Nasopharyngeal Carcinoma C666-1 Cells Research Articles

Elucidating the pharmacological effects of Compound Kushen injection on MYC-P15-CCND1 signaling pathway in nasopharyngeal carcinoma - An in vitro study

Ethnopharmacological relevanceCompound Kushen injection (CKI) is a representative medication of Chinese herbal injection and is often used in the adjuvant treatment of nasopharyngeal carcinoma (NPC), but its antitumor mechanism is poorly understood. Aim of the studyTo preliminarily elucidate the effects and possible mechanisms of CKI on NPC. MethodsIn this work, we explored the possible molecular mechanisms of CKI against NPC by using network pharmacology and molecular docking. In addition, proteomics was used to explore the localization and quantitative information of protein in NPC C666-1 cells after the intervention of CKI, and enrichment analysis was used to obtain the potential targets and pathways. Finally, the effect and the core targets of CKI in the intervention of NPC were explored in vitro experiments. ResultsNetwork pharmacology analysis identified three active components of CKI and 13 key targets. Molecular docking analysis showed that TNF, PTEN, CCND1, MAPK3, IL6, HIF1A, MYC had high affinity with corresponding components. Then the key pathway, cell cycle and the core targets MYC, CCND1, and P15 related to the key pathway were obtained. The results of in vitro experiments showed that CKI could inhibit the proliferation, migration, and invasion of NPC 5–8F cells and C666-1 cells, induce apoptosis of C666-1 cells, and arrest cell cycle G0/G1 phase. In addition, RT-qPCR and western blot showed that the expression of P15 was up-regulated and E2F4, E2F5, c-Myc, CCND1, and P107 was down-regulated in 5–8F cells and C666-1 cells intervened by CKI. ConclusionThe key pathway, cell cycle and the corresponding core targets MYC, CCND1, and P15 were obtained from network pharmacology, molecular docking, and proteomics. CKI could inhibit the proliferation, migration, and invasion of NPC cells, induce apoptosis of C666-1 cells. Especially CKI may arrest cell cycle G0/G1 phase through regulating targets MYC/P15/CCND1 of cell cycle pathway.

Geraniin inhibits cell growth and promoted autophagy-mediated cell death in the nasopharyngeal cancer C666-1 cells.

BackgroundNasopharyngeal carcinoma (NPC) is a rare malignant tumor developing from epithelial linings of nasopharynx, and 10–50 out of 100,000 NPC cases were recorded globally particularly in the Asian countries.MethodologyThe cytotoxicity of geraniin against the NPC C666-1 cells were analyzed using MTT assay. The influences of geraniin on the C666-1 cell viability with the presence of ROS and apoptosis inhibitors were also studied. The expressions of PI3K, Akt, mTOR, and autophagic markers LC3, ATG7, P62/SQSTM1 expressions in the C666-1 cells were studied by western blotting analysis. The ROS production was assayed using DCFH-DA staining. The immunofluorescence assay was performed to detect the NF-κB and β-catenin expressions in the C666-1 cells.ResultsThe cell viability of C666-1 cells were appreciably prevented by the geraniin. The geraniin treatment also inhibited the C666-1 cell growth with the presence of apoptotic inhibitor Z-VAD-FMK. The geraniin-treatment effectively improved the ROS production and inhibited the NF-κB and β-catenin expressions in the C666-1 cells. Geraniin appreciably modulated the PI3K/Akt/mTOR signaling axis and improved the autophagy-mediated cell death via improving the autophagic markers LC3 and ATG7 expressions in the C666-1 cells.ConclusionIn conclusion, our results proved that geraniin inhibits C666-1 cell growth and initiated autophagy-mediated cell death via modulating PI3K/Akt/mTOR cascade and improving LC3 and ATG7 expressions in the C666-1. Geraniin and it could be a hopeful and efficient candidate to treat the human NPC in the future.

The Therapeutic Possibility of EBV-Positive Nasopharyngeal Carcinoma through Targeting Somatostatin Receptors

Nasopharyngeal carcinoma (NPC) is a complex epithelial malignancy caused by both internal and environmental factors. Among them, Epstein–Barr virus (EBV) is a definitive etiological factor. The current therapeutic regimen for NPC is far from ideal and recurrence is commonly seen in patients after conventional radio and/or chemotherapy. We have thereby set out to develop new treatments for this deadly disease. Having analyzed a number of nasopharyngeal tissue and cell samples, we found that the level of somatostatin receptors (SSTRs) was positively correlated with EBV infection. Further, SSTR2 is the dominant SSTR isoform expressed in the EBV-positive (EBV (+)) NPC cells, a potential target for precise delivery of therapeutic agents. Cytotoxic effects of small molecule drugs including ansamitocin P-3 (AP-3), camptothecin and colchicine were subsequently evaluated in NPC cells. We found that AP-3 exhibits its strong anti-NPC activities with the IC50 values being 0.07 nM in EBV (+) NPC C666-1 cells and 0.09 nM in EBV (-) NPC CNE2 cells. In the xenograft animal model, AP-3 further shows inhibitory effects on NPC growth. These findings indicated that AP-3’s activity is not related to EBV in NPC cells. Thus, we proposed that SSTR2- specific delivery of AP-3 via linking AP-3 to somatostatin is likely a viable option to treat the EBV (+) NPC patients.

Silencing long non-coding RNA H19 combined with paclitaxel inhibits nasopharyngeal carcinoma progression

PurposeThis study aimed to study the effect of long non-coding RNA (lncRNA) H19 on proliferation, apoptosis and chemosensitivity of nasopharyngeal carcinoma (NPC) cells. MethodsNP69 and HNE3, C666-1, SUNE1, 6–10B and 5–8F cell lines were selected to detect the expression of lncRNA H19 via RT-qPCR. LncRNA H19 was overexpressed or silenced for exploring the regulatory effect of lncRNA H19 in cell proliferation, clone formation, apoptosis and drug resistance through CCK-8, clone formation experiment and flow cytometry respectively. The tumorigenic effect of lncRNA H19 silencing was verified by xenograft tumor in nude mice. LncRNA H19 was significantly up-regulated in NPC cells. ResultsSilencing lncRNA H19 inhibited the proliferation of NPC C666-1 cells and promoted apoptosis, while overexpression of lncRNA H19 promoted the proliferation of NPC C666-1 cells and inhibited apoptosis. Knockdown of lncRNA H19 in drug-resistant cells remarkably reduced their drug resistance, and overexpression of lncRNA H19 in parental cells significantly reduced their drug sensitivity. Silencing lncRNA H19 inhibits tumor growth in vivo, and silencing lncRNA H19 combined with paclitaxel can enhance tumor inhibition in vivo. ConclusionsIn NPC cells, lncRNA H19 was up-regulated, lncRNA H19 inhibited the proliferation and chemosensitivity of NPC cells, promoted apoptosis, and silencing lncRNA H19 combined with paclitaxel could enhance tumor inhibition in vivo.

FOXD3 inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma through regulation of the PI3K-Akt pathway.

FOXD3 has been found previously to positively regulate miR-26b, a tumor inhibitor of nasopharyngeal carcinoma (NPC). However, FOXD3's precise function and associated mechanism of action in NPC have not yet been investigated. In this study, the expression of FOXD3 mRNA and protein was evaluated using RT-qPCR, western blotting, and immunohistochemistry. Protein levels involved in the phosphoinositide 3-kinase - protein kinase B (PI3K-Akt) pathway were assessed by western blot, and cell proliferation was determined by MTT and colony forming assays. Additionally, cell apoptosis was assessed by flow cytometric assay. Finally, the migration and invasion capabilities of the NPC cells were determined using wound healing and Transwell assays. We found that FOXD3 levels were relatively low in NPC tissue and cells, while an increase caused the inhibition of the PI3K-Akt pathway. Functional experiments found that overexpression of FOXD3 suppressed cell proliferation, migration, and invasion and enhanced cell apoptosis in NPC C6661 cells. IGF-1, an activator of the PI3K-Akt pathway, reversed the inhibitory effect of FOXD3. Furthermore, we found upregulation of the PI3K-Akt pathway and upregulation of the inhibitory effects of FOXD3 on C6661 cellular activities. In conclusion, FOXD3 negatively affected the PI3K-Akt pathway to restrain the processes involved in C6661 cell pathology. These findings further exposed the function and downstream axis of FOXD3 in NPC and displayed a promising new target for NPC therapy.

Epstein-Barr Virus BART Long Non-coding RNAs Function as Epigenetic Modulators in Nasopharyngeal Carcinoma.

Epstein-Barr virus (EBV) establishes lifelong latent infection in humans and is associated with several lymphoid and epithelial cancers. In nasopharyngeal carcinoma (NPC), EBV expresses few viral proteins but elevated levels of Bam-HI A rightward transcripts (BARTs) RNA, which includes viral microRNAs and long non-coding RNAs (lncRNAs). BART lncRNAs localize within the nucleus of EBV-infected cells and knockdown of BART lncRNAs significantly affects the expression of genes associated with cell adhesion, oxidoreductase activity, inflammation, and immunity. Notably, downregulation of IKAROS family zinc finger 3 (IKZF3/Aiolos), which plays a role in lymphocyte development and cell attachment, occurred in NPC C666-1 cells following BART lncRNA-knockdown. Since Aiolos expression is normally restricted to lymphoid cells and rarely observed in epithelial cells, induction of Aiolos by BART lncRNA was confirmed by expressing the major BART lncRNA isoform, RPMS1, in EBV-positive and -negative cells. BART lncRNA associated with the CBP/p300 complex and RNA polymerase II (Pol II) in the nucleus, suggesting that BART lncRNAs may mediate epigenetic regulation of gene expression through interaction with the chromatin remodeling machinery. This contention is further supported by evidence that BART lncRNA appears to stall Pol II at the promoter region and may regulate IFNB1 and CXCL8 expression by inhibiting transcription by Pol II in NPC. We hypothesize that EBV BART lncRNA expression modulates host gene expression and maintains EBV latency by interfering with histone methylation and acetylation processes. Aberrant expression of affected host genes mediated by BART lncRNA may lead to immune evasion, progression, and metastasis of NPC.

PPARβ/δ Agonist GW501516 Inhibits Tumorigenesis and Promotes Apoptosis of the Undifferentiated Nasopharyngeal Carcinoma C666-1 Cells by Regulating miR-206.

In previous investigations, we reported that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activation by GW501516 inhibits proliferation and promotes apoptosis in the undifferentiated C666-1 nasopharyngeal carcinoma (NPC) cells by modulating caspase-dependent apoptotic pathway. In the present study, the mechanism by which GW501516 induces apoptosis was explored from the perspective of microRNA (miRNA) expression. Among the assayed miRNAs that were involved in regulating the expression of antiapoptotic protein Bcl-2, miR-206 was increased significantly and specifically by GW501516 in C666-1 cells at both the in vitro level and at the in vivo xenograft samples. The induction on miR-206 expression caused by GW501516 was capable of being antagonized by the PPARβ/δ antagonist GSK3787 and AMPK antagonist dorsomorphin in C666-1 cells. GW501516’s suppression on the growth and apoptosis of C666-1 cells was found to be dependent on the presence of miR-206. miR-206 overexpression resulted in suppressed proliferation and colony formation ability, and further triggered increased apoptosis in C666-1 cells in a caspase-dependent manner. The expression of cleaved caspase 3 and caspase 9, and the ratio of Bax to Bcl-2 were elevated remarkably by miR-206. Consistent with the in vitro result, miR-206 was corroborated to suppress the ectopic NPC xenograft tumorigenesis that derived from the C666-1 cells in BALB/c nu/nu mice. Taken together, the current data demonstrated that miR-206 plays a critical role in the direct apoptosis-promoting effect induced by GW501516 in C666-1 cells. Furthermore, the emphasized tumor-suppressive role of miR-206 in the C666-1 cells indicates that it has the potential to provide a new therapeutic approach for the undifferentiated NPC.

Nobiletin induces growth inhibition and apoptosis in human nasopharyngeal carcinoma C666-1 cells through regulating PARP-2/SIRT1/AMPK signaling pathway.

Background/AimNobiletin, a major polymethoxyflavones (PMFs) from citri reticulatae pericarpium (CRP), can inhibit several forms of cancer proliferation. However, the effects of nobiletin on nasopharyngeal carcinoma (NPC) C666‐1 cells remain largely unknown.Materials and MethodsCell counting kit 8 (CCK8) assay was used to measure cell vitality. Flow cytometry was performed to measure the apoptosis rate. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blot analysis were applied to determine the expression of mRNA and protein, respectively.ResultsWe showed that the proliferation rate of C666‐1 cells was inhibited and the apoptosis rate was raised after treating with nobiletin. Moreover, nobiletin inhibited the expression of poly(ADP‐ribose)polymerase‐2 (PARP‐2), and the tumor suppression effect of nobiletin on C666‐1 is associated with PARP‐2‐dependent pathway.ConclusionWe demonstrated for the first time that nobiletin inhibited the growth of C666‐1 cells, which may be relative to its regulation on PARP‐2/SIRT1/AMPK signaling pathway. Our result implied that nobiletin may serve as a strategy to treat nasopharyngeal carcinoma.

MicroRNA-663b promotes cell proliferation and epithelial mesenchymal transition by directly targeting SMAD7 in nasopharyngeal carcinoma.

MicroRNAs (miRs) serve important roles in the development of various types of human cancer, including nasopharyngeal carcinoma (NPC). In the present study, the expression levels of miR-663b in NPC were investigated and its role and underlying mechanisms were examined. Reverse transcription-quantitative polymerase chain reaction was performed to assess miR-663b expression levels in NPC tissues and C666-1 cells. TargetScan was applied to predict the putative targets of miR-663b and the dual luciferase reporter assay was used to confirm the predictions. To investigate the role of miR-663b in NPC, the NPC C666-1 cell line was transfected with miR-663b mimics, miR-663b inhibitors or negative control. The Cell Counting kit-8 assay was performed for cell proliferation detection and western blot analysis was applied to determine the expression levels of epithelial mesenchymal transition (EMT)-associated proteins. Results indicated that when compared with the adjacent normal tissues and the normal nasopharyngeal epithelial cells, miR-663b expression levels were significantly upregulated in the NPC tissues and the NPC cells (P<0.01). Notably, SMAD7 is a target gene of miR-663b and may be inhibited by miR-663b. Results indicated that NPC cell proliferation was significantly promoted by miR-663b mimics and significantly inhibited by miR-663b inhibitors (P<0.05 and P<0.01). In addition, the results indicated that, when compared with the negative control group the expression levels of E-cadherin were significantly decreased, whereas the expression levels of N-cadherin, Vimentin and matrix metalloproteinase-9 were significantly increased in the cells of the miR-663b mimics group (P<0.05 and P<0.01). However, cells in the miR-663b inhibitors group exhibited the opposite effects. In conclusion, the results of the present study indicated that miR-663b functions as a tumor promoter in NPC via promoting NPC cell proliferation and EMT by directly targeting SMAD7.

Abstract 4563: PD-L1 expression associated with treatment responses in colorectal cancer patients with XELOX/FOLFOX chemotherapy: Potential of checkpoint blockage and natural killer cell-based immunotherapy

Abstract Chemo-resistance is a major obstacle in human cancer treatment. Immunotherapies including checkpoint blockage and cell-based therapy have been clinically validated for cancer treatment. The present study aimed to investigate the role of PD-L1 and potential of immunotherapy for chemo-resistant cancers. In a pilot study, we have examined the expression of PD-L1 in colorectal cancer (CRC) patients who received XELOX (capecitabine plus oxaliplatin) or FOLFOX (fluorouracil plus oxaliplatin) chemotherapy. Detection rate of PD-L1 expression by immunohistochemistry was higher in CRC patients who demonstrated poor response (5/10, 50%) compared to good responders (0/5, 0%) on XELOX/FOLFOX chemotherapy. Chemo-resistant CRC cell lines (HT29 and HCT116) were used to examine the role of PD-L1. Cells were plated out, selected under fluorouracil and oxaliplatin in serial, and referred as fluorouracil-oxaliplatin-resistant (FOR) cells. Half maximal inhibitory concentration (IC50) for respective drug was determined by MTT assay. HT29 FOR, compared to parental cells, showed chemo-resistance (359.4 and 10.1 fold increase to fluorouracil and oxaliplatin respectively) and similarly for HCT116 FOR (8.0 and 9.0 fold increase respectively). Hepatocellular carcinoma Hep3B and nasopharyngeal carcinoma C666-1 cells that have been selected for fluorouracil chemo-resistant (FR) were included. All the chemo-resistant cell lines showed increased expression of PD-L1 by real-time qPCR compared to their respective parental cells (HT29 FOR: 6.1 fold; HCT116 FOR: 1.9 fold; Hep3B FR: 3.1 fold; C666-1 FR: 4.4 fold). To explore the potential of immunotherapy, natural killer (NK) cells were isolated from peripheral blood PBMC of healthy donors. NK cells were expanded and activated ex vivo in stem cell growth medium supplemented with interleukins. NK co-culture assay was performed and apoptosis of cancer cells examined by flow cytometry. Parental cancer cells were sensitive to cytotoxic killing by NK cells compared to control no NK treatment (36.0% vs 2.8% apoptotic cells, P=0.001), and PD1 antibody (Nivolumab) treatment promoted NK-mediated cytotoxicity (52.9%, P=0.034). Notably, chemo-resistant cancer cells were similarly sensitive to cytotoxic killing by NK cells compared to control (27.1% vs 3.4%, P=0.002), and PD1 antibody (Nivolumab) treatment further enhanced NK-mediated cytotoxicity (52.8%, P=0.013). In summary, PD-L1 expression was associated with poor response for XELOX/FOLFOX chemotherapy in CRC patients. Chemo-resistant cancer cells showed elevated PD-L1 expression, nonetheless, susceptible to NK-mediated cytotoxicity and checkpoint blockage by PD1 antibody augmented the treatment response. Further studies are warranted to examine immunotherapy for chemo-resistant cancers. Citation Format: Elaine H.L. SIU, Simon S.M. NG, Anthony W.H. CHAN, James Y.W. LAU, Kwok Wai LO, Charing C.N. CHONG, Paul B.S. LAI, Stephen L. CHAN, Marcus H.N. LAW, Linda W.C. NG, S.T. CHEUNG. PD-L1 expression associated with treatment responses in colorectal cancer patients with XELOX/FOLFOX chemotherapy: Potential of checkpoint blockage and natural killer cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4563.

