CNE2 Cells Research Articles

Timosaponin AIII Suppresses RAP1 Signaling Pathway to Enhance the Inhibitory Effect of Paclitaxel on Nasopharyngeal Carcinoma.

Although PTX has been identified as an effective drug for nasopharyngeal carcinoma (NPC) therapy, it has serious side effects in the human body. Previous studies have shown that timosaponin AIII (TSAIII) can inhibit the malignant progression of NPC cells. This study investigated the active mechanism of the combination of TSAIII and paclitaxel (PTX) on NPC. Cellular viability, apoptosis, apoptotic factors, and RAP1 signaling regulators were detected in the PNC cells (CNE-1 and HNE-2) and the subcutaneous CNE-1 transplanted nude mice treated with PTX or/and TSAIII. The results showed that TSAIII notably strengthened the inhibitory effect of PTX on the proliferation of NPC cells CNE-1 and HNE-2; upregulated the expression of Bax B-cell lymphoma 2 (Bcl-2)/Bcl-xL-associated death promoter (Bad), and Ras-associated protein1 (RAP1) GTPase activating protein (Rap1GAP); inhibited the level of Bcl-2, RAP1, and Ras guanine nucleotide releasing protein (RasGRP2); and significantly enhanced the promoting effect of PTX on apoptosis in the CNE-1 and HNE-2 cells. Besides, TSAIII strengthened the inhibitory effect of PTX on xenograft tumor in nude mice without adverse reactions. In conclusion, the combination administration of TSAIII and PTX had a significantly therapeutic effect on NPC and avoided the PTX's side effects, which may have acted as a new direction for the study of therapeutic approaches for NPC clinically.

Hyaluronic Acid-Modified Poly-Beta-Amino-Esters for Adriamycin Delivery Against Nasopharyngeal Carcinoma

We developed a delivery system for highly efficient Adriamycin (ADM) use against nasopharyngeal carcinoma (NPC), based on Hyaluronic acid (HA)-poly-beta-amino-esters (PBAE). We used two NPC cell lines, CNE1 and CNE2, and nude mice plated with CNE1 as mouse models for in vitro and in vivo testing of the characteristics and function of ADM@HAPBAE. As a result, the average size of PBAE particles is found to be approximately 100 nm, and ADM@HA-PBAE has stability in environment with changing temperature and at pH = 7.4. In addition, ADM@HA-PBAE could deliver drugs to target lesion with high efficiency and showed high permeability to target areas, facilitating enough ADM release. In NPC cell lines, ADM@HA-PBAE could inhibit cell growth with high efficiency. In NPC mouse model, ADM@HA-PBAE could alleviate NPC growth and development and maintain a healthier physiological status, including body weight recovery, decreased tumor size and NPC inhibition. All these data suggest that ADM@HA-PBAE can increase ADM function by highly efficient targeting and delivery and should be considered as a potential strategy for NPC therapy.

Effect Of Puerarin on The Growth of Nasopharyngeal Carcinoma Cells and Its Impact on Angiogenesis

Objective Puerarin is a form of isoflavones obtained from Pueraria lobata. It stimulates hepatic metabolic function and lowers serum ALT, AST, and total-bilirubin level. The purpose of this study was to examine the effect of puerarin on nasopharyngeal carcinoma (NPC) CNE1 cells and preliminarily explore its possible mechanism. Materials and Methods CCK8 method was used to detect the proliferation activity of puerarin on NPC CNE1 cells and IC50 was calculated. CNE1 cells were treated with 0 μmol/L puerarin (containing equal volume of DMSO solution) as control group and 1000 μmol/L puerarin (IC50 concentration) as experimental group. Colony formation assay, Scratch-wound test and Transwell invasion assay were used to detect the clone formation ability, migration and invasion ability of puerarin on CNE1 cells. Then, RNA Sequencing was used to detect the changes of differentially expressed genes (DEGs) and signaling pathways after puerarin was applied to CNE1 cells. Results The inhibitory effect of puerarin on the proliferation activity of CNE1 cells was enhanced with the increase of concentration, and IC50 was calculated as 1000 μmol/L. Compared with the control group, the treatment of CNE1 cells with 1000 μmol/L puerarin could inhibit the clone formation, migration and invasion of CNE1 cells (P<0.05). A total of 379 DEGs were found by RNA sequencing, including 295 down-regulated genes and 84 up-regulated genes (padj<0.05). The significant differences in biological functions of differentially expressed genes were mainly distributed in “negative regulation of growth”, “angiogenesis”, “regulation of peptidase activity”, “positive regulation of vasculature development”, “digestion”, “positive regulation of angiogenesis”, “negative regulation of peptidase activity”, “extracellular matrix” and “Golgi lumen” (padj<0.05). Conclusion Puerarin could inhibit the proliferation, migration and invasion of NPC CNE1 cells, and its mechanism might be related to the inhibition of angiogenesis and cell growth.

