Human Nasopharyngeal Carcinoma Cell Line Research Articles

Nasopharyngeal carcinoma with non-squamous phenotype may be a variant of nasopharyngeal squamous cell carcinoma after inhibition of EGFR/PI3K/AKT/mTOR pathway.

Nasopharyngeal carcinoma (NPC) is a cancerous tumor that develops in the nasopharynx epithelium and typically has squamous differentiation. The squamous phenotype is evident in immunohistochemistry, with diffuse nuclear positivity for p63 and p40. Nonetheless, a few NPCs have been identified by clinicopathological diagnosis that do not exhibit the squamous phenotype; these NPCs are currently referred to as non-squamous immunophenotype nasopharyngeal carcinomas (NSNPCs). In a previous work, we have revealed similarities between the histological appearance, etiology, and gene alterations of NSNPC and conventional NPC. According to ultrastructural findings, NSNPC still falls under the category of non-keratinized squamous cell carcinoma that is undifferentiated. NSNPC has an excellent prognosis and a low level of malignancy, according to a retrospective investigation. Based on prior research, we investigated the molecular mechanism of NSNPC not expressing the squamous phenotype and its biological behavior. IHC was used to determine the expression of EGFR, PI3K, AKT, p-AKT, mTOR, p-mTOR, Notch, STAT3 and p-STAT3 in a total of 20 NSNPC tissue samples and 20 classic NPC tissue samples. We obtained human NPC cell lines (CNE-2,5-8F) and used EGFR overexpression plasmid and shRNAs to transfect them. To find out whether mRNA and proteins were expressed in the cells, we used Western blotting and qRT-PCR. Cell biological behavior was discovered using the CCK-8 assay, cell migration assay, and cell invasion assay. EGFR, PI3K, p-AKT and p-mTOR proteins were lowly expressed in NSNPC tissues by immunohistochemistry, compared with classical NPC. In the classical NPC cell lines CNE-2 and 5-8F, overexpression EGFR can up-regulate the expression of p63 through the PI3K/AKT/mTOR pathway, and promote the proliferation, migration, and invasion of nasopharyngeal carcinoma cells. At the same time, knockout of EGFR can down-regulate p63 expression through the PI3K/AKT/mTOR pathway, and inhibit the proliferation, migration, and invasion of nasopharyngeal carcinoma cells. The lack of p63 expression in NSNPC was linked with the inhibition of the EGFR/PI3K/AKT/mTOR pathway, and NSNPC may be a variant of classical NPC.

Near-infrared-triggered Dibenzo[a,c][1,2,5]thiadiazolo[3,4-i]phenazine heterocyclic derivatives for type-I photodynamic and photothermal therapy against human nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) remains endemic in southern China. The general therapeutic consensus is to treat the disease with radiotherapy alone or together with concurrent chemotherapy. But the treatment outcome of NPC remains disappointing with bad prognoses, low overall survival rate and severe side effects. Phototherapy can be a promising approach for treating NPC due to its excellent spatiotemporal selectivity, negligible drug resistance and minimal invasiveness. In this work, two photosensitizers (DTP-Ph and DTP-Et) with similar donor-acceptor structure were designed and constructed for the treatment of NPC. Remarkably, DTP-Ph and DTP-Et show different photo-physicochemical properties as well as mechanisms of phototherapeutic actions, especially DTP-Ph, which exhibits synergistic type-I photodynamic therapy (PDT) and photothermal therapy (PTT) effect under near-infrared (NIR) activation. DTP-Et and DTP-Ph were further encapsulated with F127 to obtain nanoparticles with good water solubility. In intro and in vivo phototherapy evaluations of DTP-Ph NPs display promising phototherapeutic activity with low IC50 value and good tumor growth inhibition (TGI) against human nasopharyngeal carcinoma cell line CNE2 and tumor-bearing xenograft model.

