The Pinx1 Gene Downregulates Telomerase and Inhibits Proliferation of CD133+ Cancer Stem Cells Isolated from a Nasopharyngeal Carcinoma Cell Line by Regulating Trfs and Mad1/C-Myc/p53 Pathways.

BACKGROUND/AIMS: Cancer stem cells (CSCs) are important factors for the continuous growth, recurrence, and metastasis of malignant tumors. They are responsible for the ineffectiveness of traditional radiotherapy and chemotherapy toward malignant tumors. Currently, stem cells or side-population cells have been isolated from many cancer cell lines and malignant tumor tissues, including nasopharyngeal carcinoma. Exploring the biological characteristics of CSCs for CSC-targeted therapy has gained importance. CSCs possess higher telomerase activity; thus, the use of the gene encoding telomerase inhibitor PinX1 gene to target telomerase in CSCs and inhibit proliferation, invasion, and metastasis of CSCs has become an important means for the treatment of malignant tumors. PinX1 may regulate complex pathways, including TRF1, Mad1/c-Myc, and p53. METHODS: In this study, nasopharyngeal CD133+ CSCs were sorted using CD133 immunomagnetic beads by flow cytometry The successful isolation of CD133+ CSCs was confirmed by examining their surface markers, namely CD44, NaNOG, and SOX2 as well as their ability to undergo in vivo tumorigenesis and in vitro sphere formation, proliferation, migration, and invasion. In addition, CD133+ CSCs were transfected with the constructed PinX1 overexpression plasmid or siRNA and the resulting effects on their proliferation, migration, invasion, and apoptosis were detected using cell counting kit-8 (CCK-8), transwell assay, and scratch test, respectively. Furthermore, their effects on mRNA and protein levels of TRF1, TRF2, Mad1, c-Myc, and p53 were examined using quantitative real-time PCR and western blot, respectively. RESULTS: The overexpression of PinX1 in CD133+ CSCs significantly decreased hTERT (P < 0.001), inhibited proliferation, migration, and invasion, induced apoptosis, and significantly decreased c-Myc mRNA levels (P < 0.001), while it increased TRF1, Mad1, and p53 mRNA levels (all P < 0.001). On the other hand, PinX1 silencing in CD133+ CSCs significantly decreased TRF1, Mad1, and p53 mRNA levels (all P < 0.01), while it increased hTERT and c-Myc mRNA levels (all P < 0.05). CONCLUSION: These results indicate that PinX1 downregulates telomerase activity in CD133+ CSCs, inhibits its proliferation, migration, and invasion, and induces apoptosis possibly through TRF1, Mad1/c-Myc, and p53-mediated pathways.

Joint quantitative measurement of hTERT mRNA in both peripheral blood and circulating tumor cells of patients with nasopharyngeal carcinoma and its clinical significance.

BACKGROUND: The study was aimed to quantitatively detect mRNA levels of the catalytic subunit of telomerase (hTERT) in both peripheral blood and circulating tumor cells (CTCs) of patients with nasopharyngeal carcinoma (NPC) and explore its significance in early diagnosis and treatment of NPC. METHODS: hTERT mRNA levels in peripheral blood and CTCs of 33 NPC patients before and after treatment with intensity-modulated radiation therapy (IMRT) or/and chemotherapy and 24 healthy controls were measured using real-time quantitative PCR (qPCR) and their correlations to clinic pathological factors of NPC were analyzed. RESULTS: Peripheral hTERT mRNA content was 10.75 ± 4.29 in NPC patients and 0.95 ± 0.37 in control subjects (P < 0.05), and had a significant correlation with patients' clinical stage, T stage, and N stage (P < 0.05). Treatment of NPC patients at stages I and II with simple IMRT significantly reduced hTERT mRNA level from 5.60 ± 2.33 to 3.43 ± 1.42 (P < 0.05) and treatment of patients at advanced stage (III and IV) with induction chemotherapy followed by IMRT significantly reduced hTERT mRNA levels from 12.68 ± 3.08 to 10.68 ± 2.48 to 3.13 ± 1.69 (P < 0.05), respectively. In addition, the study also showed that hTERT mRNA content in CTCs of NPC patients was 10.65 ± 4.28, evidently higher than that of 1.09 ± 0.40 in control subjects (P < 0.05) and hTERT mRNA level in CTCs of NPC patients was obviously correlated to patients' clinical stage, T stage and N stage (P < 0.05). After treatment, hTERT mRNA level in CTCs of NPC patients lowered from 10.65 ± 4.28 to 5.59 ± 2.32 (P < 0.05). The correlation analysis found that hTERT mRNA level in peripheral blood and CTCs of NPC patients were highly correlated with a correlation coefficient of 0.981. CONCLUSIONS: hTERT mRNA levels in peripheral blood and CTCs of NPC patients were significantly enhanced compared to that in healthy controls and highly correlated. Changes in hTERT mRNA level was closely correlated to patients' clinical stage and T stage. Radiochemotherapy could effectively reduce hTERT mRNA level in peripheral blood and CTCs. Thus, it is possible using the joint detection of hTERT mRNA level in peripheral blood and CTCs as a new biomarker for early diagnosis, treatment efficacy and prognosis of NPC.

[Influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin].

OBJECTIVE: To explore the influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin. METHODS: Transfected nasopharyngeal carcinoma 5-8F cell lines with pCDH-CMV-PinX1-copGFP vector constructed by lentivirus to generate Lenti-PinX1-5-8F cells containing PinX1 gene, using Lenti-Ctrl-5-8F cell (blank vector without PinX1 gene was used to transfect 5-8F cell lines) and 5-8F cell as controls. Expression of PinX1 gene, telomerase activity, the inhibition of cancer cells proliferation, combined anticancer effect with Cisplatin and the expression of lung resistance protein (LRP) and Bcl-2 were detected with fluorescent quantitation polymerase chain reaction (PCR), flow cytometry, thiazolyl blue (MTT) method, areole test, Western blot and drug sensitivity test, respectively, in four groups (Lenti-PinX1-5-8F cell + Cisplatin, Lenti-PinX1-5-8F cell, Cisplatin and 5-8F cell) so as to explore the influence and mechanism of PinX1 gene on the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin. RESULTS: The telomerase activity in Lenti-PinX1-5-8F cell (0.146 ± 0.004) was lower than those in the other two control cells (Lenti-Ctrl-5-8F cell: 0.967 ± 0.016, 5-8F cell: 1.000 ± 0.034, both P < 0.01). The cancer cell biological activity could be intensively inhibited by 16 µg/ml Cisplatin after lower level telomerase activity induced by PinX1 gene. Proliferation index (PI) (%) in Lenti-PinX1-5-8F cell + Cisplatin (14.39 ± 3.66) was also less than the other groups (Lenti-PinX1-5-8F cell, Cisplatin and 5-8F cell groups, 32.97 ± 3.00, 31.18 ± 4.24 and 47.19 ± 4.19, all P < 0.01). And same time, the expressions of LRP (0.64 ± 0.14) and Bcl-2 (0.57 ± 0.12) protein in Lenti-PinX1-5-8F cells were obviously reduced than those in other two group cells (Lenti-Ctrl-5-8F cell: 0.84 ± 0.19 and 0.81 ± 0.16; 5-8F cell: 0.83 ± 0.35 and 0.78 ± 0.27; all P < 0.01). CONCLUSIONS: PinX1 gene can enhance the chemotherapy sensitivity of nasopharyngeal carcinoma cells in response to Cisplatin, which may be mediated by the down-regulation of telomerase activity and the inhibition of LRP and Bcl-2 gene in nasopharyngeal carcinoma cells.

PinX1-Promoted Autophagy Inhibits Cell Proliferation and Induces Cell Apoptosis by Inhibiting the NF-κB/p65 Signaling Pathway in Nasopharyngeal Carcinoma.

