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Electric pulses do not change expression profile of genes involved in development of cancer in malignant melanoma cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Damjan Glavac

PROVIDER: E-GEOD-15420 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells.

Mlakar Vid V Todorovic Vesna V Cemazar Maja M Glavac Damjan D Sersa Gregor G

BMC cancer 20090826

<h4>Background</h4>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 on human malignant melanoma cells in vitro in order to understand and predict the possible effect of electric pulses on gene expression and t ...[more]

PMID: 19709437

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Project description:Abstract; Background: Gene transfer by electroporation (electro gene transfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of electro gene transfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, as well as morphological changes evaluated by histological analysis. Electro gene transfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 @s) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. Results: Differentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1) electroporation, 2) DNA injection, and 3) time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis. Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic en-zymes such as phosphoenolpuryvate carboxykinase. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while electroporation alone caused minor loss of striation pattern but preservation of cell integrity. Conclusion: The small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that electro gene transfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the electro gene transfer to muscle for clinical use. Indeed the HV+LV pulse combination used have been optimised to ensure highly efficient and safe electro gene transfer. Experiment Overall Design: The mice did recieve to their tibialis cranialis muscle either No tretment/control (CTRL), Electroporation only (EP), Plasmid injection only (DNA) or Plasmid DNA and in vivo Electro gene transfer (EP+DNA). Experiment Overall Design: Four hrs, 48 hrs and 3 weeks after treatment the mice were euthanized and the expression profile of the treated muscles were analysed. Experiment Overall Design: The following number of mice were included: Experiment Overall Design: CTRL, 3 mice, Experiment Overall Design: EP at 4 hrs, 1 mouse, Experiment Overall Design: EP at 48 hrs, 1 mouse, Experiment Overall Design: EP at 3 weeks, 1 mouse, Experiment Overall Design: DNA at 4 hrs, 1 mouse, Experiment Overall Design: DNA at 48 hrs, 1 mouse, Experiment Overall Design: DNA at 3 weeks, 1 mouse, Experiment Overall Design: EP+DNA at 4 hrs, 2 mice, Experiment Overall Design: EP+DNA at 48 hrs, 1 mouse, Experiment Overall Design: EP+DNA at 3 weeks, 1 mouse. Experiment Overall Design: A total number of mice 13.

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:Aims/hypothesis: While lipid deposition in skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like skeletal muscle. Here we investigated the effects of PLIN5 overexpression M-bM-^@M-^S in comparison with effects of PLIN2 M-bM-^@M-^S on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in skeletal muscle promoted expression of a cluster of genes under control of PPARM-NM-1 and PGC1M-NM-1 involved in FA catabolism and mitochondrial oxidation. Rats received a high fat diet for 3 weeks; 2 weeks after start of the diet intervention Plin5 (OXPAT) or Plin2 (ADRP) were overexpressed in either the right or left tibialis anterior muscle. One week later pooled tibialis anterior muscle samples were analysed on microarrays.

Dataset Information

Electric pulses do not change expression profile of genes involved in development of cancer in malignant melanoma cells

Publications

Electric pulses used in electrochemotherapy and electrogene therapy do not significantly change the expression profile of genes involved in the development of cancer in malignant melanoma cells.

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