Activity of Colocasia esculenta (Taro) Corms against Gastric Adenocarcinoma Cells: Chemical Study and Molecular Characterization.

Colocasia esculenta (L.) Schott is a tuberous plant, also known as taro, employed as food worldwide for its renowned nutritional properties but also traditionally used in several countries for medical purposes. In this study, methanolic extracts were prepared from the corms and leaves of Colocasia, subsequently fractionated via molecular exclusion chromatography (RP-HPLC) and their anti-tumor activity assessed in an in vitro model of gastric adenocarcinoma (AGS cells). Vorm extract and isolated fractions II and III affected AGS cell vitality in a dose-dependent manner through the modulation of key proteins involved in cell proliferation, apoptosis, and cell cycle processes, such as caspase 3, cyclin A, cdk2, IkBα, and ERK. To identify bioactive molecules responsible for anti-tumoral activity fractions II and III were further purified via RP-HPLC and characterized via nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ESI-MS) techniques. The procedure enabled the identification of ten compounds including lignans and neolignans, some isolated for the first time in taro, uncommon megastigmane derivatives, and a gallic acid derivative. However, none of the isolated constituents showed efficacy equivalent to that of the fractions and total extract. This suggests that the whole Colocasia phytocomplex has intriguing anti-tumor activity against gastric cancer.

Modulation of BAG3 expression in human normal urothelial cells by Diuron.

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a substituted urea herbicide, carcinogenic for the rat urinary bladder. It has been hypothesized that Diuron cytotoxicity, resulting in regenerative proliferation, leads to urothelial hyperplasia and, finally, to bladder tumors, but molecular mechanisms of carcinogenesis have not still fully investigated. Here, we report the results of a study aimed at verifying the involvement of BAG3, an intracellular protein expressed in several tumors, in the Diuron-induced carcinogenesis. For this purpose, we analyzed the effect of Diuron on human primary urothelial cells and on human dermal fibroblasts. We found that while high concentrations of Diuron have a cytotoxic effect in human primary urothelial cells, in the same cells, noncytotoxic concentrations of the herbicide induce BAG3 expression. These findings show that BAG3 is a molecular target of Diuron and unravel the possible involvement of BAG3 protein in bladder carcinogenesis induced by the herbicide. In addition, these results suggest that BAG3 might be a potential early biomarker of damage induced by chronic exposure to Diuron.

Keratinocytes Migration Promotion, Proliferation Induction, and Free Radical Injury Prevention by 3-Hydroxytirosol.

3-hydroxytyrosol (HT) is the main phenolic compound found in olive oil with known antioxidant, anti-inflammatory, and antimicrobial properties in several dermatological conditions, both when taken in the form of olive oil or pure in cosmeceutical formulations. To date, its direct effect on the wound healing process and the molecular mechanisms involved have not yet been elucidated. Thus, in the present study, we aimed to explore its effects in vitro in epidermal keratinocyte cultures focusing on the molecular mechanism implied. HT was able to induce keratinocyte proliferation in the low micromolar range, increasing the expression of cyclin dependent kinases fundamental for cell cycle progression such as CDK2 and CDK6. Furthermore, it increased cell migration through the activation of tissue remodeling factors such as matrix metalloproteinase-9 (MMP-9) protein. Then, we evaluated whether HT also showed antioxidant activity at this concentration range, protecting from H2O2-induced cytotoxicity. The HT prevented the activation of ATM serine/threonine kinase (ATM), Checkpoint kinase 1 (Chk1), Checkpoint kinase 2 (Chk2), and p53, reducing the number of apoptotic cells. Our study highlighted novel pharmacological properties of HT, providing the first evidence of its capability to induce keratinocyte migration and proliferation required for healing processes and re-epithelialization.

Correlation of the two most frequent HLA haplotypes in the Italian population to the differential regional incidence of Covid-19.