BACKGROUND: The role of Pin2 telomeric repeat factor 1-interacting telomerase inhibitor 1 (PinX1) in tumorigenesis and development has been extensively studied. As we previously demonstrated, PinX1 plays an important role in modulating epithelial-mesenchymal transition (EMT), stemness, cell proliferation, and apoptosis in nasopharyngeal carcinoma (NPC). However, the relationship between PinX1, autophagy, and cell function in NPC remains unclear. This study aimed to investigate the mechanisms by which PinX1 regulates autophagy in NPC, and to explore its biological role and clinical significance in disease progression. METHODS: The proliferative capacity of NPC cells was assessed by MTT and xenograft tumorigenicity assays. Autophagic flux was monitored using a tandem monomeric DAPI-FITC-LC3 reporter assay. The rates of apoptosis and the cell cycle in NPC cells were analyzed using flow cytometry. The activation of autophagy and the signaling status of the AKT/mTOR and NF-κB/p65 pathways were evaluated by Western blot analysis. RESULTS: In addition to promoting autophagy and apoptosis, PinX1 overexpression suppressed proliferation, migration, invasion, and decelerated cell-cycle progression in NPC cells. These effects were reversed by inhibiting autophagy with 3-methyladenine. Mechanistic investigations clarified that PinX1 overexpression significantly reduced the expression of p-AKT, p-mTOR, p65, and p-p65. Chloroquine treatment in PinX1-overexpressing cells did not significantly alter p-AKT and p-mTOR levels, whereas 3-MA treatment in PinX1-overexpressing cells resulted in increased p65 and p-p65 expression, relative to untreated PinX1-overexpressing cells. CONCLUSIONS: It appears that PinX1 promotes autophagy by inhibiting the AKT/mTOR signaling pathway, which then inhibits NF-κB/p65 pathways, and consequently inhibiting cell proliferation and causing cell apoptosis in NPC cells.

Individualized treatment for allergic rhinitis based on key nasal clinical manifestations combined with histamine and leukotriene D4 levels.

INTRODUCTION: The types of allergic rhinitis are roughly classified based on the causative antigens, disease types, predilection time, and symptom severity. OBJECTIVE: To examine the clinical typing and individualized treatment approach for allergic rhinitis and to determine the optimal treatment method for this disease using various drug combination therapies. METHODS: A total of 108 participants with allergic rhinitis were divided into three groups based on symptoms. Subsequently, each group was further categorized into four subgroups based on the medications received. The efficacy of the treatments was evaluated using the visual analog scale VAS scores of the total and individual nasal symptoms, decline index of the symptom score, histamine and leukotriene levels, and mRNA and protein expression levels of histamine 1 and cysteinyl leukotriene 1 receptors. RESULTS: Loratadine+mometasone furoate and loratadine+mometasone furoate+montelukast significantly improved the sneezing symptom and reduced the histamine levels compared with the other combination therapies (p<0.05). Meanwhile, montelukast+mometasone furoate and montelukast+mometasone furoate+loratadine considerably improved the nasal obstruction symptom and decreased the leukotriene D4 levels compared with the other combination therapies (p<0.05). CONCLUSION: Clinical symptom evaluation combined with experimental detection of histamine and leukotriene levels can be an objective and accurate method to clinically classify the allergic rhinitis types. Furthermore, individualized treatment based on allergic rhinitis classification can result in a good treatment efficacy.

Pin2 telomeric repeat factor 1-interacting telomerase inhibitor 1 (PinX1) inhibits nasopharyngeal cancer cell stemness: implication for cancer progression and therapeutic targeting.

BACKGROUND: Recurrence and distant metastasis are still the main factors leading to treatment failure for malignant tumors including nasopharyngeal carcinoma (NPC). Therefore, elucidating the molecular mechanisms underlying nasopharyngeal carcinoma metastasis is of great clinical significance for targeted gene therapy and prognostic evaluation. PinX1, a tumor suppressor gene, was previously demonstrated to be a powerful tool for targeting telomerase in order to resist malignant tumor proliferation and migration. The aim of this study was to explore the mechanism through which PinX1 regulates epithelial-mesenchymal transition (EMT) and tumor metastasis in NPC and investigate its clinical significance and biological role with respect to disease progression. METHODS: Cell Counting Kit-8 (CCK8), Transwell assays, Colony formation analysis and Xenograft tumorigenicity assay were used to measure the nasopharyngeal CD133+ cancer stem cell proliferation, migration, and invasion abilities. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays were conducted to investigate the underlying mechanism that PinX1 inhibits cell proliferation, migration, and invasion via regulating EMT in nasopharyngeal CD133+ CSCs. RESULTS: We found that the overexpression of PinX1 and P53 inhibited cell proliferation, migration, and invasion, but that the inhibition of miR-200b blocked these effects, in nasopharyngeal CD133+ cancer stem cells (CSCs). Mechanistic investigations elucidated that PinX1 inhibits cell proliferation, migration, and invasion by regulating the P53/miR-200b-mediated transcriptional suppression of Snail1, Twist1, and Zeb1, consequently inhibiting EMT in nasopharyngeal CD133+ CSCs. CONCLUSIONS: Our findings indicate that PinX1 inhibits cell proliferation, migration, and invasion via P53/miR-200b-regulated EMT in the malignant progression of human NPC, which might suggest novel clinical implications for disease treatment.

