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Resistance to radiotherapy and chemotherapy currently represents one of the major reasons for therapeutic failure in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying resistance to chemotherapy in NPC remain unclear. In this study, cell counting assay, cell cycle assay and senescence associated β-galactosidase activity were performed to evaluate cell growth, proliferation and senescence, respectively. We found that the aberrant expression of cyclooxygenase-2 (COX-2) was associated with a poor outcome and recurrance in patients with NPC. In NPC cells, COX-2 overexpression increased cell proliferation, inhibited cellular senescence and resulted in chemoresistance, while the knockdown of COX-2 reduced cell proliferation, promoted cellular senescence and overcame chemoresistance. Furthermore, fibroblasts from COX-2 knockout mice exhibited cellular senescence, particularly when treated with chemotherapeutic agents. Mechanistically, COX-2 interacted with p53 protein and inhibited cellular senescence, which resulted in chemotherapeutic resistance. On the whole, these findings indicate that COX-2 may play a critical role in chemotherapeutic resistance in NPC via the inhibition of chemotherapy-induced senescence via the inactivation of p53. This study provides experimental evidence for the preclinical value of increasing chemotherapy-induced senescence by targeting COX-2 as an effective antitumor treatment in patients with recurrent NPC.

Sodium butyrate (NaBu), a histone deacetylase inhibitor, has demonstrated anti-tumor effects in several cancers, and is a promising candidate chemotherapeutic agent. However, its roles in nasopharyngeal carcinoma (NPC), an endemic malignant disease in Southern China and Southeast Asia, has rarely been studied. In the present study, MTT assay, colony formation assay, flow cytometry analysis and western blotting were performed to explore the influence of NaBu on NPC cells and its underlying mechanism. NaBu induced morphological changes and inhibited proliferation in 5-8F and 6-10B cells. MTT assay revealed that NaBu was cytotoxic to 5-8F and 6-10B cells in a dose- and time-dependent manner. Furthermore, flow cytometry analysis revealed that NaBu induced obvious cell apoptosis in 5-8F and 6-10B cells due to the activation of the mitochondrial apoptosis axis. In addition, flow cytometry analysis and western blotting demonstrated that NaBu could enhance the Ca2+ influx by promoting store-operated Ca2+ entry (SOCE) in 5-8F and 6-10B cells. Inhibition of SOCE by specific inhibitors or downregulated expression of calcium release-activated calcium channel protein 1 and stromal interaction molecule 1 could counteract the apoptosis of NPC cells induced by NaBu. Thus, the current study revealed that enhanced SOCE and activated mitochondrial apoptosis axis may account for the mechanisms of cytotoxicity of NaBu in NPC cells, and that NaBu serves as a promising chemotherapeutic agent in NPC therapy.

Previous studies have shown that promoter hypermethylation plays a key role in DLC-1 inactivation in nasopharyngeal carcinoma (NPC). However, DLC-1 mutation in NPC has not been reported, and there remain some discrepancies in methods and results between different groups. Here, we examined the mRNA and protein expression of DLC-1 in chronic nasopharyngitis (CN) and NPC tissues by reverse transcription-polymerase chain reaction/qPCR and immunohistochemistry, respectively. DLC-1 mRNA was undetectable in all the seven widely used NPC cell lines and absent or significantly down-regulated in 70% of NPC tissues. DLC-1 protein level was reduced in 74.3% of NPCs when compared with CN tissues, and significantly lower in NPC samples at advanced clinical stages than that at early stages. Then, we purified the same batch of specimens by microdissection and analyzed the possible mechanisms of DLC-1 downregulation with mutation and allelic loss analysis, methylation-specific PCR and bisulfite genomic sequencing. Only one mutation was detected at codon 693 of exon 8 in 3.3% of NPCs and five single nucleotide polymorphisms (SNPs) were identified. Loss of DLC-1 was detected in 23.3% of NPC tissues. The 100% of NPC cell lines, 80% of primary NPC and 22.2% of CN tissues showed methylation in DLC-1 promoter, while DLC-1 expression was recovered in seven NPC cell lines after 5-aza-dC treatment. Patched methylation assay confirmed that promoter methylation could repress DLC-1 expression. This report demonstrates that DLC-1 is negatively associated with NPC carcinogenesis, and promoter hypermethylation along with loss of heterozygosity, but not mutation, contributes to inactivation of DLC-1 in NPC.