BACKGROUND: Understanding how HLA polymorphisms may affect both susceptibility, course and severity of Covid-19 infection could help both at the clinical level to identify individuals at higher risk from the disease and at the epidemiological one to explain the differences in the epidemic trend among countries or even within a specific country. Covid-19 disease in Italy showed a peculiar geographical distribution from the northern most affected regions to the southern ones only slightly touched. METHODS: In this study we analysed the regional frequencies for the most common Italian haplotypes from the Italian Bone Marrow Donor Registry (HLA-A, -B, -C and -DRB1 at four-digit level). Then we performed Pearson correlation analyses among regional haplotypes estimated frequency in the population and Covid-19 incidence and mortality. RESULTS: In this study we found that the two most frequent HLA haplotypes in the Italian population, HLA-A*:01:01g-B*08:01 g-C*07:01g-DRB1*03:01g and HLA-A*02.01g-B*18.01g-C*07.01g-DRB1*11.04g, had a regional distribution overlapping that of Covid-19 and showed respectively a positive (suggestive of susceptibility) and negative (suggestive of protection) significant correlation with both Covid-19 incidence and mortality. CONCLUSIONS: Based on these results, in order to define such HLA haplotypes as a factor effectively associated to the disease susceptibility, the creation of national networks that can collect patients' samples from all regions for HLA typing should be highly encouraged.

3-Hydroxytyrosol Promotes Angiogenesis In Vitro by Stimulating Endothelial Cell Migration.

Cardiovascular diseases, followed by strokes, represent the leading cause of mortality worldwide. Despite its success in preventing cardiovascular diseases, the therapeutic potential of 3-Hydroxytyrosol (HT) for treating ischemic diseases is yet to be investigated in detail, especially with regard to ischemic heart disease, which is a major challenge for humans. We assessed that low concentrations (1-5 µM) of HT, generally achieved after the ingestion of olive oil, stimulate endothelial cells migration and angiogenesis in an in vitro model. At early time points (1-6 h), HT induces the expression of different proteins such as proto-oncogene tyrosine-protein kinase Src (Src), rho-associated protein kinase (ROCK) and matrix metalloproteinase-2 (MMP-2) protein influencing cell adhesion, cytoskeletal dynamics and cell migration. We observed that at the same time, HT induces prominent vascular formation in the tube formation assay, accompanied by an increase in the expression of the vascular endothelial growth factor receptor (VEGF-R2) and PI3K-Akt-eNOS protein pathways, which are recognized for their central role in angiogenesis. Therefore, in addition to the proven capability of HT to regulate reactive oxygen species (ROS) levels, through both direct scavenging properties and indirect antioxidant efficacy, our results revealed that HT promotes angiogenesis, arguing in favor of great pharma-nutritional potential in ischemic injuries.

Prosystemin Overexpression in Tomato Enhances Resistance to Different Biotic Stresses by Activating Genes of Multiple Signaling Pathways.

Systemin is a signal peptide that promotes the response to wounding and herbivore attack in tomato. This 18-amino acid peptide is released from a larger precursor, prosystemin. To study the role of systemin as a modulator of defense signaling, we generated tomato (Solanum lycopersicum) transgenic plants that overexpress the prosystemin cDNA. We carried out a transcriptomic analysis comparing two different transgenic events with the untransformed control. The Gene Ontology categories of the 503 differentially expressed genes indicated that several biological functions were affected. Systemin promotes the expression of an array of defense genes that are dependent on different signaling pathways and it downregulates genes connected with carbon fixation and carbohydrate metabolism. These alterations present a degree of overlap with the response programs that are classically associated to pathogen defense or abiotic stress protection, implying that end products of the systemin signaling pathway may be more diverse than expected. We show also that the observed transcriptional modifications have a relevant functional outcome, since transgenic lines were more resistant against very different biotic stressors such as aphids (Macrosiphum euphorbiae), phytopathogenic fungi (Botrytis cinerea and Alternaria alternata) and phytophagous larvae (Spodoptera littoralis). Our work demonstrated that in tomato the modulation of a single gene is sufficient to provide a wide resistance against stress by boosting endogenous defense pathways. Overall, the data provided evidence that the systemin peptide might serve as DAMP signal in tomato, acting as a broad indicator of tissue integrity.

A Lung Cancer Mouse Model Database.

