Nasopharyngeal Carcinoma Cell CNE2 Research Articles

Rapid intracellular pH measurement based on electroporation- surface-enhanced Raman scattering

Rapid intracellular pH measurement based on electroporation- surface-enhanced Raman scattering

Hyaluronic acid modified carbon nanotubes using for photothermal therapy by promoting apoptosis of nasopharyngeal carcinoma cells.

Background: The present work illustrates the role of multi-walled carbon nanotubes in photothermal therapy. Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck with significant ethnic and geographic differences, and conventional treatment options are no longer suitable to improve the prognosis and survival of patients. Photothermal therapy (PTT) has emerged as a new strategy for oncology treatment in recent years and is now used in the treatment of many common cancers. Multi-walled carbon nanotubes (MWCNT) have been used to advantage in several fields due to their excellent thermal conductivity. The aim of this paper is to investigate the promotion of apoptosis of nasopharyngeal cancer cells by multi-walled carbon nanotubes as an adjuvant nanomaterial for nasopharyngeal cancer photothermal therapy. Methods: Carboxylated multi-walled carbon nanotubes and prepared multi-walled carbon nanotube-hyaluronic acid (MWCNT-HA) composites were used for cell proliferation-related experiments such as CCK-8 assay, live-dead staining and flow cytometric analysis and inverted fluorescence microscopy to determine the expression level of apoptotic factors and confocal microscopy cell morphology analysis on nasopharyngeal carcinoma CNE-1 cells under near-infrared laser irradiation. The effects of multi-walled carbon nanotubes on the proliferation and apoptosis of tumor cells under NIR response were elucidated, and the mechanism of apoptosis was explored. Results: TEM and SEM demonstrated that MWCNT had good appearance morphology and the temperature rise curve indicated excellent photothermal stability. And MWCNT and MWCNT-HA could significantly inhibit the proliferation of tumor cells and change the normal morphology of cells under NIR laser irradiation. Cellular immunofluorescence analysis confirmed that MWCNT-HA significantly upregulated the expression level of apoptosis factor Caspase-3 and significantly downregulated the expression level of anti-apoptosis factor Bcl-2. Conclusion: In this study, MWCNT inhibited the proliferation of tumor cells and promoted apoptosis through the use of multi-walled carbon nanotubes as an adjuvant nanomaterial for photothermal therapy. In addition, multi-walled carbon nanotubes could inhibit the mitochondrial pathway of CNE-1 cells to cause cell death. These studies suggest that multi-walled carbon nanotubes can function as efficient photothermal conversion materials for tumor photothermal therapy.

Mechanism of lncRNA FOXD3-AS1/miR-338-3p in the malignant progression of nasopharyngeal carcinoma through ceRNA.

This work aimed to analyze the impact of lncRNA FOXD3-AS1 (F-A) on the regulation of miR-338-3p (M) on the proliferation (Pro), migration (Mig), and invasion (Inv) of nasopharyngeal carcinoma (NPC) cells. F-A and M levels in human normal nasopharyngeal (NNP) NP69 cells and NPC CNE1 and CNE2 cells were detected by real-time quantitative PCR. After transfection or cotransfection with F-A shRNA, miR-338-3p mimic (MM), and miR-338-3p inhibitor (MI), cell Pro, Mig, and Inv (PMI) were detected using CCK-8, scratch healing, and Transwell chamber experiments, respectively. As a result, relative to NP69 cells, F-A was upregulated in CNE1 and CNE2 cells, while M was downregulated (P<0.01). F-A in CNE1 and CNE2 cells was downregulated and M was upregulated relative to shF-A (P<0.01). Furthermore, shF-A and MM groups demonstrated drastically reduced cell proliferative activity, Mig, and Inv versus CNE1/CNE2 groups. The cell proliferative activity, Mig, and Inv number of the MI group increased substantially (P<0.01). The shF-A+MM group exhibited markedly reduced cell proliferative activity, Mig, and cell number of Inv relative to those of CNE1/CNE2 (P<0.01). Moreover, the shF-A+MI group exhibited greatly increased cell proliferative activity, Mig, and cell number of Inv versus the shF-A+MM group (P<0.01). In short, lncRNA F-A level was abnormally upregulated, and that of M was downregulated in NPC. Interfering with lncRNA F-A level can upregulate M expression. Silencing of lncRNA F-A can inhibit PMI of NPC cells.

