Carlo Torri

Ukrain modulates glial fibrillary acidic protein, but not connexin 43 expression, and induces apoptosis in human cultured glioblastoma cells

Glioblastoma is a highly malignant tumor, characterized by an unfavorable prognosis even in response to multidisciplinary treatment strategies, owing to its high-invasive phenotype. Ukrain, a semisynthetic thiophosphoric acid derivative of the purified alkaloid chelidonine, has been used in the therapy of several solid tumors, but little is known about its effect on glioblastoma and, in general, about the molecular mechanisms responsible for its effects. In particular, we previously demonstrated that Ukrain modulates the expression of genes and proteins involved in tumor invasion, and here we investigate some unreported effects of Ukrain on human cultured glioblastoma cells. We used morphological and molecular biology methods to analyze the expression and the intracellular distribution pattern of glial fibrillary acidic protein, the expression of the gap junction protein connexin 43 and the apoptotic effect in human glioblastoma cells treated with 0.1, 1 and 10 μmol/l Ukrain for 72 h. After treatment with 10 μmol/l Ukrain, glial fibrillary acidic protein fluorescence increased and a higher number of cells displayed glial fibrillary acidic protein organized into a filamentous state. Western blot analysis of glial fibrillary acidic protein confirmed that Ukrain tended to upregulate the protein. Connexin 43 was not modulated by Ukrain both at the mRNA and at the protein level. Ukrain-induced apoptotic rate was 4.63, 10.9 and 28.9% after 0.1, 1 and 10 μmol/l Ukrain, respectively, likely mediated by cytochrome c release in the cytoplasm. Considered as a whole, these findings provide new information to complete the understanding of the mechanisms of Ukrain antitumor and chemopreventive effect, and support the possible potential of Ukrain for the therapy of brain tumors.

Effect of Ukrain on matrix metalloproteinase-2 and Secreted Protein Acidic and Rich in Cysteine (SPARC) expression in human glioblastoma cells.

Glioblastoma is a highly malignant brain tumor with a highly invasive phenotype and hence an unfavorable prognosis even in response to multidisciplinary treatment strategies. Ukrain, a semi-synthetic thiophosphoric acid derivative of the purified alkaloid chelidonine, has been used in the therapy of several solid tumors, but little is known about its effect on glioblastoma and, in general, about the molecular mechanisms responsible for its effects. We used RT-PCR, Western blot and SDS-zymography to investigate the effects of three doses of Ukrain (0.1, 1 and 10 μmol/l) on the expression of genes and proteins involved in the extracellular matrix remodeling associated with tumor invasion in human cultured glioblastoma cells treated for 24, 48 and 72 h. We analyzed the expression of matrix metalloproteinase-2 and -9, the main mediators of glioblastoma invasiveness, and secreted protein acidic and rich in cysteine (SPARC), involved in the regulation of cell–matrix interactions. There was a significant, dose-related decrease of glioblastoma cell proliferation and a tendency to downregulation of SPARC at the protein level 72 h after 10 μmol/l Ukrain, suggesting the drug may be a useful therapeutic tool for brain tumors.

New insights in collagen turnover in orofacial cleft patients.

Objective We aimed to characterize the fibroblast phenotype of patients by analyzing gene and protein expression of cleft lip and/or cleft palate fibroblasts in relation to collagen turnover and extracellular matrix remodeling. Patients Human palatal fibroblasts were obtained from three healthy subjects without cleft lip and/or cleft palate and from three subjects with nonsyndromic cleft lip and/or cleft palate. Collagen turnover–related gene and protein expression were analyzed by real-time polymerase chain reaction, Western and dot blots, and sodium dodecyl sulfate zymography. Results Cleft lip and/or cleft palate fibroblasts, compared with controls, displayed a down-regulation of collagens type I and III messenger RNA (p < .0001 and p < .001, respectively) but an opposite tendency to increase protein levels. Cleft lip and/or cleft palate cells had higher lysyl hydroxylase-2b messenger RNA levels expressed in relation to collagen type I messenger RNA, down-regulated matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1, and Secreted Protein Acidic and Rich in Cysteine messenger RNA (p < .0001 and p < .01, respectively). Pro–matrix metalloproteinase-1 tended to decrease, and pro–matrix metalloproteinase-2 and -9 were down-regulated (p < .01, p < .05, respectively), as was Secreted Protein Acidic and Rich in Cysteine protein expression (p < .05). Conclusions Our results suggest that the cleft lip and/or cleft palate fibroblast phenotype is characterized by a tendency toward interstitial collagen deposition due to posttranslational modifications, such as decreased collagen degradation by matrix metalloproteinases and increased collagen cross-links. These findings may contribute to the knowledge of the cleft lip and/or cleft palate fibroblast phenotype and may be useful to the surgeon when considering the potential wound contraction and subsequent undesired scarring in cleft lip and/or cleft palate ocurring after the surgical closure of a cleft palate.

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.