Photodynamic effects of FosPeg®H-ALA on Epstein-Barr virus (EBV) positive and negative nasopharyngeal carcinoma cells

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Photodynamic effects of FosPeg®H-ALA on Epstein-Barr virus (EBV) positive and negative nasopharyngeal carcinoma cells

 

Author: Wu, Wing Kei
Title: Photodynamic effects of FosPeg®H-ALA on Epstein-Barr virus (EBV) positive and negative nasopharyngeal carcinoma cells
Degree: Ph.D.
Year: 2014
Subject: Photosensitizing compounds.
Epstein-Barr virus.
Nasopharynx -- Cancer.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xxiv, 186 pages : color illustrations
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2910876
URI: http://theses.lib.polyu.edu.hk/handle/200/8573
Abstract: Nasopharyngeal carcinoma (NPC) is one of the top ten cancers highly prevalent in Hong Kong with more than 800 new cases reported annually. The epidemiologic evidence implies that Epstein-Barr virus (EBV) infection is strongly associated with the NPC tumourigenesis. Among the EBV proteins expressed in NPC patients, latent membrane protein 1 (LMP1) is documented as the principal onco-protein contributes to EBV-associated oncogenesis via modulation of the intracellular signaling pathways, such as mitogen-activated protein kinases (MAPK) signaling pathways. Conventional treatment of NPC is mainly based on chemo-radiotherapy, however, the treatment outcomes of high grade (poorly differentiated and undifferentiated) NPC tumours remain poor. The extensive use of chemodrugs further complicated the situation by inducing drug resistant NPC tumours. Therefore, a novel treatment is crucial for patients suffering from high grade NPC. Photodynamic Therapy (PDT) is FDA approved cancer treatment regimen in the USA, European Union, Japan and China. It employs a combination of light-activated photosensitizer (PSs) and oxygen to selectively destroy the tumours. The PDT efficacy could be further enhanced by improved photosensitizers and novel light source. In the present work, two improved photosensitizers, namely H-ALA (5-aminolevulinic acid hexylester) and FosPeg® (liposomal formula of mTHPC) with LED light activation were comparatively studied on three NPC cells, namely EBV positive C666-1 cells (undifferentiated), EBV negative CNE2 cells (poorly differentiated) and EBV negative HK1 cells (well differentiated). Their antitumour mechanisms were investigated, including: uptake and localization of PSs, phototoxicity, regulation of EBV LMP1 related miRNAs, mRNA and protein, mode of cell death, and modulation of intracellular signaling proteins (MAPK and EGFR) and their downstream proteins on NPC. Apart from these, the action of FosPeg® PDT on the drug resistant properties in NPC cells was also addressed.
Our findings indicated that both H-ALA and FosPeg® were localized in the mitochondria of all three NPC cells. FosPeg®-PDT demonstrated a better phototoxicity in NPC cells than that of H-ALA-PDT. FosPeg®-PDT showed phototoxic effect on all three tested cell lines; whereas H-ALA-PDT was ineffective in HK1 cells. In order to achieve LD50 FosPeg®-PDT, a 48-fold lower PDT dose was needed than that of H-ALA. Similarly, a 24-fold lower PDT dose was needed for FosPeg® than that of H-ALA in CNE2 cells. No dark toxicity was identified in both PSs. EBV LMP1 miRNAs, mRNA and proteins were expressed only in EBV positive C666-1 cells among the three cell lines. FosPeg®-PDT induced a down-regulation of EBV encoded miRNAs (ebv-miR-BART1-5p, 16 and 17-5p). A significant up-regulation of LMP1 mRNA and protein were also obtained (P<0.05). H-ALA also caused a significant up-regulation of LMP1 protein in C666-1 cells. Both H-ALA and FosPeg® mediated PDT significantly provoked a down-regulation of MAPK and EGFR signal proteins in all NPC cells at LD70, thereby triggered apoptosis cell death pathway and caused cell cycle and DNA content changes in NPC cells. The PDT efficacy towards drug resistance counterpart was investigated. FosPeg®-PDT increased the P-glycoprotein (P-gp) mRNA and protein expression levels in all NPC cells. Interestingly, FosPeg® was not the substrate of P-gp transporter proteins and the PDT efficacy was not affected by the up-regulation of P-gp/MDR transporter proteins expression. In conclusion, this study offered new evidence and insights for potential PDT to EBV positive and EBV negative NPC cells. Further investigations are warranted for the benefit of PDT clinical applications in NPC patients.

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