Nasopharyngeal Carcinoma Cells Research Articles

Targeting GABARAPL1/HIF-2a axis to induce tumor cell apoptosis in nasopharyngeal carcinoma.

The primary gene mutations associated with nasopharyngeal carcinoma (NPC) are located within the phosphoinositide 3-kinase-mammalian target of rapamycin signaling pathways, which have inhibitory effects on autophagy. Compounds that target autophagy could potentially be used to treat NPC. However, autophagy-related molecular targets in NPC remain to be elucidated. We aimed to examine levels of autophagy-related genes, including autophagy-related 4B cysteine peptidase (ATG4B) and gamma-aminobutyric acid (GABA) type A receptor-associated protein-like 1 (GABARAPL1), in NPC cells and explored their potential role as novel targets for the treatment of NPC. The mRNA and protein expression of autophagy-related genes were detected in several NPC cells. Levels of GABARAPL1 were modified by either overexpression or knockdown, followed by examining downstream targets using RT-qPCR and western blotting. The role of GABARAPL1 in NPC proliferation and apoptosis was examined by flow cytometry. Furthermore, the role of GABARAPL1 was assessed in vivo using a nude mouse xenograft tumor model. The underlying mechanism by which GABARAPL1 regulated nasopharyngeal tumor growth was investigated. Autophagy-related 4B cysteine peptidase (ATG4B), GABARAPL1, and Unc-51-like kinase 1 (ULK1) were significantly down-regulated in multiple NPC cell lines. Overexpression of GABARAPL1 up-regulated the expression of autophagy-related proteins, decreased the level of hypoxia-inducible factor (HIF)-2α, and induced apoptosis in NPC cells. Importantly, overexpression of GABARAPL1 slowed tumor growth. Western blotting showed that autophagy was activated, and HIF-2α was down-regulated in tumor tissues. HIF-2α, as a substrate for autophagic degradation, may play an interesting role during NPC progression.

TRAF4 regulates ubiquitination-modulated survivin turnover and confers radioresistance.

Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. Despite continuous improvement in treatment strategies, recurrence or persistence of cancer after radiotherapy is still inevitable, highlighting the need to identify therapeutic resistance factors and develop effective methods for NPC treatment. Herein, we found that TRAF4 is overexpressed in NPC cells and tissues. Knockdown TRAF4 significantly increased the radiosensitivity of NPC cells, possibly by inhibiting the Akt/Wee1/CDK1 axis, thereby suppressing survivin phosphorylation and promoting its degradation by FBXL7. TRAF4 is positively correlated with p-Akt and survivin in NPC tissues. High protein levels of TRAF4 were observed in acquired radioresistant NPC cells, and knockdown of TRAF4 overcomes radioresistant in vitro and the xenograft mouse model. Altogether, our study highlights the TRAF4-survivin axis as a potential therapeutic target for radiosensitization in NPC.

Homeobox B2 promotes malignant behavior and contributes to the radioresistance of nasopharyngeal carcinoma by regulating forkhead box protein O1.

Background: Nasopharyngeal carcinoma (NPC) is an epithelial tumor of the head and neck with heterogeneous racial and geographical distributions. Homeobox B2 (HOXB2) is a tumor promoter in many cancers. However, the biological role of HOXB2 in NPC has not been elucidated. Methods: Bioinformatics analysis was performed to identify the differentially expressed genes (DEGs) between samples of patients with radiosensitive and radioresistant NPC. qRT-PCR, western blotting and immunohistochemistry were used to detect the expression levels of the corresponding mRNA and proteins. Cell viability was detected by CCK-8 assay and colony-forming capability was evaluated using colony formation assays. Further, migration and invasion abilities were examined using wound-healing and transwell chamber assays, respectively. Cellular apoptosis after irradiation was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Results: HOXB2 was identified as a potential regulator of radioresistance in NPC. Our in vitro results indicate that HOXB2 overexpression (HOXB2-OE) promoted malignant behaviors including invasion, migration, proliferation, and inhibited the irradiation-induced apoptosis of NPC cells. Consistent with these results, HOXB2 knockdown (HOXB2-sh) exhibited the opposite trends in these biological activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were enriched in the FOXO signaling pathway. Mechanistically, western blotting showed that HOXB2-OE inhibited forkhead box protein O1 (FOXO1) expression in NPC cells. Thereafter, we transferred the FOXO1-OE plasmid to HOXB2-OE NPC cells and found that overexpression of FOXO1 reversed cell proliferation, migration, invasion, and radioresistance profiles promoted by HOXB2 overexpression. Conclusion: Our findings showed that HOXB2 acts as a tumor promoter in NPC, activating malignant behaviors and radioresistance of tumors via FOXO1 regulation. Moreover, the inactivation of HOXB2 or activation of FOXO1 are potential strategies to inhibit tumor progression and overcome radioresistance in NPC.