MiR-25 enhances the proliferation, invasion and migration of nasopharyngeal carcinoma cells through targeted regulation of RAGE expression

Purpose: To determine the effect of microRNA-25 (miR-25) on the proliferation, invasion, and migration of nasopharyngeal carcinoma cells, and the involvement of targeted regulation of late glycation end product receptor (RAGE) in the process.Methods: Three groups of cells comprising; routinely cultured nasopharyngeal normal epithelial cell line NP69 without any treatment (blank control group), untreated human nasopharyngeal carcinoma cell line 5-8F, also cultured routinely (nasopharyngeal carcinoma group) and 5-8F human nasopharyngeal carcinoma cells infected with lentiviral vectors for miR-25 gene knockdown and cultured after stable transduction (miR-25 knockdown group). Cell proliferation was assessed using CCK-8 assay, while Western blot assay and quantitative polymerase chain reaction (qPCR) were used to measure protein and mRNA expression levels of relevant genes. Transwell assay was utilized to evaluate cell migration and invasion.Results: The miR-25 expression level was significantly increased in nasopharyngeal carcinoma group (p < 0.05). Cell proliferation, migration, and invasion rates in miR-25 knockdown group were lower than those in nasopharyngeal carcinoma group (p < 0.05). Apoptosis rate of cells and levels of apoptotic proteins were significantly elevated, whereas bcl-2 level was significantly reduced in miR-25 knockdown group when compared to nasopharyngeal carcinoma group (p < 0.05). Protein expression levels of RAGE and S100P was significantly increased in nasopharyngeal carcinoma group, but significantly down-regulated in miR-25 knockdown group (p < 0.05). Conclusion: Expression of miR-25 increases in nasopharyngeal carcinoma cells. Suppression of miR25 results in decreased viability of nasopharyngeal carcinoma cells and inhibits cell proliferation, migration, and invasion, while enhancing apoptosis. The mechanism underlying these effects was associated with modulation of protein expressions of RAGE and S100P. Targeted regulation of late glycation end-product receptors hold promising potential as prospective biomarker for therapeutic interventions targeting specific cancers.

Inhibitory mechanism of KIF3A gene on nasopharyngeal carcinoma stem cells.

To investigate the inhibitory mechanism of the KIF3A gene on nasopharyngeal carcinoma (NPC) tumor stem cells. Set up a blank control group (BCG), NPC group, and KIF3A silence (si-KIF3A) group. The BCG cells were nasopharyngeal normal epithelial cell line NP69, without any treatment, and were cultured routinely; The NPC group cells are human NPC cell line CNE2 cells, which are not subjected to any treatment and are cultured routinely; si-KIF3A group cells were cultured in the offspring of human NPC cell line CNE2 infected with Lentivirus knockdown KIF3A gene. CCK8 was used to detect the cell proliferation ability, Western blot and qPCR were used to detect protein and related mRNA expression, while cell migration and invasion were detected using Transwell. The KIF3A protein and mRNA in the NPC group were higher than those in the BCG (P<0.05), while those in the si-KIF3A group cells were lower compared to BCG cells (P<0.05). The cell proliferation, migration and invasion activity in the si-KIF3A group were reduced than those in BCG (P<0.05). Si-KIF3A group cells HIF-1, NF-κB was reduced than that of BCG (P<0.05). The expression level of HIF-1, NF-κB protein in si-KIF3A group cells was reduced compared to BCG cells (P<0.05). Knocking down the KIF3A gene can reduce the vitality of NPC stem cells, and inhibit the malignant phenotype of NPC stem cells, via inhibiting HIF-1 and NF-κB expression.

The increased expression of cytokeratin 13 leads to an increase in radiosensitivity of nasopharyngeal carcinoma HNE-3 cells by upregulating ERRFI1.

The main factors contributing to the unfavorable outcome in the clinical treatment of patients with nasopharyngeal carcinoma (NPC) patients are radiation resistance and recurrence. This study aimed to investigate the sensitivity and molecular foundation of cytokeratin 13 (CK13) in the radiotherapy of NPC. To achieve this, a human NPC cell line overexpressing CK13, HNE-3-CK13, was constructed. The effects of CK13 overexpression on cell viability and apoptosis under radiotherapy conditions were evaluated using the CCK-8 assay, immunofluorescence, and western blotting (WB). Next-generation sequencing was performed to identify the downstream genes and signaling pathways of CK13 that mediate radiotherapy response. The potential role of the candidate gene ERRFI1 in CK13-induced enhancement of radiosensitivity was investigated through rescue experiments using clone formation and WB. The effects of ERRFI1 on cell viability, cell apoptosis, cell cycle, and the related key genes were further evaluated using CCK-8, immunofluorescence, flow cytometry, quantitative polymerase chain reaction and WB. The results showed that CK13 overexpression in HNE-3 significantly inhibited cell survival under radiotherapy and promoted apoptosis marker γH2AX expression, leading to a significant increase of ERRFI1. Knockdown of ERRFI1 rescued the decreased cell viability and proliferation and the increased cell apoptosis that were caused by CK13 overexpression-mediated radiotherapy sensitization of NPC cells. In this process, EGFR, AKT, and GSK-3β were found involved. In the end, ERRFI1 was proven to inhibit expression levels of CDK1, CDK2, cyclin B1, and cyclin D1, resulting an increased G2/M cell ratio. Overexpression of CK13 enhances the radiosensitivity of NPC cells, which is characterized by decreased cell viability and proliferation and increased apoptosis. This regulation may affect the survival of HNE-3 cells by increasing the expression of ERRFI1 and activating the EGFR/Akt/GSK-3β signaling pathway, providing new potential therapeutic targets for the treatment of NPC.

Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cells.

Aloe-emodin (AE) has been shown to inhibit the proliferation of several cancer cell lines, including human nasopharyngeal carcinoma (NPC) cell lines. In this study, we confirmed that AE inhibited malignant biological behaviors, including cell viability, abnormal proliferation, apoptosis, and migration of NPC cells. Western blotting analysis revealed that AE upregulated the expression of DUSP1, an endogenous inhibitor of multiple cancer-associated signaling pathways, resulting in blockage of the extracellular signal-regulated kinase (ERK)-1/2, protein kinase B (AKT), and p38-mitogen activated protein kinase（p38-MAPK） signaling pathways in NPC cell lines. Moreover, the selective inhibitor of DUSP1, BCI-hydrochloride, partially reversed the AE-induced cytotoxicity and blocked the aforementioned signaling pathways in NPC cells. In addition, the binding between AE and DUSP1 was predicted via molecular docking analysis using AutoDock-Vina software and further verified via a microscale thermophoresis assay. The binding amino acid residues were adjacent to the predicted ubiquitination site (Lys192) of DUSP1. Immunoprecipitation with the ubiquitin antibody, ubiquitinated DUSP1 was shown to be upregulated by AE. Our findings revealed that AE can stabilize DUSP1 by blocking its ubiquitin-proteasome-mediated degradation and proposed an underlying mechanism by which AE-upregulated DUSP1 may potentially target multiple pathways in NPC cells.

MiR-137 Regulates Proliferation, Migration, Invasion and EMT of Nasopharyngeal Carcinoma Cells by Targeting TWIST1

To investigate the effects of miR-137 on biological cell function of nasopharyngeal carcinoma (NPC) cells. Totally, 31 pairs of NPC tissues and para-cancer tissues were collected. Meanwhile, human immortalized nasopharyngeal epithelial cell lines (NP69) and human NPC cell lines (6-10B) were cultured. The abilities of cell proliferation, invasion and migration were detected by CCK-8 and Transwell assay, respectively. The relative protein and mRNA expression level was detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. In quantitative real-time polymerase chain reaction (qRT-PCR) experiment, miR-137 was found widely low-expressed in clinical samples and cell lines of nasopharyngeal carcinoma (NPC). On-line target gene prediction software was applied to screen potential downstream target of miR-137 in NPC. Then, Twist Family BHLH Transcription Factor 1 (TWIST1) was verified by luciferase reporter assay and Western blot experiments as a target for negative regulation of miR-137 in NPC cells. We up-regulated the expression of miR-137 and/or TWIST1 in 6-10Bin vitro, and then examined the effects of cell function after by CCK8, Transwell, scratch-wound and Western blot experiments. The results showed that decreased expression of TWIST1 resulting from up-regulation of miR-137 in 6-10B cells could inhibit the biological functions of cells including proliferation, invasion, migration and process of epithelial-mesenchymal transition (EMT). Our research discovered the suppressor function of miR-137 on NPC cells by targeting TWIST1, suggesting that miR-137 could be used as a potential therapeutic target for NPC.

MicroRNA-142-5p promotes the proliferation and metastasis of nasopharyngeal carcinoma

Growing pieces of evidence reported abnormal expression of microRNA in various cancer. Our research aimed to ascertain the miR-142-5p expression and its potential function in the growth and metastasis of human nasopharyngeal carcinoma (NPC). In human NPC tissues and cell lines, miR-142-5p expression was quantified via the real-time qPCR assay. Functionally, the potential effect of miR-142-5p in human CNE-1 and SUNE-1 cells through MTT assay, colony formation assay, Transwell assay, and cell cycle assay. In addition, the potential target gene of miR-142-5p was determined by the dual-luciferase reporter assay. MiR-142-5p expression was remarkably elevated in human NPC tissues, CNE-1 and SUNE-1 cells. MiR-142-5p overexpression obviously enhanced the ability of cell proliferative and colony formation, and prevented G1 phase arrest in CNE-1 and SUNE-1 cells. Further, the migration number of NPC cells was increased compared to NP69 cells. BTG3 was identified as the direct target gene of miR-142-5p. Inhibition of BTG3 expression could reverse the cell proliferation by miR-142-5p-induced. Overall, miR-142-5p could strengthen the NPC cell’s proliferation and migration by directly targeting BTG3. Hence, miR-142-5p may provide a new strategy and program for future clinical treatment of NPC.

Role of SAR1B on Modulation of Nasopharyngeal Carcinoma Progression via Negative Regulation of Target of Rapamycin Complex 1 Signaling

To speculate an autophagy gene, secretion associated Ras related guanosine triphosphatase 1B related signaling pathway for nasopharyngeal carcinoma based on both in vitro and in vivo experiments. 120 nasopharyngeal carcinoma biopsies (pathologically confirmed) were analyzed and the differentially expressed genes were explored. The internal molecular mechanism was further investigated using the human nasopharyngeal carcinoma cell lines, CNE1, HONE1 and C666-1. The cell proliferation capacity examination and the metabolic assays were performed in CNE1 cell line. The subcutaneous xenograft tumor mice model was also established. Secretion associated Ras related guanosine triphosphatase 1B demonstrated a remarkable decreased activity in nasopharyngeal carcinoma tissues compared with sibling paracancerous tissues. The key components in mammalian target of rapamycin complex 1 but not mammalian target of rapamycin complex 2 were greatly enhanced in nasopharyngeal carcinoma tissues. Moreover, the secretion associated Ras related guanosine triphosphatase 1B displayed a significant decreasing expression pattern and the mammalian target of rapamycin complex 1 kept an upward trend as the tumor, node and metastases stage progressed. The clinical significances for nasopharyngeal carcinoma tumor progression were calculated based on statistical analysis. The cell proliferation assay suggested that secretion associated Ras related guanosine triphosphatase 1B manipulated nasopharyngeal carcinoma cell proliferation via mammalian target of rapamycin complex 1/p70 ribosomal protein kinase 1 dependent signaling pathway. At the same time, transfection of secretion associated Ras related guanosine triphosphatase 1B small interfering ribonucleic acid could significantly enhanced the glycolytic capacity and glycolytic reserve of nasopharyngeal carcinoma cells compared with negative control. Silencing of secretion associated Ras related guanosine triphosphatase 1B promoted xenograft tumour growth, which could be greatly suppressed by rapamycin treatment in a dosage-dependent manner. The study shed a variety of insights for nasopharyngeal carcinoma from an innovative direction.

MiR-302c-5p affects the stemness and cisplatin resistance of nasopharyngeal carcinoma cells by regulating HSP90AA1.

Nasopharyngeal carcinoma (NPC) is one of the most frequent malignant tumors diagnosed in China. Cisplatin is one of the most commonly used anticancer drugs containing platinum in combined chemotherapy. The molecular mechanism of NPC is still largely unknown, and we aim to spare no effort to elucidate it. Normal human nasopharyngeal epithelial cells and NPC cell lines were cultured. The expression levels of miR-302c-5p and HSP90AA1 were detected with quantitative real-time PCR. Western blotting was used to analyze levels of the HSP90AA1, protein kinase B (AKT), p-AKT, CD44 and SOX2 proteins. The interaction between miR-302c-5p and HSP90AA1 was detected using a luciferase reporter assay. The bicinchoninic acid assay was used to observe cisplatin resistance in NPC cells. Our records confirmed that the expression of miR-302c-5p was substantially reduced and HSP90AA1 was increased in NPC cells. Additionally, miR-302c-5p inhibited cisplatin resistance and the traits of stem cells in NPC. A luciferase assay confirmed that miR-302c-5p is bound to HSP90AA1. Overexpression of HSP90AA1 may reverse the effects of overexpressed miR-302c-5p and inhibit cisplatin resistance and stem cell traits of NPC. This study investigated whether miR-302c-5p inhibited the AKT pathway by regulating HSP90AA1 expression and altered the resistance of NPC cells to cisplatin and the traits of tumor stem cells, which has not yet been reported.

Extracellular matrix stiffness mediates radiosensitivity in a 3D nasopharyngeal carcinoma model

PurposeRadiotherapy is one of the essential treatment modalities for nasopharyngeal carcinoma (NPC), however, radioresistance still poses challenges. Three-dimensional (3D) tumor culture models mimic the in vivo growth conditions of cells more accurately than 2D models. This study is to compare the tumor biological behaviors of NPC cells in 2D, On-Surface 3D and Embedded 3D systems, and to investigate the correlation between radioresistance and extracellular matrix (ECM) stiffness.MethodsThe morphology and radioresistance of the human NPC cell line CNE-1 were observed in 2D and 3D systems. The CCK-8 assay, wounding healing assays, flow cytometry, soft agar assays, and western blot analysis were used to evaluate differences in biological behaviors such as proliferation, migration, cell cycle distribution, and stem cell activity. Different ECM stiffness systems were established by co-blending collagen and alginate in varying proportions. ECM stiffness was evaluated by compressive elastic moduli measurement and colony formation assay was used to assess radioresistance of NPC cells in systems with different ECM stiffness after irradiation.ResultsCompared to 2D models, the morphology of NPC cells in 3D culture microenvironments has more in common with in vivo tumor cells and 3D cultured NPC cells exhibit stronger radioresistance. Integrin β1 but not the epithelial-to-mesenchymal transition pathway in 3D models boost migration ability. Cell proliferation was enhanced, the proportion of tumor stem cells was increased, and G1/S phase arrest occurred in 3D models. NPC cells cultured in softer ECM systems (with low alginate proportions) exhibit striking resistance to ionizing radiation.ConclusionThe tumor biological behaviors of NPC cells in 3D groups were obviously different from that of 2D. Radioresistance of NPC cells increased with the stiffness of ECM decreasing.

Curcumin-loaded nanoparticles reversed radiotherapy-triggered enhancement of MDR1 expression of CNE-2 cells in nasopharyngeal carcinoma

This study explored the effect of nanoparticle-encapsulating curcumin on strongly expressed multidrug resistance gene 1 (MDR1) in a human low-differentiated nasopharyngeal carcinoma cell line (CNE-2). The curcumin/chitosan-deoxycholic acid nanoparticles were prepared, and cells received different treatments: radiotherapy, empty carrier, curcumin and curcumin-loaded nanoparticles, followed by analysis of cell survival using the clonogenic assay, apoptosis, MDR1 and miR-593 level. Cell survival fractions in the curcumin group and curcumin-loaded nanoparticles group were reduced significantly. Moreover, we observed a reduced cell survival fraction in the curcumin-loaded nanoparticles group (p &lt; 0.05). Remarkably, higher apoptosis rates were observed in cells receiving curcumin or curcumin-loaded nanoparticles treatments compared with radiotherapy. Moreover, the curcumin-loaded nanoparticle treatment enhanced apoptosis (p&lt;0.05). Furthermore, a decreased MDR1 level was denoted in curcumin group and curcumin-loaded nanoparticles group and a further reduced MDR1 expression in nanoparticles group (p &lt; 0.05). A higher miR-593 expression was observed in the curcumin group and curcumin-loaded nanoparticles group with a relative higher level in nanoparticles group (p&lt;0.05). MDR1 expression in inhibitor group was significantly strengthened (p&lt;0.05). Curcumin that is encapsulated in nanoparticles exhibited a stronger radio sensitizing effect. Its combination with radiotherapy can effectively inhibit NPC tumor growth, and suppress MDR1 expression while enhancing miR-593. After retarding the miR-593, the MDR1 expression was intensified. The radio sensitizing effect of curcumin-loaded nanoparticles was regulated by miR-593 but not triggered by MDR1. The curcumin-loaded nanoparticles mediated enhanced expression of miR-593, which in turn inhibited the transcription and translation of MDR1 gene, thereby reducing the radio resistance of NPC and restraining the growth of NPC more effectively.

