A. Carducci

Cytotoxic drugs up-regulate epidermal growth factor receptor (EGFR) expression in colon cancer cells and enhance their susceptibility to EGFR-targeted antibody-dependent cell-mediated-cytotoxicity (ADCC).

Cetuximab is a human-murine chimeric IgG1 monoclonal antibody to epidermal growth factor-receptor (EGFR) which exerts synergistic antitumour interactions with several cytotoxic drugs. Therefore, it is presently recommended in combination with chemotherapy in the treatment of colon, head and neck and non-small cell lung cancer. Cetuximab has been designed to inhibit EGFR signalling; however, preclinical evidence suggests that its anti-cancer effects in vivo are also related to the ability of its human IgG1 backbone to trigger immunological mechanisms. Here we have investigated whether the exposure to different cytotoxic drugs may affect the susceptibility of colon cancer cells in vitro to cetuximab immuno-targeting and related lymphokine-activated killer (LAK)-mediated antibody-dependent cell cytotoxicity (ADCC). Five colon cancer cell lines expressing a different k-ras mutational status were evaluated for: (i) EGFR-expression, (ii) susceptibility to LAK cells and (iii) cetuximab-mediated ADCC, before and after exposure to 5-flurouracil (5-FU), gemcitabine (Gem), irinotecan (Iri) alone or in multiple two/three drug combinations. These drugs were able to up-regulate EGFR expression on the surface of all the colon cancer cell lines with a maximal effect observed few hours after the exposure to GILF regimen (Gem, Iri, Levofolinic acid and 5-FU). Chemotherapy was able to greatly enhance the sensitivity to either LAK cells or cetuximab-mediated ADCC in all the colon cancer cell lines with a mechanism independent from k-ras status. The results of our study suggest that chemotherapy may enhance cetuximab-mediated immuno-targeting and ADCC thus providing the rationale to design novel immuno-biochemotherapy regimens.

The Translationally Controlled Tumor Protein Is a Novel Calcium Binding Protein of the Human Placenta and Regulates Calcium Handling in Trophoblast Cells

Abstract The translationally controlled tumor protein (TPT1, also known as TCTP) is a highly conserved, abundantly expressed protein found in mammals as well as in a wide range of other organisms of both the animal and plant kingdom. Initially considered as a growth-related protein, later studies showed TPT1 is endowed with multiple biological activities, including calcium binding. The present study aimed to evaluate the expression of TPT1 in the human placenta and to examine the functional role of the protein in the calcium binding and homeostasis of trophoblast cells. Samples were analyzed by Western blot, reverse transcription-polymerase chain reaction and immunohistochemistry. The effect of TPT1 knockdown by small interfering RNA (siRNA) on calcium uptake and buffering was assessed in the HTR-8/SVneo cell line. TPT1 protein and mRNA were detected in first-trimester and term placenta. In the tissue, TPT1 was localized in the villous trophoblast. TPT1 expression significantly increased during gestation, with the higher protein and mRNA levels reached at term. Recombinant placental TPT1 bound calcium in vitro, while downregulation of the protein levels by siRNA in HTR-8/SVneo cells was associated with a reduced cellular calcium-uptake activity and buffering capacity. These data demonstrate, for the first time, the expression of TPT1 in the human placenta and support a direct role of the protein in placental calcium transport.

11β-Hydroxysteroid dehydrogenase expression in first trimester human trophoblasts

This study evaluated the levels and the enzymatic characteristics of 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) of chorionic villi isolated from first trimester human placenta. The results demonstrated a predominant expression of the NAD-dependent dehydrogenase isoform (11beta-HSD2) over the NADP-dependent oxoreductase (11beta-HSD1). Thus, in tissue homogenates exogenous NAD increased the conversion of corticosterone to 11-dehydrocorticosterone of about 14-fold while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in villous tissue while 11beta-HSD1 mRNA levels were undetectable. In addition, immunohistochemical staining localized the 11beta-HSD2 protein to syncytiotrophoblasts and cell columns of the chorionic villi. These results suggest roles for the trophoblast-associated 11beta-HSD2 oxidative activity in modulating the exposure of the embryo to active glucocorticoids in the early gestation and in regulating trophoblasts invasion of the uterine wall.

Progestin regulation of 11β-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells

Abstract This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11β-HSD2 in T-47D cells, while 11β-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11β-HSD catalytic activity was elevated 11-fold, while estrone (E 1 ), estradiol (E 2 ) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11β-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11β-HSD2 gene expression, increasing the steady-state levels of 11β-HSD2 mRNA. Taken together, these results demonstrate that 11β-HSD2 is the 11β-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.

Sheep (Ovis aries) Macrophage Migration Inhibitory Factor: molecular cloning, characterization, tissue distribution and expression in the ewe reproductive tract and in the placenta.

Macrophage Migration Inhibitory Factor (MIF) is a pivotal regulator of innate and acquired immunity affecting the response and behavior of macrophages and lymphocytes. However, a number of studies indicated wider physiological functions for this cytokine to include key-roles in reproductive biology. The present study was designed to clone the coding sequence of sheep MIF, to examine the characteristics of the protein in vitro, and to evaluate its expression in sheep tissues and in the ewe reproductive tract in vivo. Ovine MIF cDNA consisted of 348 nucleotides encoding a 115 amino acids protein with an estimated molecular mass of 12,343 Da and an isoelectric point of 7.68. Sheep MIF shared high amino acid identity with the other mammalian MIF family members and showed parallel functions to human MIF, displaying enzymatic oxoreductase activity and inducing monocyte transmigration. Expression studies detected a MIF transcript in all the sheep tissues examined. Among reproductive tissues, MIF mRNA and protein were detected in the ovary, oviduct, uterus and placenta. These results indicate that sheep MIF shares crucial features with other MIF family members and delineate its potential involvement in several aspects of ovine physiology.

Platelet Activation in Hypercholesterolemic Patients Submitted to Therapeutic Plasmapheresis. An Ultrastructural Study.

Therapeutic plasmapheresis has been recommended as the choice therapy in patients with familial hypercholesterolemia. Little is known about the effect of plasmapheresis on platelet behavior. By means of electron microscopy we have studied the platelet plasma membrane of 4 patients with familial hypercholesterolemia who were submitted to repeated plasmaphereses. After each procedure of plasmapheresis, at the 15th day, morphometrical studies revealed a statistically significant increase in the surface density of the Open Canalicular System, which is considered a marker of platelet activation. However, during 12 months of plasmapheresis, a significant mean decrease in the morphometric parameters was observed. On one hand, these results indicate the necessity to consider the blood hemocoagulatory state in the patient who has been submitted to the above mentioned treatment, since the variation of these parameters after the therapeutic procedure, in a short time, could be potentially harmful to the patient; on the other hand, these results indicate, for the first time, the ability of the procedure to improve platelet behavior after repeated treatments on long term basis.