lactoferrin and Nasopharyngeal-Neoplasms

It is reported that LTF had a radiation resistance effect, and its expression in nasopharyngeal carcinoma (NPC) was significantly down-regulated. However, the mechanism of down-regulated LTF affecting the sensitivity of radiotherapy has remained elusive.. We re-analyzed the microarray data GSE36972 and GSE48503 to find differentially expressed genes (DEGs) in NPC cell line 5-8 F transfected with LTF or vector control, and the DEGs between radio-resistant and radio-sensitive NPC cell lines. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein-protein interaction network (PPI) analysis of DEGs were performed to obtain the node genes. The target genes of miR-214 were also predicted to complement the mechanism associated with radiotherapy resistance because it could directly target LTF.. This study identified 1190 and 1279 DEGs, respectively. GO and KEGG analysis showed that apoptotic process and proliferation, PI3K-Akt signaling pathway were significantly enriched pathways. Four nodes (DUSP1, PPARGC1A, FOS and SMARCA1) associated with LTF were screened. And 42 target genes of miR-214 were cross-linked to radiotherapy sensitivity.. The present study demonstrates the possible molecular mechanism that the down-regulated LTF enhances the radiosensitivity of NPC cells through interaction with DUSP1, PPARGC1A, FOS and SMARCA1, and miR-214 as its superior negative regulator may play a role in regulating the radiotherapy effect.

Topics: Cell Line, Tumor; Computational Biology; Databases, Genetic; Down-Regulation; Humans; Lactoferrin; MicroRNAs; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Protein Interaction Maps; Radiation Tolerance; X-Rays

Topics: Autoantigens; Fatty Acid-Binding Proteins; Glycoproteins; Humans; Immunity, Innate; Lactoferrin; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Phosphoproteins; Proteins

Lactotransferrin (LTF), also known as lactoferrin, is a key component of innate immune defense. We previously reported that LTF was downregulated in nasopharyngeal carcinoma (NPC) and could suppress NPC cell proliferation. However, the relevance of the relationship between LTF expression and NPC clinical outcome has not been reported. This study aims to assess the possible correlations between LTF expression and clinical parameters and its potential prognostic predictive ability in the outcomes of patients with NPC. Complementary DNA (cDNA) microarray, quantitative real-time PCR (qRT-PCR), and immunohistochemistry (IHC) results suggested that LTF expression was significantly downregulated in NPC tissues compared to non-NPC tissues. LTF was negatively correlated with lymph node metastasis (P = 0.042), T stage (P < 0.001), clinical tumor-node-metastasis (TNM) stage (P = 0.022), and EBV-encoded RNA 1 (EBER-1) expression (r = -.167, P = 0.016). A survival analysis of 108 patients with NPC revealed that positive expression of LTF could predict a good prognosis [disease-free survival (DFS): P = 0.043, overall survival (OS): P = 0.040]. Multivariable analysis revealed that LTF could independently predict prognosis (DFS: HR = 0.414, P = 0.003; OS: HR = 0.309, P = 0.005). These observations indicated that LTF is a potential prognostic factor of NPC.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Carcinoma; Cell Proliferation; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Lactoferrin; Lymphatic Metastasis; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Prognosis

LTF (lactotransferrin, or lactoferrin) plays important role in innate immunity, and its anti-tumor function has also been reported in multiple cancers. We previously reported that LTF is significantly down-regulated in nasopharyngeal carcinoma (NPC) and acts as a tumor suppressor by suppressing AKT signaling. However, the exact mechanism of the down-regulation of LTF in NPC has not been revealed. In the current study, we screened and identified LTF is a bona fide target of miR-214 in NPC cells. miR-214 mimics significantly suppressed LTF mRNA and protein expression levels in NPC cells. miR-214 not only can promote NPC cell proliferation and invasion abilities in vitro, but also can accelerate tumor formation and lung metastasis in a mouse xenograft model. The pro-tumor function of miR-214 was depended on LTF suppression since LTF re-expression can reverse it. miR-214 can also activate AKT signaling by suppressing LTF expression. Furthermore, miR-214 expression level was up-regulated in NPC especially in metastasis-prone NPC tumor tissues compared with normal nasopharyngeal epithelial tissues, while the LTF expression level was negatively correlated with miR-214, suggesting that miR-214 targeting is partly responsible for LTF down-regulation in NPC specimens.