Sevoflurane inhibits the malignant behavior of nasopharyngeal carcinoma cells by regulating mitochondrial membrane potential.

This study aims to determine the effect of sevoflurane (Sev) on nasopharyngeal carcinoma (NPC) in malignant behavior and mitochondrial membrane potential (MMP). NPC cells (5-8F and CNE2) were exposed to Sev at different concentrations and then tested for proliferation by CCK-8 and colony formation assays, apoptosis by flow cytometry, and invasion and migration by Transwell assays. In addition, the Warburg effect was examined by measurements of glucose consumption, lactic acid production, and adenosine triphosphate (ATP). Mitochondrial function was evaluated by reactive oxygen species (ROS) production, oxidative stress-related indexes, and mitochondrial membrane potential. Sev suppressed 5-8F and CNE2 cell proliferation, invasion, and migration, and enhanced apoptosis. Moreover, Sev dampened the Warburg effect by reducing glucose consumption, lactic acid production, and ATP, as well as decreasing hexokinase 2 and pyruvate kinases type M2 protein expressions. Also, Sev induced ROS production and malondialdehyde content and reduced superoxide and glutathione peroxidase levels. Finally, Sev caused damage to mitochondrial homeostasis through induction of cleaved caspase-3, cleaved caspase-9, and cytochrome c protein expression and reduction of MMP. Sev inhibits the malignant behavior of NPC cells by regulating MMP.

CCDC86 promotes the aggressive behavior of nasopharyngeal carcinoma by positively regulating EGFR and activating the PI3K/Akt signaling.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor of the head and neck. A number of studies have confirmed that coiled-coil domain-containing protein 86 (CCDC86) plays an important role in the pathogenesis of lymphoma but the role of CCDC86 in NPC has not yet been reported. Here, in vivo and in vitro experiments were conducted to explore whether CCDC86 plays a role in the pathogenesis of NPC and to identify the specific mechanism. We found that CCDC86 was highly expressed in NPC tissues and cells, and the expression level of CCDC86 was correlated with the prognosis of patients with advanced NPC. CCDC86 promoted the proliferation, invasion, and migration of NPC cells in vivo and in vitro by promoting the EMT process and upregulating the expression of MMPs. Then, we confirmed that EGFR is a downstream target gene of CCDC86 and that CCDC86 can promote the proliferation, invasion, and migration of NPC cells by upregulating the expression of EGFR and activating downstream PI3K/Akt. Furthermore, we confirmed that CCDC86 did not directly bind to EGFR but positively regulated EGFR by binding to NPM1. CCDC86 is expected to be used as a novel biomarker and therapeutic target for predicting the prognosis of NPC.

Mechanism of luteolin induces ferroptosis in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) originates from the nasopharynx epithelium, and luteolin is recognized as an important anti-cancer agent. This study investigated the effects of luteolin on ferroptosis in NPC cells. NPC cells were cultured and exposed to varying concentrations of luteolin. Cell viability, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, glutathione (GSH) levels, Fe2+ concentration, and glutathione peroxidase 4 (GPX4) protein level were assessed. Additionally, SRY-related high-mobility-group box 4 (SOX4) expression was measured. Subsequently, the binding of SOX4 to the growth differentiation factor-15 (GDF15) promoter and GDF15 mRNA levels were evaluated. The impact of the SOX4/GDF15 axis on luteolin-induced ferroptosis in NPC cells was assayed. Luteolin treatment induced cell ferroptosis, evidenced by decreased cell viability, increased MDA and Fe2+ levels, and reduced SOD, GSH, and GPX4 levels. Furthermore, luteolin downregulated SOX4 expression, while overexpression of SOX4 reversed luteolin's pro-ferroptotic effects in NPC cells. SOX4 was found to up-regulate GDF15 transcription by directly binding to its promoter. Conversely, overexpression of GDF15 mitigated the ferroptotic effects induced by luteolin in NPC cells. Therefore, luteolin induces ferroptosis in NPC cells via modulation of the SOX4/GDF15 axis. In conclusion, luteolin reduces the binding of SOX4 to the GDF15 promoter by suppressing SOX4 expression, thereby down-regulating GDF15 transcription levels and inducing ferroptosis in NPC cells.