Lung cancer, the leading cause of cancer mortality, exhibits diverse histological subtypes and genetic complexities. Numerous preclinical mouse models have been developed to study lung cancer, but data from these models are disparate, siloed, and difficult to compare in a centralized fashion. Here we established the Lung Cancer Mouse Model Database (LCMMDB), an extensive repository of 1,354 samples from 77 transcriptomic datasets covering 974 samples from genetically engineered mouse models (GEMMs), 368 samples from carcinogen-induced models, and 12 samples from a spontaneous model. Meticulous curation and collaboration with data depositors have produced a robust and comprehensive database, enhancing the fidelity of the genetic landscape it depicts. The LCMMDB aligns 859 tumors from GEMMs with human lung cancer mutations, enabling comparative analysis and revealing a pressing need to broaden the diversity of genetic aberrations modeled in GEMMs. Accompanying this resource, we developed a web application that offers researchers intuitive tools for in-depth gene expression analysis. With standardized reprocessing of gene expression data, the LCMMDB serves as a powerful platform for cross-study comparison and lays the groundwork for future research, aiming to bridge the gap between mouse models and human lung cancer for improved translational relevance.

Transcriptomic and proteomic analysis of a compatible tomato-aphid interaction reveals a predominant salicylic acid-dependent plant response.

BACKGROUND: Aphids are among the most destructive pests in temperate climates, causing significant damage on several crops including tomato. We carried out a transcriptomic and proteomic study to get insights into the molecular mechanisms and dynamics of the tomato response to the Macrosyphum euphorbiae aphid. RESULTS: The time course analysis of aphid infestation indicated a complex, dynamic pattern of gene expression. Several biological functions were affected and genes related to the stress and defence response were the most represented. The Gene Ontology categories of the differentially expressed genes (899) and identified proteins (57) indicated that the tomato response is characterized by an increased oxidative stress accompanied by the production of proteins involved in the detoxification of oxygen radicals. Aphids elicit a defense reaction based on the cross-communication of different hormone-related signaling pathways such as those related to the salicylic acid (SA), jasmonic acid (JA), ethylene and brassinosteroids. Among them, the SA-signaling pathway and stress-responsive SA-dependent genes play a dominant role. Furthermore, tomato response is characterized by a reduced accumulation of photosynthetic proteins and a modification of the expression of various cell wall related genes. CONCLUSIONS: Our work allowed a more comprehensive understanding of the signaling events and the defense dynamics of the tomato response to aphids in a compatible interaction and, based on experimental data, a model of the tomato-aphid molecular interaction was proposed. Considering the rapid advancement of tomato genomics, this information will be important for the development of new protection strategies.

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia.

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Couroupita guianensis bark decoction: From Amazonian medicine to the UHPLC-HRMS chemical profile and its role in inflammation processes and re-epithelialization.

ETHNOPHARMACOLOGICAL RELEVANCE: In the Amazon rainforest, the shamans of the Mayantuyacu site use the healing virtues of decoctions and teas from different parts of the Couroupita guianensis Aubl. (Lecythidaceae) trees as remedies in Ashaninka medicine. However, composition of the remedy and the underlying mechanism remain unclear. AIM OF THE STUDY: This study was designed to compare the metabolite profile of Couroupita guianensis bark decoction produced by Amazonian shamans with that obtained under standardised laboratory conditions and to investigate biological properties of both decoction and isolated constituents in skin wound healing process and inflammation. MATERIALS AND METHODS: The chemical analyses were carried out by Ultra-High-Performance Liquid Chromatography coupled with UV and High-Resolution Mass Spectrometry detectors (UHPLC-UV-HRMS). 1D and 2D-NMR experiments were performed to identify the main decoction constituents. The decoction and pure compound effect on keratinocyte migration was determined by the in vitro wound healing model; the mechanism of action was elucidated by western blot analysis. RESULTS: UHPLC-UV-HRMS analysis revealed the occurrence of polyphenolic compounds as catechins, ellagitannins and, notably, of unusual sulphated derivatives of ellagic acid isolated for the first time from Couroupita guianensis bark. A new natural sulphated molecule [4-(2″-O-sulphate- ß-D-glucuronopyranosyl) ellagic acid] was identified as the potential active compound responsible for the efficacy of bark decoction stimulating wound healing in human HaCaT keratinocytes. The molecular mechanism involved the induction of pro-migratory pathways mediated by ERK and AKT phosphorylation and the increase of MMP2 expression in HaCaT cells. At the same time, the treatment inhibited inflammation interfering with NFkB activation. CONCLUSION: Beyond identifying a new bioactive compound, the overall results scientifically validate the traditional use of Couroupita guianensis bark decoction as an anti-inflammatory remedy. Moreover, the beneficial effects on keratinocytes suggest promising therapeutic applications in skin diseases.

Prenylation Defects and Oxidative Stress Trigger the Main Consequences of Neuroinflammation Linked to Mevalonate Pathway Deregulation.

The cholesterol biosynthesis represents a crucial metabolic pathway for cellular homeostasis. The end products of this pathway are sterols, such as cholesterol, which are essential components of cell membranes, precursors of steroid hormones, bile acids, and other molecules such as ubiquinone. Furthermore, some intermediates of this metabolic system perform biological activity in specific cellular compartments, such as isoprenoid molecules that can modulate different signal proteins through the prenylation process. The defects of prenylation represent one of the main causes that promote the activation of inflammation. In particular, this mechanism, in association with oxidative stress, induces a dysfunction of the mitochondrial activity. The purpose of this review is to describe the pleiotropic role of prenylation in neuroinflammation and to highlight the consequence of the defects of prenylation.

Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: a comparative proteomic approach.

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kappaB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.

Involvement of aryl hydrocarbon receptor signaling in the development of small cell lung cancer induced by HPV E6/E7 oncoproteins.

BACKGROUND: Lung cancers consist of four major types that and for clinical-pathological reasons are often divided into two broad categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). All major histological types of lung cancer are associated with smoking, although the association is stronger for SCLC and squamous cell carcinoma than adenocarcinoma. To date, epidemiological studies have identified several environmental, genetic, hormonal and viral factors associated with lung cancer risk. It has been estimated that 15-25% of human cancers may have a viral etiology. The human papillomavirus (HPV) is a proven cause of most human cervical cancers, and might have a role in other malignancies including vulva, skin, oesophagus, head and neck cancer. HPV has also been speculated to have a role in the pathogenesis of lung cancer. To validate the hypothesis of HPV involvement in small cell lung cancer pathogenesis we performed a gene expression profile of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins. METHODS: Gene expression profile of SCLC has been performed using Agilent whole mouse genome (4 × 44k) representing ~ 41000 genes and mouse transcripts. Samples were obtained from two HPV16-E6/E7 transgenic mouse models and from littermate's normal lung. Data analyses were performed using GeneSpring 10 and the functional classification of deregulated genes was performed using Ingenuity Pathway Analysis (Ingenuity® Systems, http://www.ingenuity.com). RESULTS: Analysis of deregulated genes induced by the expression of E6/E7 oncoproteins supports the hypothesis of a linkage between HPV infection and SCLC development. As a matter of fact, comparison of deregulated genes in our system and those in human SCLC showed that many of them are located in the Aryl Hydrocarbon Receptor Signal transduction pathway. CONCLUSIONS: In this study, the global gene expression of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins led us to identification of several genes involved in SCLC tumor development. Furthermore, our study revealed that the Aryl Hydrocarbon Receptor Signaling is the primarily affected pathway by the E6/E7 oncoproteins expression and that this pathway is also deregulated in human SCLC. Our results provide the basis for the development of new therapeutic approaches against human SCLC.

Gene Expression Analysis of Mevalonate Kinase Deficiency Affected Children Identifies Molecular Signatures Related to Hematopoiesis.

Mevalonate kinase deficiency (MKD) is a rare autoinflammatory genetic disorder characterized by recurrent fever attacks and systemic inflammation with potentially severe complications. Although it is recognized that the lack of protein prenylation consequent to mevalonate pathway blockade drives IL1ß hypersecretion, and hence autoinflammation, MKD pathogenesis and the molecular mechanisms underlaying most of its clinical manifestations are still largely unknown. In this study, we performed a comprehensive bioinformatic analysis of a microarray dataset of MKD patients, using gene ontology and Ingenuity Pathway Analysis (IPA) tools, in order to identify the most significant differentially expressed genes and infer their predicted relationships into biological processes, pathways, and networks. We found that hematopoiesis linked biological functions and pathways are predominant in the gene ontology of differentially expressed genes in MKD, in line with the observed clinical feature of anemia. We also provided novel information about the molecular mechanisms at the basis of the hematological abnormalities observed, that are linked to the chronic inflammation and to defective prenylation. Considering the broad and unspecific spectrum of MKD clinical manifestations and the difficulty in its diagnosis, a better understanding of MKD molecular bases could be translated to the clinical level to facilitate diagnosis, and improve management and therapy.

FKBP51 Affects TNF-Related Apoptosis Inducing Ligand Response in Melanoma.

Melanoma is one of the most immunogenic tumors and has the highest potential to elicit specific adaptive antitumor immune responses. Immune cells induce apoptosis of cancer cells either by soluble factors or by triggering cell-death pathways. Melanoma cells exploit multiple mechanisms to escape immune system tumoricidal control. FKBP51 is a relevant pro-oncogenic factor of melanoma cells supporting NF-κB-mediated resistance and cancer stemness/invasion epigenetic programs. Herein, we show that FKBP51-silencing increases TNF-related apoptosis-inducing ligand (TRAIL)-R2 (DR5) expression and sensitizes melanoma cells to TRAIL-induced apoptosis. Consistent with the general increase in histone deacetylases, as by the proteomic profile, the immune precipitation assay showed decreased acetyl-Yin Yang 1 (YY1) after FKBP51 depletion, suggesting an impaired repressor activity of this transcription factor. ChIP assay supported this hypothesis. Compared with non-silenced cells, a reduced acetyl-YY1 was found on the DR5 promoter, resulting in increased DR5 transcript levels. Using Crispr/Cas9 knockout (KO) melanoma cells, we confirmed the negative regulation of DR5 by FKBP51. We also show that KO cells displayed reduced levels of acetyl-EP300 responsible for YY1 acetylation, along with reduced acetyl-YY1. Reconstituting FKBP51 levels contrasted the effects of KO on DR5, acetyl-YY1, and acetyl-EP300 levels. In conclusion, our finding shows that FKBP51 reduces DR5 expression at the transcriptional level by promoting YY1 repressor activity. Our study supports the conclusion that targeting FKBP51 increases the expression level of DR5 and sensitivity to TRAIL-induced cell death, which can improve the tumoricidal action of immune cells.

Nutrigenomic Effect of Hydroxytyrosol in Vascular Endothelial Cells: A Transcriptomic Profile Analysis.

Hydroxytyrosol (HT), a peculiar olive and olive oil phenolic antioxidant, plays a significant role in the endothelial and cardiovascular protection associated with olive oil consumption. However, studies examining the effects of HT on the whole-genome expression of endothelial cells, which are prominent targets for vasculo-protective effects of olive oil polyphenols, have been lacking. This study aims to comprehensively evaluate the genomic effects exerted by HT, at the transcriptional level, in endothelial cells under resting or proinflammatory conditions. Human umbilical vein endothelial cells (HUVECs) were treated with 10 µmol/L HT for 1 h and then stimulated with 5 ng/mL interleukin (IL)-1ß for 3 h. Total RNA was extracted, and gene expression profile assessed with microarray analysis. Functional enrichment analysis and pathway analysis were performed by Ingenuity Pathways Analysis. Microarray data were validated by qRT-PCR. Fixing a significance threshold at 1.5-fold change, HT affected the expression of 708 and 599 genes, respectively, in HUVECs under resting and IL-1ß-stimulated conditions; among these, 190 were common to both conditions. Unfolded protein response (UPR) and endoplasmic reticulum stress resulted from the two top canonical pathways common between HT and HT-IL-1ß affected genes. IL-17F/A signaling was found in the top canonical pathways of HT modified genes under resting unstimulated conditions, whereas cardiac hypertrophy signaling was identified among the pathways affected by HT-IL-1ß. The transcriptomic analysis allowed pinpointing immunological, inflammatory, proliferative, and metabolic-related pathways as the most affected by HT in endothelial cells. It also revealed previously unsuspected genes and related gene pathways affected by HT, thus broadening our knowledge of its biological properties. The unbiased identification of novel genes regulated by HT improves our understanding of mechanisms by which olive oil prevents or attenuates inflammatory diseases and identifies new genes to be enquired as potential contributors to the inter-individual variation in response to functional food consumption.

Tumor-Associated Macrophage Status in Cancer Treatment.

Tumor-associated macrophages (TAMs) represent the most abundant innate immune cells in tumors. TAMs, exhibiting anti-inflammatory phenotype, are key players in cancer progression, metastasis and resistance to therapy. A high TAM infiltration is generally associated with poor prognosis, but macrophages are highly plastic cells that can adopt either proinflammatory/antitumor or anti-inflammatory/protumor features in response to tumor microenvironment stimuli. In the context of cancer therapy, many anticancer therapeutics, apart from their direct effect on tumor cells, display different effects on TAM activation status and density. In this review, we aim to evaluate the indirect effects of anticancer therapies in the modulation of TAM phenotypes and pro/antitumor activity.

Different mechanisms underlie IL-6 release in chemosensitive and chemoresistant ovarian carcinoma cells.

Interleukin (IL)-6 has been detected in serum and ascites from patients affected by epithelial ovarian cancers, and also in some human ovarian cancer cell lines. To investigate the role of IL-6 in ovarian lesions, we first measured its levels in serum samples of 24 healthy donors and in 17 and 9 patients affected by ovarian carcinomas and ovarian benign cysts respectively. IL-6 levels were significantly higher than healthy donors in serum samples from ovarian cancer patients, but not in benign ovarian cysts. We then investigated the mechanism of IL-6 production in two cell lines obtained from the same patient with high grade serous ovarian carcinoma before (PEA1) and after (PEA2) development of cisplatinum resistance. The levels of intracellular IL-6, analysed by western blotting, did not relevantly differ in the two cell lines, and they did not change after the cell treatment with an AKT inhibitor. Although the interleukin was present in supernatants from both cell lines, its concentration in the supernatant of chemoresistant cells was significantly higher than chemosensitive cells. Interestingly, exposure to the AKT inhibitor resulted in a reduced IL-6 release in PEA1, but not in PEA2 cells. These results let infer different mechanisms of IL-6 release in chemoresistant and chemosensitive cell lines, and contribute new insights in ovarian cancer biology that suggest more in depth studies about the role of AKT in IL-6 release and in development of chemoresistance.

Establishment and characterization of murine small cell lung carcinoma cell lines derived from HPV-16 E6/E7 transgenic mice.

We have established two murine cell lines derived from Small Cell Lung Carcinomas (SCLCs) developed by HPV-E6/E7 transgenic mice. These cells named PPAP-9 and PPAP-10 were isolated from mice bearing tumors, 9 and 10 months old, respectively. The cells, 5 microm in diameter, express HPV oncoproteins and sustain tumor formation after subcutaneous injection in syngenic mice. A detailed analysis indicated the epithelial origin and the neuroendocrine differentiation of these cells. We showed by confocal immunofluorescence the expression of the epithelial marker cytokeratin 5, whose gene promoter was used to direct the expression of HPV E6/E. Cells express several neuroendocrine markers such as CGRP, MAP-2, Ash1, CgrA, Scg2. The neuroendocrine differentiation of these cells was further confirmed by electron microscopy demonstrating neuropeptides secreting granules in their cytoplasm. Furthermore, in agreement with the altered expression observed in the majority of human SCLC we showed in these cells the absence of both p53 and pRB and a dramatic reduction in the expression of Caveolin-1.

Short and highly efficient synthetic promoters for melanoma-specific gene expression.

Here, we report the construction and functional analysis of synthetic promoters designed for gene therapy applications requiring strong and specific gene expression in melanoma cell lines. We have analysed the transcriptional activity of different combinations of two transcriptional regulatory modules, a melanocyte-specific element from the human tyrosinase promoter and a cell-cycle-specific element from the human alpha-fetoprotein promoter. Transient expression assays in different cell lines show that several of these composite synthetic promoters can drive a strong and selective expression of a reporter gene in melanoma cell, providing us with a new powerful tool for gene therapy of melanomas.