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.

Salidroside suppresses the activation of nasopharyngeal carcinoma cells via targeting miR-4262/GRP78 axis

ABSTRACT To study the effect of Salidroside on nasopharyngeal carcinoma (NPC) cells and its mechanism. NPC cells were cultured, MTT was used to detect the effect of Salidroside on cell proliferation, apoptosis detected by flow cytometry assay, Western blot was used to detect the related protein expression. MiR-4262 and GRP78 used qRT-PCR for evaluation. Mimics/mimic NC and miR-4262 inhibitor/inhibitor NC were transfected into CNE2 and HONE1 cell lines, and cell viability was detected by MTT. Caspase-3, −8 and −9 activities were detected by caspase colorimetric assay kit. Targetscan predicted that downstream target of miR-4262. Relative luciferase activity was detected by luciferase assay. The effect of Salidroside on the growth of transplanted tumor in nude mice was observed. After Salidroside treatment, cell proliferation decreased and apoptosis increased, Bax protein expression increased and Bcl-2 decreased; miR-4262 expression level in nasopharyngeal carcinoma tissues was lower than that in adjacent tissues. GRP78 was the target of miR-4262 and downregulate the expression of miR-4262 in NPC cells can increase the expression of GRP78, and the expression of GRP78 decreased after upregulating the expression of miR-4262. Salidroside could inhibit the growth of NPC xenografts in nude mice. The level of Bax was increased and Bcl-2 was decreased in Salidroside group. Salidroside can significantly inhibit the proliferation and promote the apoptosis of NPC cells via regulating miR-4262/GRP78 signal axis.

Molecular Assessment of Scutellaria barbata D. Don in the Treatment of Nasopharyngeal Carcinoma Based on Network Pharmacology and Experimental Verification.

Objective To predict the molecular mechanisms behind the benefits of Scutellaria barbata D. Don (S. barbata) in nasopharyngeal carcinoma (NPC) by network pharmacology and experimental verification. Methods The active ingredients and targets of S. barbata were searched in the traditional Chinese medicine system pharmacology database and analysis platform, and the disease targets of NPC were obtained by searching the GeneCards database. A common target protein-protein interaction network was constructed by STRING, and then, an active ingredients-NPC-target interaction network map was constructed by Cytoscape 3.7.2 software. The functional enrichment analyses of Gene Ontology and KEGG pathway data were carried out by R software programming. Finally, cell proliferation was assessed by CCK8, apoptosis was detected by Annexin V-FITC/PI double fluorescence staining, and protein expression was analyzed by Western blotting. Results In this study, 29 active ingredients were found in S. barbata. Among these, the main targets for NPC were baicalein, wogonin, luteolin, and quercetin. The main molecular targets of S. barbata on NPC were EGFR, MYC, CASP3, CCND1, and ESR1. The main biological processes involved the binding of DNA-binding transcription factors, RNA polymerase II-specific DNA-binding transcription factors, ubiquitin-like protein ligases, and ubiquitin-protein ligases. S. barbata mainly affects NPC through the PI3K-Akt, p53, and MAPK signaling pathways. The experimental results showed that baicalein and wogonin could inhibit proliferation and induce apoptosis of NPC cells and downregulate the expression of PI3K, AKT, and p53, the key proteins of the PI3K/AKT and p53 signaling pathway in CNE2 cells. Conclusion Baicalein and wogonin, the main active ingredients of S. barbata, inhibited the proliferation and induced apoptosis of NPC cells through the PI3K/AKT and p53 signaling pathways.