PPARβ/δ Agonist GW501516 Inhibits Tumorigenicity of Undifferentiated Nasopharyngeal Carcinoma in C666-1 Cells by Promoting Apoptosis.

Activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) had been linked to inhibition on the proliferation and apoptosis in a few cancer cell lines. However, limited data exists regarding the role of PPARβ/δ in nasopharyngeal carcinoma (NPC). This study was undertaken to determine the effect of PPARβ/δ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the human NPC cell lines. Gene and protein expression of PPARβ/δ were reduced specifically in the poor- and un-differentiated NPC cell lines as compared with the control NP-69 cells. Ligand activation of PPARβ/δ by GW501516, a specific PPARβ/δ selective agonist, inhibited cell proliferation and colony formation strikingly, and induced a G2/M phase arrest in the EBV positive undifferentiated NPC C666-1 cells relative to the control cells. Moreover, GW501516 induced C666-1 cell apoptosis in a caspase and BAX dependent manner. In accordance with the in vitro result, GW501516 significantly suppressed the ectopic NPC xenograft tumorigenicity that derived from the C666-1 NPC cells in BALB/c nu/nu mice. This effect is greatly associated with its inhibition on the gene and protein expression of integrin-linked kinase (ILK) through activation of the AMPKα-dependent signaling pathways. Collectively, we showed that PPARβ/δ expression is in reverse correlation with the degree of differentiation in the NPC cell lines, and revealed the anti-tumorigenic effects of GW501516 in NPC cells by activation of AMPKα. This study suggested that PPARβ/δ targeting molecules may be useful for the poor-, and particularly un-differentiated NPC chemoprevention.

MicroRNA-19a promotes nasopharyngeal carcinoma by targeting transforming growth factor β receptor 2.

MicroRNA (miR), a class of small non-coding RNA, function as key regulators in gene expression through directly binding to the 3′ untranslated region of their target mRNA, which further leads to translational repression or mRNA degradation. miR-19a, a member of miR-17-92 cluster, has an oncogenic role in a variety of malignant tumors. However, the exact role of miR-19a in nasopharyngeal carcinoma (NPC) has not previously been studied. The present study aimed to investigate the function and mechanism of miR-19a in regulating the viability and invasion of NPC cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) data indicated that the expression levels of miR-17-92 cluster members (miR-17, miR-18a, miR-19a and miR-20a) were frequently increased in NPC tissues compared to the normal tissues. It was also demonstrated that miR-19a was significantly upregulated in NPC C666-1 cells compared to NP69 cells (P<0.01). Knockdown of miR-19a led to a significant decrease in the viability and invasion of NPC C666-1 cells (P<0.01), and induced increased protein expression levels of transforming growth factor β receptor 2 (TGFβR2), which was further identified as a direct target gene of miR-19a by using a luciferase reporter assay. Overexpression of TGFβR2 also suppressed the viability and invasion of C666-1 cells, similar to the effects of miR-19a inhibition. Furthermore, knockdown of TGFβR2 reversed the suppressive effects of miR-19a inhibition on C666-1 cell viability and invasion, suggesting that the role of miR-19a in mediating cell viability and invasion is through directly targeting TGFβR2 in NPC cells. In addition, RT-qPCR data demonstrated that the mRNA expression level of TGFβR2 was markedly reduced in NPC tissues and C666-1 cells. In summary, the present study demonstrated an oncogenic role of miR-19a in NPC via mediation of TGFβR2. Therefore, miR-19a may be a potential therapeutic target for NPC.

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma.

ABSTRACTEpstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins.

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

Epigenetic inactivation of inositol polyphosphate 4-phosphatase B (INPP4B), a regulator of PI3K/AKT signaling pathway in EBV-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common viral-associated neoplasm in which multiple signaling cascades are interfered with by Epstein-Bar virus (EBV) latent proteins and various genetic alterations. Aside from the previously reported PIK3CA amplification, we examined the role of INPP4B, a negative regulator of the PI3K/AKT signaling pathway in the development of NPC. By RT-PCR and Western blotting, we revealed that the expression of INPP4B was down-regulated in all five established EBV-positive tumor lines. While INPP4B was consistently expressed in normal nasopharyngeal epithelial cells, downregulation of INPP4B was found in 32/65 (49.2%) of primary tumors by immunohistochemistry. Furthermore, our study also demonstrated the hypermethylation of the 5′CpG island of INPP4B in the tumors in which INPP4B transcription was downregulated. Notably, the re-expression of INPP4B was detected in the NPC cells treated with the demethylation agent (5-aza-2′deoxycytidine). Our study showed that promoter hypermethylation was the major mechanism for transcriptional silencing of INPP4B in NPC. Furthermore, restoration of INPP4B expression significantly suppressed PI3K/AKT downstream signals in the NPC C666-1 cells. In vivo growth inhibition was clearly demonstrated in the tumor cells stably expressing INPP4B. The findings indicate that epigenetic inactivation of INPP4B is one of the key mechanisms in activating PI3K/AKT signaling cascade and playing a role in the tumorigenesis of NPC.

Constitutive Interferon-Inducible Protein 16-Inflammasome Activation during Epstein-Barr Virus Latency I, II, and III in B and Epithelial Cells

Epstein-Barr virus (EBV), etiologically linked with human B-cell malignancies and nasopharyngeal carcinoma (NPC), establishes three types of latency that facilitate its episomal genome persistence and evasion of host immune responses. The innate inflammasome responses recognize the pathogen-associated molecular patterns which lead into the association of a cytoplasmic sensor such as NLRP3 and AIM2 proteins or nuclear interferon-inducible protein 16 (IFI16) with adaptor ASC protein (apoptosis-associated speck-like protein with a caspase recruitment domain) and effector procaspase-1, resulting in active caspase-1 formation which cleaves the proforms of inflammatory interleukin-1β (IL-1β), IL-18, and IL-33 cytokines. Whether inflammasome responses recognize and respond to EBV genome in the nuclei was not known. We observed evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β, -IL-18, and -IL-33, in EBV latency I Raji cells, latency II NPC C666-1 cells, and latency III lymphoblastoid cell lines (LCL). Interaction between ASC with IFI16 but not with AIM2 or NLRP3 was detected in all three latencies and during EBV infection of primary human B cells. IFI16 and cleaved caspase-1, IL-1β, IL-18, and IL-33 were detected in the exosomes from Raji cells and LCL. Though EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) are common to all forms of EBV latency, caspase-1 cleavage was not detected in cells expressing EBNA1 alone, and blocking EBER transcription did not inhibit caspase-1 cleavage. In fluorescence in situ hybridization (FISH) analysis, IFI16 colocalized with the EBV genome in LCL and Raji cell nuclei. These studies demonstrated that constant sensing of latent EBV genome by IFI16 in all types of latency results in the constitutive induction of the inflammasome and IL-1β, IL-18, and IL-33 maturation.

EBV-LMP1-targeted DNAzyme restrains nasopharyngeal carcinoma growth in a mouse C666-1 xenograft model

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been demonstrated to be linked to the pathogenesis of nasopharyngeal carcinoma (NPC), one of the most common cancers in southeast Asia as well as in the areas of southern China. RNA-cleaving DNAzymes are catalytic nucleic acids that bind and cleave a target RNA and have been increasingly used to inhibit gene expression. In this study, we aimed to explore the effects of down-regulation of LMP1 by DNAzymes on the NPC growth using a mouse xenograft model derived from human NPC C666-1 cells that constitutively express the LMP1. A specific LMP1-targeted DNAzyme, named DZ509, was synthesized, showing high activities in the inhibition of LMP1 expression, while the control DNAzyme (mutDZ509) had little effect on LMP1 expression. Knockdown of the LMP1 expression by DZ509 reduced cell proliferation and increased apoptosis compared to the cells transfected with mutDZ509. Intratumoral injections of DZ509 into C666-1 xenografts caused a significant suppression of tumor growth compared to mutDZ509-treated xenografts. Examination of the LMP1 expression in the xenografts demonstrated a marked inhibition of LMP1 by DZ509. An antisense oligonucleotide targeting the same site of the LMP1 transcripts was employed in parallel and produced a comparable inhibition on cell proliferation in vitro and tumor growth in vivo. These data demonstrate that LMP1-targeted DNAzyme is efficient in controlling tumor growth in vivo, and thus may have therapeutic implications in the treatment of NPC.

Silencing of LMP1 Induces Cell Cycle Arrest and Enhances Chemosensitivity Through Inhibition of AKT Signaling Pathway in EBV-Positive Nasopharyngeal Carcinoma Cells

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). In this study, we investigated that the effect of silencing LMP1 on cell cycle distribution and chemosensitivity in EBV-positive nasopharyngeal carcinoma C666-1 cells. Silencing of LMP1 by specific siRNA induced G1 arrest in C666-1 cells. The protein expression of CDK4 and cyclin D1 decreased and P27 was upregulated following LMP1 knockdown. Phosphorylation of AKT and its downstream targets IКB, FKHR was inhibited by LMP1 siRNA. The chemosensitivity of C666-1 cells to bleomycin and cisplatin was enhanced by siRNA targeting LMP1. The cells treated with LMP1 siRNA showed enhanced cleavage of the effector caspase3 and PARP, and Bax had the tendency to exhibit higher expression. Also, co-transfection of constitutive active AKT plasmid with LMP-1 siRNA plasmid abrogates sensitivity of C666-1 to bleomycin and cisplatin. It is reported for the first time that AKT signaling pathway was directly involved in the effects induced by siRNA targeting LMP1. Our findings confirm LMP1 as a rational therapeutic target in NPC.

Recombinant adeno-associated virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by suppression of Epstein-Barr virus encoded LMP-1

Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma characterized by consistent association with Epstein-Barr virus (EBV). Of the EBV-encoded product, latent membrane protein-1 (LMP-1) is considered to be an onco-protein playing an essential role in cell transformation and metastasis. In this study, we used a recombinant adeno-associated virus type 2 vector (rAAV-2) to deliver small hairpin RNA (shRNA) targeting EBV LMP-1 into the EBV-positive human NPC C666-1 cells and evaluated the effect of long-term suppression of LMP-1 on NPC growth and metastasis in vivo and in vitro. An NPC metastasis nude mouse model with NPC xenograft transplanted in liver was established. The NPC C666-1 cells infected with rAAV-shRNA-LMP-1 or rAAV-EGFP were inoculated in the livers of nude mice. Formation of liver and lung metastasis was evaluated at day 14 after tumor inoculation. Our results demonstrate that rAAV-shRNA-LMP-1 effectively infected C666-1 cells and suppressed LMP-1 expression. Such suppression, in turn, did not significantly inhibit tumor growth, but prevented NPC metastasis in the liver as well as in the lung. Consistent with in vivo data, the in vitro studies in NPC C666-1 cell cultures showed that suppression of LMP-1 by rAAV-shRNA-LMP-1 significantly reduced cell mobility and transmembrane invasion ability. These results demonstrated for the first time that long-term suppression of EBV-encoded LMP-1 in vivo is an effective means for preventing NPC metastasis.