Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor.

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA- was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.

In vitro and in vivo evaluation of the radiosensitizing effect of a selective FGFR inhibitor (JNJ-42756493) for rectal cancer.

BACKGROUND: We examined the anti-tumor effect and radiosensitizing potential of a small molecule inhibitor of fibroblast growth factor receptor (FGFR) in colorectal cancer (CRC) in vitro and in vivo. METHODS: Effects of in vitro drug treatment on cell survival, proliferation, FGFR signaling, cell cycle distribution, apoptosis and radiosensitivity were assessed using various CRC cell lines with FGFR wild type (Caco2 and HCA7) and FGFR2 amplification (HCT116, NCI-H716). In vivo tumor responses to FGFR inhibition with and without radiation therapy were evaluated by growth delay assays in two colorectal xenograft mouse models (NMRI nu/nu mice injected with NCI-H716 or CaCo2 cells). Mechanistic studies were conducted using Western blot analysis, immunohistochemistry and qPCR. RESULTS: In the tested cell lines, the FGFR inhibitor (JNJ-42756493) was effective in vitro and in vivo in CRC tumors with highest expression of FGFR2 (NCI-H716). In vitro, cell proliferation in this line was decreased, associated with increased apoptotic death and decreased cell survival. In vivo, growth of NCI-H716 tumors was delayed by 5 days by drug treatment alone, although when drug delivery was stopped the relative tumor volume increased compared to control. The FGFR inhibitor did not radiosensitize NCI-H716 tumors either in vitro or in vivo. CONCLUSIONS: Among tested CRC cell lines, the growth inhibitory activity of this FGFR inhibitor was evident in cell lines with high constitutive FGFR2 expression, suggesting that FGFR addiction may provide a window for therapeutic intervention, though caution is advised. Preclinical study with NCI-H716 and Caco2 tumor demonstrated that continued presence of drug could be essential for tumor growth control, especially in cells with aberrant FGFR expression. In the tested set-up, the inhibitor showed no radiosensitizing effect.

Investigation of possible endogenous hypoxia markers in colorectal cancer.

OBJECTIVE: We evaluated the potential of some recently proposed hypoxia markers, being monocarboxylic acid transporter 1 (MCT1), MCT4 and prolyl hydroxylase 2 (PHD2); and a more established hypoxia marker, glucose transporter-1 (GLUT-1), by testing the association with the exogenous marker pimonidazole. MATERIALS AND METHODS: Paraffin embedded tumour sections of 20 colorectal cancer patients were stained for blood vessels together with either pimonidazole or carbonic anhydrase-IX (CA-IX) and single stained for MCT1, MCT4, GLUT-1, and PHD2. Expression of all markers was compared with expression of pimonidazole and micro-vessel density (MVD) and with disease-free survival (DFS) and overall survival (OS). RESULTS: No correlation was found between the different intrinsic hypoxia markers tested and pimonidazole. A trend for high MCT1 expression in biopsies with low CA-IX expression was found (R = -0.45, p = 0.06) and also the expression of MCT1 was higher in tumours with a high MVD (R = 0.49, p = 0.04). The more advanced tumours showed a higher expression of GLUT-1 (p = 0.03). A low CA-IX expression in the tumour correlated with better DFS (p = 0.03) and related to better OS (p = 0.07). CONCLUSION: Although none of the tested intrinsic hypoxia markers correlated with pimonidazole staining, we confirmed the important role of both GLUT-1 and CA-IX for a more advanced pTNM (pathological tumour-node-metastasis) stage and DFS respectively.