Regenerating gene IA predicts radiosensitivity and survival in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common malignancy in Southern China and Southeast Asia. Radiotherapy is the main treatment option. However, radiotherapy does not benefit all patients because there is no known precise biomarker that can be used for screening radioresistant patients. Genetic predisposition is closely related to tumor development, therapeutic response, and prognosis. The relationship between regenerating gene IA (REGIA) and NPC is unclear. This study aimed to retrospectively analyze the association between REGIA expression and metastasis, radiosensitivity, and survival in patients with NPC as well as assess the effect of radiation on REGIA expression in vitro. Immunohistochemical staining was used to detect REGIA. The relationship between REGIA expression in radioresistant NPC and the prognosis of CNE1 NPC cells were analyzed using quantitative real-time polymerase chain reaction and Western blotting. We found that increased doses of radiation in CNE1 cells significantly decreased REGIA expression (P<0.05). The overall rate of REGIA-positive expression was 47.15% in NPC tissues and 45.00% and 61.02% in radiosensitive and radioresistant cases, respectively, showing significant differences (P<0.05). A REGIA-positive protein expression rate had a negative correlation with radiosensitivity in NPC (r= -0.109, P=0.047). Both REGIA-positive and REGIA-negative expression strongly predicted the overall survival rate and progression-free survival of NPC patients (P<0.01). A multivariate analysis indicated that REGIA was an inverse prognostic factor in NPC patients (REGIA-positive expression: hazard ratio (HR)=2.139, 95% confidence interval (CI)=1.56-2.94, P<0.001 and REGIA-negative expression: HR=1.958, 95% CI=1.42-2.69, P<0.001). In conclusion: Radiation can affect REGIA expression. The REGIA expression level correlated with radioresistance and a poor prognosis. In addition, REGIA expression might act as a potential therapeutic target and prognostic predictor in NPC patients.

Optimization of endothelial growth factor receptor monoclonal antibody-gold nanorods photothermal therapy for laryngeal squamous cell carcinoma

ABSTRACT It reported that heat generated by near-infrared laser irradiation of gold nanorods (AuNRs) effectively inhibited tumor cells, and the conjugate of epidermal growth factor receptor monoclonal antibody (EGFRmAb) and gold nanorods could selectively binded to the surface of cancer cell membrane expressing EGFR. However, there are few research reports on EGFRmAb-AuNRs in laryngeal squamous cell carcinoma. Therefore, our study aimed to investigate the photothermal effect of EGFRmAb modified AuNRs in inducing tumor cell death in an animal model of laryngeal squamous cell carcinoma. We showed that the conjugates of EGFRmAb and AuNRs selectively entered laryngeal squamous cell carcinoma cells. We analyzed the parameters of laser irradiation by controlling the near-infrared to optimize the condition and procedure of targeted treatment in nude mice treated with EGFRmAb and AuNRs. In addition, we examined the safety of the combined therapy. Test results showed that EGFRmAb-AuNRs inhibited the growth of Hep-2 and CNE-2 cells but not normal epithelial cells, and the semi-inhibitor concentration of EGFRmAb in Hep-2 and CNE-2 cells was 4 pmol/ml and 2 pmol/ml, respectively. AuNRs injected into the tumor and irradiated by near-infrared laser effectively inhibited tumor growth in nude mice without toxic effect in mice. We further confirmed that the apoptosis and necrosis rates of tumor cells in mice were highest under 3 W/cm2 near-infrared laser irradiation and AuNRs minimum concentration of 280 μg/kg. In conclusion, we developed a new method of targeting EGFRmAb combined with AuNRs to achieve photothermal effect in the treatment of laryngeal squamous cell carcinoma.

Theaflavin promoted apoptosis in nasopharyngeal carcinoma unexpectedly via inducing autophagy in vitro

Objective(s):This study aimed to investigate the mechanism of the anticancer effect of theaflavin (TF) in nasopharyngeal carcinoma.Materials and Methods:CNE2 cells were used to study the anticancer effect of TF. This study used Cell Counting Kit-8 (CCK8) assay on proliferation and used flow cytometry to detect apoptosis. The protein expression of Bcl-2, Bax, caspase 3, and caspase 9 was detected by Western blot, and autophagy-related proteins were also detected.Results:TF inhibited proliferation of CNE2 cells, promoted apoptosis, and up-regulated the expression of caspase 3, caspase 9, and Bax, and decreased the level of Bcl-2. Unexpectedly, TF induced autophagy rather than inhibiting autophagy through up-regulating the levels of the autophagy marker light chain 3 (LC3) and Lysosomal-associated membrane protein 1 (LAMP1) and reducing levels of the autophagosome cargo protein p62, and the effect was via the mTOR pathway. Besides, autophagy inhibitor Chloroquine (CQ) suppressed the effect of TF on Bax, Bcl-2 and activation of caspase 3 and caspase 9.Conclusion:TF promoted apoptosis of nasopharyngeal carcinoma cells, the mechanism was unexpectedly involved in inducing autophagy.

Icotinib Enhanced Radiosensitization of Nasopharyngeal Carcinoma by Inhibiting the Expression of Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR) is frequently overexpressed in multiple malignancies. Icotinib (IH), a new EGFR tyrosine kinase inhibitor, enhances radiosensitivity in various types of cancer, but its effect on nasopharyngeal carcinoma (NPC) remains unclear. Total 115 NPC tissue sections and 30 nasopharyngitis tissue sections were enrolled. The correlation of EGFR expression and clinicopathologic features of NPC was analyzed. Survival analysis was calculated by using univariate and multivariate regression analysis. A radioresistant NPC cell line, CNE-2R, was established with a gradient irradiation schedule. Cell viability, colony formation and EGFR expression of CNE-2/2R cells were examined. Significant higher expression of EGFR was observed in NPC tissues than chronic nasopharyngitis lesions. EGFR expression was significantly correlated with both tumor stage (P < 0.001) and tumor-node-metastasis stage of NPC (P = 0.006). EGFR expression was an independent prognostic factor of disease-free survival (P = 0.047) and the overall survival of NPC (P = 0.016). Cell viability was higher in CNE-2R than CNE-2 on days 1, 2, 4, and 6 after radiation of 4 Gy. The colony number of CNE-2R was significantly higher than that of CNE-2 (P < 0.05), while IH enhanced the radiosensitizing effect of CNE-2R with lower survival fraction (P < 0.05). EGFR mRNA and protein expression levels were significantly higher in CNE-2R cells compared to CNE-2 cells, but significantly decreased after IH treatment (all P < 0.05). In conclusion, high EGFR expression is a poor prognostic factor for NPC patients. IH enhances the radiosensitivity of CNE-2R cells and reduce EGFR expression.

Establishment of a Zebrafish Xenograft Model for in Vivo Investigation of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a unique malignant tumor of the head and neck. Despite higher survival rates by the combination of radiotherapy and chemotherapy, the recurrence or metastasis of NPC still occurs at about 10%. Therefore, there is urgent demand to develop more effective in vivo models for preclinical trials to investigate the mechanisms of NPC development and progression and to explore better treatment approaches. In this study, we transplanted human NPC CNE1 cells into zebrafish embryos to establish a xenograft model of NPC, where the proliferation and invasion behaviors of NPC cells were investigated in vivo. Combining in vitro and in vivo analyses, we found that activating transcription factor 7 (ATF7) was involved in the occurrence and development of NPC regulated by peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). The zebrafish NPC xenograft model established here thereby provides an in vivo tool for exploring the occurrence and development of NPC, which may help to identify new tumor markers and develop new therapeutic strategies for the treatment of NPC.

Identification of limonoids from Walsura yunnanensis and evaluation of their cytotoxicity against cancer cell lines

Three new limonoids, walsurauias A-C (1–3), along with four known ones, were isolated from the leaves and twigs of Walsura yunnanensis C. Y. Wu. Their structures were determined on the basis of comprehensive spectroscopic data analysis. The new limonoids were screened for their cytotoxic activity (IC50 0.81–5.73 μM) against four human cancer cell lines, including A549, HepG2, HCT116 p21KO and CNE-2. And α,β-unsaturated ketone moieties in rings A and B are essential for their cytotoxic activity. Selected compounds were further investigated. Compounds 1–3 effectively induced G2/M cell cycle arrest and apoptosis in a dose-dependent manner in cancer cells. In addition, compounds 1–3 inhibited the colony formation and compounds 2 and 3 suppressed the migration of cancer cells.

Aaptamine derivatives with CDK2 inhibitory activities from the South China Sea sponge Aaptos suberitoides

Three new aaptamines (1–3) together with two known derivatives (4–5) were isolated from the South China Sea sponge Aaptos suberitoides. The structures of all compounds were unambiguously elucidated by spectroscopic analyses as well as the comparison with literature data. All the compounds were evaluated for their cytotoxic activities against five human cancer cell lines including H1299, H520, SCG7901, CNE-2 and SW680 cells. As a result, compounds 3–5 showed moderate cytotoxicities against H1299 and H520 cells with IC50 values ranging from 12.9 to 20.6 μg/mL. Besides, compounds 3–5 also showed potent inhibitory activities toward cyclin-dependent kinase-2 (CDK2) with IC50 values of 14.3, 3.0 and 6.0 μg/mL, respectively. In addition, compounds 3–5 significantly induced G1 arrests of H1299 cells at low concentrations. Drug affinity responsive target stability (DARTS) experiments were carried out and further demonstrated that compound 3 could effectively bind with CDK2 protein and protect it from the degradation by pronase.

The expression of clock gene CLOCK and its clinical significance in nasopharyngeal carcinoma

Objective: To explore the relationship between expression levels of CLOCK mRNA and protein and the clinical characteristics of patients with nasopharyngeal carcinoma. Methods: The frozen tissue specimens from 33 patients with nasopharyngeal carcinoma in the Affiliated Tumor Hospital of Guizhou Medical University from 2018 to 2019 were collected. Seventeen cases of tissue specimens from patients with nasopharyngeal chronic inflammation in the Affiliated Hospital of Guizhou Medical University in 2019 were collected. From 2008 to 2014, 68 cases of formalin-fixed paraffin-embedding (FFPE) nasopharyngeal carcinoma tissue and 37 cases of FFPE nasopharyngeal chronic inflammation tissue were collected from the Affiliated Tumor Hospital of Guizhou Medical University. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB) were used to detect the mRNA and protein expression levels of CLOCK. The nasopharyngeal carcinoma cells including CNE1, CNE2, 5-8F and the normal nasopharyngeal epithelial cell NP69 were cultured. qRT-PCR was used to detect the expression level of CLOCK mRNA in each cell line at the time points of ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22. The cosine method was used to fit the rhythm of CLOCK gene in nasopharyngeal carcinoma. The protein expression of CLOCK protein was detected by using immunohistochemical method in 68 cases of nasopharyngeal carcinoma and 37 cases of nasopharyngeal chronic inflammation tissue. Survival was analyzed by Kaplan-Meier method and Log rank test, and the influencing factors was analyzed by Cox regression model. Results: The expression levels of CLOCK mRNA in CNE1, CNE2 and 5-8F cells (0.63±0.07, 0.91±0.02 and 0.33±0.04, respectively) were lower than that in NP69 cell (1.00±0.00, P<0.05). The expression levels of CLOCK protein in CNE1, CNE2 and 5-8F cells (0.79±0.06, 0.57±0.05 and 0.74±0.10, respectively) were lower than that of NP69 cells (1.00±0.00, P<0.05). The expressions of CLOCK mRNA in nasopharyngeal carcinoma cells including CEN1, CNE2, 5-8F and normal nasopharyngeal epithelial cell NP69 were different at different time points, with temporal fluctuations. The fluctuation periods of CLOCK mRNA in CNE1, CNE2, 5-8F, and NP69 cells were 16, 14, 22 and 24 hours, respectively. The peak and trough times were ZT10: 40 and ZT18: 40, ZT10 and ZT3, ZT14: 30 and ZT3: 30, ZT12: 39 and ZT0: 39, respectively. CLOCK mRNA and protein expression levels in nasopharyngeal carcinoma tissues (0.37±0.20 and 0.20±0.26, respectively) were lower than those in nasopharyngeal chronic inflammation tissues (1.00±0.00 and 0.51±0.41, respectively, P<0.05). The 1, 3, and 5-year survival rates of patients in the CLOCK protein high expression group (CLOCK protein expression level ≥ 0.178) were 96.2%, 92.1%, and 80.1%, respectively, which were higher than those in the low expression group (CLOCK protein expression level <0.178, 92.9% , 78.6% and 57.1%, respectively, P=0.009). The 1, 3, and 5-year progression-free survival (PFS) rates of patients in the CLOCK protein high expression group were 96.2%, 87.8%, and 87.7%, respectively, which were higher than those in the low expression group (92.7%, 82.2%, and 70.8%, respectively, P=0.105). Compared with the low-expression group (100.0%, 96.9%, and 90.0%, respectively), the 1, 3, and 5-year recurrence-free survival rates of patients in the CLOCK protein high expression group (100.0%, 95.7%, and 95.7%, respectively) were not statistically significant (P=0.514). Compared with the low-expression group (92.7%, 82.2%, and 79.3%), the 1, 3, and 5-year survival rates without metastasis in the CLOCK protein high expression group (96.2%, 92.0%, and 92.0%, respectively) were not statistically significant (P=0.136). CLOCK protein expression and T stage were independent prognostic factors of overall survival (P<0.05). Conclusions: The expression of CLCOK is downregulated in the nasopharyngeal carcinoma cell and nasopharyngeal carcinoma tissues. Clock gene CLOCK is rhythmically expressed in the nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells. Compared with normal nasopharyngeal epithelial cells, the fluctuation period of CLOCK in nasopharyngeal carcinoma cells is shortened. The overall survival of patients in the CLOCK protein high expression group is better than that of low expression group. The expression of CLOCK protein is an independent influencing factor for overall survival. CLOCK gene may be a potential tumor suppressor gene in the nasopharyngeal carcinoma.

Synthesis and Antiproliferative Activity of Ester Derivatives of Mogrol through JAK2/STAT3 Pathway.

In attempt to enhance the antiproliferative activity of mogrol, two series of ester derivatives modified at C3 -OH and C11 -OH were designed and synthesized. The activity against human cancer cells including A549, NCI-H460 and CNE1 was screened by Cell Counting Kit-8 (CCK8) assay. According to the results, modifications of the mogrol core through introduction of different ester scaffolds drastically improved the cytotoxicity, and some of the derivatives exhibited even higher activity than the positive drug. Among them, compound M2h exhibited nearly 4 times more cytotoxic than 5-Fu against CNE1 cells, derivative M6c showed ten times higher activity with the IC50 value of 10.59 μM than mogrol against NCI-H460 cells, and compound M6a which contained one 1,2,3-triazole motif showed the strongest activity with an three folds lower IC50 value than mogrol. Furthermore, the most potent compound M2h could lead to cell cycle arrest at G2 phase on CNE1 cell lines and M6a induced G1 phase arrest on A549 cell lines. It was noteworthy that both M2h and M6a regulated signal transducer and activator of transcription 3 (STAT3) signal pathway through inhibiting phosphorylation of Janus Kinase 2 (JAK2) and STAT3, and simultaneously increasing the protein level of downstream cyclin p21.

Mechanistic Study of Irradiated Dying NPC Cells Promoting the Metastatic Ability of Surviving NPC Cells After Radiotherapy

<h3>Purpose/Objective(s)</h3> Distant metastasis remained the major cause of mortality in nasopharyngeal carcinoma (NPC) receiving radiotherapy despite favorable locoregional control in the past decades. Tumor microenvironment plays a critical role in tumor progression by secreting factors that regulate cancer cell metastasis. However, the underlying mechanisms remain elusive. The aim of this study was to elucidate the role and mechanism of necrotic or apoptotic NPC cells killed by radiotherapy in distant metastasis. We hypothesize that necrotic or apoptotic NPC cells killed by radiotherapy could release metastasis-stimulating signals to the microenvironment and promote the metastatic ability of the surviving cancer cells. <h3>Materials/Methods</h3> Based on the co-culture system, cell migration assay and cell invasion assay were used to analyze the effect of lethally irradiated NPC CNE1 cell line on migration and invasion abilities of radioresistant NPC cell line CNE1-IR. Subsequently, ITRAQ was used to screen the key factors in this process, and ELISA, qRT-PCR and Western blot were used to detect the expression of slit2 in lethally irradiated CNE1 cells. CNE1-IR cells were infected with Slit2 shRNA lentivirus (LV3-shSlit2) or LV3-NC. Cell migration and invasion assay as well as tumor metastasis experiment in vivo were used to examine the role of Slit2 in promoting metastasis of NPC cell lines. Lastly, Cell migration, invasion assay and Co-IP assay were used to explore which Robo receptors was involved in Slit2 mediated metastasis of NPC cell lines. <h3>Results</h3> The migration and invasion abilities of CNE1-IR cells were significantly promoted (<i>P</i> < 0.01) when CNE1-IR was co-cultured with lethally irradiated CNE1 cells. ITRAQ found that the expression of Slit2 was upregulated in the supernatant of lethally irradiated CNE1 cells. ELISA assay confirmed that Slit2 in the supernatant of lethally irradiated CNE1 cells was significantly higher than control cells. qRT-PCR analysis and Western blot analysis also showed that the expressions of Slit2 was significantly overexpressed in irradiated NPC cells compared with unirradiated control CNE1 cells. Knockdown of Slit2 significantly suppressed lung and liver metastasis in vivo. The expressions of both Robo1 and Robo2 could be detected in CNE1 and CNE1-IR. Subsequently, addition of rSlit2 could enhance cell migration ability. In contrast, Robo1-depleted cells showed decreased migration ability and did not respond to rSlit2. When rSlit2 was added to CNE1-IR/shRobo2 cells, the number of trans-membrance CNE-IR cells was increased by about 26% compared with CNE1-IR/NC cells. Co-IP assay with anti-Robo1 antibody indicated that Robo1 was immunoprecipitated with Slit2, indicating the interaction of Robo1 and Slit2. <h3>Conclusion</h3> Lethally irradiated NPC cells could promote migration and invasion of radioresistant NPC cell lines. Slit2, a secreted factor released from irradiated dying NPC cells could promote metastasis of living/resistant NPC cells via Robo1 receptor.