Heavy-atom-free BODIPY dendrimer: utilizing the spin-vibronic coupling mechanism for two-photon photodynamic therapy in zebrafish.

In this study, the heavy-atom-free BODIPY dendrimer TM4-BDP was synthesized for near-infrared photodynamic therapy, and was composed of a triphenylamine-BODIPY dimer and four 1-(2-morpholinoethyl)-1H-indole-3-ethenyl groups. The TM4-BDP could achieve near-infrared photodynamic therapy through two different photosensitive pathways, which include one-photon excitation at 660 nm and two-photon excitation at 1000 nm. In the one-photon excitation pathway, the TM4-BDP could generate singlet oxygen and superoxide radicals under 660 nm illumination. In addition, the one-photon PDT experiment in human nasopharyngeal carcinoma (CNE-2) cells also indicated that the TM4-BDP could specifically accumulate in lysosomes and show great cell phototoxicity with an IC50 of 22.1 μM. In the two-photon excitation pathway, the two-photon absorption cross-section at 1030 nm of TM4-BDP was determined to be 383 GM, which means that it could generate reactive oxygen species (ROS) under 1000 nm femtosecond laser excitation. Moreover, the two-photon PDT experiment in zebrafish also indicated the TM4-BDP could be used for two-photon fluorescence imaging and two-photon induced ROS generation in biological environments. Furthermore, in terms of the ROS generation mechanism, the TM4-BDP employed a novel spin-vibronic coupling intersystem crossing (SV-ISC) process for the mechanism of ROS generation and the femtosecond transient absorption spectra indicated that this novel SV-ISC mechanism was closely related to its charge transfer state lifetime. These above experiments of TM4-BDP demonstrate that the dendrimer design is an effective strategy for constructing heavy-atom-free BODIPY photosensitizers in the near-infrared region and lay the foundation for two-photon photodynamic therapy in future clinical trials.

Establishment and Application of Novel Hypoxia-driven Dual-reporter Model to Investigate Hypoxic Impact on Radiation Sensitivity in Human Nasopharyngeal Carcinoma Xenografts.

Background: Tumor hypoxia has been frequently detected in nasopharyngeal carcinoma (NPC) and is intently associated with therapeutic resistance. The aim of the study is to establish a clonogenically stable hypoxia-inducible dual reporter model and apply it to investigate the effect of tumor hypoxia on DNA double strand break (DSB) and synergistic effect of irradiation in combination with chemotherapy or targeted therapy. Methods: The plasmid vector consisting of hypoxia response elements to regulate HSV1-TK and GFP genes, was constructed and stably transfected into human NPC cells. The expected clone was identified and validated by in vivo and in vitro assay. DSB repair was measured by γH2AX foci formation. Tumor growth delay assay and spatial biodistribution of various biomarkers was designed to investigate the anti-tumor effect. Results: The system has the propensity of high expression of reporter genes under hypoxia and low to no expression under normoxia. Intratumoral biodistributions of GFP and classic hypoxic biomarkers were identical in poor-perfused region. Upon equilibration with 10% O2, the xenografts showed higher expression of hypoxic biomarkers. Cisplatin radiosensitized SUNE-1/HRE cells under hypoxia by suppressing DSB repair while the addition of PI3K/mTOR inhibitor further enhanced the anti-tumoral therapeutic efficacy. Combination of IR, DDP and NVP-BEZ235 exhibited most effective anti-tumor response in vivo. These observations underline the importance of dual reporter model for imaging tumor hypoxia in therapeutic study. Conclusions: Our preclinical model enables the investigation of heterogeneous tumor hypoxic regions in xenograft tissues and explores the treatment efficacy of combinations of various therapeutic approaches to overcome hypoxia.