Different Effects of Reversible Electroporation-Mediated Fe3O4@AuNPs Delivery as Radiosensitizing Agents on Cancer Cells versus Normal Cells

Aim: In the present study, we sought to enhance the efficacy of radiotherapy via increasing the cellular uptake of Fe3O4@AuNPs as radiosensitizing agents using reversible electroporation, attaining a higher sensitivity for cancer cells to therapy-induced damage. Methods: The KB human nasopharyngeal carcinoma cell line was treated with different concentrations of gold-coated magnetic nanoparticles. The cells then received electroporation (750[Formula: see text]V/cm, 8 pulses for a duration 100[Formula: see text][Formula: see text]s with 100[Formula: see text]ms intervals) and radiotherapy (6MV X-ray, 2[Formula: see text]Gy). Control groups were carefully considered to assess the pure effect of both single and combinational therapeutic protocols. The MTT test and flow cytometry assay using Annexin V-PI kit were performed to evaluate potential effects on cell survival rate and to determine the induced level of apoptosis. Additionally, cellular uptake of nanoparticles was assessed and quantified using ICP-OES analysis. Results: Our study showed that nanoparticles and the applied procedure of electroporation had no considerable effects when utilized separately, but their combined application induced significant cell death ([Formula: see text]45%) and apoptosis ([Formula: see text]17%). Besides, the application of Fe3O4@AuNPs in the presence of electric field, 24[Formula: see text]h before the radiotherapy of tumor cells enhanced the cancer cell death ([Formula: see text]53%) as well as apoptosis rate ([Formula: see text]33%). The same scenario was also observed with normal HGF human gingival fibroblasts, confirmed by significantly lower levels of cell death and apoptosis. Conclusion: It can be concluded that electric fields at low voltage levels may significantly and selectively enhance the entry of nanoparticles into cancer cells, thereby accentuating the sensitivity of these cells to nanoparticle-mediated radiation therapy.

MiRNA profiling of human nasopharyngeal carcinoma cell lines HONE1 and CNE2 after X-ray therapy.

Radiotherapy is the main treatment for nasopharyngeal carcinoma. The radioresistance mechanism of cells is related to miRNAs. To investigate the miRNA profiling of HONE1 and CNE2 after X-ray therapy. The HONE1 and CNE2 cells were treated with X-ray at 4 Gy, 8 Gy, 16 Gy, and 20 Gy doses. The cell lines CNE2 with the best therapy effects and HONE1 with the worst therapy effects were screened out. Apoptosis and cell viability were detected with flow cytometry and Cell Counting Kit-8 (CCK-8). High-throughput sequencing was performed. A miRNA library was constructed. The miRNA annotation expression distribution, family prediction and target gene interaction, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted. The 24-hour 20 Gy dose X-rays were selected as the optimal therapy conditions. The CNE2_C, CNE2_M, HONE1_C and HONE1_M miRNAs accounted for 26.5%, 31.7%, 21.3%, and 22.9% of the Cleandata reads count, respectively, and the contents of rRNAs accounted for 24.9%, 14.7%, 25.1%, and 25.1% of the Cleandata reads count, respectively. The miRNAs with differential expression between the HONE1 and CNE2 cell lines including hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-125a-5p, hsa-miR-26a-5p, hsa-let-7f-5p, hsa-miR-20a-5p, and hsa-miR-24a-3p. There were also differentially expressed miRNAs in HONE1_C vs. HONE1_M, such as hsa-miR-21-5p and hsa-let-7i-5p. The differentially expressed miRNA in CNE2_C vs. CNE2_M was hsa-miR-148b-3p. The Gene Ontology analysis showed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were mainly enriched in biological process such as negative and positive regulation of transcription from RNA polymerase II promoter, cellular component such as cytosol and molecular function such as protein binding factor. The KEGG pathway analysis revealed that the differentially expressed miRNA interacting genes in HONE1_M vs. CNE2_M were enriched in the cancer-related pathways, such as pathways in cancer, MAPK signaling pathway and Wnt signaling pathway. Twelve miRNAs and 9 genes which contribute to X-ray radiation resistance were identified. Among those with differential expression between the HONE1 and CNE2 cell lines, which played a regulatory role in multiple pathways, were hsa-miR-20a-5p, hsa-let-7a-5p, hsa-let-7f5p, hsa-let-7i-5p, hsa-miR-30e-5p, hsa-miR-148b-3p, and hsa-miR-200c-3p. The corresponding genes were MAPK1, SOS1, TGFBR1, TGFBR2, TP53, CASP3, CCNE2, PTEN, and CDK2.