The Correlation Study of Circadian Clock Gene BMAL1 Regulates the Biological Behavior of Human Nasopharyngeal Carcinoma Cell After Radiotherapy

To investigate the effect of circadian clock gene BMAL1 on proliferation and cell cycle of Nasopharyngeal Carcinoma(NPC) CNE1 cells with different expression status, compare the expression level of pathway related proteins before and after radiotherapy, to further understand the mechanism of BMAL1 gene regulating the biological behavior of NPC cells, aim to explore the mechanism of BMAL1 gene regulating the expression of cell cycle related proteins through ATM/ATR pathway at the molecular level.Constructed CNE1 cell lines with BMAL1 gene overexpression group, control group, inhibitor group, blank group. Using Western blot (WB) to detect the overexpression of BMAL1 gene at protein level and the expression of ATM/ATR pathway related proteins. The absorbance of cells with the same number of plates was determined by a cell counting kit for 6 consecutive days. The effect of BMAL1 gene on the proliferation rate of CNE1 was analyzed. The effect of BMAL1 gene on cell cycle of CNE1 was studied by flow cytometry. Co-immunoprecipitation (co-IP) was used to detect the interaction between BMAL1 gene and ATM/ATR checkpoints.WB showed BMAL1 gene had a higher expression in the overexpression group. In the BMAL1 gene overexpression group, the absorbance values of CNE1 cells were lower at 1 to 6 days, the proportion of G0/G1 phase cells was higher, the relative protein expression of P21,P27 were higher. Co-IP confirmed the interaction between BMAL1 gene and the checkpoints of ATM/ATR pathway before and after radiotherapy. The protein expression of upstream genes of ATM/ATR pathway in the overexpression group was significantly higher before radiotherapy (P < 0.05). The protein expression of the upstream genes of ATM/ATR pathway increased significantly, while the expression of the downstream genes decreased significantly after radiotherapy. Comparing with the overexpression group and inhibitor group showed when the ATM/ATR pathway blocked by inhibitor KU55933, the expression of ATM/ATR pathway upstream genes decreased, and the expression of downstream genes increased. The expression of p-chk1 in blank group, control group and overexpression group after radiotherapy was significantly higher than before, all the difference were statistically significant.The circadian clock gene BMAL1 inhibits the proliferation of NPC cell line CNE1, arrests cell cycle in the G0/G1 phase by up-regulate the expression of P21,P27 and P53.The response of ATM/ATR pathway could be activated after radiotherapy, the overexpression of Bmal1 gene could up-regulate the expression of ATM/ATR pathway upstream genes such as p-chk1 and p-chk2,then inhibited the activity of downstream Cyclin-CDK complexes by phosphorylated CDC25A and CDC25C,finally achieved the function of blocking cell cycle after radiotherapy.

We Hypothesize That BMAL1 Has a Positive Regulatory Effect on the Radiosensitivity of Nasopharyngeal Carcinoma

<h3>Purpose/Objective(s)</h3> BMAL1 is a core biological clock gene, which is expressed rhythmically in a variety of tumor cells, and is related to cancer cell proliferation and chemotherapy sensitivity. Radiotherapy is the main treatment for nasopharyngeal carcinoma (NPC). However, the role of baml1 in radiotherapy for nasopharyngeal carcinoma is unclear. <h3>Materials/Methods</h3> We used 5 types of nasopharyngeal carcinoma cells (CNE1, CNE2, HONE1, C666-1, 5-8F) and normal nasopharyngeal epithelial cells NP69 as the research objects, and detected the transcription level changes of BMAL1 mRNA within 24 hours by RT-PCR. Furthermore, after overexpression and knockdown of BMAL1 in nasopharyngeal carcinoma CNE1 cells and transplanted tumors in nude mice, the effects of BMAL1 on the proliferation of CNE1 cells and subcutaneous transplanted tumors and the changes after radiotherapy were observed. Finally, RT-PCR and Western Blot were used to detect BMAL1, P53, P21 gene and protein expression in tumor tissues. <h3>Results</h3> The results suggest that the expression level of the circadian clock gene BMAL1 in NPC cell lines and NP69 cells fluctuates with time. Overexpression of BMAL1 can inhibit the proliferation of NPC cells, while knockdown promotes the proliferation of NPC cells. In the xenograft model, we observed that up-regulation of BMAL1 inhibited tumor growth and enhanced the sensitivity of NPC cells to radiotherapy, accompanied by increased expression of P53 and P21. Down-regulating BMAL1 promotes tumor growth, reduces the sensitivity of radiotherapy, and decreases the expression of P53 and P21. <h3>Conclusion</h3> BMAL1 showed rhythmic expression in NPC cells and NP69. BMAL1 plays a tumor suppressor effect in NPC, and can increase the sensitivity of NPC to radiotherapy, which may be related to the up-regulation of P53 and P21 protein expression. It is speculated that BMAL1 may become a new prognostic biomarker and new therapeutic target for NPC.

Pheophorbide co-encapsulated with Cisplatin in folate-decorated PLGA nanoparticles to treat nasopharyngeal carcinoma: Combination of chemotherapy and photodynamic therapy

The adverse effect and drug resistance of Cisplatin (CDDP) could be potential reduced by delivering in targeted nanoparticles and by combining with adjuvant therapy such as photodynamic therapy. In this study, F/CDPR-NP was formulated and characterized for all the physicochemical, biological and in vivo analysis. The results obtained from various in vitro and biological studies showed that encapsulation of CDDP and PBR in PLGA nanoparticles results in controlled release of encapsulated drugs and exhibited significantly low cell viability in CNE-1 and HNE-1 cancer cells. F/CDPR-NP significantly prolonged the blood circulation of the encapsulated drugs. The AUC of CDDP from F/CDPR-NP (4-fold) was significantly higher compared to that of free CDDP and similarly significantly higher t1/2 for CDDP from F/CDPR-NP was observed. F/CDPR-NP in the presence of laser irradiation showed significant reduction in the tumor burden with low tumor cell proliferations compared to either CDPR-NP or free CDDP indicating the potential of targeted nanoparticles and photodynamic therapy. Overall, combination of treatment modalities and active targeting approach paved way for the higher antitumor activity in nasopharyngeal carcinoma model. The positive results from this study will show new horizon for the treatment of other cancer models.

8-Formylophiopogonanone B induces ROS-mediated apoptosis in nasopharyngeal carcinoma CNE-1 cells.

Cancer is one of the leading causes of death in the world. It is very important to find drugs with high efficiency, low toxicity, and low side effects for the treatment of cancer. Flavonoids and their derivatives with broad biological functions have been recognized as anti-tumor chemicals. 8-Formylophiopogonanone B (8-FOB), a naturally existed homoisoflavonoids with rarely known biological functions, needs pharmacological evaluation. In order to explore the possible anti-tumor action of 8-FOB, we used six types of tumor cells to evaluate in vitro effects of this agent on cell viability and tested the effects on clone formation ability, scratching wound-healing, and apoptosis. In an attempt to elucidate the mechanism of pharmacological action, we examined 8-FOB-induced intracellular oxidative stress and -disrupted mitochondrial function. Results suggested that 8-FOB could suppress tumor cell viability, inhibit cell migration and invasion, induce apoptosis, and elicit intracellular ROS production. Among these six types of tumor cells, the nasopharyngeal carcinoma CNE-1 cells were the most sensitive cancer cells to 8-FOB treatment. Intracellular ROS production played a pivotal role in the anti-tumor action of 8-FOB. Our present study is the first to document that 8-FOB has anti-tumor activity in vitro and increases intracellular ROS production, which might be responsible for its anti-tumor action. The anti-tumor pharmacological effect of 8-FOB is worthy of further investigation.