MiR-205-5p promotes the proliferation, migration and invasion of nasopharyngeal carcinoma cells by regulating CALM1

miR-205-5p promotes the proliferation, migration and invasion of nasopharyngeal carcinoma cells by regulating CALM1

Targeting Cancer Mitochondria by Inducing an Abnormal Mitochondrial Unfolded Protein Response Leads to Tumor Suppression.

The mitochondrial unfolded protein response (UPRmt) is a pivotal cellular mechanism that ensures mitochondrial homeostasis and cellular survival under stress conditions. This study investigates the role of UPRmt in modulating the response of nasopharyngeal carcinoma cells to cisplatin-induced stress. We report that the inhibition of UPRmt via AEB5F exacerbates cisplatin cytotoxicity, as evidenced by increased lactate dehydrogenase (LDH) release and apoptosis, characterized by a surge in TUNEL-positive cells. Conversely, the activation of UPRmt with oligomycin attenuates these effects, preserving cell viability and reducing apoptotic markers. Immunofluorescence assays reveal that UPRmt activation maintains mitochondrial membrane potential and ATP production in the presence of cisplatin, countering the rise in reactive oxygen species (ROS) and inhibiting caspase-9 activation. These findings suggest that UPRmt serves as a cytoprotective mechanism in cancer cells, mitigating cisplatin-induced mitochondrial dysfunction and apoptosis. The data underscore the therapeutic potential of modulating UPRmt to improve the efficacy and reduce the side effects of cisplatin chemotherapy. This study provides a foundation for future research on the exploitation of UPRmt in cancer treatment, with the aim of enhancing patient outcomes by leveraging the cellular stress response pathways.

Allicin inhibits the growth of HONE-1 and HNE1 human nasopharyngeal carcinoma cells by inducing ferroptosis.

Allicin (AL) is one of garlic-derived organosulfides and has a variety of pharmacological effects. Studies have reported that AL has notable inhibitory effects on liver cancer, gastric cancer, breast cancer, and other cancers. However, there are no relevant reports about its role in human nasopharyngeal carcinoma. Ferroptosis is an iron-dependent form of non-apoptotic regulated cell death. Increasing evidence indicates that induction of ferroptosis can inhibit the proliferation, migration, invasion, and survival of various cancer cells, which act as a tumor suppressor in cancer. In this study, we confirmed that AL can inhibit cell proliferation, migration, invasion, and survival in human nasopharyngeal carcinoma cells. Our finding shows that AL can induce the ferroptosis axis by decreasing the level of GSH and GPX4 and promoting the induction of toxic LPO and ROS. AL-mediated cytotoxicity in human nasopharyngeal carcinoma cells is dependent on ferroptosis. Therefore, AL has good anti-cancer properties and is expected to be a potential drug for the treatment of nasopharyngeal carcinoma.

Houttuyn Berberine Promotes Apoptosis of Nasopharyngeal Carcinoma Cells and Inhibits Proliferation, Migration and Invasion by Regulating the circ-SMARCA5/microRNA-346 pathway

Houttuyn Berberine Promotes Apoptosis of Nasopharyngeal Carcinoma Cells and Inhibits Proliferation, Migration and Invasion by Regulating the circ-SMARCA5/microRNA-346 pathway

Induction of interleukin-6 by SPZ1-mediated Wnt5a signaling boosts progression of nasopharyngeal carcinoma cells

Induction of interleukin-6 by SPZ1-mediated Wnt5a signaling boosts progression of nasopharyngeal carcinoma cells

Network pharmacology, molecular docking and experimental study of CEP in nasopharyngeal carcinoma

