BackgroundRecent 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/β-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.ResultsColonospheres 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 ≤ 1% in the corresponding parental cells. Additionally, colonospheres showed reduced membrane bound β-catenin but had increased levels of total β-catenin, cyclin-D1 and c-myc and down regulation of axin-1 and phosphorylated β-catenin. Increased expression of β-catenin was associated with a marked transcriptional activation of TCF/LEF. The latter was greatly decreased following down regulation of β-catenin by the corresponding siRNA, leading to a marked reduction in CD44 positive cells as well as colonospheres formation. In contrast, upregulation of c-myc, a down-stream effector of TCF/LEF greatly augmented the formation of colonospheres.ConclusionOur data suggest that colonospheres formed by colon cancer cell lines are highly enriched in CSCs and that Wnt/β-catenin pathway plays a critical role in growth and maintenance of colonospheres.

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes ϳ130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21

5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics but with limited success. This could partly be due to the enrichment of cancer stem cells (CSCs) that are resistant to conventional chemotherapy. Therefore, validation of a nontoxic agent that can either cause reversal of chemoresistance or promote the killing of CSCs would be highly desirable. The current study examines whether curcumin, the major active ingredient of turmeric, either alone or together with FOLFOX, would be an effective strategy to eliminate colon CSCs. Exposure of colon cancer HCT-116 or HT-29 cells to FOLFOX that inhibited their growth led to the enrichment of CSC phenotype as evidenced by increased proportion of CD133-, CD44-, and/or CD166-positive cells and epidermal growth factor receptor (EGFR) levels. Treatment of FOLFOX-surviving colon cancer cells with either curcumin alone or together with FOLFOX resulted in a marked reduction in CSCs, as evidenced by the decreased expression of CD44 and CD166 as well as EGFR and by their ability to form anchorage-dependent colonies. They also caused disintegration of colonospheres. Increased expression of EGFR in FOLFOX-surviving cells could be attributed to hypomethylation of the EGFR promoter, whereas an opposite phenomenon was observed when the FOLFOX-surviving cells were treated with curcumin and/or FOLFOX. These changes were accompanied by parallel alterations in the levels of DNA methyltransferase 1. In conclusion, our data suggest that curcumin by itself or together with the conventional chemotherapeutic could be an effective treatment strategy for preventing the emergence of chemoresistant colon cancer cells by reducing/eliminating CSCs.

Curcumin (diferuloylmethane) is the major active ingredient of turmeric (Curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiologic conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the NH 2 -terminal threonine of the proteasomal chymotrypsin-like (CT-like) subunit. Consistently, curcumin potently inhibits the CT-like activity of a purified rabbit 20S proteasome (IC 50 = 1.85 Mmol/L) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor-bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression, and apoptosis induction in tumor tissues. Our study shows that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early-stage and late-stage/refractory colon cancer. [Cancer Res 2008;68(18):7283-92]

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 (TGFβR2). In contrast, the levels of β-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 TGFβR2 could be attributed to decreased expression of the receptor as evidenced by reduction in the activity of the luciferase gene construct comprising TGFβR2-3' untranslated region (UTR) sequence that binds to miR-21. Moreover, we observed that downregulation of miR-21 enhances luciferase-TGFβR2-3' UTR activity suggesting TGFβR2 as being one of the direct targets of miR-21. Further support is provided by the observation that transfection of TGFβR2 in HCT-116 cells attenuates TCF/LEF luciferase activity, accompanied by decreased expression of β-catenin, c-Myc and Cyclin-D1. Our current data suggest that miR-21 plays an important role in regulating stemness by modulating TGFβR2 signaling in colon cancer cells.

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Curcumin (diferuloylmethane), which has been shown to inhibit growth of transformed cells, has no discernible toxicity and achieves high levels in colonic mucosa. 5-fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but with limited success. The present investigation was, therefore, undertaken to examine whether curcumin in combination with conventional chemotherapeutic agent(s)/regimen will be a superior therapeutic strategy for colorectal cancer. Indeed, results of our in vitro studies demonstrated that curcumin together with FOLFOX produced a significantly greater inhibition (p < 0.01) of growth and stimulated apoptosis (p < 0.001) of colon cancer HCT-116 and HT-29 cells than that caused by curcumin, 5-FU, curcumin 1 5-FU or FOLFOX. These changes were associated with decreased expression and activation (tyrosine phosphorylation) of EGFR, HER-2, HER-3 (72-100%) and IGF-1R (67%) as well as their downstream effectors such as Akt and cycloxygenase-2 (51-97%). Furthermore, while these agents produced a 2-3-fold increase in the expression of IGF-binding protein-3 (IGFBP-3), curcumin together with FOLFOX caused a 5-fold increase in the same, when compared to controls. This in turn led to increased sequestration of IGF by IGFBP-3 rendering IGF-1 unavailable for binding to and activation of IGF-1R. We conclude that the superior effects of the combination therapy of curcumin and FOLFOX are due to attenuation of EGFRs and IGF-1R signaling pathways. We also suggest that inclusion of curcumin to the conventional chemotherapeutic agent(s)/regimen could be an effective therapeutic strategy for colorectal cancer. ' 2007 Wiley-Liss, Inc.Key words: colorectal cancer; EGFR; IGF-1R; curcumin; chemotherapy; IGFBP3There will be an estimated 148,610 new cases and 55,170 deaths due to colorectal caner (CRC) in 2006 in the USA. 1 CRC is estimated to be the second and third leading cause of cancerrelated deaths in men and women, respectively, in 2006. 1 Surgery and subsequent chemotherapy can cure over 75% colon cancer patients, but more than 30% of these patients develop new neoplastic polyps, and 10% progress to frank second malignancy. [2][3][4] The risk of second malignancy is higher for microsatellite instable tumor (MSI). 5 Metastatic colorectal cancer has poor a prognosis with 5-year survival of less than 10%. 6 As a result of great efforts are being spent on improving chemotherapeutic interventions for metastatic colon cancer, and the median survival has improved to over 20 months of this group of patients. 7 However, this comes at a cost of additional toxicities, some of which are even fatal. 7 The validation of a nontoxic agent that could improve upon the current chemotherapeutic regimen would therefore be highly desirable.Accumulating evidence suggests that the development and progression of many malignancies, including colorectal cancer, are associated with constitutive activation of multiple signaling pathways that promote proliferation, inhibit apoptosis an...

CARP-1, a novel apoptosis inducer, regulates apoptosis signaling by diverse agents, including adriamycin and growth factors. Epidermal growth factor receptor (EGFR)-related protein (ERRP), a panErbB inhibitor, inhibits EGFR and stimulates apoptosis. Treatments of cells with ERRP or Iressa (an EGFR tyrosine kinase inhibitor) results inProgrammed cell death (apoptosis) is essential for the development and maintenance of cellular homeostasis. The pathways regulating apoptosis serve as important targets for many anti-cancer agents currently utilized for treatment of diverse malignancies. Recently, we reported identification of a novel perinuclear protein CARP-1, also known as CCAR1 (1). CARP-1 regulates apoptosis signaling by chemotherapeutic agent adriamycin and by a novel retinoid [3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437). [3-(1-adamantyl)-4-Hydroxyphenyl]-2-naphthalene carboxylic acid belongs to a novel class of apoptogenic adamantyl retinoids that induce growth arrest and stimulate apoptosis in a wide variety of malignant cell types, including breast and prostate cancer and leukemia, by an retinoic acid receptor/retinoid X receptor-independent mechanism (2-4). Expression of CARP-1, on the other hand, induces apoptosis while causing elevated levels of CDKI p21 WAF1CIP1 and inhibiting the expression of a number of cell cycle-regulatory proteins such as c-Myc, cyclin B, and topoisomerase II␣ (1). Whereas it is evident that CARP-1 is an important apoptosis signal transducer, it is unclear how CARP-1-dependent apoptosis is accomplished. Understanding the apoptosis-inducing pathways utilized by CARP-1 will define mechanism(s) of action of agents such as adriamycin. Members of the EGFR 2 family of receptor tyrosine kinases, which includes EGFR, ErbB-2/HER-2, ErbB-3/HER-3, and ErbB-4/HER-4, collectively referred to as EGFRs, serve as critical mediators of the cellular communication network regulating complex biological processes such as growth, differentiation, motility, or death (5, 6). Since deregulated signaling by EGFRs is frequently noted in a variety of human cancers (7-9), interference with growth factor receptor activation and/or with intracellular growth factor-activated signal transduction pathways represents a promising strategy for the development of novel and selective anti-cancer therapies (10 -12). Small molecule inhibitors of EGFR, such as gefitinib (Iressa) and erlotinib (Tarceva; OSI-774), monoclonal antibodies to EGFR (cetuximab/IMC-C225/Erbitux), and HER-2 (trastuzumab/Herceptin) are being utilized as anti-cancer therapeutics.ERRP, a recently isolated pan-EGFR inhibitor, is a 53-55-kDa protein that possesses substantial homology to the extracellular ligand-binding domain of EGFR and its family members (13). ERRP is a secretory protein that forms an inactive heterodimer with EGFR, causing inhibition of EGFR-dependent signaling events (14). ERRP inhibits proliferation and induces apoptosis as well as attenuates ligand-induced activation of EGFR and HER-2 in cancer cells th...