Plasmid injection and application of electric pulses alter endogenous mRNA and protein expression in B16.F10 mouse melanomas

Abstract

The application of electric pulses to tissues causes cell membrane destabilization, allowing exogenous molecules to enter the cells. This delivery technique can be used for plasmid gene therapy. Reporter gene expression after plasmid delivery with eight representative published protocols was compared in B16.F10 mouse melanoma tumors. This expression varied significantly based on the pulse parameters utilized for delivery. To observe the possible influence of plasmid injection and/or pulse application on endogenous gene expression, levels of stress related mRNAs four and 24 hours after delivery were determined by PCR array. Increases in mRNA levels for several inflammatory chemokines and cytokines were observed in response to plasmid injection, electric pulses alone, or the combination. This upregulation was confirmed by individual real-time reverse transcription TaqMan PCR assays. Proteins were extracted at the same time points from identically treated tumors and inflammatory protein levels were assayed by ELISA and by a custom multiplex bead array. Increases in inflammatory protein levels generally paralleled mRNA levels. Some differences were observed, which may have been due to differing expression kinetics. The observed upregulated expression of these cytokines and chemokines may aid or inhibit the therapeutic effectiveness of immune-based cancer gene therapies.

Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response

“…The PI-positive and Annexin-V-negative region is recognized as a commonly accepted region for necrotic cells and the cells in such a region increased from 10.66% to 22.70% in BAL fluid as detected after 4 h. This indicates that an early lung damage involving cell necrosis was caused by injection of cationic nanocarriers. It has been known that plasmid DNA alone may induce inflammatory response 40 . To investigate the possible mechanisms of the toxicities caused by free cationic nanocarriers, we use the free cationic carriers alone but not plasmids/carriers complexes in most experiments.…”

Delivery of Interleukin-15 to B16 Melanoma by Electroporation Leads to Tumor Regression and Long-term Survival

“…These antitumor effects could be attributed to the CpG motifs that are present on the plasmid DNA as these sequences can cause tumor-specific immune responses ( 47 – 50 ). Plasmid delivery by EP alters endogenous mRNA and protein expression of cytokines and chemokines in melanoma based on the pulse parameters used ( 10 ). EP itself has also been shown to activate proinflammatory chemokine and stress genes and cause an influx of inflammatory cells to the site of administration ( 51 , 52 ).…”

“…EP for gene delivery has been extensively studied in a number of preclinical melanoma models ( 8 ) which lead to the first-in-man clinical trial that utilized EP to deliver inter leukin-12 (IL-12) directly to tumors ( 9 ). Studies have also shown that delivery of non-coding plasmid DNA using EP can stimulate inflammatory as well as anti-tumor responses ( 10 , 11 ) and EP can recruit immune cells to the site of administration ( 12 ). This provides an added advantage when using this delivery method for cancer therapy.…”

Assessment of the tumourigenic and metastatic properties of SK-MEL28 melanoma cells surviving electrochemotherapy with bleomycin

“…In our in vitro study, there was no change in expression of genes involved in inflammation as only tumour cells were included for gene expression analysis. On the other hand, in vivo study of mouse melanoma demonstrated that plasmid DNA encoding reporter luciferase gene alone or in combination with electric pulses and different electroporation protocols affects endogenous gene expression 46. Increased levels of mRNA and protein levels for inflammatory chemokines and cytokines were observed 4 h after gene electrotransfer and by 24 h, the expression levels of mRNAs and proteins were already considerably reduced 46…”