ID: 2182

• Title:
  Electrochemotherapy affects early myogenesis: a study on skeletal muscle cells in vitro
  
  Authors:
  

  Cemazar, Maja - Institute of Oncology Ljubljana, 
  Kranjc Brezar, Simona - Institute of Oncology Ljubljana, 
  Medved, Ajda - Institute of Oncology Ljubljana, 
  Matkovic, Urska - Institute of Oncology Ljubljana, 
  Sersa, Gregor - Institute of Oncology Ljubljana, 
  Markelc, Bostjan - Institute of Oncology Ljubljana,  
  Bozic, Tim - Institute of Oncology Ljubljana, 
  Jurdana, Mihaela - University of Primorska

  
  
  Abstract:
  Electrochemotherapy (ECT) is a local ablative therapy for the treatment of various skin and subcutaneous tumours of different histologic origins, as well as certain tumours in internal organs, such as colorectal liver metastases and hepatocellular carcinoma. The use of reversible electroporation with a small number of high-voltage electric pulses allows the application of low doses of cytostatic drugs (bleomycin and cisplatin) with high therapeutic efficiency and minimal systemic toxicity. Skeletal muscle, along with skin, vascular, endothelial, and nervous tissue, is an important tumour-surrounding tissue exposed to the side effects of ECT. These side effects are instantaneous muscle contractions that occur during the delivery of electric pulses. At the cellular level, the side effects of ECT on skeletal muscle and the underlying mechanisms have not yet been studied. Therefore, our aim was to determine the effect of ECT using bleomycin or cisplatin in the mouse muscle cell line C2C12 during in vitro myogenesis.
  
  To obtain myotubes, C2C12 cells were plated, and after three days, when myoblasts had reached 85-90% confluence, the growth medium was replaced with differentiation medium containing horse serum, which was renewed every two days until myotubes formed after approximately six days. Experiments on myotubes were performed when 80-85% of myotube area was reached.
  
  First, the electroporation efficiency and viability of the differentiated C2C12 myotubes were evaluated at increasing voltages from 200 to 1300 V/cm using propidium iodide, a non-permeant dye that emits fluorescence when bound to a DNA molecule. The percentage of propidium iodide-positive cells was voltage dependent and was approximately 40% positive cells at 500 V/cm, 74% at 900 V/cm to nearly 100% efficiency at 1300 V/cm. In parallel, we measured cell viability using a cell viability assay based on the reduction of a resazurin-based permeable solution. High cell survival and myotube integrity were maintained until day 4 after electroporation. Then, using an electroporation protocol that permeabilized nearly 100% of cells with minimal toxicity, ECT was assessed with cytostatic agents, bleomycin and cisplatin. Survival of myoblasts and myotubes, secretion of interleukin-6, and expression and distribution of nicotinic acetylcholine receptors (nAChRs) after different doses of bleomycin or cisplatin were studied. In combination with electroporation, increased doses of bleomycin (from 1400 nM to 0.14 nM) and cisplatin (from 150 µM to 5µM) were tested. Electroporation with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. ECT with bleomycin or cisplatin significantly increased IL -6 expression in 3 days in vitro myoblasts and early myotubes, whereas expression decreased in 5 days in vitro and 7 days in vitro treated myotubes, confirming the effects on early myogenesis. Untreated control myotubes exhibited aggregation of nAchRs into clusters, indicating fully differentiated myotubes after day 11. The clusters were absent in the treated groups, suggesting that ECT affects myotube differentiation.
  
  In conclusion, ECT affects early C2C12 myogenesis, whereas it has little or no effect on myotubes, i.e., differentiated skeletal muscle cells, so special attention should be paid to tumours located in muscle. In such cases, the inclusion of muscle in the treatment area should be kept to a minimum.
  
  
  
  Keywords:
  electrochemotherapy, muscle cell line, myogenesis
  
  Refs:
  
  
  Topic 1:
  1. Biological responses (molecular, subcellular, cellular and intercellular)
  
  Topic 2:
  6. Cancer treatment and tumor ablation