Epigenetic mechanisms underlying the dynamic expression of cancer-testis genes, PAGE2, -2B and SPANX-B, during mesenchymal-to-epithelial transition.

Cancer-testis (CT) genes are expressed in various cancers but not in normal tissues other than in cells of the germline. Although DNA demethylation of promoter-proximal CpGs of CT genes is linked to their expression in cancer, the mechanisms leading to demethylation are unknown. To elucidate such mechanisms we chose to study the Caco-2 colorectal cancer cell line during the course of its spontaneous differentiation in vitro, as we found CT genes, in particular PAGE2, -2B and SPANX-B, to be up-regulated during this process. Differentiation of these cells resulted in a mesenchymal-to-epithelial transition (MET) as evidenced by the gain of epithelial markers CDX2, Claudin-4 and E-cadherin, and a concomitant loss of mesenchymal markers Vimentin, Fibronectin-1 and Transgelin. PAGE2 and SPAN-X up-regulation was accompanied by an increase in Ten-eleven translocation-2 (TET2) expression and cytosine 5-hydroxymethylation as well as the disassociation of heterochromatin protein 1 and the polycomb repressive complex 2 protein EZH2 from promoter-proximal regions of these genes. Reversal of differentiation resulted in down-regulation of PAGE2, -2B and SPANX-B, and induction of epithelial-to-mesenchymal transition (EMT) markers, demonstrating the dynamic nature of CT gene regulation in this model.

The NF-κB target genes ICAM-1 and VCAM-1 are differentially regulated during spontaneous differentiation of Caco-2 cells.

Intestinal epithelial differentiation entails the formation of highly specialized cells with specific absorptive, secretory, digestive and immune functions. Cell-cell and cell-microenvironment interactions appear to be crucial in determining the outcome of the differentiation process. Using the Caco-2 cell line, which undergoes spontaneous re-differentiation when grown past confluency, we observed a loss of VCAM-1 (vascular cell adhesion molecule 1) mRNA expression, while ICAM-1 (intercellular cell adhesion molecule 1) mRNA expression was seen to increase over the course of differentiation. Protein kinase Cθ (PKCθ) acted downstream of protein kinase Cα (PKCα) to inactivate inhibitor of κB (IκB) and activate nuclear factor κB (NF-κB) in undifferentiated cells, and this pathway was inhibited in the differentiated cells. The increase in ICAM-1 mRNA expression in the differentiated cells was due to increased promoter recruitment of C/EBPß, which transcriptionally up-regulated ICAM-1 mRNA. However, protein expression of ICAM-1 was found to decrease over the course of differentiation due to degradation in the proteasome and lysosome. Immunohistochemistry using tumor samples from colon cancer patients indicated that non-transformed matched normal cells (well-differentiatied) showed no ICAM-1 expression, but the poorly differentiated tumor cells showed higher expression. Functionally, a decrease in adhesion to human umbilical vein endothelial cells was observed in the differentiated Caco-2 cells. Thus, regulation of ICAM-1 and VCAM-1, although both NF-κB target genes, appears to be different over the course of epithelial differentiation in Caco-2 cells.

Celecoxib reduces fluidity and decreases metastatic potential of colon cancer cell lines irrespective of COX-2 expression.

CLX (celecoxib), a selective COX-2 (cyclo-oxygenase-2) inhibitor, has numerous pleiotropic effects on the body that may be independent of its COX-2 inhibitory activity. The cancer chemopreventive ability of CLX, particularly in CRC (colorectal cancer), has been shown in epidemiological studies. Here we have, for the first time, examined the biophysical effects of CLX on the cellular membranes of COX-2 expressing (HT29) and COX-2 non-expressing (SW620) cell lines using ATR-FTIR (attenuated total reflectance-Fourier transform IR) spectroscopy and SL-ESR (spin label-ESR) spectroscopy. Our results show that CLX treatment decreased lipid fluidity in the cancer cell lines irrespective of COX-2 expression status. As metastatic cells have higher membrane fluidity, we examined the effect of CLX on the metastatic potential of these cells. The CLX treatment efficiently decreased the proliferation, anchorage-independent growth, ability to close a scratch wound and migration and invasion of the CRC cell lines through Matrigel. We propose that one of the ways by which CLX exerts its anti-tumorigenic effects is via alterations in cellular membrane fluidity which has a notable impact on the cells' metastatic potential.

Concentration-dependent differing actions of the nonsteroidal anti-inflammatory drug, celecoxib, in distearoyl phosphatidylcholine multilamellar vesicles.

The interactions of the nonsteroidal anti-inflammatory drug, celecoxib, with 1,2-distearoyl-sn-glycero-3-phosphocholine multilamellar vesicles were studied as a function of temperature and different drug concentrations, using Fourier transform infrared spectroscopy, differential scanning calorimetry, and turbidity technique at 440 nm. Our studies reveal that celecoxib lowers the main phase-transition temperature and decreases the fluidity of the membranes at all concentrations. Celecoxib induced opposing effects on molecular order at different concentrations by increasing the ordering of the system at low concentrations and disordering it at high concentrations. Further, the drug increases the number of hydrogen bonds around the carbonyl groups at low concentrations in both phases, whereas the degree of dehydration increases at high concentrations in the gel phase. An evidence of phase separation has also been clearly observed at high concentrations. Thus, depending on the concentration used, celecoxib induces significant changes in the biophysical properties of membranes that may aid in understanding its mechanism of action.

Celecoxib-loaded liposomes: effect of cholesterol on encapsulation and in vitro release characteristics.

CLX (celecoxib) is a highly hydrophobic non-steroidal anti-inflammatory drug with high plasma protein binding. We describe here the encapsulation of CLX in MLVs (multilamellar vesicles) composed of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and variable amounts of cholesterol. The effects of cholesterol content on liposome size, percentage drug loading and in vitro drug release profiles were investigated. Differential scanning calorimetry and FTIR (Fourier-transform infrared) spectroscopy were used to determine molecular interactions between CLX, cholesterol and DSPC. The phase transition temperature (Tm) of vesicles was reduced in a synergistic manner in the presence of both CLX and cholesterol. Encapsulation efficiency, loading and release of CLX decreased with increasing cholesterol content. FTIR results indicated that this decrease was due to a competition between CLX and cholesterol for the co-operativity region of the phospholipids. In the presence of cholesterol, CLX was pushed further into the hydrophobic core of the bilayer. However, MLVs prepared with DSPC only (without cholesterol) exhibited the lowest ability for drug retention after 72 h. Our results indicated that CLX, without the requirement of modifications to enhance solubilization, can be encapsulated and released from liposomal formulations. This method of drug delivery may be used to circumvent the low bioavailability and systemic side effects of oral CLX formulations.