Microarry Analysis Identifies Activation Of Multiple Signalling Pathways In Primary Prostate Epithelial Cells By Low Temperature Plasma

Abstract

We have previously reported the killing effects of Low Temperature Plasma (LTP) on primary prostate epithelial cells, of both normal and cancerous origin, via necrosis [1] following initial DNA damage. However it was evident that a population of cells was resistant to LTP treatment. To discern possible survival mechanisms in treated cells; the gene expression of 6 samples (4 cancers and 2 matched normal) was analysed 2 hours post-treatment, on a transcriptome wide level, by microarray. Initial analysis found alteration in the expression of 646 transcripts with significant activation of Notch, NF-kB, Nrf2 and AP-1 signalling in our primary cells. Pathway activation was subsequently validated by qRT-PCR in four different patient-derived prostate cultures to give a robust LTP-induced expression signature.

Upstream effector activation was then confirmed by immunofluorescence and Western blot. We found as other groups have; [2, 3] accumulation of Nrf2 in response to LTP, alongside AP-1 activation [4, 5] – through phosphorylation of both JNK and Jun. The Notch signalling pathway is typically described in relation to stem cell maintenance and we observed LTP induced Notch activation through release of its intracellular domain (NICD) that then acts as a nuclear transcription factor.

Tumours are innately heterogeneous and contain multiple cell populations, both intrinsic and recruited. We have established that our cultures contain three readily identifiable epithelial cell pools that can be separated to permit further analysis.[6] Preliminary investigation has found that the progenitor epithelial cells respond in greater measure than their more differentiated progeny, suggesting that they are more resistant to plasma. Similar observations of prostate stem cell resistance have been made with radiotherapy.[7] However, if these stem-like cells can be targeted – LTP may have a greater killing effect if utilized in prostate cancer treatments.

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Figure – Pathway activation in primary prostate cancer epithelial culture; Nrf2 accumulation, AP-1 activation by JNK and Jun phosphorylation and Notch cleavage, with release of NICD by LTP. U – Untreated, T- Treated. Samples taken 0.5 and 2 hours following treatment with 3 minutes LTP.