RESULTS: We found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial-mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF. CONCLUSIONS: Our results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/ß-catenin signaling and thus could be considered promoters of colon cancer.

Role of cancer stem cells in racial disparity in colorectal cancer.

Although African-Americans (AAs) have a higher incidence of colorectal cancer (CRC) than White people, the underlying biochemical mechanisms for this increase are poorly understood. The current investigation was undertaken to examine whether differences in self-renewing cancer stem/stem-like cells (CSCs) in the colonic mucosa, whose stemness is regulated by certain microRNAs (miRs), could partly be responsible for the racial disparity in CRC. The study contains 53 AAs and 47 White people. We found the number of adenomas and the proportion of CD44(+) CD166(-  ) CSC phenotype in the colon to be significantly higher in AAs than White people. MicroRNAs profile in CSC-enriched colonic mucosal cells, expressed as ratio of high-risk (≥3 adenomas) to low-risk (no adenoma) CRC patients revealed an 8-fold increase in miR-1207-5p in AAs, compared to a 1.2-fold increase of the same in White people. This increase in AA was associated with a marked rise in lncRNA PVT1 (plasmacytoma variant translocation 1), a host gene of miR-1207-5p. Forced expression of miR-1207-5p in normal human colonic epithelial cells HCoEpiC and CCD841 produced an increase in stemness, as evidenced by morphologically elongated epithelial mesenchymal transition( EMT) phenotype and significant increases in CSC markers (CD44, CD166, and CD133) as well as TGF-ß, CTNNB1, MMP2, Slug, Snail, and Vimentin, and reduction in Twist and N-Cadherin. Our findings suggest that an increase in CSCs, specifically the CD44(+) CD166(-) phenotype in the colon could be a predisposing factor for the increased incidence of CRC among AAs. MicroRNA 1207-5p appears to play a crucial role in regulating stemness in colonic epithelial cells in AAs.

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.

Inhibition of insulin-like growth factor receptor/AKT/mammalian target of rapamycin axis targets colorectal cancer stem cells by attenuating mevalonate-isoprenoid pathway in vitro and in vivo.

We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an 'in vivo' HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer.

Omega-3 fatty acid is a potential preventive agent for recurrent colon cancer.

Increasing evidence supports the contention that many malignancies, including sporadic colorectal cancer, are driven by the self-renewing, chemotherapy-resistant cancer stem/stem-like cells (CSC/CSLC), underscoring the need for improved preventive and therapeutic strategies targeting CSCs/CSLCs. Omega-3 polyunsaturated fatty acids (ω-3 PUFA), have been reported to inhibit the growth of primary tumors, but their potential as a preventive agent for recurring cancers is unexplored. The primary objectives of this investigation are (i) to examine whether eicosapentaenoic acid (EPA; one of the ω-3 PUFA) synergizes with FuOx (5-FU+Oxaliplatin), the backbone of colon cancer chemotherapy, and (ii) whether EPA by itself or in combination with conventional chemotherapy prevents the recurrence of colon cancer via eliminating/suppressing CSCs/CSLCs. FuOx-resistant (chemoresistant; CR) colon cancer cells, highly enriched in CSCs, were used for this study. Although EPA alone was effective, combination of EPA and FuOx was more potent in (i) inhibiting cell growth, colonosphere formation, and sphere-forming frequency, (ii) increasing sphere disintegration, (iii) suppressing the growth of SCID mice xenografts of CR colon cancer cells, and (iv) decreasing proinflammatory metabolites in mice. In addition, EPA + FuOx caused a reduction in CSC/CSLC population. The growth reduction by this regimen is the result of increased apoptosis as evidenced by PARP cleavage. Furthermore, increased pPTEN, decreased pAkt, normalization of ß-catenin expression, localization, and transcriptional activity by EPA suggests a role for the PTEN-Akt axis and Wnt signaling in regulating this process. Our data suggest that EPA by itself or in combination with FuOx could be an effective preventive strategy for recurring colorectal cancer.

Metformin: a potential therapeutic agent for recurrent colon cancer.

Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties. However, most of the studies to evaluate therapeutic efficacy of metformin have been on primary cancer. No information is available whether metformin could be effectively used for recurrent cancer, specifically colorectal cancer (CRC) that affects up to 50% of patients treated by conventional chemotherapies. Although the reasons for recurrence are not fully understood, it is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs/CSLCs). Therefore, development of non-toxic treatment strategies targeting CSCs would be of significant therapeutic benefit. In the current investigation, we have examined the effectiveness of metformin, in combination with 5-fluorouracil and oxaliplatin (FuOx), the mainstay of colon cancer therapeutics, on survival of chemo-resistant colon cancer cells that are highly enriched in CSCs/CSLCs. Our data show that metformin acts synergistically with FuOx to (a) induce cell death in chemo resistant (CR) HT-29 and HCT-116 colon cancer cells, (b) inhibit colonospheres formation and (c) enhance colonospheres disintegration. In vitro cell culture studies have further demonstrated that the combinatorial treatment inhibits migration of CR colon cancer cells. These changes were associated with increased miRNA 145 and reduction in miRNA 21. Wnt/ß-catenin signaling pathway was also down-regulated indicating its pivotal role in regulating the growth of CR colon cancer cells. Data from SCID mice xenograft model of CR HCT-116 and CR HT-29 cells show that the combination of metformin and FuOX is highly effective in inhibiting the growth of colon tumors as evidenced by ∼ 50% inhibition in growth following 5 weeks of combination treatment, when compared with the vehicle treated controls. Our current data suggest that metformin together with conventional chemotherapy could be an effective treatment regimen for recurring colorectal cancer (CRC).

Cancer stem cells biomarkers in gastric carcinogenesis.

BACKGROUND: Gastric carcinogenesis is a multistep process, involving multiple molecular alterations, including changes in cancer stem cells (CSCs). The present investigation was undertaken to determine whether changes in cancer stem cells could be utilized as a marker of progression of gastric carcinogenesis by examining the expression of gastric CSCs at different stages of carcinogenesis. METHODS: Ninety-three cases with 31 in each group of chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), or gastric cancer (GC) were analyzed immunohistochemically for proliferating cell nuclear antigen (PCNA) and Bcl-xl as biomarkers of proliferation and apoptosis, respectively, and CD44, CD166, and LGR5 levels by qRT-PCR as markers of gastric CSCs. Additionally, the levels of P53 and phosphorylated form of epidermal growth factor receptor (p-EGFR) were examined. RESULTS: While the levels of each of these biomarkers were found to be low to moderate in CSG and CAG patients, they were markedly increased in GC patients, in whom co-expression of CD44 with LGR5 and CD166 with p-EGFR was found to be the highest. We have also observed that although the expression of different CSC markers as well as the levels of p-EGFR were increased in precancerous lesions (CSG and CAG), they are further augmented in GC suggesting that they may play a pivotal role in the development and progression of gastric cancer. CONCLUSIONS: Our observations suggest that the progression to gastric carcinogenesis from preneoplastic lesions such as superficial gastritis and chronic atrophic gastritis is associated with induction of CSCs together with increase in cell proliferation and inhibition of apoptosis.

Difluorinated-curcumin (CDF) restores PTEN expression in colon cancer cells by down-regulating miR-21.

Despite recent advancement in medicine, nearly 50% of patients with colorectal cancer show recurrence of the disease. Although the reasons for the high relapse are not fully understood, the presence of chemo- and radiotherapy-resistant cancer stem/stem-like cells, where many oncomirs like microRNA-21 (miR-21) are upregulated, could be one of the underlying causes. miR-21 regulates the processes of invasion and metastasis by downregulating multiple tumor/metastatic suppressor genes including PTEN (phosphatase and tensin homolog). Tumor suppressor protein PTEN controls self-renewal of stem cells. Indeed, our current data demonstrate a marked downregulation of PTEN in SCID mice xenografts of miR-21 over-expressing colon cancer HCT116 cells. Colonospheres that are highly enriched in cancer stem/stem like cells reveal increased miR-21 expression and decreased PTEN. Difluorinated curcumin (CDF), a novel analog of the dietary ingredient curcumin, which has been shown to inhibit the growth of 5-Flurouracil + Oxaliplatin resistant colon cancer cells, downregulated miR-21 in chemo-resistant colon cancer HCT116 and HT-29 cells and restored PTEN levels with subsequent reduction in Akt phosphorylation. Similar results were also observed in metastatic colon cancer SW620 cells. Since PTEN-Akt confers drug resistance to different malignancies including colorectal cancer, our observation of normalization of miR-21-PTEN-Akt pathway by CDF suggests that the compound could be a potential therapeutic agent for chemotherapy-resistant colorectal cancer.

