Project description:Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses in human malignant melanoma cells in vitro in order to understand and predict possible effect of electric pulses on gene expression and their possible effect on cell behaviour. We used microarrays with 2698 different oligonucleotides to obtain expression profile of genes involved in apoptosis and development of cancer in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Cells exposed to ECT pulses showed 68.8% average survival rate and 31.4% average survival rate in cells exposed to EGT pulses. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis. Our results show that electroporation does not significantly change expression profile of major tumour suppressor genes or oncogenes of cell cycle. Moreover, electroporation also does not changes the expression of genes involved in stability of DNA, supporting the current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method it does to some extent induce stress that resulted in expression of environmental stress response mechanism, HSP70. The difference in expression of genes involved in development of cancer was obtained by comparison of malignant melanoma cells exposed to EGT or ECT pulses against the same untreated malignant melanoma cells. In our experimental design, microarrays with 2698 different genes were used as a dual colour system in which exposed and non-exposed cellsâ mRNA were separately labelled, mixed and hybridised together on each array. Only microarrays expressing at least 50% of genes were used for further analysis. All oligonucleotides on the same array were spotted in quadruplicates and each microarray analysis was performed in duplicates, therefore obtaining eight measurements of the same oligonucleotide. The acquired data were analysed with Acuity 4.0 to select reliable signals. Only genes, present in both duplicated microarrays were considered for further processing.

Project description:In this project we aimed at deciphering modulation in genes expression induced by external pulsed electric fields applied in reversible electroporation-based treatments. Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel extracellular matrix, which was the point of interest in our to be published study. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute, a tissue model rich in endogeneous extracellular matrix such as collagens, was used to demonstrate the effects of microsecond and millisecond pulsed electric fields applied respectively in electrochemotherapy (ECT) treatment and gene electrotransfer (GET) strategy. Our analyses, focused on matrisome genes and extracellular matrix remodeling, underpin that pulsed electric fields, a technology already approved for clinical use combined with anti-cancer agents, are particularly promising to provide local and effective treatment of abnormal extracellular matrix. Part of this dataset was used to describe how pulsed electric field on its own (with no addition of external drugs) induce extracellular matrix (ECM) remodeling at human dermal scale, by focusing at genes related to matrisome subset. In this manuscript to be published, electrochemotherapy (ECT) parameters were named SP for "short pulses" and gene electrotransfer (GET) parameters were named LP for "long pulses". W demonstrated that these both types of electric parameters inducing reversible electroporation of the cells whitin the dermal tissue substitute induced 1) a rapid modulation (4h after electrostimulation) of mRNA’s genes composing the matrisome, particularly a down-regulation of pro-collagens and ECM maturation’s enzymes such as transglutaminase TG2 and LOX-like; 2) a transient decrease in pro-collagens production and hydroxyproline tissue content within a week after electrostimulation; 3) a long-lasting ROS-dependent over-activation of MMPs for at least 48h and 4) a down-regulation at both mRNA and protein level of pro-fibrotic TGF-β.

Project description:Pappalardo2016 - PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer This model is described in the article: Computational Modeling of PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer. Pappalardo F, Russo G, Candido S, Pennisi M, Cavalieri S, Motta S, McCubrey JA, Nicoletti F, Libra M. PLoS ONE 2016; 11(3): e0152104 Abstract: Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches. Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways. Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention. According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease.Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development. Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents. The source code of the various versions of the model are available as S1 Archive. This model is hosted on BioModels Database and identified by: MODEL1609190000. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:A new approach to treating solid tumours (both operable and inoperable) has been carried out by the Cork Cancer Research Centre (CCRC) at the Mercy University Hospital, Cork, Ireland since 2002. The approach simply allows a greater concentration of chemotherapy drugs to enter the tumour cells rather than healthy cells. The uptake of the chemotherapeutic drug directly by the tumour is aided through applying short electric pulses to the tumor mass (referred to as - Electrochemotherapy or ECT). The pulses make the tumour more porous which allows the drug easier access into the cancer cells, whereas other tissues and organs in the body remain relatively poor at absorbing the drug, thereby reducing the potential side effects on healthy tissues. This approach to date has been limited to skin based tumours due to the requirement for the electrodes to be placed directly in contact with the tumour. Procedures with electrochemotherapy have been applied to human patients in other countries of the EU, the US and Japan. The drug concentration used is significantly reduced due to the more targeted absorption by the tumor and this significantly reduces side effects normally associated with chemotherapy. A large number of preclinical and clinical Phase I and I/II studies have demonstrated the efficiency and safety of ECT. These studies have included patients with melanoma, head and neck squamous cell carcinoma, merkel cell carcinomas, basal cell carcinoma and adenocarcinoma nodules. Case reports concerning other primary tumours have also been reported. The investigators have developed an endoscopic approach (EndoVe system) for delivering the electric pulses to internal cancers and are currently seeking to evaluate its efficacy in the treatment of inoperable colorectal cancer. The treatment procedure is similar to standard endoscopic colorectal examination (colonoscopy) with the added element of an intravenous injection of bleomycin followed after eight minutes by the delivery of electric pulses (each one less than 1msec in duration). The pulses are endoscopically delivered directly to the tumour mass. The entire procedure is minimally invasive and does not require intensive care follow up or stitches. If the treatment is successful the tumour will shrink in size in the weeks following the procedure. The objective of this study is to investigate the efficacy and safety of this approach in reducing the size of the tumour.

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