Lucialdehyde B suppresses proliferation and induces mitochondria-dependent apoptosis in nasopharyngeal carcinoma CNE2 cells

Context Lucialdehyde B (LB), an effective triterpenoid isolated from Ganoderma lucidum (Leyss. ex Fr.) Karst. (Polyproraceae), exerts cytotoxic activity against nasopharyngeal carcinoma CNE2 cells. Objective To investigate the antiproliferative and pro-apoptotic effects of LB on CNE2 cells and explore its underlying mechanisms. Materials and methods LB concentrations of 5–40 μg/mL were used. Cell proliferation was determined using MTT, CFSE, and colony formation assays. LB-induced apoptosis and cell cycle arrest were measured by flow cytometry after 48-h LB treatments. Fluorescence microscopy and flow cytometry were performed to measure the alteration of MMP, mPTP opening, ROS level, and Ca2+ content in CNE2 cells. Western blotting was performed to evaluate the expression of mitochondrial apoptosis-related and Ras/ERK signaling proteins. Results IC50 values of LB against CNE2 cells for 24, 48, and 72 h were 25.42 ± 0.87, 14.83 ± 0.93, and 11.60 ± 0.77 μg/mL, respectively. The CFSE assay showed that the cell proliferation index was 12.70 in the LB treatment group and 31.44 in the control group. LB significantly reduced clonogenic capacity, promoted cell apoptosis and induced cell cycle arrest at the G2/M phase. Our observations also revealed that LB induced ROS and calcium aggregation, opening of mPTP, MMP reduction, upregulation of mitochondrial apoptosis-related protein expression and inhibition of Ras/ERK signaling cascades. Discussion LB suppresses proliferation and induces mitochondrial-dependent apoptosis in nasopharyngeal carcinoma CNE2 cells. Conclusions LB may have a potential use as a clinical drug candidate for nasopharyngeal carcinoma treatment.

Maackiain inhibits proliferation and promotes apoptosis of nasopharyngeal carcinoma cells by inhibiting the MAPK/Ras signaling pathway

Nasopharyngeal carcinoma (NPC) is the third most common malignancy with a high recurrence and metastasis rate in South China. Natural compounds extracted from traditional Chinese herbal medicines have been developed and utilized for the treatment of a variety of cancers with modest properties and slight side effects. Maackiain (MA) is a type of flavonoid that was first isolated from leguminous plants, and it has been reported to relieve various nervous system disorders and exert anti-allergic as well as anti-inflammatory effects. In this study, we demonstrated that MA inhibited proliferation, arrested cell cycle and induced apoptosis in nasopharyngeal carcinoma CNE1 and CNE2 cells in vitro and in vivo. The expression of the related proteins associated with these processes were consistent with the above effects. Moreover, transcriptome sequencing and subsequent Western blot experiments revealed that inhibition of the MAPK/Ras pathway may be responsible to the anti-tumor effect of MA on NPC cells. Therefore, the effects of MA and an activator of this pathway, tertiary butylhydroquinone (TBHQ), alone or combination, were investigated. The results showed TBHQ neutralized the inhibitory effects of MA. These data suggest that MA exerts its anti-tumor effect by inhibiting the MAPK/Ras signaling pathway and it has the potential to become a treatment for patients with NPC.

Super Enhancer Driven Hyaluronan Synthase 3 Promotes Malignant Progression of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck with high metastatic and invasive nature. Super enhancers (SEs) control the expression of cell identity genes and oncogenes during tumorigenesis. As a glycosaminoglycan in the tumor microenvironment, hyaluronan (HA) is associated with cancer development. High expression of hyaluronan synthase 3 (HAS3) resulted in HA deposition, which promoted the growth of cancer cell. However, its role in NPC development remains elusive. We demonstrated that the levels of HAS3 mRNA or protein were increased in NPC cell lines. Transcription of HAS3 is associated with SE. Disruption of SE by bromodomain containing 4 (BRD4) inhibitor JQ1 resulted in downregulation of HAS3 and inhibition of cell proliferation and invasiveness in NPC cells. Inhibition of HA synthesis by HAS inhibitor 4-MU suppressed cell growth and invasion of NPC cells, whereas HA treatment exerted opposite effects. Genetically silencing HAS3 in HK1 and FaDu NPC cells attenuated cell proliferation and mobility, while re-expression of HAS3 enhanced malignant potential of CNE1 and CNE2 NPC cells. Furthermore, loss of HAS3 impaired metastatic potential of HK1 cells in nude mice. Mechanistically, inhibition of HA synthesis by chemical inhibitor or silencing HAS3 led to reduction of the levels of phosphorylation of EGFR, AKT, and ERK proteins. In contrast, exogenous HA treatment or forced expression of HAS3 activated EGFR/AKT/ERK signaling cascade. This study suggested that HAS3 is driven by SE and overexpressed in NPC. High expression of HAS3 promotes the malignant features of NPC via activation of EGFR/AKT/ERK signaling pathway.

