Interleukins, laminin and Epstein - Barr virus latent membrane protein 1 (EBV LMP1) promote metastatic phenotype in nasopharyngeal carcinoma.
BACKGROUND
Nasopharyngeal carcinoma (NPC) is a type of neoplasm that is highly prevalent in East Asia and Africa with Epstein-Barr virus (EBV), genetic, and dietary factors implicated as possible aetiologic factors. Previous studies suggested the association of certain cytokines with the invasion and metastatic properties of NPC. The present study examined the roles of EBV latent membrane protein-1 (LMP1), interleukin-6 (IL-6), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-β1) and laminin in the regulation of matrix-metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) in NPC. The effects of these factors on bmi-1, an oncogene, and ngx6, a tumour suppressor gene, were also investigated.


METHODS
TW01 cells expressing LMP1 (TW01-LMP1) were established via transfection with the B95.8 EBV LMP1 gene. Both TW01 and TW01-LMP1 cells were treated with 100 pg/ml IL-6, 1000 pg/ml IL-10 and 100 pg/ml TGF-β1, separately and also in combination at their respective concentration for 48 hours. Treated cells were subjected to laminin adherence assay. The cells were also cultured with and without laminin and assayed for MMP-3, MMP-9 and VEGF production using enzyme-linked immunosorbent assay (ELISA). The cellular apoptotic property was analysed using caspase-3 apoptosis assay. The expression of bmi-1 and ngx6 gene was investigated using real time reverse transcriptase polymerase chain reaction.


RESULTS
LMP1 was found to reduce the adherence of NPC cells towards laminin (p < 0.05) as compared to control. Treatment with IL-6 at 100 pg/ml enhanced the production of MMP-9 in both TW01 and TW01-LMP1 cells (p < 0.05). When cultured on laminin, the levels of MMP-3 and VEGF were significantly increased (p < 0.05) in TW01-LMP1 cells. TW01-LMP1 cells had relatively greater resistance to apoptosis as compared to TW01 cells (p < 0.05). Laminin, IL-6 and LMP1 were found to up-regulate the expression of bmi-1 and suppressed the expression of ngx6.


CONCLUSIONS
We conclude that IL-6 reduced cell adherence towards laminin and increased MMP-9 production in NPC cells. Our data suggested that EBV LMP1 was able to confer resistance of apoptosis and increased MMP-9 production in NPC cells. When cultured on laminin, TW01 cells expressing the EBV LMP1 (TW0-LMP1) that were treated with IL-6 at 100 pg/ml displayed increased MMP-9 production, up-regulation of bmi-1 oncogene expression and down-regulation of ngx6 tumour suppressor gene expression. These findings implicate the roles of EBV LMP1, laminin and IL-6 in the promotion of invasion and metastasis in NPC.
Department of Research and Postgraduate Studies, International Medical University, No. 126, Jalan 19/155B, Bukit Jalil, Kuala Lumpur, Malaysia.
Michelle M S
MM
Chew
Sook-Yee
SY
Gan
Alan S B
AS
Khoo
Eng-Lai
EL
Tan
D002277:Carcinoma; D053148:Caspase 3 / Q000378:metabolism; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D015967:Gene Expression Regulation, Viral; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000378:metabolism / Q000821:virology; D009362:Neoplasm Metastasis; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000378:metabolism*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D015850:Interleukin-6; D007378:Interleukins; D007797:Laminin; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D014763:Viral Matrix Proteins; D016753:Interleukin-10; D053148:Caspase 3; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9