Topics: Animals; Apoptosis; Blotting, Western; Case-Control Studies; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lactoferrin; Luciferases; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Nasopharyngeal Neoplasms; Nasopharynx; Neoplasm Staging; Prognosis; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

LTF (lactotransferrin, also known as lactoferrin) is a key component of innate immune defense. It has recently been found to have anti-tumor and anti-metastatic activity in different cancers. We previously reported LTF to be the most significantly downregulated gene in nasopharyngeal carcinoma (NPC) specimens relative to normal nasopharyngeal epithelial tissues, and it was also negatively associated with the progression and metastasis of NPC. However, the mechanism underlying this remains unclear. In the current study, we revealed that LTF can suppress 3-phosphoinositide-dependent protein kinase 1 expression via the mitogen-activated protein kinase/c-Jun pathway and thus repress AKT signaling. We also showed that LTF interacts with keratin 18 (K18) and so blocks the formation of the K18-14-3-3 complex, leading to downregulation of K18-mediated AKT activation. Thus, LTF suppresses AKT signaling by two separate mechanisms, leading to inhibition of NPC tumorigenesis. This is the first report on the tumor suppressive effects of LTF through repression of AKT signaling in NPC. It suggests that both LTF and AKT signaling merit further study in the field of NPC research.

Topics: 14-3-3 Proteins; Adult; Aged; Animals; Biomarkers, Tumor; Carcinoma; Cell Line, Tumor; Cell Proliferation; Exonucleases; Exoribonucleases; Female; Gene Expression; Humans; Lactoferrin; Male; Mice; Middle Aged; Mitogen-Activated Protein Kinases; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; Neoplasm Staging; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction

Lactotransferrin (LTF) is a component of the nonspecific immune system, having antimicrobial properties against bacteria, fungi, and several viruses. The gene coding for LTF is polymorphic, with the occurrence of several common alleles in the general population. Our previous study found that LTF inhibited nasopharyngeal carcinoma (NPC) cell proliferation in vitro and in vivo. To better understand one possible mechanism of LTF-mediated antitumor activity in NPC cells, in the present study, we investigated the distribution of LTF gene polymorphisms (rs1126477, rs1126478, rs2073495, and rs9110) in NPC and revealed whether these polymorphisms were associated with LTF gene expression. It was found that rs2073495 and rs9110 were correlated significantly with NPC. The frequency of CC genotype was higher and GG or TT genotype was lower, in NPC patients compared with that in the control group (P < 0.05, χ(2) = 8.73 and 9.33, respectively). CC genotype is the risk factor for NPC. Haplotype analyses indicated that NPC patients had lower rate of 'A-G-G-T' haplotype (constructed with rs1126477, rs1126478, rs2073495, and rs9110) compared with controls (P = 4.12 × 10(-6) < 0.001, χ(2) = 21.25). The population with 'A-G-G-T' haplotype had 0.322-fold risk to be NPC. The expression of LTF gene was high in NPC tissues and control tissues with 'A-G-G-T' haplotype compared with these without its. These findings suggested that rs2073495 and rs9110 could play important roles in NPC physiological processes.

Topics: Blotting, Western; Carcinoma; Female; Genetic Predisposition to Disease; Haplotypes; Humans; Lactoferrin; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Polymorphism, Single Nucleotide; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors

Lactoferrin (LF) is a multifunctional glycoprotein that plays an important role in native immune defense against infections, including human herpetic viruses, such as cytomegalovirus and herpes simplex virus types 1 and 2. However, its anti-Epstein-Barr virus (EBV, a γ-herpesvirus) function has not been reported in the literature. EBV is widespread in all human populations and is believed to be linked to tumorigenesis, such as lymphomas and nasopharyngeal carcinoma (NPC). We previously reported that LF expressed a significantly lower level in NPC tissues and was a likely tumor suppressor. Since EBV infection is a major carcinogen of NPC development, we investigated the effect of LF on EBV infection and found that LF could protect human primary B lymphocytes and nasopharyngeal epithelial cells from EBV infection, but had no effect on EBV genome DNA replication. LF prevented EBV infection of primary B cells mediated by its direct binding to the EBV receptor (CD21) on the B-cell surface. Tissue array immunohistochemistry revealed that LF expression was significantly downregulated in NPC specimens, in which high EBV viral capsid antigen-IgA levels were observed. These data suggest that LF may inhibit EBV infection and that its downregulation could contribute to NPC development.

Topics: Antigens, Viral; B-Lymphocytes; Capsid Proteins; Carcinoma; Cell Line, Tumor; Cells, Cultured; DNA Replication; DNA, Viral; Epithelial Cells; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Immunoglobulin A; Lactoferrin; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Nasopharynx; Virus Replication

The lactoferrin (LTF) gene, located at 3p21.3, behaves like a tumor suppressor gene in diverse tumors. To elucidate the exact role of LTF in NPC, we first detected its expression level in seven NPC cell lines by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed the mRNA level of LTF was nearly undetectable in all the seven NPC cell lines, while it could be detected in chronic nasopharyngitis tissues. Subsequently, we used methylation-specific PCR (MSP), microsatellite assay, PCR-single-strand conformation polymorphism (PCR-SSCP) and sequencing methods to examine the promoter methylation, loss of heterozygosity (LOH) and gene mutation of LTF in NPC cell lines respectively. Consequently, we found that 100% (7 of 7) of NPC cell lines were methylated in LTF promoter, only one cell line (14%, 1 of 7) had LOH and gene mutation of LTF, respectively, while LTF exhibited re-expression in all cell lines after 5-aza-dC treatment, indicating promoter methylation should be the key mechanism causing LTF downregulation in NPC cell lines. Furthermore, patched methylation assay confirmed that promoter methylation could down-regulate LTF gene expression in NPC cells. Finally, we investigated the function of LTF in NPC cell lines by gene transfection. Restoration of LTF expression in NPC cells resulted in blockage of cell cycle progression, significant inhibition of cell growth and a reduced colony-formation capacity in vitro and obviously weaker tumor formation potential in vivo. In conclusion, our data indicate LTF may participate in NPC carcinogenesis as a negative effector, that is, a tumor suppressor gene.

Topics: Animals; Base Sequence; Carcinoma; Cell Line, Tumor; Cell Proliferation; DNA Methylation; DNA Mutational Analysis; Female; G1 Phase; Humans; Lactoferrin; Loss of Heterozygosity; Male; Mice; Mice, Nude; Molecular Sequence Data; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Promoter Regions, Genetic; Recombinant Proteins; Transcription, Genetic; Tumor Burden

To investigate the expression of LTF mRNA in several nasopharyngeal cancer (NPC) cell lines, and analyze the relationship between the genetic and epigenetic changes and expression of LTF gene.. The expression level of LTF was detected in NPC cell lines HNE1, HNE2, HNE3, CNE1, CNE2, 5-8F, 6-10B cells and tissues of 15 cases of chronic nasopharyngitis by RT-PCR. The LTF protein level was analyzed by Western blotting in 6-10B cells. Then LOH, mutation and methylation status of LTF was examined by microsatellites analysis, PCR-SSCP, MSP and bisulfite genomic sequencing, respectively.. 15 chronic nasopharyngitis tissues showed stable LTF expression, while there were weak expression in 6-10B cells and absent expression in remaining detected NPC cell lines. There was a significantly lower LTF expression in chronic nasopharyngitis tissues (Z = -3.738, P = 0.000). No LTF protein expression was observed in 6-10B cells. LOH analysis demonstrated that allele loss of LTF wasn't found in NPC cell lines. LTF mutation was noted in 14.3% (1/7) of NPC cell lines. DNA sequencing confirmed the mutation point in the promoter region (-305 bp to -50 bp) was at -218 bp (del T) of LTF gene in the HNE1 cell line. Methylation of LTF gene was not found in chronic nasopharyngitis. However, methylation of LTF promoter was detected in all NPC cell lines. LTF mRNA expression was increased in 5-8F and 6-10B cell lines after treatment with 5-aza-2-deoxycytidine.. There is an inactivation of expression of LTF gene in the NPC cell lines. Its molecular mechanism may be related with methylation of promoter region and deletion mutation.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Deletion; Humans; Lactoferrin; Loss of Heterozygosity; Nasopharyngeal Neoplasms; Nasopharyngitis; Promoter Regions, Genetic; RNA, Messenger

