Letizia Pettinari

Ukrain affects pancreas cancer cell phenotype in vitro by targeting MMP-9 and intra-/extracellular SPARC expression.

Background/Aims: We investigated whether the anticancer drug Ukrain (UK) is able to modulate the expression of some of the key markers of tumor progression in pancreatic cell carcinoma, in order to assess its potential therapeutic effect. Methods: Three cell lines (HPAF-II, PL45, HPAC) were treated with UK (5, 10 and 20 µM) for 48 h, or left untreated. Secreted protein acidic and rich in cysteine (SPARC) mRNA levels were assessed by real-time PCR. Matrix metalloproteinases (MMP)-2 and -9 activity was analyzed by SDS zymography; SPARC protein levels in cell lysates and supernatants were determined by Western blot. Cell cycle was determined by flow cytometric analysis, and invasion by matrigel invasion assay. Results: UK down-regulated MMP-2 and MMP-9, suggesting that UK may decrease pancreatic cancer cell invasion, as confirmed by the matrigel invasion assay. SPARC protein down-regulation in supernatants points to an inhibition by UK of extracellular matrix remodeling in the tumor microenvironment. At the same time, SPARC mRNA and cellular protein level up-regulation suggests that UK can affect cell proliferation by cell cycle inhibition, showing a cell cycle G2/M arrest in UK-treated cells. Conclusion: Our results suggest that UK modulates two major aspects involved in tumorigenesis of pancreatic cancer cells, such as extracellular matrix remodeling and cell proliferation.

Human palatal and tuberosity mucosa as donor sites for ridge augmentation

Since different clinical outcomes of periodontal bilaminar surgeries using the palate or the maxillary tuberosity as connective tissue (CT) donor sites have been observed, tissues grafted with CT from the palate or the tuberosity 1 year after surgical procedures for ridge augmentation were compared with nongrafted tissues by using morphologic and molecular methods. Collagen content and matrix metalloproteinases 1 and 2 expression were similar in tissues and cultured fibroblasts from the palate and tuberosity, although with interindividual differences. In contrast, differences in collagen cross-linking and maturation in the tuberosity fibroblasts were observed, suggesting a possible role in determining hyperplastic responses in some patients.

Tendon structure and extracellular matrix components are affected by spasticity in cerebral palsy patients

We studied the effect of spasticity-induced overload on tendons from the gracilis and semitendinosus muscles from cerebral palsy (CP) and healthy subjects (CT) stained with haematoxylineosin, Sirius red and Alcian blue. Vascularity was also characterized using an anti-CD34 antibody. Light microscopy analysis of haematoxylin-eosin stained sections revealed that the overall structure of tendons was maintained, characterized by parallel and slightly wavy collagen fibers in both CT and CP tendons. However, hypercellularity, cell rounding, increased vascularity and lipoid degeneration were observed in CP samples. Sirius red stained collagen fibers were more evident in CP tendons, suggesting an increased collagen content induced by spasticity. Alcian blue staining revealed an overall increase of glycosaminoglycans in CP tendons as observed in tendinopathy. Our results suggest that CP-induced spasticity may be considered as a chronic, persisting and repetitive loading of tendons, inducing ECM remodeling as adaptive response to increased functional demand. At the same time, the evidence of some tendinopathic-like markers in CP tendons suggests that the chronic nature of the CP condition could represent a pathologic condition, possibly leading to a transient weakness of the tissue making it more susceptible to damage from cumulative loading until an overt tendinopathy develops.

Malignant phenotype of renal cell carcinoma cells is switched by Ukrain administration in vitro

We investigated whether Ukrain modulates the malignant phenotype of clear cell renal cell carcinoma (ccRCC) cells Caki-1, Caki-2, and ACHN treated with four doses (5, 10, 20, and 40 &mgr;mol/l) for 24 and 48 h. The epithelial-to-mesenchymal transition markers E-cadherin, &bgr;-catenin, and vimentin were analyzed by immunofluorescence as well as actin and tubulin; matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was analyzed by SDS-zymography, intracellular and secreted SPARC levels by western blot, and cell cycle by flow cytometry. Ukrain did not induce E-cadherin/&bgr;-catenin immunoreactivity at the cell–cell boundary, although it determined the actin cortical expression in Caki-2 and ACHN, and did not affect vimentin organization; however, in some Caki-1 and ACHN cells the perinuclear concentration of vimentin was consistent with its downregulation. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activity was significantly downregulated 48 h after 20 &mgr;mol/l Ukrain administration. At this time point, Ukrain significantly decreased migration and invasion, and downregulated SPARC levels in cell supernatants at all doses in Caki-2, and at 20 &mgr;mol/l in Caki-1 and ACHN cells. Concomitantly, SPARC was upregulated in all ccRCC cells, suggesting that Ukrain could also affect cell proliferation by cell cycle inhibition, as supported by the cell cycle analysis, as SPARC also acts as a cell cycle inhibitor. Our results suggest that Ukrain may switch the epithelial-to-mesenchymal transition-related phenotype of ccRCC cells, and targets the two major aspects involved in RCC progression, such as tumor invasion/microenvironment remodeling and cell proliferation.

Pancreatic cancer cells retain the epithelial-related phenotype and modify mitotic spindle microtubules after the administration of ukrain in vitro

The aim of this study is to characterize the phenotype of pancreatic ductal adenocarcinoma (PDAC) cells in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers and determine whether ukrain, an anticancer drug based on the alkaloids extracted from greater celandine, modulates in vitro the malignant behavior of PDAC cells in order to extend our understanding of its therapeutic potential. Three cell lines (HPAF-II, HPAC, and PL45) were treated with ukrain (5, 10, and 20 &mgr;mol/l) for 48 h or left untreated (control). Cell proliferation was assessed by growth curves. Apoptosis was determined by Hoechst nuclear staining and by cytochrome c and caspase-8 expressions. The EMT markers E-cadherin, &bgr;-catenin, and vimentin, as well as actin and tubulin cytoskeletons, were analyzed by immunofluorescence. Interphase and mitotic microtubules as well as abnormal mitotic figures were studied by fluorescence microscopy after tubulin immunolabeling. Ukrain strongly suppressed cell proliferation and induced apoptosis possibly through an extrinsic pathway as cytochrome c immunoreactivity suggested that the integrity of the mitochondria was not affected. Tubulin expression indicated an antiproliferative effect of ukrain on the basis of alterations in mitotic spindle microtubule dynamics, leading to abnormal mitosis. Membranous E-cadherin/&bgr;-catenin immunoreactivity was similarly expressed in control-treated and ukrain-treated cells, although the drug upregulated E-cadherin in cell lysates. Our results suggest that ukrain exerts its chemotherapeutic action on PDAC cells targeting mitotic spindle microtubules, leading to abnormal mitosis and apoptosis, and favoring cell cohesiveness. The differentiated epithelial phenotype of HPAF-II, HPAC, and PL45 cell lines concomitant with a highly invasive potential suggests that further experiments will be necessary to definitively clarify the role of EMT in PDAC progression.

A Therapeutic Dose of FK506 Does Not Affect Collagen Turnover Pathways in Healthy Human Gingival Fibroblasts

We previously demonstrated that a high dose of tacrolimus (1 mumol/L) induced expression of matrix metalloproteinase (MMP) proteins in human cultured gingival fibroblasts, suggesting a molecular mechanism maintaining gingival collagen homeostasis in tacrolimus-treated patients. Herein we have analyzed whether the effect on collagen turnover might be influenced by a therapeutic tacrolimus dose. Human gingival fibroblasts were incubated for 72 hours with 10 nmol/L, 100 nmol/L, and 1 mumol/L tacrolimus, or left untreated (CT). Collagen type I and III (COL-I, COL-III), lysyl hydroxylase 2b (LH2b), MMP-1 and -2, tissue inhibitor of MMP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA levels were assayed by reverse-transcriptase polymerase chain reaction, collagen protein levels by dot blot, and MMP activity by sodium dodecyl sulfate zymography. Tacrolimus did not affect COL-I, COL-III, or MMP gene expression, while LH2b and TGF-beta1 tended to be down-regulated after 1 mumol/L FK506. Conversely, protein levels of MMP-1 (P = NS) and MMP-2 (P < .05 vs CT, 10 nmol/L, 100 nmol/L) were up-regulated after 1 mumol/L tacrolimus. Our findings confirmed that a high dose of tacrolimus does not induce interstitial collagen overexpression by gingival fibroblasts and induces up-regulation of MMPs protein levels. Interestingly, at doses corresponding to whole blood trough levels, tacrolimus did not exert any evident effect on collagen turnover pathways, suggesting that tacrolimus is likely to not affect collagen homeostasis in the gingival connective tissue compartment of FK506-immunosuppressed subjects. This effect did not seem to be dose-dependent.

Synchronized human osteoblasts exhibit circadian rhythmicity in expression of genes involved in collagen turnover

The human circadian system modulates cell activities along the 24-h time scale to assure predictive adaptation of individuals to the 24-h day/night alternations; it operates through interlaced molecular loops involving the recurrent expression of clock genes within each cell, and a central synchronization system in the suprachiasmatic nuclei of the hypothalamus. In cell cultures, exposure to serum treatment allows to obtain in vitro models of synchronized cells to investigate circadian periodicity in cell morphology and activities. Osteoblasts play a key role in regulating bone matrix collagen turnover; since biochemical markers of bone formation/resorption exhibit circadian variations in healthy humans, we investigated the circadian pattern of collagen turnover-related genes in primary cultures of synchronized osteoblasts. Cells obtained from the mandibular bone of a healthy 20-year-old woman were grown with 199 medium plus 10% fetal bovine serum (FBS) and antibiotics. At the 4th passage, fully confluent cultures were treated with 50% FBS for 2 hours and then maintained in 199 medium containing 10% FBS. From the end of the serum shock, at 4 hour intervals for 48 hours, cells were harvested and the extracted total RNA was analysed for the expression of clock genes (Bmal1, Cry1) and collagen turnover-related genes (COL-I, MMP-1, MMP-2, TIMP-1, LH2b, SPARC) by real-time PCR. Rhythmometric analysis of the data was performed by the cosinor method. In osteoblasts exposed to serum shock, the expression of clock genes exhibited circadian rhythmicity (p < 0.05) starting from the end of the serum treatment for 28 hours. Also the expression of genes related to collagen synthesis and maturation (COL-I and LH2b), and degradation (MMP-2, TIMP-1) showed circadian rhythmicity (p < 0.05), every gene being rhythmically expressed for 24 hours, starting 4, 8, or 16 hours from the end of the synchronizing stimulus. The results suggest a role of clock genes in controlling the circadian expression of downstream genes related to the collagen turnover in bone and rise the hypothesis that responses to substances affecting bone remodelling might display variations depending upon time of treatment, leading to different outcomes.

Extracellular matrix variation along the human aorta: an autoptic observational study focusing on highly sulphated proteoglycans

Purpose Differences in the distribution of elastic and collagenic components between the proximal and the distal aorta are well known. We sought to determine the changes in content and organization of non-collagenic constituents of the extracellular matrix, focusing in particular on highly sulphated proteoglycans (PGs) throughout the normal human aorta. Methods Histochemical staining with Alcian Blue pH 0.2 allowed for tinctorial distinction of highly sulphated proteoglycans on complete 1-cm transverse rings removed from the ascending and descending thoracic aorta and abdominal supraceliac, suprarenal, and infrarenal aorta of young subjects at autopsy. Counterstaining with Orcein and Sirius Red allowed comparing with the total content of elastin and collagen. Light microscopy and image analysis were used to determine changes in total and relative content of highly sulphated proteoglycans at each level, along with the variation of collagen and elastin. Results Collagen/elastin ratio increases from the proximal to distal aorta as the elastin content decreases from the supradiaphragmatic to the abdominal aorta. The proportion of highly sulphated proteoglycans does not change significantly throughout the thoracic and thoraco-abdominal aorta, while a significant increase in total highly sulphated proteoglycans content is observed at the infrarenal site. Conclusion The infrarenal aorta differs histologically and biochemically from the remainder of the aorta. Highly sulphated proteoglycans in the distal aorta bear an increased load as compared to the proximal aorta. A decrease in infrarenal elastin without a corresponding decrease in collagen may effect the compliance and integrity of the distal aorta. These anatomic differences may be important in predisposing the infrarenal aorta to atherosclerosis and aneurysm formation.

Extracellular matrix remodeling and invasive potential but not epithelial-to-mesenchymal transition markers are targeted in vitro in renal cell carcinoma cells by Ukrain administration

1 Department of Human Morphology and Biomedical Sciences “Citta Studi”, School of Medicine, University of Milan, Italy 2 Department of Medical Chemistry, Biochemistry and Biotechnology, School of Medicine, University of Milan, Italy 3 Laboratory of Immunology, Department of Science and Biomedical Technologies, School of MedicineUniversity of Milan, Italy 4 Department of Biology, University of Milan, Italy