Effects of Oxaliplatin Treatment on the Enteric Glial Cells and Neurons in the Mouse Ileum.

Oxaliplatin, currently used for treatment of colorectal and other cancers, causes severe gastrointestinal side effects, including nausea, vomiting, diarrhea, and constipation that are attributed to mucosal damage. However, delayed onset and long-term persistence of these side effects suggest that damage to the enteric nervous system (ENS) regulating physiological function of the gastrointestinal tract may also occur. The ENS comprises myenteric and submucosal neurons and enteric glial cells (EGCs). This study aimed to investigate the effects of oxaliplatin treatment on enteric neurons and EGCs within the mouse ileum. BALB/c mice received repeated intraperitoneal injections of oxaliplatin (3 mg/kg, 3 injections/week). Tissues were collected 3, 7, 14, and 21 days from the commencement of treatment. Decreases in glial fibrillary acidic protein-immunoreactive (IR) EGCs and protein gene product 9.5/ß-Tubulin III-IR neurons as well as increase in s100ß-IR EGCs after chronic oxaliplatin administration were observed in both the myenteric and submucosal plexi. Changes in EGCs were further observed in cross-sections of the ileum at day 14 and confirmed by Western blotting. Alterations in EGCs correlated with loss of myenteric and submucosal neurons in the ileum from oxaliplatin-treated mice. These changes to the ENS may contribute to the mechanisms underlying gastrointestinal side effects associated with oxaliplatin treatment.

Leukocyte populations and IL-6 in the tumor microenvironment of an orthotopic colorectal cancer model.

Colorectal cancer (CRC) is a major health problem worldwide. It is often diagnosed late due to its asymptomatic nature. As with all cancers, an immune reaction is involved; however, in CRC, it is unknown if this immune response is favorable or unfavorable for disease progression. In this study, the immune response in mesenteric lymph nodes (MLNs) and Peyer's patches was investigated during development of CRC in an orthotopic mouse model. CRC was induced by injecting CT26 cells into the cecum wall of BALB/c mice. Flow cytometry was used to analyze leukocyte populations involved in tumor immunity in MLNs and Peyer's patches. Cryostat sections for immunohistochemistry were prepared from the caecum and colon from CRC-induced and sham-operated animals. Cytokines produced by mouse CT26 cell line were measuredin vitroandin vivo Significant increases in the number of CD8(+)/TCR(+)and CD49b(+)/TCR(-)(natural killer) cells were found in MLNs and Peyer's patches in the CRC group. In addition, γδT cells were present in the lamina propria of the colon tissues from sham-operated mice, but absent in the colon tissues from mice with CRC. Immunohistochemical analysis of tumorous tissues showed eosinophil, CD69(+)T cell, and CD11b(+)cell infiltration. Bothin vitroandin vivoCT26 tumor cells were interleukin (IL)-6 positive. In addition, tumor-infiltrating CD45(+)cells were also IL-6 positive. In summary, the kinetics of the immune response to CRC and the key effector lymphocytes that are implicated in tumor immunity are demonstrated. Furthermore, IL-6 is a key cytokine present within the tumor microenvironment.

Loss of cell-surface receptor EphB2 is important for the growth, migration, and invasiveness of a colon cancer cell line.

PURPOSE: In normal colonic epithelium, the receptor tyrosine kinase, EphB2 interacts with ephrinB1 ligand to maintain the integrity and architecture of the colonic crypt. Loss of EphB2 is seen in most colorectal cancers and correlates with poor prognosis. In this study, we investigated the effects of two levels of EphB2 expression on cell migration and invasion in a colon cancer cell line and on the growth of tumour xenografts. METHODS: An EphB2-negative colon cancer cell line (LIM2405) was transfected with a full-length EphB2 cDNA in a vector designed to respond to the drug tetracycline. The effect of two levels of EphB2 expression on the ability of cells to migrate through a porous barrier in response to a chemo-attractant and to invade through artificial basement membranes was tested in vitro. Finally, the effects of two expression levels of EphB2 on tumour growth using an in vivo model of colonic tumour xenograft in a mouse model were assessed. RESULTS: Expression of moderate levels of EphB2 significantly reduced the migration of tumour cells compared to control (p < 0.05, Kruskal-Wallis test). Expression of high levels of EphB2 further reduced migration of tumour cells (p < 0.05, Kruskal-Wallis test). Similarly, expression of EphB2 markedly reduces the invasive ability of tumour cells. The in vivo model of tumour growth showed that tumours with the highest level of EphB2 expression had a reduced risk of reaching a 10-mm size (defined event) compared with the control group (Cox regression, hazard ratio (HR) = 0.052, 95% CI 0.005-0.550; p = 0.014). Tumours derived from EphB2 expressing cells had a significantly reduced number of mitotic figures (p < 0.05) and an increased number of apoptotic cells (p < 0.05) compared to tumours from control cells. CONCLUSION: Even a moderate level of EphB2 expression has effects on tumour cells which results in reduced migration and invasiveness and slows the growth of colonic tumour implants in an in vivo model.

Stromal interaction molecule 1 (STIM1), a transmembrane protein with growth suppressor activity, contains an extracellular SAM domain modified by N-linked glycosylation.

Stromal interaction molecule 1 (STIM1) is a cell surface transmembrane glycoprotein implicated in tumour growth control and stromal-haematopoietic cell interactions. A single sterile alpha motif (SAM) protein-protein interaction domain is modelled within its extracellular region, a subcellular localisation not previously described for other SAM domain-containing proteins. We have defined the transmembrane topology of STIM1 by determining the sites of N-linked glycosylation. We have confirmed that STIM1 is modified by N-linked glycosylation at two sites within the SAM domain itself, deduced as asparagine residues N131 and N171, demonstrating that STIM1 is translocated across the membrane of the endoplasmic reticulum such that the SAM domain resides within the endoplasmic reticulum (ER) lumen. Both N-linked oligosaccharides remain endoglycosidase H-sensitive, indicating absence of full processing within the ER and Golgi. This immature modification is nevertheless sufficient and critical for cell surface expression of STIM1. We show that STIM1-STIM1 homotypic interactions are mediated via the cytoplasmic rather than the extracellular region of STIM1, excluding an essential role for the SAM domain in these protein interactions. These studies provide the first evidence for an extracellular localisation of a SAM domain within any protein, and the first example of a SAM domain modified by N-linked glycosylation.