J. Thomas Pento

Time-dependent deterioration of active transport in duodenal segments of rat intestine

In this study, a time-dependent deterioration of the active transport in duodenal segments of rat intestine was observed. The everted gut-sac technique was used to quantitate both calcium and glucose transport in intestinal segments. The results indicate that both calcium and glucose transport decreased significantly in intestinal segments of animals killed by cervical dislocation 10-20 min prior to tissue removal. It was further determined that animal anesthesia permitted excision of intestinal segments prior to death and thus avoided transport deterioration.

Selective Growth Inhibition of Cancer Cells by L-Methioninase-Containing Fusion Protein Targeted to the Urokinase Receptor

Background: We have reported the development of a novel fusion protein (FP) consisting of an amino-terminal fragment of urokinase linked to the amino terminus of the enzyme L-methioninase (L-M). The present study compared the effect of this novel FP on the proliferation of human ovarian, skin, breast endometrial and pancreatic cancer cell lines. Methods: The FP, L-M and a mutated FP, with reduced L-M activity, were produced by recombinant methods. The effect of treatment with FP, L-M and mutated FP on the proliferation of the cancer cells was measured in vitro using an MTS assay. Results: The inhibitory effect of the FP was found to be significantly greater than that of L-M alone or the mutated FP. In addition, the FP produced a greater inhibitory effect on an ovarian cancer cell line than on comparable normal, non-cancerous cells. Further, the FP produced a dose-dependent inhibition of the proliferation of pancreatic cancer cell lines. Conclusion: These results suggest that this FP is a potent and selective inhibitor of the proliferation of various cancer cell lines and has potential as a therapeutic agent for the treatment of various methionine-dependent cancers.

Targeting of a novel fusion protein containing methioninase to the urokinase receptor to inhibit breast cancer cell migration and proliferation.

It has been shown that methionine depletion inhibits tumor cell growth and reduces tumor cell survival. A novel fusion protein targeted specifically to tumor cells was developed. The fusion protein contained two components: the amino terminal fragment of human urokinase (amino acids 1–49) that binds to the urokinase receptor protein expressed on the surface of invasive cancer cells, and the enzyme l-methioninase (containing 398 amino acids) which depletes methionine and arrests the growth of methionine-dependent tumors. The influence of the fusion protein on the growth and motility of human breast cancer cells was examined using a culture wounding assay. It was determined that MCF-7 breast cancer cells, used in this study, were methionine-dependent and that the fusion protein bound specifically to urokinase receptors of the surface of the cancer cells. Further treatment of the cancer cells with fusion protein over the concentration range 10−8 to 10−6xa0M produced a dose-dependent inhibition of both the migration and proliferation index of MCF-7 cells in the culture wounding assay over a period of 1 to 3xa0days. The results of this study suggest that this novel fusion protein may serve as a prototype for specific targeting of methioninase and perhaps other cytotoxic agents to cancer cells.

Synthesis and in vivo evaluation of N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide for inhibition of intestinal tumorigenesis in APCMin/+ mice

A selective KGFR tyrosine kinase inhibitor, N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide, was synthesized and its possible inhibitory effects on the development of colon polyps and colorectal tumors was examined in APC(Min/+) mice, a mouse model of human intestinal familial adenomatous polyposis. The present study shows for the first time that a dietary administration of a selective KGFR tyrosine kinase inhibitor lacks the overt-toxicities and significantly reduced the growth of small intestinal polyps in both male and female APC(Min/+) mice. This inhibition of polyp growth appears to occur at a greater extent in female mice.

Expression of prohibitin 3? untranslated region suppressor RNA alters morphology and inhibits motility of breast cancer cells

The prohibitin 3′ untranslated region (3′UTR) belongs to a novel class of non-coding regulatory RNAs. It arrests cell cycle progression by blocking G1-S transition in breast and other cancers. Our previous studies comparing MCF7 derived clones constitutively expressing a common allelic form of prohibitin RNA (UTR/C) to various controls demonstrated that it functions as a tumor suppressor. Here, we further characterized the morphology and motility of these transgenic breast cancer cells when grown in cell culture and on nude mice. In contrast to empty vector (EV) cells, UTR/C cells were observed to grow in an organized manner with more cell-cell contact and differentiate into structures with a duct-like appearance. Computer assisted cytometry to evaluate differences in nuclear morphology was performed on UTR/C and EV tissues from nude mice. Receiver operator curve areas generated using a logistic regression model were 0.8, indicating the ability to quantitatively distinguish UTR/C from EV tissues. Keratinocyte growth factor-induced motility experiments showed that migration of UTR/C cells was significantly reduced (80–90%) compared to EV cells. Together, these data indicate that this novel 3′UTR influences not only the tumorigenic phenotype but also may play a role in differentiation and migration of breast cancer cells.