Identification and verification of aging-related lncRNAs for prognosis prediction and immune microenvironment in patients with head and neck squamous carcinoma.

Aging is highly associated with tumor formation and progression. However, little research has explored the association of aging-related lncRNAs (ARLs) with the prognosis and tumor immune microenvironment (TIME) of head and neck squamous cell carcinoma (HNSCC). RNA sequences and clinicopathological data of HNSCC patients and normal subjects were downloaded from The Cancer Genome Atlas. In the training group, we used Pearson correlation, univariate Cox regression, least absolute shrinkage/selection operator regression analyses, and multivariate Cox regression to build a prognostic model. In the test group, we evaluated the model. Multivariate Cox regression was done to screen out independent prognostic factors, with which we constructed a nomogram. Afterward, we demonstrated the predictive value of the risk scores based on the model and the nomogram using time-dependent receiver operating characteristics. Gene set enrichment analysis, immune correlation analysis, and half-maximal inhibitory concentration were also performed to reveal the different landscapes of TIME between risk groups and to predict immuno- and chemo-therapeutic responses. The most important LINC00861 in the model was examined in HNE1, CNE1, and CNE2 nasopharyngeal carcinoma cell lines and transfected into the cell lines CNE1 and CNE2 using the LINC00861-pcDNA3.1 construct plasmid. In addition, CCK-8, Edu, and SA-β-gal staining assays were conducted to test the biofunction of LINC00861 in the CNE1 and CNE2 cells. The signature based on nine ARLs has a good predictive value in survival time, immune infiltration, immune checkpoint expression, and sensitivity to multiple drugs. LINC00861 expression in CNE2 was significantly lower than in the HNE1 and CNE1 cells, and LINC00861 overexpression significantly inhibited the proliferation and increased the senescence of nasopharyngeal carcinoma cell lines. This work built and verified a new prognostic model for HNSCC based on ARLs and mapped the immune landscape in HNSCC. LINC00861 is a protective factor for the development of HNSCC.

Luteolin Isolated from Polygonum cuspidatum Is a Potential Compound against Nasopharyngeal Carcinoma.

To reveal the mechanisms by which luteolin, the major bioactive component of the Traditional Chinese Medicine (TCM) Polygonum cuspidatum, inhibits proliferation and promotes apoptosis in nasopharyngeal carcinoma (NPC) CNE2 cells. Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), bioactive compounds of P. cuspidatum, potential target genes and NPC disease targets of TCMSP were screened, relationship networks were constructed using these potential targets of NPC, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. The predicted compounds, targets and pathways were corroborated using in vitro experiments, such as MTT, Cytation™ 5 real-time cell monitoring, cell cycle detection, Annexin V-FITC/PI double staining, Hoechst 33342 staining, and mitochondrial membrane potential (ΔΨm) detection. The results showed that 10 bioactive compounds (OB ≥30% and DL ≥0.18), 157 potential target genes from P. cuspidatum, and 56 common targets related to NPC were found. These included important bioactive compounds such as luteolin, quercetin, and beta-sitosterol. Key common targets included EGFR, MYC, AKT1, CASP3, CCND1, ERBB2, and common targets were enriched for the PI3K-AKT, JAK/STAT, MAPK, and C-type lectin receptor signaling pathways. The binding energy of luteolin for six common targets was less than -5.0 kcal·mol-1. After luteolin (20 μM, and 40 μM) treatment to CNE2 cells for 36 h, cell survival rates decreased, accompanied by cell morphology changes, inhibition of the cell cycle at G2/M phase, and an induction of apoptosis. The expression of the cell proliferation related protein PCNA, the antiapoptosis protein XIAP, and the PI3K-AKT pathway diagram related proteins p-ERK1/2, ERK1/2, AKT, and PI3K, all decreased. Luteolin derived from P. cuspidatum inhibited the proliferation of NPC CNE2 cells and promoted cell apoptosis through the PI3K-AKT signal pathway.

Fabrication of metformin and survivin siRNA encapsulated into polyethyleneimine-altered silk fibroin nanoparticles for the treatment of nasopharyngeal carcinoma

Metformin (MET) and survivin siRNA were delivered using silk fibroin nanoparticles (SFNs) modified with polyethyleneimine (PEI). Nanoparticle functional groups, structural, stability and physicochemical characteristics of the nanoparticles were determined. SFNs loaded with siRNA that had appropriate size, zeta potential, and stability were confirmed by various analytical and spectroscopical methods. The vitro cytotoxicity analysis was performed using CNE2 nasopharyngeal carcinoma cells with different compositions, and the results showed that the newly fabricated nanoparticles effectively triggered apoptosis in CNE2. Afterward, the mice analysis was used to investigate the in vivo therapeutic efficacy. With no apparent weight loss, the co-delivery systems dramatically slowed nasopharyngeal tumor development in mice. Survivin siRNA and MET increased the number of apoptotic tumor cells on histopathology slides. Apoptosis induction in cancer cells and therapeutic effectiveness were satisfactory with the drug delivery method. In this way, it might be used as a therapeutic agent in the fight against nasopharyngeal cancer.

Autophagy promotes recurrence of nasopharyngeal carcinoma via inducing the formation of dormant polyploid giant cancer cells

Objective: To explore the effect of dormant polyploid giant cancer cells (PGCC) on nasopharyngeal carcinoma (NPC) recurrence and to clarify the role of inhibition of autophagy in inhibiting NPC-PGCC formation and preventing NPC recurrence. Methods: NPC cells-derived PGCC (NPC-PGCC) were induced by paclitaxel (PTX), and the morphology, polyploid characteristics and cell activity of PGCC were identified by light microscopy, immunofluorescence and Live/Dead cell double staining assays. RNA-seq was used to analyze the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. Functional enrichment and pathway annotation analysis of differentially expressed genes were performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). The level of autophagy in NPC-PGCC cells was assessed by Western Blot and transmission electron microscopy analysis. The role of autophagy in the formation of NPC-PGCC and the effect of NPC-PGCC on the recurrence of nasopharyngeal carcinoma were studied using a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model. Statistical analysis was performed using GraphPad Prism 6 and P-values<0.05 were considered statistically significant. Results: NPC-PGCC induced by paclitaxel had the characteristics of burst-like division after dormancy. GO enrichment and KEGG pathway analyses identified the significant biological processes and pathways mainly concentrated in autophagy and related pathways involving the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. The autophagy level was significantly enhanced in NPC-PGCC cells. In a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model, the number of PGCC in the primary tumor of the nude mice treated with cisplatin were higher than those of the other groups. In nude mice pretreated with autophagy inhibitor and then co-treatment with autophagy inhibitor and cisplatin, the number of PGCC in primary tumors was less and the recurrence rate was significantly lower than in other groups. Conclusions: The mechanism of dormant polyploid giant cancer cells formation is related to autophagy. Inhibition of autophagy can inhibit the formation of PGCC and thus prevent the recurrence of nasopharyngeal carcinoma.

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&lt;0.05). A total of 379 DEGs were found by RNA sequencing, including 295 down-regulated genes and 84 up-regulated genes (padj&lt;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&lt;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.

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.

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.

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.

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.

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.

Cephalosporin antibiotics specifically and selectively target nasopharyngeal carcinoma through HMOX1-induced ferroptosis

AimsMany antibiotics derived from mold metabolites have been found to possess anticarcinogenic properties. We aimed to investigate whether they may elicit anticancer activity, especially against nasopharyngeal carcinoma. Main methodsThe response of nasopharyngeal and other carcinoma cell lines to cephalosporin antibiotics was evaluated in vitro and in vivo. MTT and clonogenic colony formation assays assessed the viability and proliferation of cultured cells. Flow cytometry was used to assess cell cycle parameters and apoptotic markers. Tumor growth was determined using a xenograft model in vivo. Microarray and RT-qPCR expression analyses investigate differential gene expression. Mechanistic assessment of HMOX1 in cefotaxime-mediated ferroptosis was tested with Protoporphyrin IX zinc. Key findingsCephalosporin antibiotics showed highly specific and selective anticancer activity on nasopharyngeal carcinoma CNE2 cells both in vitro and vivo with minimal toxicity. Cefotaxime sodium significantly regulated 11 anticancer relevant genes in CNE2 cells in a concentration-dependent manner. Pathway analyses indicate apoptotic and the ErbB-MAPK-p53 signaling pathways are significantly enriched. HMOX1 represents the top one ranked upregulated gene by COS and overlaps with 16 of 42 enriched apoptotic signaling pathways. Inhibition of HMOX1 significantly reduced the anticancer efficacy of cefotaxime in CNE2 cells. SignificanceOur discovery is the first to highlight the off-label potential of cephalosporin antibiotics as a specific and selective anticancer drug for nasopharyngeal carcinoma. We mechanistically show that induction of ferroptosis through HMOX1 induction mediates cefotaxime anticancer activity. Our findings provide an alternative treatment for nasopharyngeal carcinoma by showing that existing cephalosporin antibiotics are specific and selective anticancer drugs.

Effects of S100 calcium-binding protein A8 (S100A8) and S100 calcium-binding protein A9 (S100A9) on matrix metalloproteinase (MMP) expression in nasopharyngeal carcinoma CNE-2 cells.

BackgroundStudies have shown that S100A8 and S100A9 are highly expressed in a variety of tumors, including NPC, and are associated with tumor invasion and migration. MMPs are associated with the invasion and migration of tumor cells. To further investigate the mechanism by which S100A8 and S100A9 affect the invasion and migration of NPC cells, the present study examined the effects of S100A8 and S100A9 on MMPs in NPC CNE-2 cells.MethodsRecombinant pEGFP-N1-S100A8 and recombinant pEGFP-N1-S100A9 overexpression vectors and S100A8 and S100A9 RNA interference (RNAi) vectors were constructed and transfected into NPC CNE-2 cells. The transfection efficiency in each group of cells was assessed, and the gene and protein expression of MMP7, MMP9 and MMP12 were determined.ResultsThe transfection efficiency was approximately 60–70%. Compared with those in the control group, the expression levels of MMP7, MMP9 and MMP12 in the S100A8 and S100A9 overexpression groups was significantly higher (P<0.05), and the expression levels of MMP7, MMP9 and MMP12 in the S100A8-RNAi and S100A9-RNAi groups were significantly lower (P<0.05). The number of cells in S100A8 overexpression group and S100A9 overexpression group at 24, 48 and 72 h was higher than that in RNAi group, RNAi control group, overexpression control group and normal control group, with statistical significance; The cell doubling time in S100A8 and S100A9 overexpression group was significantly shorter than that in RNAi control group, overexpression control group and normal control group, with statistical significance.ConclusionsHigh S100A8 and S100A9 expression may promote the expression of MMP7, MMP9 and MMP12, which are related to the invasion and metastasis of NPC cells.

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

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

Overexpression of Notch2 enhances radiosensitivity via inhibition of the AKT/mTOR signaling pathway in nasopharyngeal carcinoma

ABSTRACT Our previous study found that in nasopharyngeal carcinoma (NPC) cells, overexpression of Notch2 can inhibit epithelial-mesenchymal transition (EMT), which plays a vital role in mediating radiosensitivity. The purpose of this study was to explore the radiosensitizing efficacy of the Notch2 gene in NPC cells and its potential mechanism. We used the recombinant plasmid transfection technique to establish Notch2-overexpressing 5–8 F and CNE-2 NPC cells. Cell proliferation, radiosensitivity, apoptosis and cell cycle distribution were assessed by cell counting kit-8 (CCK-8) experiments, colony formation experiments and flow cytometry. The levels of proteins related to cell cycle, apoptosis, and the AKT/mTOR signaling pathway were evaluated by using Western blotting. The results suggested that Notch2 overexpression increased the radiosensitivity of NPC cells, with sensitizing enhancement ratios (SERs) of 1.24 (5–8 F cells) and 1.34 (CNE-2 cells). Flow cytometry indicated that the level of apoptosis and percentage of cells in G2/M-phase were highest in NPC cells overexpressing Notch2 and treated with radiotherapy compared to cells overexpressing Notch2 alone or administered radiotherapy alone. Western blotting showed that compared to that of cells treated with Notch2 overexpression or radiotherapy alone, the levels of γH2AX, Bax, Bcl-2, Cyclin D1 and AKT/mTOR signaling pathway-related proteins were modified in NPC cells overexpressing Notch2 and treated with radiotherapy. These findings showed that overexpression of Notch2 can increase the radiosensitivity of NPC cells by inhibiting the AKT/mTOR pathway. Abbreviations NPC: Nasopharyngeal carcinoma; EMT: Epithelial-mesenchymal transition; CCK8: Cell counting kit-8; EBV: Epstein-Barr virus; FBS: Fetal bovine serum; PE: Plating efficiency; SF: Survival fraction; SER: Sensitizing enhancement ratio; DSBs: DNA double-strand breaks