Xanthohumol targets the JNK1/2 signaling pathway in apoptosis of human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is one of the most aggressive malignant tumors of the head and neck. Xanthohumol (Xn) is a compound extracted in a high concentration from the hard resin of hops (Humulus lupulus L.), the basic raw material of beer. This study investigated the apoptotic effect and anticancer properties of Xn in human NPC cell lines. Our study demonstrated that at the concentration 40 μM, Xn significantly reduced cell viability and promoted cell cycle arrest in the G2/M phase in two cell lines. The results indicated that Xn induced apoptosis in NPC cell lines through annexin V/propidium iodide staining, chromatin condensation, and apoptosis-related pathways. Xn upregulated the expression of apoptosis-related proteins, namely DR5, cleaved RIP, caspase-3, caspase-8, caspase-9, PARP, Bim, and Bak, and it downregulated the expression of Bcl-2. Xn upregulated the c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK), and the inhibition of JNK clearly resulted in decreasing expression of Xn-activated cleaved caspase-3 and PARP. Our research provides sufficient evidence to confirm that Xn induces the MAPK JNK pathway to promote apoptosis of NPC and is expected to become a safe and acceptable treatment option for human NPC.

Hyperthermia inhibits growth of nasopharyngeal carcinoma through degradation of c-Myc

Background Hyperthermia is a widely used adjunct treatment for different cancers including nasopharyngeal carcinoma (NPC). The protooncogene c-Myc is up-regulated in NPC and its expression is associated with poor prognosis. Objective We hypothesized that c-Myc constitutes an important hyperthermia treatment target, and we investigated its contribution to hyperthermia responses in NPC. Methods The growth of the human NPC cell lines CNE1 and CNE2 was analyzed using CCK-8 and clonogenicity assays after 43 °C hyperthermia, knockdown or overexpression of c-Myc. Flow cytometry measurements assessed cell cycle parameters and apoptosis, while levels of c-Myc together with key transcriptional targets were determined using qPCR and Western blotting. Parallel experiments were undertaken using NPC xenografts in nude mice and lastly, global transcriptomic changes were determined using ‘RNAseq’. Results Hyperthermia increased the ubiquitination and proteasomal destruction of c-Myc, causing a rapid decline in c-Myc protein levels in NPC cells. Similar to c-Myc knockdown, NPC cells treated with hyperthermia showed growth inhibition associated with the downregulation of c-Myc target genes. Moreover, low levels of c-Myc could be sustainably repressed in NPC cells through repeated hyperthermia treatments. Importantly, the key findings of growth inhibition and decreased c-Myc protein levels were reproduced in NPC tumor xenografts. Bioinformatic analyses showed that downregulation of c-Myc constituted a central node in the hyperthermia response of NPC cells. Conclusion Our study reveals that hyperthermia can readily destabilize c-Myc levels in NPC cells and inhibit tumor growth. This proposes new strategies for implementing hyperthermia to target c-Myc-driven cancers to improve therapeutic efficacy.

Dihydromyricetin inhibits cancer cell migration and matrix metalloproteinases-2 expression in human nasopharyngeal carcinoma through extracellular signal-regulated kinase signaling pathway.

Nasopharyngeal carcinoma (NPC) is endemic in Southeast Asia and the main cause of treatment failure is metastasis. A lot of biological and pharmacological actions of dihydromyricetin (DHM) have been reported such as regulating glucose and anti-cancer effects. The effects of DHM on the cancer invasion and migration of NPC, however, are still unclear. We therefore investigated the in vitro anti-metastatic properties of DHM on three human NPC cell lines (HONE-1, NPC-39, and NPC-BM), as well as the underlying signaling pathways. Our study revealed that DHM could suppress the migration and invasion in NPC cells. Gelatin zymography assay and western blotting assays demonstrated that DHM suppressed the enzyme activity and protein expression of matrix metalloproteinases-2 (MMP-2). Mitogen-activated protein kinases were also investigated to elucidate the signaling pathway, which showed that phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) was inhibited after the treatment of DHM. In conclusion, our data revealed that DHM inhibited the migration and invasion of NPC cells by suppressing the expression of MMP-2 via down regulating the ERK1/2 signaling pathway.

MiR-26a-5p suppresses nasopharyngeal carcinoma progression by inhibiting PTGS2 expression

ABSTRACT Nasopharyngeal carcinoma (NPC) has a low five-year survival rate, and its pathogenesis remains unclear. There is an urgent need to improve our understanding of the genetic regulation of NPC tumorigenesis and development. The role of miR-26a-5p in NPC growth regulation and the expression of its target, PTGS2, was analyzed. Quantitative Real-time PCR assay was used to detect miR-26a-5p and PTGS2 expression in human NPC tissues and cell lines. The RNA pull-down dual-luciferase reporter assay was used to determine the association between miR-26a-5p and PTGS2. The effects of miR-26a-5p and PTGS2 on NPC cell viability, proliferation, migration, and invasion were measured by CCK-8, BrdU, and Transwell assays. miR-26a-5p expression in NPC tissues and cell lines was significantly decreased. The overexpression of miR-26a-5p inhibited the viability, proliferation, migration, and invasion of NPC cells. miR-26a-5p bound to the 3-ʹuntranslated region of PTGS2, thus reducing PTGS2 protein levels. miR-26a-5p inhibited NPC development by reducing the expression of its target PTGS2.