Lactotransferrin (LTF) has been shown to regulate tumorogenesis. However, little is known about the role of LTF in regulating the development of human nasopharyngeal carcinoma (NPC). The aim of our study was to investigate whether LTF could regulate the development of NPC by characterizing the pattern of LTF expression in human NPC tissues using cDNA and tissue microarrays. Loss of LTF expression was observed in a significantly higher frequency of NPC tissues compared to that in nontumor nasopharyngeal epithelial tissues. While 61.25% of NPC tissues at the T1/T2 stage were positive for LTF expression, only 40.82% of NPC at the T3/T4 stage were stained by anti-LTF. Similarly, 41.58% of NPC with local lymph node metastasis displayed LTF expression, a value significantly lower than the 46.36% in primary tumors (p < 0.05). These findings suggest that LTF may negatively regulate the development and metastasis of NPC in vivo. Furthermore, overexpression of or treatment with LTF inhibited the proliferation of NPC cells and promoted cell cycle arrest at the G(0)/G(1) phase in vitro. While LTF treatment downregulated expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb), expression of p21 and p27 in 5-8F NPC cells was enhanced. Moreover, LTF treatment modulated the mitogen-activated protein kinase (MAPK) pathway, but did not affect p53 and STAT3 expression in 5-8F NPC cells. Thus LTF is likely to be a candidate tumor suppressor and downregulates the development of NPC by inhibiting NPC proliferation through induction of cell cycle arrest and modulation of the MAPK signaling pathway. Therefore, our findings provide new insights in understanding the mechanism(s) underlying the action of LTF in regulating the development of human NPC.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 3; Cyclin D1; Humans; Lactoferrin; MAP Kinase Signaling System; Nasopharyngeal Neoplasms; Oligonucleotide Array Sequence Analysis; Phosphorylation; Retinoblastoma Protein; Tissue Array Analysis; Tumor Suppressor Proteins

To investigate the roles of lactotransferrin gene (LTF, also referred to as the lactoferrin gene, LF), located at 3p21.3 within the common minimal deletion region, in the pathogenesis of nasopharyngeal carcinoma (NPC), we first detected its expression level in 33 primary NPC tissues and 15 chronic nasopharyngitis tissues. Absent expression or downregulation of LTF were observed in 76% (25 of 33) of primary NPC tissues. We further found that 25% (5 of 20) of NPC specimens had loss of heterozygosity (LOH) at the LTF locus. LTF mutation assessed by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing was noted in 30% (6 of 20) of primary NPC tissues. In addition, hyper-methylation of LTF promoter region was found in 63.6% (21 of 33) of primary NPC samples but not in chronic nasopharyngitis tissues. The LTF transcripts in NPC cell lines increased upon treatment with the demethylation compound, 5-aza-2-deoxycytidine. In conclusion, our data indicate that two-hit silencing of LTF through genetic and epigenetic changes may be a common and important event in the carcinogenesis of NPC.

Topics: Azacitidine; Base Sequence; Carcinoma; Cell Line, Tumor; Chromosomes, Human, Pair 3; CpG Islands; Decitabine; DNA Methylation; DNA Modification Methylases; DNA Mutational Analysis; Down-Regulation; Enzyme Inhibitors; Epigenesis, Genetic; Exons; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lactoferrin; Loss of Heterozygosity; Male; Middle Aged; Molecular Sequence Data; Mutation; Nasopharyngeal Neoplasms; Polymorphism, Single-Stranded Conformational; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger