Nasopharyngeal Carcinoma Cell Line C666-1 Research Articles

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

Hsa_circ_0136839 regulates the malignant phenotypes of nasopharyngeal carcinoma via the Wnt/β-catenin signaling pathway.

With the development of RNA sequencing (RNA-seq) technology, circular RNA (circRNA), a new class of RNA, has received much attention in cancer research. However, information available on the biogenesis and functional value of circRNAs in nasopharyngeal carcinoma (NPC) is scarce. In the present study, we screened the circRNA profile of the NPC cell line C666-1 compared with that of the normal control NP69 by RNA-seq and identified a novel and relatively higher expressed circRNA, hsa_circ_0136839. Hsa_circ_0136839 was markedly downregulated in NPC tissues, as confirmed by quantitative reverse transcription polymerase chain reaction. Functional in vitro studies revealed that hsa_circ_0136839 knockdown in C666-1 cell notably promoted cell proliferation, migration, and invasion abilities, as well as affected cell cycle distribution with an S-phase arrest. However, hsa_circ_0136839 overexpression in CNE2 cells resulted in an opposite response. Mechanistically, we demonstrated that aberrant hsa_circ_0136839 expression might affect the malignant phenotypes of NPC cells by activating the wnt/β-catenin signaling pathway. Thus, our findings contribute to further the understanding of NPC pathogenesis and provide new ideas for NPC clinical diagnosis and treatment.

MiR-138-1-3p alters the stemness and radiosensitivity of tumor cells by targeting CRIPTO and the JAK2/STAT3 pathway in nasopharyngeal carcinoma.

BackgroundTumor resistance to radiotherapy is one of the main obstacles to the clinical treatment of nasopharyngeal carcinoma (NPC). Improving the radiosensitivity of tumor cells has an important clinical significance in treatment of clinical NPC. This study aimed to identify that miR-138-1-3p as a novel therapeutic target in radioresistant NPC cells and found its targets, CRIPTO and the JAK2/STAT3 pathway.MethodsRadioresistant C666-IR and HK-1R cells were derived from the NPC cell lines C666-1 and HK-1. The different microRNAs (miRNAs) and their targeting genes were analyzed between C666-1 and C666-IR cells using microarray bioinformatics. Western blot, qRT-PCR, gene transfection, Luciferase reporter assay, and confocal laser scanning microscopy were applied for the analysis of the different genes.ResultsMiR-138-1-3p was found to target CRIPTO, which involved in the epithelial-mesenchymal transition (EMT) and JAK2/STAT3 signaling pathways. The luciferase reporter assay confirmed that miR-138-1-3p targeted CRIPTO and downregulated the expression of CRIPTO. Furthermore, miR-138-1-3p affected the stability of the CRIPTO-GRP78 complex on the cell membrane and also reversed the radioresistant characteristics of NPC stem cells, which affected EMT and the JAK2/STAT3 signaling pathway.ConclusionsThe miR-138-1-3p is a small molecule that can modulate radiosensitivity in the radioresistant C666-IR and HK-1R NPC cell lines by inhibiting EMT and targeting CRIPTO to reduce the activation of the JAK2/STAT3 pathway.

Abstract 65: Anti-apoptotic proteins BCL-XL and MCL-1 are crucial for nasopharyngeal carcinoma (NPC) cell survival

Abstract Patients diagnosed with Nasopharyngeal carcinoma (NPC) initially respond well to chemo- and radiotherapy but eventually develop resistance to these treatments. Hence, novel and improved treatment strategies are needed for NPC. The BCL-2 family proteins are critical regulators of the intrinsic apoptosis pathway. These proteins are up-regulated in many cancers hence they have become attractive therapeutic targets. Given that different cell population rely on different anti-apoptotic proteins for survival, it is crucial to determine which proteins are important for NPC survival. A human apoptosis RT2 Profiler PCR Array was first employed to profile the anti-apoptotic gene expressions in both the NPC cell lines. The HK1 cells expressed all the anti-apoptotic genes (MCL-1, BFL-1, BCL-2, BCL-XL, and BCL-w). On the other hand, C666-1 expressed all except for BFL-1 (undetectable level). Given that there are no specific BFL-1 inhibitors, the role of BFL-1 in NPC cell survival was determined by deleting the gene using the CRISPR/Cas9 technique. The BFL-1 single guide RNAs were cloned into the PX459 plasmid (pSpCas9(BB)-2A-Puro) and transfected into the NPC cells to establish BFL-1 knock-out cells. Parallel to this experiment, the contributions of the other anti-apoptotic proteins in NPC survival was determined by treating NPC cell lines HK1, C666-1 and C17 with BH3 mimetics ABT-199, A-1331852 and S63845 which inhibit BCL-2, BCL-XL and MCL-1, respectively, alone and in combination, in both 2D and 3D cell culture models. The HK1 and C666-1 cells were resistant to single agent treatment of all three drugs, suggesting that none of these anti-apoptotic proteins singly mediate survival of the cells. The NPC C17 cells however were sensitive to single agent treatment of S63845 indicating that these cells rely on MCL-1 for survival. Co-inhibition of BCL-XL and MCL-1 with A1331852 and S63845 significantly inhibited cell proliferation in all three cell lines. Similar data were obtained with 3-dimensional spheroid cell culture models. Treatment with a combination of A1331852 and S63845 resulted in inhibition of growth and invasion of the 3D spheroids generated from the HK1 cells. Co-inhibition of BCL-2 and MCL-1 with ABT-199 and S63845, also inhibited cell proliferation of the NPC cell lines. However, the effect of the combination was not as pronounced as combination of A1331852 and S63845. Collectively, our data demonstrate that BCL-XL and MCL-1 are crucial for NPC survival and targeting these proteins with selective inhibitors may be potentially useful as treatment strategies for the management of NPC. Citation Format: Siti Fairus Abdul Rahman, Kalaivani Muniandy, Ghows Azzam, Nethia Mohana Kumaran. Anti-apoptotic proteins BCL-XL and MCL-1 are crucial for nasopharyngeal carcinoma (NPC) cell survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 65.

Silibinin down-regulates PD-L1 expression in nasopharyngeal carcinoma by interfering with tumor cell glycolytic metabolism

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

CBP-mediated acetylation of NF-κB p65 contributes to the proliferation of human nasopharyngeal carcinoma cells

IntroductionReportedly, activation of the NF-B signaling pathway is related to the proliferation of nasopharyngeal carcinoma (NPC) cells; however, the underlying mechanism by which NF-B is activated in NPC cells is yet to be addressed. In our present studies, CREB-binding protein (CBP)-mediated acetylation of the NF-B p65 subunit and its effect on activation of NF-B p65 as well as its role in the proliferation of NPC cells were explored.Material and methodsHuman NPC cell lines CNE1 and C666-1 were cultured in vitro. The acetylation (Lys310) and phosphorylation (Ser536) levels of NF-B p65 in NPC cell lines were detected by Western blot. Then, the mRNA levels of CBP, p300, PCAF and GCN5 in NPC cell lines were determined by real-time PCR. Subsequently, the interaction of p65 with CBP in NPC cell lines was detected by a co-immunoprecipitation experiment. Furthermore, an NF-B p65 mutant (at Lys310), NF-B inhibitor and CBP shRNA were used to study the effect of CBP-mediated acetylation of NF-B p65 on the proliferation of NPC cell lines by CCK-8 assay.ResultsNF-B p65 was markedly acetylated at the site of Lys310 in NPC cells. Moreover, the mutation of NF-B p65 at the acetylation site markedly reduced proliferation of human NPC cells. Further investigation revealed that CBP was up-regulated and physically associated with NF-B p65 at the protein level in NPC cells. CBP-mediated NF-B p65 acetylation enhanced its phosphorylation and further contributed to the proliferation of NPC cells.ConclusionsThis study indicated that CBP-mediated NF-B p65 acetylation promotes the proliferation of NPC cells.

Brevilin A Induces Cell Cycle Arrest and Apoptosis in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant cancers in Southeast Asia and Southern China. Centipeda minima extract (CME) had previously demonstrated anti-cancer effects in human NPC. Brevilin A, a sesquiterpene lactone isolated from C. minima, has been reported to exhibit biological activities. In this study, we investigated its anti-NPC effect and further explored its molecular mechanisms. The effects of brevilin A were tested in the NPC cell lines CNE-1, CNE-2, SUNE-1, HONE1, and C666-1. Effects of brevilin A on cell viability were determined by MTT assay, and cell cycle and apoptosis were detected by flow cytometry. The molecular mechanism of cell cycle regulation and apoptosis were investigated via Western blot. Results showed that brevilin A inhibited NPC cell viability in a concentration- and time-dependent manner. Brevilin A induced cell cycle arrest at G2/M and induced apoptosis. Western blot results demonstrated that brevilin A could down-regulate cyclin D3, cdc2, p-PI3K, p-AKT, p-mTOR, and p-STAT3, while up-regulating cleaved PARP, cleaved caspase 9, and Bax. Regulation of cyclin B1, cdk6, and Bcl-2 expression by brevilin A showed dynamic changes according to dose and time. In the tumor xenograft model, brevilin A could reduce tumor growth, at a similar magnitude to cisplatin. However, notably, whereas cisplatin treatment led to significant weight loss in treated mice, treatment with brevilin A did not, indicating its relative lack of toxicity. Taken together, brevilin A regulated cell cycle, activated the caspase signaling pathway, and inhibited PI3K/AKT/mTOR and STAT3 signaling pathways in vitro, and exhibited similar efficacy to the common chemotherapeutic cisplatin in vivo, without its associated toxicity. These findings provide a framework for the preclinical development of brevilin A as a chemotherapeutic for NPC.

Inhibition of Ezrin suppresses cell migration and invasion in human nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most severe types of malignant cancer of the head and neck as it is difficult to treat. Ezrin is highly expressed in numerous types of cancer. However, the role of Ezrin in NPC has not been fully investigated and further studies are required in order to uncover its therapeutic potential in the treatment of NPC. The aim of the present study was to investigate the expression of Ezrin in human NPC and to evaluate the effect of knockdown of Ezrin using small interfering (si)-RNA on NPC cell migration and invasion. The expression levels of Ezrin were determined using reverse transcription-quantitative polymerase chain reaction, immunohistochemical staining and western blotting. Following transfection of Ezrin-siRNA into NPC cells, cell invasion and migration were analyzed and the mRNA expression levels of matrix metalloproteinase(MMP)-2 and MMP9 were determined. The results revealed that the expression of Ezrin was markedly increased in human NPC tissue samples compared with normal adjacent nasopharyngeal tissue samples. Ezrin was also highly expressed in the NPC cell lines 6–10B and C6661 when compared with the normal nasopharyngeal cell line NP69. Transfection of NPC cell lines with siRNA targeting Ezrin significantly inhibited NPC cell migration and invasion, and downregulated the mRNA expression level of MMP2; however, no effect was observed on MMP9 mRNA expression. At the same time, knockdown of Ezrin significantly decreased the expression levels of phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (Akt), which downregulated the mRNA expression of MMP2. In conclusion, the results revealed that knockdown of Ezrin suppressed NPC migration and invasion by reducing the mRNA expression of MMP2 via the PI3K/Akt signaling pathway. These results highlight the important role of Ezrin in NPC cell migration and invasion. In addition, they indicate that silencing of Ezrin may serve as a potential therapeutic strategy to treat human NPC.

Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient

ObjectivesA combination of platinum-based chemotherapy and radiotherapy is the standard treatment for nasopharyngeal carcinoma (NPC). However, the efficacy of chemotherapy has reached a plateau. Many autophagy studies suggest that autophagy can either promote or suppress to cancer progression. Thus, a role of autophagy in the acquisition of chemoradioresistance has recently been a notable event. Therefore, we examined the relationship between autophagy and chemotherapy in NPC. MethodsThe expression of Beclin 1 and microtubule-associated protein light chain 3 (LC3), a marker of autophagy, was determined by immunohistochemistry in the biopsy samples of patients with NPC before and after the first course of chemotherapy. Additionally, to investigate in the effect of autophagy suppression in chemotherapy, NPC cell line C666-1 cells were treated with cisplatin and/or chloroquine, an inhibitor of autophagy. ResultsThe expression of Beclin 1 increased after chemotherapy in all patients. In NPC cell line C666-1, compared to cisplatin alone, combination therapy (cisplatin and chloroquine) reduced cell viability, and promoted cell apoptosis. ConclusionsThese results suggest that autophagy, represented by Beclin 1, is upregulated after chemotherapy in both in vitro and in vivo NPC studies. Inhibition of autophagy could therefore be new strategy for NPC treatment.

Andrographolide inhibits proliferation and induces apoptosis of nasopharyngeal carcinoma cell line C666-1 through LKB1-AMPK-dependent signaling pathways.

Andrographolide (Andro) belongs to the main bioactive ingredients of Andrographis paniculata. Many studies have shown that andro has a variety of pharmacological activities such as anti-inflammatory, anti-bacterial, anti-virus, anti-oxidant, immune regulation and liver protective effects. Moreover andro has been reported to have anticancer activity in multiple types of cancer, including gastric cancer, breast cancer, lung cancer and so on. However, there is no report about the effect of andro on the human NPC cell line C666-1 and the molecular mechanisms of andro-mediated apoptosis in C666-1 cells remain to be clarified. Cell proliferation was measured by a CCK8 assay, cell apoptosis rate was evaluated by flow cytometric analysis, and the protein expression of LKB1/AMPK signaling pathways was detected by Western blotting. Treatment with andro inhibited cell proliferation and induced apoptosis of C666-1 cells. Moreover, andro could activate LKB1-AMPK signaling. We also demonstrated that Ca2+/calmodulin-dependent protein kinase kinaseβ (CaMKKβ) was not involved in the regulation of andro on AMPK activation in C666-1 Cells. Andro suppressed proliferation and induced apoptosis of C666-1 cells through regulating the LKB1/AMPK/mTOR signal pathway.

IMB-6G induces endoplasmic reticulum stress-mediated apoptosis in human nasopharyngeal carcinoma cells.

IMB-6G is a novel N-substituted sophoridine acid that has been reported to have anticancer effects. The purpose of the present study was to investigate the effect and underlying mechanism of IMB-6G on human nasopharyngeal carcinoma (NPC) cells. The NPC cell line C666-1 was used in the present study and treated with different concentrations of IMB-6G (0, 1, 2 and 5 µM) for 24 h. Subsequently, cell viability was determined using the Cell Counting kit-8 assay and cell apoptosis was analyzed by performing flow cytometry. The expression levels of genes and proteins in the current study were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Results indicated that IMB-6G dose-dependently inhibited C666-1 cell viability and induced apoptosis. It was also revealed that IMB-6G induced apoptosis via inducing endoplasmic reticulum (ER) stress activation. Notably, IMB-6G administration enhanced the expression levels of Binding immunoglobulin protein and CCAAT-enhancer-binding protein homologous protein in C666-1 cells. Further analysis suggested that IMB-6G treatment activated inositol-requiring enzyme 1α (IRE1α) and PKR-like ER kinase (PERK) signaling pathways in C666-1 cells. In addition, silencing of IRE1α and PERK significantly reversed IMB-6G-induced cell growth inhibition and apoptosis. In conclusion, the present findings indicated that IMB-6G induced ER stress-mediated apoptosis through activating IRE1α and PERK signaling pathways. The present study suggests that IMB-6G may be a promising agent for NPC treatment.

Inhibiting eEF-2 kinase-mediated autophagy enhanced the cytocidal effect of AKT inhibitor on human nasopharyngeal carcinoma.

AimOur previous research showed that AKT inactivation via small molecule inhibitors did not induce significant apoptosis, but rather markedly increased autophagy in nasopharyngeal carcinoma (NPC). The purpose of the current study was to determine whether autophagy inhibition can enhance the anticancer efficacy of an AKT inhibitor (MK-2206).Materials and methodsNPC cell lines CNE-2 (Epstein–Barr virus negative) and C666-1 (Epstein–Barr virus positive) were used to conduct the research. Autophagy induction effects were evaluated via Western blotting. Eukaryotic elongation factor-2 (eEF-2) kinase was specifically and stably knocked down using shRNA. The growth and proliferation of the cells were assessed by Cell Counting Kit-8. In CNE-2 xenograft tumors, the antitumor effects of an AKT inhibitor (MK-2206) combined with an eEF-2 kinase inhibitor (NH125) were tested.ResultsMK-2206 induced eEF-2 kinase-dependent autophagy in NPC cell lines. Knockdown of eEF-2 kinase using shRNA blunted the autophagy activated by MK-2206. Compared with treatment with MK-2206 alone, shRNA or NH125 suppressed eEF-2 kinase and increased the growth-inhibitory effect of MK-2206 on the human NPC cell lines. The synergistic effects of eEF-2 kinase inhibition and MK-2206 were similar to those of the combination of hydroxychloroquine and MK-2206. Moreover, NH125 showed good synergistic effects with MK-2206 in vivo.ConclusioneEF-2 kinase-mediated autophagy induced by AKT inhibition played a protective role in NPC cells. Inhibition of eEF-2 kinase may be an effective method for increasing the efficacy of an AKT inhibitor such as MK-2206 in NPC.

Pan-CDK inhibition augments cisplatin lethality in nasopharyngeal carcinoma cell lines and xenograft models

In addition to their canonical roles in regulating cell cycle transition and transcription, cyclin-dependent kinases (CDKs) have been shown to coordinate DNA damage response pathways, suggesting a rational pairing of CDK inhibitors with genotoxic chemotherapeutic agents in the treatment of human malignancies. Here, we report that roniciclib (BAY1000394), a potent pan-CDK inhibitor, displays promising anti-neoplastic activity as a single agent and potentiates cisplatin lethality in preclinical nasopharyngeal carcinoma (NPC) models. Proliferation of the NPC cell lines HONE-1, CNE-2, C666-1, and HK-1 was effectively curbed by roniciclib treatment, with IC50 values between 11 and 38 nmol/L. These anticancer effects were mediated by pleiotropic mechanisms consistent with successful blockade of cell cycle CDKs 1, 2, 3, and 4 and transcriptional CDKs 7 and 9, ultimately resulting in arrest at G1/S and G2/M, downregulation of the transcriptional apparatus, and repression of anti-apoptotic proteins. Considerably enhanced tumor cell apoptosis was achieved following combined treatment with 10 nmol/L roniciclib and 2.0 μmol/L cisplatin; this combination therapy achieved a response over 250% greater than either drug alone. Although roniciclib chemosensitized NPC cells to cisplatin, it did not sensitize untransformed (NP69) cells. The administration of 0.5 mg/kg roniciclib to BALB/c xenograft mice was well tolerated and effectively restrained tumor growth comparable to treatment with 6 mg/kg cisplatin, whereas combining these two agents produced far greater tumor suppression than either of the monotherapies. In summary, these data demonstrate that roniciclib has strong anti-NPC activity and synergizes with cisplatin chemotherapy at clinically relevant doses, thus justifying further evaluation of this combinatorial approach in clinical settings.

MiR-100 inhibits the migration and invasion of nasopharyngeal carcinoma by targeting IGF1R

Nasopharyngeal carcinoma (NPC) is a cancer pattern that often develops in the epithelial cells of the nasopharynx. miR-100 is a miRNA that has been identified in a number of cancers. The aim of the present study was to investigate whether miR-100 can affect cell migration and proliferation of NPC by targeting insulin-like growth factor 1 receptor (IGF1R). Western blot analysis was used to determine the protein levels of genes. The reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression level of miR-100 and IGF1R. Transwell assay was used to detect the migration and invasion of cell lines. The luciferase reporter assay was employed to confirm the target gene of miR-100. miR-100 expression was highly reduced in NPC tissues compared with non-cancerous tissues. Overexpression of miR-100 significantly inhibited the migration and invasion of NPC cell lines C666-1 and SUNE1. IGF1R was a downstream target of miR-100 and was downregulated by miR-100. Knockdown of IGF1R by siRNA suppressed cell proliferation of the C666-1 cell line. The newly identified miR-100/IGF1R axis offers novel biomarkers for the therapeutic intervention of NPC treatment. As a result, our findings suggest that miR-100 plays an important role in suppressing migration and invasion of NPC cells and suppresses IGF1R expression by directly targeting its 3′-UTR. It is suggested that miR-100 may be a novel therapeutic target of microRNA-mediated suppression of cell migration and invasion in NPC. However, the role of the miR-100/IGF1R axis in NPC progression needs further investigation.

Slug inhibition increases radiosensitivity of nasopharyngeal carcinoma cell line C666-1.

Slug is associated with the radioresistance of nasopharyngeal carcinoma (NPC) and the main current approach of treatment for NPC is radiotherapy. Hence, the aim of the current study was to determine the effect of Slug silencing on the radiosensitivity of NPC cells. Lentiviral-mediated transfection of Slug RNA interference (RNAi) in NPC cell line C666-1 was performed in vitro. Following Slug inhibition, its expression was detected using western blotting. A clonogenic survival assay and flow cytometry were then performed to evaluate the clonogenic cell survival, cell cycle distribution and apoptosis of C666-1 cells following irradiation. The results indicated that Slug RNAi decreased cell proliferation, and increased cell apoptosis and G0/G1 arrest. Thus, lentiviral-mediated transfection of Slug RNAi enhanced the radiosensitivity of the NPC cell line C666-1, and Slug may therefore be a potential target to improve radiotherapy in treatment of NPC and reduce the radioresistance of NPC.

MiR-101 promotes nasopharyngeal carcinoma cell apoptosis through inhibiting Ras/Raf/MEK/ERK signaling pathway.

Extra-cellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway is widely involved in cell proliferation and apoptosis. MAPK kinase 1 (MEK1) is the upstream protein kinase of ERK that can activate ERK/MAPK signaling pathway. microRNA-101 (MiR-101) down-regulation is found to be associated with nasopharyngeal carcinoma (NPC) pathogenesis. Bioinformatics analysis shows the complementary targeted relationship between miR-101 and the 3'-UTR of MEK1 mRNA. This study explores the role of miR-101 in regulating MEK1 expression, ERK/MAPK signaling pathway activation, and NPC pathogenesis. Dual luciferase assay confirmed the targeted relationship between miR-101 and MEK1. MiR-101 and MEK1 expressions were compared in inflammatory nasopharynx tissue and NPC tissue. MiR-101, MEK1, phosphorylated ERK 1/2 (p-ERK1/2), survivin expressions in NP69, CNE-1, HONE1, and C666-2 cell lines were detected. NPC cell line C666-1 was cultured in vitro and divided into four groups, including miR-NC, miR-101, si-NC and si-MEK1. Cell apoptosis was determined by flow cytometry. Cell proliferation was evaluated by EdU staining. MiR-101 targeted inhibited MEK1 expression. MiR-101 was significantly down-regulated, while MEK1 was significantly elevated in NPC tissue compared with inflammatory nasopharynx tissue. MiR-101 was markedly declined, whereas MEK1, p-ERK1/2, and survivin were apparently increased in CNE-1, HONE1, and C666-1 cells compared with NP69 cells. MiR-101 mimic and/or si-MEK1 transfection significantly reduced MEK1, p-ERK1/2, and survivin levels, attenuated cell proliferation, and enhanced cell apoptosis. Down-regulation of miR-101 was related to NPC pathogenesis. MiR-101 elevation suppressed NPC cell proliferation and promoted apoptosis through targeted inhibiting MEK1 expression to alleviate ERK/MAPK signaling pathway and survivin expression.

KAI1 overexpression promotes apoptosis and inhibits proliferation, cell cycle, migration, and invasion in nasopharyngeal carcinoma cells

ObjectivesThe purpose of this study is to characterize the effect of KAI1 overexpression on the biological behavior of nasopharyngeal carcinoma (NPC) cells. BackgroundNasopharyngeal carcinoma is a highly malignant tumor with a high rate of incidence in China. Currently, there are no ideal therapeutic options for patients with NPC, but a targeted therapy would have great potential for treating it. Therefore, there is an urgent need for novel therapeutic targets to provide new options for treating NPC. The KAI1 gene was originally identified as a metastasis suppressor gene for advanced human cancer. In NPC cell lines and tissues, the expression of KAI1 decreased as the metastatic potential of cells increased, but its potential as a therapeutic target has not been elucidated. MethodsNon-transformed nasopharyngeal epithelium cell NP69 and NPC cell line C666-1 were cultured and KAI1 expression in these cells was detected by qRT-PCR and Western blot. After the transfection of KAI1-pCDNA3.1 to NP69 and C666-1, the KAI1 expression in these cells was detected by qRT-PCR and Western blot, the proliferation was performed by MTS, the cell cycle and apoptosis were performed by flow cytometry, the migration and invasion were examined by transwell. ResultsOur results showed that KAI1 was significantly upregulated in C666-1 cells compared to that in NP69 cells. In addition, KAI1 overexpression significantly inhibited the proliferation, cell cycle, migration, and invasion, and promoted apoptosis of C666-1 cells, but had no significant effect on NP69 cells. ConclusionOur findings suggest that KAI1 overexpression promotes apoptosis and inhibits proliferation, cell cycle, migration, and invasion in NPC cells. We hypothesize that KAI1 overexpression could be a potential therapeutic target for NPC.

Activation of miR-21 by STAT3 induces proliferation and suppresses apoptosis in nasopharyngeal carcinoma by targeting PTEN gene.

The present study is to investigate the role of microRNA-21 (miR-21) in nasopharyngeal carcinoma (NPC) and the mechanisms of regulation of PTEN by miR-21. Fifty-four tissue samples were collected from 42 patients with NPC and 12 healthy controls. Human NPC cell lines CNE-1, CNE-2, TWO3 and C666-1 were used for cell assays. To investigate the expression of miR-21, RT-PCR was employed. RT-PCR, Western blotting, and immunohistochemistry were used to measure the expression of STAT3 mRNA and STAT3 protein. To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. TUNEL assay was used to detect DNA fragmentation. To validate whether miR-21 directly recognizes the 3′-UTRs of PTEN mRNA, luciferase reporter assay was employed. miR-21 expression was increased in NPC tissues compared with control and the same result was found in NPC cell lines. Notably, increased expression of miR-21 was directly related to advanced clinical stage and lymph node metastasis. STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines. Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity. Both in vitro and in vivo assays revealed that miR-21 enhanced NPC cell proliferation and suppressed apoptosis. miR-21, activated by STAT3, induced proliferation and suppressed apoptosis in NPC by targeting PTEN-AKT pathway.

Lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of nasopharyngeal carcinoma cell line C666-1 in vitro

Latent membrane protein 2A (LMP2A) is found to play a key role in the development of nasopharyngeal carcinoma (NPC). However, the role of LMP2A silencing in the inhibition of cell growth of NPC has not been clarified. In this study, we inhibited LMP2A gene expression by lentivirus-mediated RNAi, to explore the effects of LMP2A silencing on the growth of NPC cell line in vitro. A lentivirus-mediated RNAi technology was employed to specifically knock down the LMP2A gene in NPC cell line C666-1. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and colony formation assays were performed to evaluate the expression of LMP2A and biological behavior of cell line C666-1 in vitro. We successfully construct a highly efficient and stable lentivirus vector, which efficiently downregulate the expression of LMP2A gene in infected cell line C666-1. Down-regulation of the expression of LMP2A significantly inhibits the proliferation and colony formation of C666-1 cells. In addition, the specific down-regulation of LMP2A arrests cells in G0/G1 phase of cell cycle and increases apoptosis rate. Our findings suggest that lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of NPC cell line C666-1 in vitro, and LMP2A may be a potential target for gene therapy in treatment of NPC.

Fluorescence Visualization Screening for EBV‐LMP1‐Targeted DNAzymes

To develop a novel screening method for DNAzymes targeting the LMP1 carboxy region. To design a method to screen special DNAzymes toward the Epstein-Barr virus (EBV)-associated carcinoma before clinic use. Key Laboratory of the Ministry of Education-Molecular Biology of Infectious Diseases in Chongqing Medical University. Four novel 10-23 DNAzymes (DZ509, DZ1037, DZ893, and DZ827) targeting the EBV-LMP1 gene were designed and evaluated by detecting enhanced green fluorescence protein (EGFP) expression of LMP1 mRNA and the protein in the nasopharyngeal carcinoma (NPC) cell line CNE2 transfected with the pEGFP-C1-LMP1c vector. The screened specific DNAzymes were then transfected into NPC cell lines C666-1 while a mutant oligonucleotide mutDZ509 and an antisense oligonucleotide ASODN509 were designed as positive and negative controls. Cell proliferation, cell apoptosis, LMP1 mRNA, and the protein were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V-fluorescence isothiocyanate (FITC), reverse transcription polymerase chain reaction (RT-PCR), and Western blots. The inhibition rates of fluorescence expression of the DNAzymes DZ509, DZ1037, DZ893, and DZ827 were 91.25%, 65.84%, 49.02%, and 44.56%, respectively. The results were in accordance with the inhibition effects of mRNA and protein expression. The screened DZ509 could effectively knock down endogenous LMP1 expression in C666-1 cells, inhibit cell proliferation, and induce cell apoptosis compared with mutDZ509 and ASODN509. LMP1 could present a potential target for DNAzymes toward the EBV-associated carcinoma, and the EGFP expression vector could be a visible method for screening special DNAzymes before clinic use.