Allelic loss of chromosome 3p, including the 3p21.3 region, is found in 95-100% of primary nasopharyngeal carcinoma (NPC) biopsies, suggesting that this region should harbor some tumor suppressor genes (TSGs) closely related to NPC development. Several TSGs located at 3p21.3, such as RASSF1A, LTF and BLU, have been demonstrated to be involved in NPC development. LARS2 (leucyl-tRNA synthetase 2, mitochondrial) is another gene located in the chromosome 3 common eliminated region-1 (C3CER1) at 3p21.3. In this study, we focussed on the epigenetic and genetic alterations of LARS2 in NPC. The mRNA expression of LARS2 was detected in 36 NPC and 8 chronic nasopharyngitis (NP) tissues by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Subsequently, the mutation, allelic loss, and methylation status of LARS2 were analysed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), homozygous deletion (HD) analysis and methylation-specific polymerase chain reaction in primary NPC tissues. No expression or downregulation of LARS2 was observed in 78% of primary NPC tissues. No mutations, assessed by PCR-SSCP and DNA sequencing, were found in the promoter region and exon 1 of LARS2 in NPC tissues, whereas HD was detected in 28% of NPC specimens at the LARS2 locus. In addition, hypermethylation of LARS2 was found in 64% of NPC samples but only in 12.5% of NP biopsies. Our data indicate that inactivation of LARS2 by both genetic and epigenetic mechanisms may be a common and important event in the carcinogenesis of NPC.

Background: Loss of heterozygosity (LOH) at 13q22 is a common event in nasopharyngeal carcinoma (NPC). EDNRB gene located at 13q22 has been demonstrated to be hypermethylated in some kinds of tumors. In the current study, we focused on the epigenetic and genetic alterations of EDNRB in NPC. Methods: The mRNA expression of EDNRB was detected by semiquantitative RT-PCR and real-time quantitative PCR in 49 NPC and 12 chronic nasopharyngitis biopsies. The methylation and LOH status of EDNRB were examined by methylation-specific polymerase chain reaction, microsatellite PCR and sequencing. We also examined the mRNA expression of EDNRB in four NPC cell lines after 5-aza-2′-deoxycytidine treatment. Results:EDNRB was downregulated in primary NPC tissues and NPC cell lines, and a relatively higher methylation level of EDNRB was found in NPC biopsies (84%) compared to that in chronic nasopharyngitis biopsies (42%). Treatment of NPC cell lines with 5-aza-2′-deoxycytidine activated EDNRB expression. LOH of EDNRB gene was also found at two microsatellite sites with ratios of 6.25 and 16.67% in NPC. Conclusion: Our results suggested that EDNRB expression may be affected by aberrant promoter methylation and gene deletion and may play a role in the development of NPC.