Ethnopharmacological relevanceThe Stephania cephalantha Hayata is an important traditional medicinal plant widely used in traditional medicine to treat cancer. Cepharanthine (CEP) was extracted from the roots of Stephania cephalantha Hayata. It has been found to exhibit anticancer activity in different types of cancer cells. Nevertheless, the activity of CEP against nasopharyngeal carcinoma (NPC) and its underlying mechanism warrant further investigation. Aims of the studyNPC is an invasive and highly metastatic malignancy that affects the head and neck region. This research aimed to investigate the pharmacological properties and underlying mechanism of CEP against NPC, aiming to offer novel perspectives on treating NPC using CEP. Materials and methodsIn vitro, the pharmacological activity of CEP against NPC was evaluated using the CCK-8 assay. To predict and elucidate the anticancer mechanism of CEP against NPC, we employed network pharmacology, conducted molecular docking analysis, and performed Western blot experiments. In vivo validation was performed through a nude mice xenograft model of human NPC, Western blot and immunohistochemical (IHC) assays to confirm pharmacological activity and the mechanism. ResultsIn a dose-dependent manner, the proliferation and clonogenic capacity of NPC cells were significantly inhibited by CEP. Additionally, NPC cell migration was suppressed by CEP. The results obtained from network pharmacology experiments revealed that anti-NPC effect of CEP was associated with 8 core targets, including EGFR, AKT1, PIK3CA, and mTOR. By performing molecular docking, the binding capacity of CEP to the candidate core proteins (EGFR, AKT1, PIK3CA, and mTOR) was predicted, resulting in docking energies of −10.0 kcal/mol for EGFR, −12.4 kcal/mol for PIK3CA, −10.8 kcal/mol for AKT1, and -8.6 kcal/mol for mTOR. The Western blot analysis showed that CEP effectively suppressed the expression of EGFR and the phosphorylation levels of downstream signaling proteins, including PI3K, AKT, mTOR, and ERK. After CEP intervention, a noteworthy decrease in tumor size, without inducing any toxicity, was observed in NPC xenograft nude mice undergoing in vivo treatment. Additionally, IHC analysis demonstrated a significant reduction in the expression levels of EGFR and Ki-67 following CEP treatment. ConclusionCEP exhibits significant pharmacological effects on NPC, and its mechanistic action involves restraining the activation of the EGFR/PI3K/AKT pathway. CEP represents a promising pharmaceutical agent for addressing and mitigating NPC.

TSPAN1 inhibits metastasis of nasopharyngeal carcinoma via suppressing NF-kB signaling.

Nasopharyngeal carcinoma (NPC) originates in the epithelial cells of the nasopharynx and is a common malignant tumor in southern China and Southeast Asia. Metastasis of NPC remains the main cause of death for NPC patients even though the tumor is sensitive to radiotherapy and chemotherapy. Here, we found that the transmembrane protein tetraspanin1 (TSPAN1) potently inhibited the in vitro migration and invasion, as well as, the in vivo metastasis of NPC cells via interacting with the IKBB protein. In addition, TSPAN1 was essential in preventing the overactivation of the NF-kB pathway in TSPAN1 overexpressing NPC cells. Furthermore, reduced TSPAN1 expression was associated with NPC metastasis and the poor prognosis of NPC patients. These results uncovered the suppressive role of TSPAN1 against NF-kB signaling in NPC cells for preventing NPC metastasis. Its therapeutic value warrants further investigation.

USP7 inhibits the progression of nasopharyngeal carcinoma via promoting SPLUNC1-mediated M1 macrophage polarization through TRIM24

Reprogramming of macrophages toward an M1 phenotype is a novel strategy to induce anticancer immunity. However, the regulatory mechanisms of M1 macrophage polarization and its functional roles in nasopharyngeal carcinoma (NPC) progression need to be further explored. Here we found that SPLUNC1 was highly expressed and responsible for M1 macrophage polarization. JAK/STATs pathway activation was involved in SPLUNC1-mediated M1 macrophage polarization. Importantly, regulation of SPLUNC1 in macrophages affected CM-mediated influence on NPC cell proliferation and migration. Mechanistically, USP7 deubiquitinated and stabilized TRIM24, which promoted SPLUNC1 expression via recruitment of STAT3 in M1 macrophages. Depletion of TRIM24 inhibited M1 macrophage polarization, which facilitated NPC cell growth and migration. However, over-expression of USP7 exhibited the opposite results and counteracted the tumorigenic effect of TRIM24 silencing. Finally, the growth and metastasis of NPC cells in vivo were repressed by USP7-induced M1 macrophage polarization via modulating TRIM24/SPLUNC1 axis. USP7 delayed NPC progression via promoting macrophage polarization toward M1 through regulating TRIM24/SPLUNC1 pathway, providing evidence for the development of effective antitumor immunotherapies for NPC.

