Riccardo Spizzo

Increased blood volume and CD34+CD38- progenitor cell recovery using a novel umbilical cord blood collection system

A major problem with the use of umbilical cord/placental blood (UCB) is the limited blood volume that can be collected from a single donor. In this study, we evaluated a novel system for the collection of UCB and analyzed the kinetics of output of hematopoietic stem cells in the collected blood.

Expression of thymidine phosphorylase and cyclooxygenase-2 in melanoma.

Several studies have reported an increase in vascular structures in malignant melanoma. Neovascularization can be enhanced by several factors. Among them, thymidine phosphorylase (TP) and cyclooxygenase-2 (COX-2) have been reported to play a role. The expressions of TP and COX-2 were evaluated trough immunohistochemistry in a series of 78 primary cutaneous melanomas diagnosed between 2000 and 2004. The expressions of TP and COX-2 through mRNA and western blot analysis were also evaluated in several melanoma cell lines. TP expression and COX-2 expression were considered positive in 25 cases (32%) and 22 cases (28.2%), respectively. TP-positive melanomas showed a lower mitotic rate (P=0.008), smaller thickness (P=0.01), and absence of lymphovascular invasion (P=0.04). COX-2-positive melanomas showed a higher mitotic rate (P=0.01) and higher thickness (P=0.03). COX-2 expression was associated with reduced disease-free survival (P=0.01). COX-2-positive cases showed a trend toward reduced survival, whereas TP was not correlated with overall survival. COX-2 expression was detected in four of 11 melanoma cell lines both by mRNA and by western blot analysis. Our data show that TP expression is associated with more favorable prognostic factors (such as thin melanoma, low mitotic count, and absence of lymphovascular invasion), whereas COX-2 expression is associated with poor prognostic factors (thicker melanoma and high mitotic count).

BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress

Rs3814113 is the single-nucleotide polymorphism (SNP) showing the strongest association with high-grade serous ovarian carcinoma (HGSOC) incidence and is located in an intergenic region about 44u2009kb downstream of basonuclin 2 (BNC2) gene. Lifetime number of ovulations is associated with increased risk to develop HGSOC, probably because of cell damage of extrauterine Müllerian epithelium by ovulation-induced oxidative stress. However, the impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on the damage induced by oxidative stress remains unclear. Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact on cell survival after oxidative stress. To do this, we analyzed gene expression levels of BNC2 first in HGSOC data sets and then in an isogenic cell line that we engineered to carry a 5u2009kb deletion around rs3814113. Finally, we silenced BNC2 and measured surviving cells after hydrogen peroxide (H2O2) treatment to simulate oxidative stress after ovulation. In this paper, we describe that BNC2 expression levels are reduced in HGSOC samples compared with control samples, and that BNC2 expression levels decrease following oxidative stress and ovulation in vitro and in vivo, respectively. Moreover, deletion of 5u2009kb surrounding rs3814113 decreases BNC2 expression levels in an isogenic cell line, and silencing of BNC2 expression levels increases cell survival after H2O2 treatment. Altogether, our findings suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that probably, in the initial phases of oncogenic transformation, conferred resistance to oxidative stress and ultimately reduced the clearance of cells with oxidative-induced damages.

Exploring the Role of Fallopian Ciliated Cells in the Pathogenesis of High-Grade Serous Ovarian Cancer

High-grade serous epithelial ovarian cancer (HGSOC) is the fifth leading cause of cancer death in women and the first among gynecological malignancies. Despite an initial response to standard chemotherapy, most HGSOC patients relapse. To improve treatment options, we must continue investigating tumor biology. Tumor characteristics (e.g., risk factors and epidemiology) are valuable clues to accomplish this task. The two most frequent risk factors for HGSOC are the lifetime number of ovulations, which is associated with increased oxidative stress in the pelvic area caused by ovulation fluid, and a positive family history due to genetic factors. In the attempt to identify novel genetic factors (i.e., genes) associated with HGSOC, we observed that several genes in linkage with HGSOC are expressed in the ciliated cells of the fallopian tube. This finding made us hypothesize that ciliated cells, despite not being the cell of origin for HGSOC, may take part in HGSOC tumor initiation. Specifically, malfunction of the ciliary beat impairs the laminar fluid flow above the fallopian tube epithelia, thus likely reducing the clearance of oxidative stress caused by follicular fluid. Herein, we review the up-to-date findings dealing with HGSOC predisposition with the hypothesis that fallopian ciliated cells take part in HGSOC onset. Finally, we review the up-to-date literature concerning genes that are located in genomic loci associated with epithelial ovarian cancer (EOC) predisposition that are expressed by the fallopian ciliated cells.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O 2ˉ · production in cancer cells

BackgroundThe TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy.MethodsA panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection.ResultsWe demonstrate that oncogenic mutant p53 isoforms are able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1α/UCP2 axis and mitochondrial O2ˉ· production. We also show a correlation between the decrease of reduced thiols with a poorer clinical outcome of CLL patients bearing mutant TP53 gene. The restoration of the mitochondrial uncoupling protein 2 (UCP2) expression, as well as the addition of the radical scavenger N-acetyl-l-cysteine, reversed the oncogenic effects of mutant p53 as cellular hyper-proliferation, antiapoptotic effect, and resistance to drugs.ConclusionsThe inhibition of the SESN1/AMPK/PGC-1α/UCP2 axis contributes to the pro-oxidant and oncogenic effects of mutant p53, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing mutant TP53 gene.