Loss of deleted in liver cancer-1 (DLC-1) can induce apoptosis and inhibit the mobility, migration and metastasis in several cancers. Previously, we revealed that ectopic expression of DLC-1 can suppress proliferation, mobility, migration and tumorigenesis in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms accounting for the roles of DLC-1 in NPC are still obscure. In the present work, we attempted to study and uncover the mechanisms underlying the functions of DLC-1 in NPC. The apoptosis of 5-8F-DLC-1 cells, established previously, was analyzed by mitochondrial membrane potentials assay and flow cytometer analysis. And the antibodies involving pathways such as mitochondrial-associated apoptosis, epithelial mesenchymal transition and metastasis were applied to detect and compare the expression level of targeted proteins. The obvious apoptosis of 5-8F-DLC-1 cells was observed reflected by mitochondrial depolarization and lower ratio in cell viability. Subsequently, the activation of mitochondrial apoptosis was verified by the increased expressions of Bax, Apaf1, cleave-caspases and cleave-PARP, etc, and the decreased expressions of Bcl-2, Bcl-xL, Mcl-1, Survivin, etc, in 5-8F-DLC-1 cells. Then, the inhibited epithelial mesenchymal transition of 5-8F-DLC-1 cells was validated by upregulated expression of E-cadherin and downregulated expression of N-cadherin, Snail, Vimentin. Subsequently, downregulated expressions of proteins such as FAK, RhoA, ROCK1 and cdc25 related to cell adhesion and cytoskeleton organization were also observed. And expressions of MMPs were inhibited in 5-8F-DLC-1 cells. At last, the inhibited activity of EGFR/Akt/NF-κB axis was revealed by the decreased expressions of phosho-EGFR, phosho-Akt, phosho-p38MAPK, phosho-IKKα and phosho-p65. Here, we systematically explored the mechanisms underlying the negative roles of DLC-1 in NPC cells. For the first time, we confirmed that the ectopic expression DLC-1 can induce mitochondrial apoptosis, inhibit EMT and related processes by targeting the EGFR/Akt/NF-κB pathway, which, beyond all doubt, offered beneficial guidelines for other studies and laid a good foundation for its clinical applications ultimately.

Lipid raft proteins have been confirmed to be important in cell signal transduction. Some reports have shown that the aberrant expression of lipid raft proteins is associated with malignant phenotypes in some cancers. However, the role of the lipid raft protein flotillin-2 (Flot-2) in nasopharyngeal carcinoma (NPC) remains to be comprehensively characterized. Here, overexpression of Flot-2 in NPC tissues and cell lines was detected by immunostaining and Flot-2 expression was found to be positively associated with NPC metastasis. Furthermore, inhibiting Flot-2 expression impaired the malignancy of the highly metastatic NPC cell line 5-8F by constraining its growth and proliferation, mobility and migration and decreasing the capacity of 5-8F cells to metastasize in nude mice. In contrast, forced overexpression of Flot-2 increased the malignancy of 6-10B, a non-metastatic NPC cell line that weakly expresses Flot-2. Moreover, in 5-8F-shFlot-2 cells, which have inhibited Flot-2 expression, the NF-κB and PI3K/Akt3 pathways were inactivated. Subsequently, MMPs expression were decreased and Foxo1 activity was increased. In addition, enhanced NF-κB and PI3K/Akt3 activities were observed in Flot-2 overexpressing 6-10B cells. Thus, Flot-2 exerts a pro-neoplastic role in NPC and is involved in tumor progression and metastasis. Moreover, Flot-2 exerts its role through NF-κB and PI3K/Akt3 signaling.

Objective: To compare gene expression profiles of nasopharyngeal carcinoma (NPC) tissue with that of control tissue by cDNA Array and to discuss possible reasons of TP53 accumulation in NPC tissue. Methods: (1) hybridization of Atlas Human Cancer cDNA Expression Array 7742-1; (2) analysis of Atlas Arrays using Atlasimage 1.01a; (3) verification of results of array by RT-PCR; (4) verification of protein expression alterations by immunohistochemistry. Results: (1) Of 588 tumor-related genes, 134 genes were upregulated, 88 downregulated; (2) Of 32 TP53-regulated genes, 13 genes were shown differential expression, 11 upregulated, 2 downregulated; (3) ATM and JNK2 were upregulated; (4) mRNA expression of ubiquitin-conjugating enzyme E2 (M74524) and ubiquitin-conjugating enzyme E2 (L22005) has no evident changes; Conclusion: (1) TP53 dysfunction exists in NPC tissues; (2) ATM and JNK might be the important causes of TP53 accumulation.

Background Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet. Methods Immunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi). Results The positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities. Conclusions Our study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.

In our previous study we demonstrated the downregulation or loss of deleted in liver cancer‑1 (DLC-1) gene expression in nasopharyngeal carcinoma (NPC). In this study, we report the effects of the DLC-1 gene on NPC cells and its mechanisms of action. DLC-1 expression was restored in the 5-8F NPC cell line, which lacks DLC-1 expression, and the biological characteristics of 5-8F-DLC‑1 cells were analyzed by MTT assay, colony formation assay, flow cytometry (FCM), tumorigenesis analysis in nude mice, as well as invasion and migration assay. Differentially expressed genes in response to DLC-1 expression were screened using microarray analysis and identified by RT-PCR. The re-expression of DLC-1 in the NPC cells attenuated the proliferation and colony formation ability of the cells in vitro, blocked NPC cells at the G0/G1 phase, reduced tumorigenicity potential in vivo, inhibited the invasion and migration ability of NPC cells and resulted in the reorganization of the actin cytoskeleton. DLC-1 altered the gene expression profile in 5-8F cells. Some tumor suppressor genes (TSGs) were upregulated and some oncogenes were downregulated. These results demonstrate that DLC-1 gene can partially reverse the malignant phenotype of NPC cells by changing the tumor-related gene expression profile, and may be a candidate tumor suppressor gene and a promising diagnostic and therapeutic target in NPC.

Previous studies have shown that promoter hypermethylation plays a key role in DLC-1 inactivation in nasopharyngeal carcinoma (NPC). However, DLC-1 mutation in NPC has not been reported, and there remain some discrepancies in methods and results between different groups. Here, we examined the mRNA and protein expression of DLC-1 in chronic nasopharyngitis (CN) and NPC tissues by reverse transcription-polymerase chain reaction/qPCR and immunohistochemistry, respectively. DLC-1 mRNA was undetectable in all the seven widely used NPC cell lines and absent or significantly down-regulated in 70% of NPC tissues. DLC-1 protein level was reduced in 74.3% of NPCs when compared with CN tissues, and significantly lower in NPC samples at advanced clinical stages than that at early stages. Then, we purified the same batch of specimens by microdissection and analyzed the possible mechanisms of DLC-1 downregulation with mutation and allelic loss analysis, methylation-specific PCR and bisulfite genomic sequencing. Only one mutation was detected at codon 693 of exon 8 in 3.3% of NPCs and five single nucleotide polymorphisms (SNPs) were identified. Loss of DLC-1 was detected in 23.3% of NPC tissues. The 100% of NPC cell lines, 80% of primary NPC and 22.2% of CN tissues showed methylation in DLC-1 promoter, while DLC-1 expression was recovered in seven NPC cell lines after 5-aza-dC treatment. Patched methylation assay confirmed that promoter methylation could repress DLC-1 expression. This report demonstrates that DLC-1 is negatively associated with NPC carcinogenesis, and promoter hypermethylation along with loss of heterozygosity, but not mutation, contributes to inactivation of DLC-1 in NPC.

Tumor stem cells are a small subset of tumor cells with the ability of self-renewal and differentiation and are regarded as a cause of tumor growth and recurrence. Previously we have shown that stem-like label-retaining cells (LRCs) can be detected in nasopharynx, tongue, esophagus and xenograft tumors formed by nasopharyngeal carcinoma (NPC) cell lines (5–8F, 6–10B and TMNE). The present study aimed to identify ABCG2+ cells in 5–8F NPC cells and compare their tumorigenic potential with ABCG2− cells, expecting that we can obtain insight into the mechanism of the differential phenotypes of ABCG2+ and ABCG2− cells. By using magnetic cell sorting (MACS) method, we isolated ABCG2+ cells and ABCG2− cells from 5–8F cells. Among these two subpopulations and unsorted 5–8F cells, the rate of ABCG2+ cells at G1 phase was highest, while the rate of ABCG2− cells at S phase was highest, indicating that ABCG2+ cells were mostly quiescent. However, ABCG2+ cells showed lower cloning efficiency and tumorigenicity than ABCG2− cells. We also used Affymetrix U133 plus 2.0 human whole genome expression chip to identify the gene expression profile of ABCG2+ and ABCG2− cells and found that both subpopulations expressed some stem cell associated genes, e.g., PSCA, ABCG2 and ALPI were expressed in ABCG2+ cells, and K19, integrin α6, integrin β4, CD44 and K14 were expressed in ABCG2− cells, suggesting there were stem cells in both ABCG2+ and ABCG2− cells. Our data demonstrated that there exist ABCG2+ cells in NPC cells, but ABCG2 alone is not sufficient for isolating cancer stem cells in 5–8F NPC cells.

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. J. Cell. Biochem. 112: 1832–1843, 2011. © 2011 Wiley-Liss, Inc.

Deleted in liver cancer-1 (DLC-1), encoding a Rho GTPase-activating protein (GAP), is considered as a promising candidate tumor suppressor gene in nasopharyngeal carcinoma (NPC). The single-nucleotide polymorphism (SNP) −29A/T upstream of ATG start codon was found when gene mutation profile of DLC-1 in NPC was analyzed. To evaluate the correlation between SNP −29A/T in the promoter region of DLC-1 gene and risk of NPC, a total of 521 samples from a Chinese population, including 320 healthy individuals and 201 NPC patients, were collected for SNP analysis by PCR–single-strand conformation polymorphism and sequencing. The differences in allele and genotype frequencies between NPC patients and controls were tested using logistic regression statistical method. No significant differences were found in allele or genotype frequencies between NPC patients and controls or among different NPC clinical stages. Hence, our data indicate that the SNP −29A/T of DLC-1 gene is not associated with NPC susceptibility.

Nasopharyngeal carcinoma (NPC) is a malignancy that is prevalent among populations from Southeast Asia. The carcinogenesis of NPC is thought to be a multistep process involving several genetic changes. Our previous study based on distance and branching-tree models for NPC carcinogenesis indicated +12p11-p12 was an early event and should play an important role in NPC development. To understand the role of +12p11-p12 as the tree model predicted and evaluate which gene located at 12p11-p12 might be involved in NPC development, semiquantitative RT-PCR was applied to examine the expression status of 18 genes selected from 12p11-p12 in 36 NPC and 8 normal nasopharynx (NP) biopsies. The results revealed that BCAT1, KCNJ8, PTX1, and KRAS2 genes were overexpressed in NPC tissues and BCAT1 was of particular interest based on its function reported in other tumors. To further elucidate the function of BCAT1 gene in NPC, BCAT1 expression was specifically suppressed in 5-8F NPC cell line by RNA interference (RNAi), confirmed by RT-PCR and Western blotting. As expected, the depletion of BCAT1 could effectively block the proliferation of NPC cells. The BCAT1 identified in the amplified 12p11-p12 region may play a certain role in NPC development.

We examined the promoter hypermethylation of tumor-suppressor genes RASSF1A and TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC) tissues (T tissues), and matched tumor-adjacent tissues outside 0.5 cm (P tissues) and outside 1.0 cm (Z tissues) to evaluate the role of promoter hypermethylation of RASSF1A and TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR) for RASSF1A and TSLC1 and quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, and Z tissues (n = 28) as well as chronic nasopharyngitis tissues (n = 8). Hypermethylated RASSF1A was frequently detected in the T (82%) and P tissues (75%), but less frequently in Z tissues (46%). he average quantities of EBV DNA (copies/microg DNA) in matched T, P, and Z tissues were 673,000, 90,000, and 7000. The differences of promoter hypermethylation of RASSF1A and EBV viral load among T, P, and Z tissues were statistically significant, with more frequent methylation and higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A and high EBV load might be important events in NPC pathogenesis, and they may be useful molecular diagnostic markers for this cancer.