Mariarosaria Boccellino

Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

ABSTRACT HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified α- and β-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of α-, β-, and γ-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in theregulation of cell motility by modulating microtubule stability.

Methylation Induced Gene Silencing of HtrA3 in Smoking-Related Lung Cancer

Purpose: Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer. Experimental Design: We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment. Results: We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2′-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history. Conclusions: Collectively, these results suggest that cigarette smoke–induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer. Clin Cancer Res; 16(2); 398–409

Apoptosis induced by interferon-α and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells

We have previously reported that interferon‐α (IFNα) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNα and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNα induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C‐terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNα‐induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNα induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNα‐treated transfected cells caused a recovery of the apoptosis. Notably, IFNα and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNα induced also gelsolin cleavage that occurred together with caspase‐3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNα‐treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase‐3 with 20 μM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNα. In conclusion, our data are the first demonstration that IFNα can induce morphological cell changes that are peculiar of apoptosis onset through the caspase‐3‐mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase‐3 activation. J. Cell. Physiol. 201: 71–83, 2004.

New evidence of the presence of endometriosis in the human fetus.

The aetiology of endometriosis, a gynaecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity, is still open to debate. Research has recently found evidence for endometriosis in human female fetuses at different gestational ages. This paper reports a new case of fetal endometriosis in a 25-week female fetus, deceased due to placental pathology, from a series of 13 female fetuses analysed at autopsy. The exact anatomical localization of this misplaced endometrium, as well as its histopathological and immunohistochemical characteristics are illustrated. The case suggests that endometriosis can be caused by dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis.

3‐O‐methylfunicone produced by penicillium pinophilum affects cell motility of breast cancer cells, downregulating αvβ5 integrin and inhibiting metalloproteinase‐9 secretion

Recent evidence assigns integrins and metalloproteinases (MMPs) an important role in regulating tumor cell progression. Here, we demonstrate that 3‐O‐methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility of breast cancer MCF‐7 cells, downregulating αvβ5 integrin, and inhibiting MMP‐9 secretion. This effect was absent when the non‐tumoral MCF‐10 cell line was used. Inhibition of cell motility was also associated to modifications in cell shape and in the distribution of tubulin fibers of OMF‐treated MCF‐7 cells. In addition, a possible effect on survivin and hTERT was also investigated. We found that OMF strongly inhibits survivin and hTERT gene expression. The results of this study indicate that OMF‐induced inhibition of cell motility may be mediated through the modulation of αvβ5 integrin and MMP‐9 secretion. In addition, the inhibition of typical markers of tumor progression such as hTERT and survivin in MCF‐7 and their inactivity towards MCF10 provide strong evidence for a potential use of OMF in anticancer therapy.

Interaction between combustion-generated organic nanoparticles and biological systems: in vitro study of cell toxicity and apoptosis in human keratinocytes.

Abstract The purpose of this work was to evaluate the effect of flame-generated nucleation mode particles with an organic carbon structure on growth and apoptosis in immortalized human keratinocytes. In this study, cells were stimulated with nanoparticles collected from flames that produce only nucleation mode particles operating with a fuel:air mixture typical of low-emission combustion systems. Cytotoxicity as a function of particle concentration was monitored by fluorescence-activated cell sorting (FACS) analysis, and apoptosis was observed by FACS using DNA fragmentation and hypodiploidism and confirmed by annexin assay. A dose-dependent reduction in cell viability by apoptosis in incubation periods of 48 and 72 hours was observed with a statistically significant increase in apoptosis over controls for a dose larger than 7 μg/mL (1.4 μg/cm2). The results presented here may be relevant for understanding the association between exposure to traffic-generated particulate pollution and enhanced skin aging reported in epidemiology studies.

Altered oxido-reductive state in the diabetic heart: loss of cardioprotection due to protein disulfide isomerase

Diabetes is associated with an increased risk of heart failure, in part explained by endoplasmic reticulum stress and apoptosis. Protein disulfide isomerase (PDI) prevents stressed cardiomyocytes apoptosis. We hypothesized that diabetes impairs PDI function by an alteration in its oxido-reductive state. Myocardial biopsies harvested from the anterolateral left ventricular wall from diabetic (n = 7) and nondiabetic (n = 8) patients were used to assess PDI expression and cardiomyocyte death. A mouse model of diabetes (streptozotocin injection, 130 mg/mL) was used to study PDI expression and its redox state after ischemia/reperfusion injury induced by 30-min occlusion of the left anterior coronary artery followed by reperfusion. Transthoracic echocardiography was performed to assess cardiac remodeling after 1 wk. Western blot analysis was used to analyze PDI expression, and methoxypolyethyleneglycol-maleimide was used to assess its redox state. Dehydroascorbate (DHA) administration was used to restore the PDI redox state. Diabetic patients had a greater number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells than nondiabetic patients despite a greater myocardial PDI expression suggesting altered PDI function. Diabetic mice had a worse postinfarction remodeling associated with an altered PDI redox state. DHA treatment restored functional PDI redox state and ameliorated post-myocardial infarction remodeling. An increase in PDI levels with a paradoxical decrease of its active form occurs in the diabetic heart after ischemia and may explain the lack of protective effects of PDI in diabetes. Restoration of PDI redox state prevents adverse remodeling. The potential significance of these findings deserves to be validated in a clinical setting.

Antagonistic effects of chloroquine on autophagy occurrence potentiate the anticancer effects of everolimus on renal cancer cells

Renal cell carcinoma is an aggressive disease often asymptomatic and weakly chemo-radiosensitive. Currently, new biologic drugs are used among which everolimus, an mTOR inhibitor, that has been approved for second-line therapy. Since mTOR is involved in the control of autophagy, its antitumor capacity is often limited. In this view, chloroquine, a 4-alkylamino substituted quinoline family member, is an autophagy inhibitor that blocks the fusion of autophagosomes and lysosomes. In the present study, we evaluated the effects of everolimus alone or in combination with chloroquine on renal cancer cell viability and verified possible synergism. Our results demonstrate that renal cancer cells are differently sensitive to everolimus and chloroquine and the pharmacological combination everolimus/chloroquine was strongly synergistic inducing cell viability inhibition. In details, the pharmacological synergism occurs when chloroquine is administered before everolimus. In addition, we found a flow autophagic block and shift of death mechanisms to apoptosis. This event was associated with decrease of Beclin-1/Bcl-2 complex and parallel reduction of anti-apoptotic protein Bcl-2 in combined treatment. At last, we found that the enhancement of apoptosis induced by drug combination occurs through the intrinsic mitochondrial apoptotic pathway activation, while the extrinsic pathway is involved only partly following its activation by chloroquine. These results provide the basis for new therapeutic strategies for the treatment of renal cell carcinoma after appropriate clinical trial.

Platelet-Activating Factor Regulates Cadherin-Catenin Adhesion System Expression and β-Catenin Phosphorylation during Kaposi’s Sarcoma Cell Motility

In the present study, we evaluated whether motility of Kaposis sarcoma (KS) cells induced by platelet-activating factor (PAF) is dependent on the regulation of adherens junctions components. The results obtained indicate that PAF dose and time dependently reduced the endogenous expression of the main components of the adherens junctions: VE-cadherin, alpha-catenin, and beta-catenin. In addition, PAF initiated events that directly or indirectly up-regulated both the tyrosine and serine/threonine phosphorylation pathways, and both types of phosphorylation of beta-catenin were involved in the motility of KS cells. This motility was abrogated by addition of the tyrosine kinase inhibitor genistein, suggesting that this phosphorylation is an important signal responsible for breaking down the adherens junctions and diminishing the ability of neighboring cells to interact. Furthermore, immunofluorescence analysis showed that beta-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of controls to a diffuse pattern with gap formation in PAF-treated KS cells. In conclusion, the data presented here indicate that PAF induces tumor cell motility by altering cell-cell adhesion through beta-catenin phosphorylation.

In vitro model of stromal and epithelial immortalized endometriotic cells.

Endometriosis is a relatively common chronic gynecologic disorder that usually presents with chronic pelvic pain or infertility. It results from implantation of endometrial tissue outside the uterine cavity. Despite its frequency and its impact on quality of life, the understanding of pathogenesis of endometriosis remains incomplete and its treatment remains controversial. In this work, we established a suitable in vitro model system of immortalized human endometriotic cell line taking advantage of the human telomerase reverse transcriptase. The results demonstrate that these cells retain the natural characteristics of endometrial cells in term of phenotype and of functional expression of estrogen and progesterone receptors, without chromosomal abnormalities. In conclusion, these cells are potentially useful as an experimental model to investigate endometriosis biology. J. Cell. Biochem. 113: 1292–1301, 2012.