Recent Advances in Assessing the Clinical Implications of Epstein-Barr Virus Infection and Their Application to the Diagnosis and Treatment of Nasopharyngeal Carcinoma.

Reports about the oncogenic mechanisms underlying nasopharyngeal carcinoma (NPC) have been accumulating since the discovery of Epstein-Barr virus (EBV) in NPC cells. EBV is the primary causative agent of NPC. EBV-host and tumor-immune system interactions underlie the unique representative pathology of NPC, which is an undifferentiated cancer cell with extensive lymphocyte infiltration. Recent advances in the understanding of immune evasion and checkpoints have changed the treatment of NPC in clinical settings. The main EBV genes involved in NPC are LMP1, which is the primary EBV oncogene, and BZLF1, which induces the lytic phase of EBV. These two multifunctional genes affect host cell behavior, including the tumor-immune microenvironment and EBV behavior. Latent infections, elevated concentrations of the anti-EBV antibody and plasma EBV DNA have been used as biomarkers of EBV-associated NPC. The massive infiltration of lymphocytes in the stroma suggests the immunogenic characteristics of NPC as a virus-infected tumor and, at the same time, also indicates the presence of a sophisticated immunosuppressive system within NPC tumors. In fact, immune checkpoint inhibitors have shown promise in improving the prognosis of NPC patients with recurrent and metastatic disease. However, patients with advanced NPC still require invasive treatments. Therefore, there is a pressing need to develop an effective screening system for early-stage detection of NPC in patients. Various modalities, such as nasopharyngeal cytology, cell-free DNA methylation, and deep learning-assisted nasopharyngeal endoscopy for screening and diagnosis, have been introduced. Each modality has its advantages and disadvantages. A reciprocal combination of these modalities will improve screening and early diagnosis of NPC.

Nasopharyngeal carcinoma cell screening based on the electroporation-SERS spectroscopy

Nasopharyngeal carcinoma (NPC) is a malignant tumor in head and neck. Early diagnosis can effectively improve the survival rate of patients. Nasopharyngeal exfoliative cytology, as a convenient and noninvasive auxiliary diagnostic method, is suitable for the population screening of NPC, but its diagnostic sensitivity is low. In this study, an electroporation-based SERS technique was proposed to detect and screen the clinical nasopharyngeal exfoliated cell samples. Firstly, nasopharyngeal swabs was used to collected the nasopharyngeal exfoliated cell samples from NPC patients (n = 54) and healthy volunteers (n = 60). Then, gold nanoparticles, as the Raman scattering enhancing substrates, were rapidly introduced into cells by electroporation technique for surface-enhanced Raman scattering (SERS) detection. Finally, SERS spectra combined with principal component analysis (PCA) and linear discriminant analysis (LDA) were employed to diagnose and distinguish NPC cell samples. Raman peak assignments combined with spectral differences reflected the biochemical changes associated with NPC, including nucleic acid, amino acid and carbohydrates. Based on the PCA-LDA approach, the sensitivity, specificity and accuracy of 98.15 %, 96.67 % and 97.37 %, respectively, were achieved for screening NPC. This study offers valuable assistance for noninvasive NPC auxiliary diagnosis, and has grate potential in expanding the application of the SERS technique in clinical cell sample testing.

Expressions of NLRP3, Caspase-1, and GSDMD in nasopharyngeal carcinoma tissue and association with recurrence and metastasis

