| BackgroundCancer-related inflammation is recognised as the seventh hallmarks of cancer. Chronic inflammation plays an important role in the initiation and progression of various types of cancer, including nasopharyngeal carcinoma (NPC), and these cancers are regarded as inflammation-related cancer. NPC is a malignant tumour with a characteristic geographic and racial distribution. Additionally, nasopharyngeal epithelial cells are continuously exposed to environmental challenges, such as bacteria, fungi and viruses, during breathing and digestion. When these stimuli destroy the epithelial surface barriers, it can potentially activate the immune system and trigger a pro-inflammatory response, which lead to an inflammation response and recruit more inflammatory cells infiltrating in the nasopharyngeal path, and further result in nasopharyngeal carcinoma. However, the initial trigger for nasopharyngeal inflammation resulting in nasopharyngeal carcinoma remains unclear. Using suppression subtractive hybridisation (SSH) and cDNA microarray, we previously identified the LPLUNC1gene (C20orf114) as a tissue-specific gene in nasopharyngeal epithelia that is down-regulated in NPC. Recently studies have shown that LPLUNC1is a secreted protein and can function in host defence by the BPI-domain which inhibiting the proliferation and metastasis of cancer. Thus, we investigated the role and mechanism of LPLUNC1on inhibiting the initiation and progression of NPC when encounter chronic inflammation.[Nasopharyngeal epithelial cancer cells involved in inflammation stimulated by inflammatory cytokines]A low dose of LPS can induce the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-lβ and IL-8) in D-THP-1cells, thus, the culture supernatants from untreated and LPS-treated D-THP-1cells were collected and used as "D-THP-1conditioned media" in subsequent experiments. To investigate whether nasopharyngeal epithelial cells themselves can similarly produce pro-inflammatory cytokines, we examined the production of TNF-α, IL-6, IL-1β and IL-8in5-8F cells treated with conditioned media from D-THP-1cells. Exposure of5-8F cells to the conditioned medium from LPS-treated D-THP-1cells significantly induced production of these pro-inflammatory cytokines and triggered significant activation of NF-κB and Stat3, which correlated with a concomitant degradation of IicBa and an increase of Jak2phosphorylation. Moreover, the LPS-treated D-THP-1conditioned media promoted the proliferation of5-8F cells. These results showed nasopharyngeal epithelial cancer cells may play a significant role in maintaining and amplifying the inflammation process via activation of NF-κB and the Stat3pathway and through the local production of pro-inflammatory cytokines, which recruit and activate additional immune cells in the nasopharyngeal path and promote tumour progression.[IL-6promoted the initiation and progression of nasopharyngeal carcinoma]IL-6mainly secreted by macrophages, is a pleiotropic cytokine, also considered a key growth-promoting and anti-apoptotic factor. The macrophages within the tumor, referring to as tumour-associated macrophages (TAMs), are the pivotal member of tumour microenvironment. TAMs constitute an important interface between tumour cells and the immune system and that they might influence neoplastic growth and progression in several ways. IL-6and TAMs are associated with an adverse prognosis in cancer. In order to explore the correlation of IL-6expression and macrophages infiltration, and whether their status correlated with the patients’survival in NPC, the IL-6and CD68protein expression (CD68positive expression could show the status of TAMs infiltrating.) in NPC were examined using tissues microarray by immunohistochemical analysis. High protein expression of IL-6and CD68were seen in NPC tissues. Significant and positive correlation was found between IL-6expression and infiltrating macrophage density. High IL-6protein expression and infiltration of CD68+macrophages were significantly correlated with poor patients’ survival. IL-6significantly promoted the proliferation of NPC cells via activation of NF-κB and Stat3pathways.[LPLUNC1inhibited the initiation and progression of NPC]The protein expression of LPLUNC1was examined by immunohistochemical analysis in NPC tissues microarray. LPLUNC1expression was significantly lower in the NPC compared to normal tissues. Negative LPLUNC1protein expression was significantly correlated with poor patients’survival in univariate analysis and multivariate analysis. To further study the effects of LPLUNC1on nasopharyngeal carcinoma, we stably transfected the full length of LPLUNC1expression plasmid into the NPC cell lines, and found that LPLUNC1inhibited the NPC cell growth and cell proliferation. Further studies revealed that over-expression of LPLUNC1in NPC cells inhibited tumour formation in nude mice; flow cytometry analysis indicated that LPLUNC1arrested the NPC cell cycle in G0/G1phase, and induced apoptosis. Furthermore, LPLUNC1suppressed the activation of NF-κB and Stat3pathways, followedly decreased the expression of CDK4, cyclin D1, and Bcl-2, increased the expression of Bax and p21in vitro and in vivo, thereby resulted in inhibiting the NPC cell growth.[LPLUNC1suppressed cell proliferation via inhibiting IL-6-activation of NF-κB and Stat3pathways]We analysed the relationships between LPLUNC1expression and macrophage infiltration density or IL-6expression in the NPC tissues, and found significant and negative correlation was found between LPLUNC1protein expression and macrophage infiltration density or IL-6expression, and through obtaining pure nasopharyngeal epithelium tissues by Laser Capture Microdissection, we confirmed the significant negative correlation between the expression of LPLUNC1and IL-6on mRNA level. Additionally, LPLUNC1could decrease the pro-inflammatory cytokines (TNF-α, IL-6, IL-1β and IL-8) expression in HNE2cells and macrophages regardless of whether LPS was present at both the protein and mRNA levels. These results showed that LPLUNC1was involved in inflammation and performs anti-inflammatory host defence functions. Furthermore, through suppressing the IL-6-activation of NF-κB and Stat3pathways, which resulted in decreasing the expression of cyclin D14, and Bcl-2, increasing the expression of Bax and p21, LPLUNC1inhibited the cell proliferation and cell cycle progression caused by IL-6in HNE2cells, which arrested the NPC cell cycle in G0/G1phase, and induced cell apoptosis.[Effect of LPLUNC1on protein expression profiling in NPC5-8F cells]We used differential in-gel electrophoresisi (DIGE) and MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry MALD1-TOF) to compare the protein expression profiling in NPC5-8F cells transfected with or without LPLUNC1.44different proteins were identified, including19up-regulated proteins,25down-regulated proteins. The functional classification of these different proteins was divided into chaperone, cytoskeleton, metabolism, signal transduction and other functions protein. It showed that via regulating the expression of these differentially proteins, LPLUNC1was involved in diverse biology events to inhibit the initiation and progression of NPC, including cell metabolism, proliferation, translation, signal transduction et al. In addition, we found the expression of Prohibitin (PHB, up-regulated different protein by LPLUNC1) was significantly down-regulated in NPC tissue specimens compared to normal samples both at mRNA and protein levels, closely associated with the advanced clinical stage and metastasis in NPC lesions, and the negative PHB protein expression significantly correlated with poor prognosis. Therefore, we favour the hypothesis that the expression levels of PHB could be used as potential prognostic biomarkers for NPC. |
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