Identification of genes and pathways in nasopharyngeal carcinoma by bioinformatics analysis.

Nasopharyngeal carcinoma is a metastatic malignant tumor originating from nasopharyngeal epithelium. Lacking or nonspecific symptoms of patients with early stage nasopharyngeal carcinoma have significantly reduced the accuracy of diagnosing and predicting nasopharyngeal carcinoma development. This study aimed to identify gene signatures of nasopharyngeal carcinoma and uncover potential mechanisms. Two gene expression profiles (GSE12452 and GSE13597) containing 56 nasopharyngeal carcinoma samples and 13 normal control samples were analyzed to identify the differentially expressed genes. In total, 179 up-regulated genes and 238 down-regulated genes were identified. Functional and pathway enrichment analysis showed that up-regulated genes were significantly involved in cell cycle, oocyte meiosis, DNA replication and p53 signaling pathway, while down-regulated genes were enriched in Huntington's disease,metabolic pathways. Subsequently, the top 10 hub genes, TOP2A (topoisomerase (DNA) II alpha), CDK1 (cyclin-dependent kinase 1), CCNB1 (cyclin B1), PCNA (proliferating cell nuclear antigen), MAD2L1 (mitotic arrest deficient 2 like 1), BUB1 (budding uninhibited by benzimidazoles 1 homolog), CCNB2 (cyclin B2), AURKA (aurora kinase A), CCNA2 (cyclin A2), CDC6 (cell division cycle 6 homolog), were identified from protein-protein interaction network. Furthermore, Module analysis revealed that the ten hub genes except TOP2A were belonged to module 1, indicating the upregulation of these hub genes associated molecular pathways in nasopharyngeal carcinoma might activate nasopharyngeal carcinoma pathogenesis. In conclusion, this study indicated that the identified differentially expressed genes and hub genes enrich our understanding of the molecular mechanisms of nasopharyngeal carcinoma, which could eventually translate into additional biomarkers to facilitate the early diagnosis and therapeutic approaches.

[Clinical features of advanced adenoid cystic carcinoma in the nasal cavity and paranasal sinuses: analysis of 21 cases].

OBJECTIVE: To evaluate the clinical characteristics, treatment and prognosis of advanced adenoid cystic carcinoma (ACC) in the nasal cavity and paranasal sinuses. METHODS: Twenty-one patients with advanced ACC in the nasal cavity and paranasal sinuses were treated in our department between February, 2007 and May, 2016. The clinical manifestations, T-stage, N-status, treatment, histological grade, recurrence and distant metastasis of the tumors were analyzed. Univariate survival analysis was performed with Kaplan-Meier method and Log-rank test, and the factors affecting the prognosis of the patients were explored using multivariate analysis with Cox proportional hazard model. RESULTS: Among the 21 patients, 10 (47.6%) had ACC containing less than 30% of solid tumor tissues and their overall survival rates at 1, 3, and 5 years were 100%, 100% and 70%, respectively; in the 11 cases (52.4%) with solid tumor tissues no less than 30%, the overall survival rates at 1, 3, and 5 years were 70%, 40% and 10%, respectively, showing significant differences between the two groups (P=0.02). The Log-rank test and survival analysis using the covariate variable model curve indicated a significant impact of the pathological classification on the patients' prognosis. The patients in T3 stage had slightly better prognosis than those in T4 stage; tumors originating from the maxillary sinus had a slightly better prognosis than those from the sphenoid sinus. Surgery combined with radiotherapy resulted in better outcomes of the patients than surgery or radiotherapy alone. Multiariable Cox regression model analysis showed that the pathological classification (P=0.045) and the disease course (P=0.028) were closely related with the prognosis of the patients. CONCLUSION: ACC in the nasal cavity and paranasal sinuses has a low incidence without specific symptoms. Its early diagnosis can be difficult, and most of the patients are in advanced stage upon diagnosis. We recommend comprehensive treatments combining surgery, postoperative radiotherapy and chemotherapy for these patients. The pathological classification, disease course, lesion site, clinical stage, treatment approache, compromise of the peripheral nerves, status at the edge of resection, and postoperative radiotherapy dose can all be factors affecting the prognosis of patients with advanced ACC.