Down-regulation of miR-21 Induces Differentiation of Chemoresistant Colon Cancer Cells and Enhances Susceptibility to Therapeutic Regimens.

MicroRNAs are endogenous posttranscriptional modulators that negatively control the expression of their target genes and play an important role in the development and progression of many malignancies, including colorectal carcinoma. In particular, expression of microRNA-21 (miR-21) is greatly increased in chemotherapy-resistant (CR) colon cancer cells that are enriched in undifferentiated cancer stem/stem-like cells (CSCs/CSLCs). We hypothesize that miR-21 plays a critical role in regulating differentiation of CR colon cancer cells. Indeed, we observed that downregulation of miR-21 in CR colon cancer cells (HCT-116 or HT-29) by antisense miR-21 induced differentiation, as evidenced by marked increases in cytokeratin-20 (CK-20) expression and alkaline phosphatase activity. These changes were accompanied by a significant reduction in the expression of colon CSC/CSLC marker CD44, colonosphere formation, and T-cell factor/lymphoid enhancer factor (TCF/LEF) activity but increased the expression of proapoptotic programmed cell death 4 gene. Induction of differentiation greatly increased sensitivity of CR colon cancer cells to the growth inhibitory properties of all three regimens tested: 5-fluorouracil + oxaliplatin (FUOX), difluorinated curcumin (CDF), and the combination of CDF and FUOX. However, the magnitude of inhibition of growth by either CDF (75%) alone or CDF + FUOX (80%) was much higher than that observed with only FUOX (40%). Growth inhibition by CDF and CDF + FUOX in differentiating CR colon cancer cells was associated with a 98% to 99% reduction in the expression of CD44 and epidermal growth factor receptor (EGFR). However, down-regulation of CK-20 in CR colon cancer cells produced no significant change in cellular growth in the absence or presence of FUOX, when compared with the corresponding controls. The current observation suggests that CDF and CDF + FUOX are highly effective in inhibiting growth and reducing colon CSCs/CSLCs in anti-miR-21-induced differentiating CR colon cancer cells and supports our contention that differentiation enhances susceptibility of CR cancer cells to conventional and nonconventional therapeutic regimen.

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.

Expression of miR-34 is lost in colon cancer which can be re-expressed by a novel agent CDF.

BACKGROUND: Colorectal Cancer (CRC) is one of the leading causes of death worldwide. Numerous cellular events, including deregulated expression of microRNAs (miRNAs), specifically the family of miR-34 consisting of miR-34a, b and c, is known to regulate the processes of growth and metastasis. METHODS: We evaluated the expression of miR-34 in formalin-fixed paraffin-embedded (FFPE) human colon cancer tissue specimens compared to normal colonic mucosa. Moreover, we also assessed the expression of miR-34 in colon cancer cell lines treated with our newly developed synthetic analogue of curcumin referred as difluorinated curcumin (CDF) compared to well known inhibitor of methyl transferase. RESULTS: We found that the expression of miR-34a and miR-34c was down-regulated in colon cancer specimens compared to normal colonic mucosa and the loss of expression was also consistent with data from colon cancer cell lines. This down-regulation was attributed to promoter hypermethylation, because we found that the treatment of colon cancer cells with 5-aza-2´-deoxycytidine, a methyltransferase inhibitor, markedly induced the levels of miR-34a and miR-34c expression. Likewise, CDF was very effective in the re-expression of miR-34a and miR-34c, which was consistent with inhibition of cell growth of both chemo-sensitive and chemo-resistant colon cancer cells. The re-expression of miR-34 led to a marked reduction in the expression of its target gene, Notch-1. CONCLUSION: The loss of expression of miR-34 in colon cancer is in part due to promoter hypermethylation of miR-34, which can be re-expressed with our novel agent CDF, suggesting that CDF could be a novel demethylating agent for restoring the expression of miR-34 family, and thus CDF could become a newer therapeutic agent for the treatment of colon cancer.

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.

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.

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.