Cyclic Peptides from the Soft Coral-Derived Fungus Aspergillus sclerotiorum SCSIO 41031.

Three novel cyclic hexapeptides, sclerotides C–E (1–3), and a new lipodepsipeptide, scopularide I (4), together with a known cyclic hexapeptide sclerotide A (5), were isolated from fermented rice cultures of a soft coral-derived fungus: Aspergillus sclerotiorum SCSIO 41031. The structures of the new peptides were determined by 1D and 2D NMR spectroscopic analysis, Marfey’s method, ESIMS/MS analysis, and single crystal X-ray diffraction analysis. Scopularide I (4) exhibited acetylcholinesterase inhibitory activity with an IC50 value of 15.6 μM, and weak cytotoxicity against the human nasopharyngeal carcinoma cell line HONE-EBV with IC50 value of 10.1 μM.

Abstract P186: Preclinical evaluation of the proteasome inhibitor ixazomib (MLN2238) on nasopharyngeal cacinoma (NPC)

Abstract Background: Nasopharyngeal carcinoma (NPC) is a common cancer in the South Asia and is closely associated with EBV infection. The standard treatment for newly diagnosed NPC is radiotherapy alone or in combination with adjuvant chemotherapy and there is currently no targeted therapy available for NPC. About 30% of the NPC patient will progress within 5 years after treatment, so study on new therapeutic agents is in need. Proteasome inhibitor is emerged as an effective antitumor therapeutic agent in both haematological and solid tumour. Studies have reported that proteasome inhibitor can inhibit nuclear factor-kB (NF-kB) signalling pathway which was found constitutively activated in NPC. Here, we examined the antiproliferation effect and molecular mechanisms of the second-generation, selective and reversible proteasome inhibitor ixazomib (MLN2238) on NPC in-vitro and in-vivo. Ixazomib is the first oral proteasome inhibitor approved by the FDA for multiple myeloma treatment and has shown less side effect and more selective to proteasome than the first-generation proteasome inhibitor, bortezomib. Method: Three human EBV positive NPC cell lines (C666-1, NPC43 and C17) and two PDXs (Xeno113 and Xeno76) PDXs were used. Results: In our study, we showed that Ixazomib can inhibit cell viability and proliferation in EBV-associated NPC cells C666-1, NPC43 and C17 in a time- and dose-dependent manner. The IC50 of 72h treatment of ixazomib in NPC cells is 11-40nM which is more sensitive than other solid tumour, e.g. hepatocellular carcinoma (260-428nM) and breast cancer (43-1007nM). Induction of apoptosis by ixazomib was confirmed by flow cytometry analysis of Annexin V staining and western blotting of caspase-3/9 activation and PARP cleavage. S and G2/M cell cycle arrest was observed after Ixazomib treatment, which is demonstrated by flow cytometry analysis of PI staining. Evaluation of the expression level of EBV lytic genes by RT-qPCR and western blot showed that Ixazomib associates with EBV lytic cycle activation in NPC. Consistent with the previous studies, inhibition of canonical NF-kB signalling pathway was seen after 12h of ixazomib treatment in NPC cells. In animal study, 4 or 8mg/kg of ixazomib was administered orally 2 times per week for 3 weeks on NPC cells/PDX transplanted BALD/c-nu/nu and NOD/SCID mice and all showed statistical significant inhibition on NPC growth. Combination treatment of ixazomib and cisplatin showed additive effect in both in-vitro and in-vivo studies. Conclusions: Ixazomib is a potential therapeutic agent for NPC treatment and the preclinical data supports further evaluation and clinical trial of ixazomib for NPC. Citation Format: Ka Yee Li, Victor Ho Fun Lee, George Sai Wah Tsao. Preclinical evaluation of the proteasome inhibitor ixazomib (MLN2238) on nasopharyngeal cacinoma (NPC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P186.