Tiziana Marrocco

The Antiproliferative Effect of Synthetic Peptidyl GH Secretagogues in Human CALU-1 Lung Carcinoma Cells

The specific binding of [125I]Tyr-Ala-hexarelin, a radiolabeled peptidyl GH secretagogue (GHS), has been investigated in nontumoral and neoplastic human lung tissues. This binding was very marked in nonendocrine lung carcinomas with values that were greater than found in either normal lung or in endocrine lung neoplasms. Tyr-Ala-hexarelin binding was also present in a human lung carcinoma cell line (CALU-1). [125I]Tyr-Ala-hexarelin binding to tumor membranes was displaced by peptidyl GHS (GHRP-6, hexarelin) and EP-80317, an hexarelin analog devoid of GH-releasing activity in vivo. In contrast, no competition was observed in the presence of the nonpeptidyl GHS MK-0677 and the endogenous ligand of the GHS-R1a ghrelin. GHS-R1a mRNA expression was found in 50% of endocrine lung tumors but was never seen in other nontumoral and neoplastic lung tissues nor in CALU-1. In these cells, hexarelin and EP-80317, but not ghrelin or MK-0677, caused a dose-dependent inhibition of IGF-II-stimulated thymidine incorporatio...

Inhibition of erythropoiesis in malaria anemia: role of hemozoin and hemozoin-generated 4-hydroxynonenal.

Severe malaria anemia is characterized by inhibited/altered erythropoiesis and presence of hemozoin-(HZ)-laden bone-marrow macrophages. HZ mediates peroxidation of unsaturated fatty acids and production of bioactive aldehydes such as 4-hydroxynonenal (HNE). HZ-laden human monocytes inhibited growth of cocultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts. Cocultivation with HZ or treatment with low micromolar HNE inhibited growth of erythroid cells interfering with cell cycle without apoptosis. After HZ/HNE treatment, 2 critical proteins in cell-cycle regulation, p53 and p21, were increased and the retinoblastoma protein, central regulator of G₁-to-S-phase transition, was consequently hypophosphorylated, while GATA-1, master transcription factor in erythropoiesis was reduced. The resultant decreased expression of cyclin A and D2 retarded cell-cycle progression in erythroid cells and the K562 cell line. As a second major effect, HZ and HNE inhibited protein expression of crucial receptors (R): transferrinR1, stem cell factorR, interleukin-3R, and erythropoietinR. The reduced receptor expression and the impaired cell-cycle activity decreased the production of cells expressing glycophorin-A and hemoglobin. Present data confirm the inhibitory role of HZ, identify HNE as one HZ-generated inhibitory molecule and describe molecular targets of HNE in erythroid progenitors possibly involved in erythropoiesis inhibition in malaria anemia.

Oxytocin Induces Proliferation and Migration in Immortalized Human Dermal Microvascular Endothelial Cells and Human Breast Tumor-Derived Endothelial Cells

Oxytocin either increases or inhibits cell growth in different cell subtypes. We tested here the effect of oxytocin on cell proliferation and migration of human dermal microvascular endothelial cells (HMEC) and tumor-associated endothelial cells purified from human breast carcinomas (B-TEC). Oxytocin receptors were expressed in both cell subtypes at mRNA and protein levels. Through oxytocin receptor, oxytocin (1 nmol/L-1 μmol/L) significantly increased cell proliferation and migration in both HMEC and B-TEC, and addition of a selective oxytocin antagonist fully reverted these effects. To verify whether a different expression of adhesion molecule-related genes could be responsible for the oxytocin-induced cell migration, untreated and treated cells were compared applying a microarray technique. In HMEC, oxytocin induced the overexpression of the matrix metalloproteinase (MMP)-17, cathepsin D, and integrin β6 genes. In B-TEC, oxytocin significantly switched on the gene profile of some MMP (MMP-11 and MMP-26) and of integrin β6. The up-regulation of the integrin β6 gene could be involved in the oxytocin-induced cell growth, because this subunit is known to determine activation of mitogen-activated protein kinase-extracellular signal-regulated kinase 2, which is involved in the oxytocin mitogenic effect. In B-TEC, oxytocin also increased the expression of caveolin-1 at gene and protein levels. Because oxytocin receptor localization within caveolin-1-enriched membrane domains is necessary for activation of the proliferative (instead of the inhibitory) response to oxytocin, its enhanced expression can be involved in the oxytocin-induced B-TEC growth as well. Altogether, these data indicate that oxytocin contributes to cell motility and growth in HMEC and B-TEC. (Mol Cancer Res 2006;4(6):351–9)

Oxytocin and Oxytocin Receptors in Cancer Cells and Proliferation

The hypothalamic nonapeptide oxytocin plays a crucial role in many reproductive and behavioural functions. However, in recent years, an additional new role for oxytocin has been identified in neoplastic pathology. In tumours, oxytocin acts as a growth regulator, through the activation of a specific G‐coupled transmembrane receptor, the oxytocin receptor. In vitro, oxytocin inhibits proliferation of neoplastic cells of either epithelial (mammary and endometrial), nervous or bone origin, all expressing oxytocin receptor. Furthermore, an oxytocin growth‐inhibiting effect was also tested and confirmed in vivo in mouse and rat mammary carcinomas. In neoplastic cells derived from two additional oxytocin target tissues, trophoblast and endothelium, oxytocin was found to promote cell proliferation, an effect opposite to that previously described in all other neoplastic oxytocin‐responsive cells. The signal transduction pathways coupled to the biological effects of oxytocin are different in oxytocin growth‐inhibited or growth‐stimulated cells, and may depend on the membrane localization of the oxytocin receptor itself. The inhibitory effect of oxytocin is apparently mediated by activation of the cAMP‐protein kinase A pathway, a nonconventional oxytocin signalling pathway, whereas the mitogenic effect is coupled to the increase of intracellular [Ca2+] and tyrosine phosphorylation, ‘classical’ oxytocin transducers. Moreover, the oxytocin receptor localization in lipid rafts enriched in caveolin‐1 turns the inhibition of cell growth into a proliferative response, eliciting different epidermal growth factor receptor/mitogen‐activated protein kinase activation patterns. This unexpected role of oxytocin (and oxytocin analogues) in regulating cell proliferation, as well as the widespread expression of oxytocin receptors in neoplastic tissues of different origin, opens up new perspectives on the biological role of the oxytocin–oxytocin receptor system in cancer.

Ghrelin and cortistatin in lung cancer: expression of peptides and related receptors in human primary tumors and in vitro effect on the H345 small cell carcinoma cell line.

Ghrelin, a natural GH secretagog (GHS) acylated peptide, and cortistatin (CST), a natural SRIF-like peptide, interfere with neoplastic growth in different cancers. We tested forty-one lung carcinomas and the H345 small cell lung carcinoma (SCLC) cell line by RT-PCR to investigate the presence of ghrelin and CST and related receptors, including type 1a GHS receptor (GHS-R1a), all SRIF-receptor subtypes (sst 1–5) and MRGX2. Moreover, the presence of ghrelin and CST peptides was studied in both tumors and H345 cells. Ghrelin and CST mRNA were present in the majority of tested tumors, but ghrelin and CST proteins were revealed only in tumors with a neuroendocrine phenotype. All the receptors mRNA had a heterogeneous expression without correlation between ghrelin (or CST) and their receptor distribution. All the transcripts, but not GHS-R1a, were expressed in H345 cells. However, ghrelin and desacyl ghrelin induced in vitro a dose-dependent inhibition on the H345 cell proliferation and increased apoptosis. Conversely, neither CST nor SRIF affected H345 cell growth, despite the presence of their specific receptors. The anti-proliferative and the pro-apoptotic effects of ghrelin were consistent with binding experiments on H345 cell, where either acylated or des-acylated ghrelin recognized a common binding site. In conclusion, the present study indicates that: a) ghrelin and CST mRNAs are expressed in lung cancers, although some neuroendocrine tumors contain detectable amounts of the peptides; b) GHSR-1a mRNA is present exclusively in neuroen-docrine tumors, whereas MRGX2 mRNA (but not peptide) is expressed in all histological types; c) both ghrelin forms inhibit H345 cell proliferation, both directly and enhancing apoptosis, despite the absence of GHS-R1a, whereas CST and its receptors do not interfere with cell growth.

Cortistatin-14 inhibits cell proliferation of human thyroid carcinoma cell lines of both follicular and parafollicular origin.

Cortistatin (CST-14, Pro-c[Cys-Lys- Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys]-Lys- NH2), a neuropeptide member of the SRIH family, binds to all 5 SRIH receptor (sst) subtypes, but also possesses a significant binding affinity to GH secretagogue receptors (GHS-R), which have been reported to mediate the antiproliferative activity of GHS on thyroid cancer cells. The effect of CST-14 on cell proliferation was studied in 3 different human thyroid carcinoma cell lines of follicular origin (N-PAP, WRO, ARO) and in one thyroid medullary carcinoma cell line (TT). CST-14 1 μM determined a significant inhibition of cell proliferation in TT, N-PAP and WRO cells and this effect was dose-dependent and more pronounced than that displayed by SRIH-14 (Ala- Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr- Ser-Cys]-OH) treatment. To a minor extent, CST- 14, but not SRIH-14, also temporary inhibited ARO cell proliferation. By immunofluorescence, sst2, sst3 and sst5 have been demonstrated in TT cells, whereas types 3 and 5 only were expressed in N-PAP and WRO cells, and no sst subtype was found in ARO cells. The presence of both GHS-R1a and 1b mRNA has been studied and demonstrated in the TT medullary carcinoma cell line, whereas follicular derived cell lines were already known to express GHS binding sites. Addition of EP-80874 (D-Mrp-c[D-CyspyridilalanyI3- D-Trp-Lys-Val-Cys]-Mrp-NH2), a synthetic peptide that binds to SRIH and GHS-R, completely abolished the antiproliferative effects of CST-14 or SRIH-14 on sst/GHS-R positive thyroid carcinoma cell lines (WRO, N-PAP and TT). EP-80874 was also able to antagonize the inhibitory activity of CST-14 on the growth of cells (ARO) expressing GHS-R but not sst. Taken together, these data firstly demonstrate that EP- 80874 has a mixed SRIH/CST antagonist activity and suggest that the oncostatic effect of CST-14 on thyroid cancer cells could be mediated by both sst and/or GHS-R.

Blood oxidative stress markers and Plasmodium falciparum malaria in non-immune African children.

Converging in vitro evidence and clinical data indicate that oxidative stress may play important roles in Plasmodium falciparum malaria, notably in the pathogenesis of severe anaemia. However, oxidative modifications of the red blood cell (RBC)‐membrane by 4‐hydroxynonenal (4‐HNE) and haemoglobin‐binding, previously hypothesized to contribute mechanistically to the pathogenesis of clinical malaria, have not yet been tested for clinical significance. In 349 non‐immune Mozambican newborns recruited in a double‐blind placebo‐controlled chemoprophylaxis trial, oxidative markers including 4‐HNE‐conjugates and membrane‐bound haemoglobin were longitudinally assessed from 2·5 to 24 months of age, at first acute malaria episode and in convalescence. During acute malaria, 4‐HNE‐conjugates were shown to increase significantly in parasitized and non‐parasitized RBCs. In parallel, advanced oxidation protein products (AOPP) rose in plasma. 4‐HNE‐conjugates correlated with AOPP and established plasma but not with RBC oxidative markers. High individual levels of 4‐HNE‐conjugates were predictive for increased malaria incidence rates in children until 2 years of life and elevated 4‐HNE‐conjugates in convalescence accompanied sustained anaemia after a malaria episode, indicating 4‐HNE‐conjugates as a novel patho‐mechanistic factor in malaria. A second oxidative marker, haemoglobin binding to RBC‐membranes, hypothesized to induce clearing of RBCs from circulation, was predictive for lower malaria incidence rates. Further studies will show whether or not higher membrane‐haemoglobin values at the first malaria episode may provide protection against malaria.

Oxyphilic and non-oxyphilic thyroid carcinoma cell lines differ in expressing apoptosis-related genes

Oxyphilic tumors of the thyroid are characterized by mitochondrion-rich cells and extensive DNA fragmentation. In order to clarify if a different expression of apoptosis-related genes could be responsible for DNA fragmentation in oxyphilic cell tumors, two thyroid follicular carcinoma-derived cell lines, having oxyphilic (XTC.UC1) and non-oxyphilic (WRO) features, were compared applying a gene array technique. Under basal culture conditions, several pro-apoptotic genes [caspases 3 and 10, Fas and the tumor necrosis factor-related apoptosis-inducing ligand (trail) genes] were switched on in oxyphilic, but not in non-oxyphilic cells. No difference in the mitochondrial apoptosis-related genes (bax, bad, bcl family etc.) was observed. Using the ISEL technique, the extent of DNA fragmentation did not differ under basal conditions in the two cell lines. Conversely, following an oxidative pro-apoptotic stress (6-h methylene blue treatment and light exposure), XTC.UC1 cells showed an extensive DNA fragmentation (up to 70% of cells), dramatically exceeding that observed in WRO cells (up to 20% of cells). In contrast, the oxidative stimulus induced a remarkable apoptosis gene activation in non-oxyphilic WRO cells only. These results suggest that oxyphilic cells may have a unique silent activation of a pro-apoptotic phenotype, which could be responsible for DNA instability and lead to cell death as the consequence of an increased sensitivity to ischemic stresses, as frequently observed in vivo.

No red blood cell damage and no hemolysis in G6PD-deficient subjects after ingestion of low vicine/convicine Vicia faba seeds

TO THE EDITOR: Favism, or “favic crisis,” is a potentially life-threatening acute hemolysis elicited in carriers of low-activity glucose 6-phosphate dehydrogenase (G6PD) variants by ingestion of raw faba bean ( Vicia faba L) (FB) seeds.[1][1],[2][2] In 2 surveys of hemolytic crises because of