Objective: To investigate the expressions of Nod-like receptor protein 3 (NLRP3), cysteine-aspartic acid protease 1 (Caspase-1), and Gasdermin D (GSDMD) in nasopharyngeal carcinoma (NPC), and their relationships with the recurrence and metastasis of NPC. Methods: A retrospective study was conducted on 421 patients diagnosed with NPC between December 2014 and January 2020. The expressions of NLRP3, Caspase-1, and GSDMD in pathological specimens were examined with immunohistochemistry and multiplex immunofluorescence staining. Univariate and multivariate Cox regression analyses were applied to identify the factors influencing NPC recurrence and metastasis. In vitro experiments with NPC cell line HNE-2 were used to explore the functional mechanisms of NLRP3, Caspase-1, and GSDMD. Results: Multivariate Cox analysis revealed that tumor staging of Ⅲ-Ⅳ(HRrecurrence=2.74, 95%CIrecurrence: 1.61-4.65; HRmetastasis=1.90, 95%CImetastasis: 1.04-3.49) and pre-treatment plasma EBV-DNA levels≥1 500 copies/ml (HRrecurrence=1.91, 95%CIrecurrence: 1.13-3.23; HRmetastasis=2.07, 95%CImetastasis: 1.23-3.50)were independent risk factors for NPC recurrence and metastasis, while positive expression of NLRP3(HRrecurrence=0.17, 95%CIrecurrence: 0.08-0.35; HRmetastasis=0.30, 95%CImetastasis: 0.15-0.59), Caspase-1(HRrecurrence=0.32, 95%CIrecurrence: 0.18-0.59; HRmetastasis=0.43, 95%CImetastasis: 0.25-0.76), and GSDMD(HRrecurrence=0.48, 95%CIrecurrence: 0.25-0.91; HRmetastasis=0.96, 95%CImetastasis: 0.53-1.74) served as independent protective factors. Age (HR=1.02, 95%CI: 1.01-1.04) and intensity-modulated radiotherapy (HR=0.51, 95%CI: 0.30-0.88) were independent factors for NPC recurrence, whereas chemotherapy (HR=0.50, 95%CI: 0.29-0.88) acted as an independent protective factor for NPC metastasis (all P<0.05). NPC patients with positive expressions of the three proteins had higher locoregional recurrence-free survival, distant metastasis-free survival, and overall survival compared to those with negative expressions (all P<0.05). In vitro experiments revealed that the overexpression of NLRP3 activated the NLRP3/Caspase-1/GSDMD signaling pathway, as evidenced by Western Blot analysis. Enzyme-linked immunosorbent assay and scanning electron microscopy demonstrated that overexpression of NLRP3 promoted pyroptosis in HNE-2 cells. Cellular functional assays further confirmed that overexpression of NLRP3 significantly inhibited the proliferation, invasion, and migration of HNE-2 cells. Conclusion: Positive expressions of NLRP3, Caspase-1, and GSDMD serves as independent protective factors for recurrence and metastasis of NPC, potentially by promoting cell pyroptosis and thus inhibiting NPC cell proliferation, invasion, and migration.

Overexpressed Gαi1 exerts pro-tumorigenic activity in nasopharyngeal carcinoma

The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate that Gαi1 transcripts’ number in NPC tissues is significantly higher than that in the normal nasal epithelial tissues. Its overexpression correlates with poor survival in certain NPC patients. Moreover, Gαi1 is significantly upregulated in NPC tissues of local primary patients and in different primary human NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in primary nasal epithelial cells. Genetic silencing (via shRNA strategy) or knockout (via CRISPR-sgRNA method) of Gαi1 substantially suppressed viability, proliferation, cell cycle progression, and migration in primary NPC cells, causing significant caspase-apoptosis activation. Contrarily, ectopic Gαi1 expression exerted pro-tumorigenic activity and strengthened cell proliferation and migration in primary NPC cells. Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated after Gαi1 shRNA or KO in primary NPC cells, but strengthened following Gαi1 overexpression. In Gαi1-silenced primary NPC cells, a S473D constitutively-active mutant Akt1 (caAkt1) restored Akt-S6K phosphorylation and ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Bioinformatics analyses proposed zinc finger protein 384 (ZNF384) as a potential transcription factor of Gαi1. In primary NPC cells, ZNF384 shRNA or knockout (via CRISPR-sgRNA method) decreased Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated it. Importantly, there was an increased binding between ZNF384 protein and the Gαi1 promoter in human NPC tissues and different NPC cells. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing adeno-associated virus (AAV) impeded subcutaneous NPC xenograft growth in nude mice. Gαi1 downregulation, Akt-mTOR inactivation, and apoptosis induction were detected in Gαi1-silenced NPC xenograft tissues. Gαi1 KO also effectively inhibited the growth of NPC xenografts in nude mice. Together, overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation.