Colon epithelial cell-specific Bmal1 deletion impairs bone formation in mice

The circadian clock system regulates multiple metabolic processes, including bone metabolism. Previous studies have demonstrated that both central and peripheral circadian signaling regulate skeletal growth and homeostasis. Disruption in central circadian rhythms has been associated with a decline in bone mineral density and the global and osteoblast-specific disruption of clock genes in bone tissue leads to lower bone mass. Gut physiology is highly sensitive to circadian disruption. Since the gut is also known to affect bone remodeling, we sought to test the hypothesis that circadian signaling disruption in colon epithelial cells affects bone. We therefore assessed structural, functional, and cellular properties of bone in 8 week old Ts4-Cre and Ts4-Cre;Bmal1fl/fl (cBmalKO) mice, where the clock gene Bmal1 is deleted in colon epithelial cells. Axial and appendicular trabecular bone volume was significantly lower in cBmalKO compared to Ts4-Cre 8-week old mice in a sex-dependent fashion, with male but not female mice showing the phenotype. Similarly, the whole bone mechanical properties were deteriorated in cBmalKO male mice. The tissue level mechanisms involved suppressed bone formation with normal resorption, as evidenced by serum markers and dynamic histomorphometry. Our studies demonstrate that colon epithelial cell-specific deletion of Bmal1 leads to trabecular and cortical bone loss in male mice.

Cell-matrix adhesion contributes to permeability control in human colon organoids

Background and aims: Cell-cell adhesion structures (desmosomes and, especially, tight junctions) are known to play important roles in control of transepithelial permeability in the colon. The involvement of cell-matrix interactions in permeability control is less clear. The goals of the present study were to: i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control and ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes improves permeability control and mitigates the effects of cell-matrix disruption. Methods: Human colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions (0.25 mM calcium) and in the presence of Aquamin®, a multi-mineral product, at a level providing 1.5 mM calcium. The effects of Aquamin® on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. In parallel, TEER was assessed in the presence of a function-blocking antibody directed at an epitope in the C-terminal region of laminin α3 chain. Results: Treatment of colon organoids with Aquamin® increased the expression of multiple basement membrane and hemidesmosomal proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. Anti-laminin antibody reduced TEER values under all conditions but was most effective in the absence of Aquamin®, where laminin expression was low and TEER values were lower to begin with. Conclusions: These findings provide evidence that cell-matrix interactions contribute to permeability control in the colon. They suggest that the elaboration of proteins important to cell-matrix interactions can be increased in human colon organoids by exposure to a multi-mineral natural product. Increasing the elaboration of such proteins may help to mitigate the consequences of disrupting cell-matrix interactions on permeability control.

Anti-cancer effects of polyphenol-rich sugarcane extract

Plant polyphenols have an array of health benefits primarily thought to be related to their high content of anti-oxidants. These are commonly undervalued and knowledge of their biological properties have grown exponentially in the last decade. Polyphenol-rich sugarcane extract (PRSE), a natural extract from sugar cane, is marketed as high in anti-oxidants and polyphenols, but its anti-cancer activity has not been reported previously. We show that, PRSE exerts anti-cancer properties on a range of cancer cells including human (LIM2045) and mouse (MC38, CT26) colon cancer cells lines; human lung cancer (A549), human ovarian cancer (SKOV-3), pro-monocytic human leukemia (U937) and to mouse melanoma (B16) cell lines; whereas no effects were noted on human breast (ZR-75-1) and human colon (HT29) cancer cell lines, as well as to human normal colon epithelial cell line (T4056). Anti-proliferative effects were shown to be mediated via alteration in cytokines, VEGF-1 and NF-κB expression.

MiR-566 mediates cell migration and invasion in colon cancer cells by direct targeting of PSKH1

Background Colorectal cancer (CRC), a common malignancy worldwide, and microRNAs (miRs) have been suggested to play roles in the disease. MiR-566 expression has been shown to be reduced in CRC, but its functions and mechanisms are still unclear. Methods Cell viability was assessed by using the CellTiter 96 AQueous One Solution Cell Proliferation kit. Cell proliferation was measured with MTT assay. Cell metastasis were measured by transwell assay. Luciferase reporter assays was used to confirm the target of MiR-566. PSKH1 expression was measured by RT-PCR and western blot. Results In the present study, we first observed that miR-566 was expressed in several CRC cell lines (SW480, SW620, LoVo, HT29 and Caco-2) at low levels compared to control colon epithelial cell lines (FHC). Further study showed that miR-566 overexpression suppressed cell survival and impeded cell proliferation, whereas inhibition of its expression enhanced cell survival and proliferation. Transwell assays showed that cell invasion and migration were reduced in cells overexpressing miR-566 and increased in those with inhibition of miR-566. Further analysis confirmed that PSKH1 is a target of miR-566. MiR-566 overexpression significantly inhibited PSKH1 expression and reintroduction of PSKH1 partially reversed the effects of miR-566 on CRC cell growth and metastasis in SW480 and Caco-2 cells. Conclusions Taken together, the data show that CRC cell growth and metastasis can be significantly suppressed by miR-566 through targeting PSKH1.

Role of tropomyosin 4 gene misregulation in colon epithelial cell tumorigenesis.

e15184 Background: Ordered migration of colon epithelial cells from the bottom to the top of the crypt is required for their differentiation. Because aberrant colon cell migration may contribute to early stages of colon tumorigenesis, elucidation of how this migration is regulated provides insight into how colon cancer can be prevented and treated before malignancy. Certain actin-binding tropomyosins, including tropomyosin 4 (TPM4), regulate cytoskeletal contractility and migration in many different cell types. Methods: To determine whether aberrant TPM4 regulation may be an early event in colon cell tumorigenesis, we measured TPM4 RNA expression in uninvolved, grossly normal colon tissue from 10 human familial adenomatous polyposis (FAP) patients genetically predisposed to colon cancer versus 7 patients not genetically predisposed (normal). We also examined TPM4 expression in a colon adenocarcinoma cell line (Caco2). These are highly proliferative, undifferentiated cells but can be induced to growth arrest and differentiate, thus acquiring many characteristics of normal absorptive colon epithelial cells. Reporter assays were used to identify sequences that regulate TPM4 gene expression in Caco2 cells. Results: Average TPM4 RNA expression is 2.59 fold higher in the FAP versus normal tissue. TPM4 RNA and protein are highly expressed in proliferating, undifferentiated Caco2 cells but are severely reduced in growth arrested, differentiated cells. Reporter assays have identified genetic regions, near the TPM4 transcriptional start site, that may regulate TPM4 gene expression in proliferating Caco2 cells. Conclusions: This data suggests that aberrantly upregulated TPM4 may be an early event in colon tumorigenesis and that normal TPM4 expression, by fostering ordered colon epithelial cell migration, may promote normal cell differentiation and mitigate adoption of a premalignant state.