[Effect of adipose-derived mesenchymal stem cells (ADSC) on the T cell immune status of allergic rhinitis mouse model].

OBJECTIVE: To investigate the regulation of adipose-derived mesenchymal stem cells (ADSC) on helper T cells and regulatory T cells in allergic rhinitis(AR) mouse model and the underlying mechanisms. METHODS: Using random number table, 60 Balb/c mice were divided into 6 groups (represented by: sensitized/challenged/treated ), they were the experimental group 1(OVA/OVA/high dose ADSC), the experimental group 2(OVA/OVA/low dose ADSC), the experimental group 3(OVA/OVA/PBS), the experimental group 4(OVA/OVA/0), the control group 1(PBS/PBS/0) and the control group 2(0/0/0). The mouse ADSC were isolated and cultured through conventional method, and AR mouse model was built with OVA and aluminum. The mice were injected with high (3×10(6)), low (1×10(6)) ADSC respectively labeled by CM-Dil for 3 consecutive days via tail-vein injection and sacrificed 48 hours later. Finally, levels of IL-4, IL-6, IL-10 and IFN -γ in serum were examined by ELISA; expressions of the four cytokines in spleen were examined by q RT-PCR; migration of ADSC to mouse model nasal mucosa were observed through fluorescence microscope; eosinophil infiltration were observed by the nasal HE staining. RESULTS: Mouse ADSC was isolated, cultured and identified successfully. There was significant difference in symptom scores of AR models (compared with 0/0/0 group, P<0.01). The IL-4 and IL-6 levels of OVA/OVA/high ADSC group were significantly lower than OVA/OVA/0 group (group 1: (17.95±7.78), (27.51±5.93) pg/ml; group 4: (56.82±9.12), (70.03±7.22) pg/ml), the IFN-γ and IL-10 levels increased significantly (group 1: (367.74±13.79), (417.10±72.40) pg/ml; group 4: (199.46±11.25), (122.50±15.57) pg/ml) in serum. These differences were statistically significant(P<0.01). Compared with OVA/OVA/low ADSC group, the IL-4 and IL-6 levels decreased significantly (group 1: (17.95±7.78), (27.51±5.93) pg/ml; group 2: (41.57±12.27), (56.21±9.23)pg/ml) of OVA / OVA / high ADSC group, and the IFN-γ and IL-10 increased significantly (group 1: (367.74±13.79), (417.10±72.40)pg/ml; group 2: (281.77±30.41), (203.45±87.10) pg/ml). These differences were statistically significant(P<0.01). At the same time, the corresponding changes observed at the levels of the cytokines' mRNA. ADSC labeled by CM-Dil could migrate to the mouse nasal mucosa. OVA/OVA/high ADSC group showed the more red fluorescence than the OVA/OVA/low ADSC group. The eosinophils in nasal mucosa of the two groups reduced compared with the normal control. CONCLUSION: ADSC injected via tail-vein can migrate to nasal mucosa and play non-specific immune effects, that may to effect the releases of some cytokines then to regulate the Th1/Th2 imbalance and the function of Treg cell, finally that be dose-related in a certain extent.

[Quantitative detection of plasma level of human telomerase reverse transcriptase mRNA in patients with nasopharyngeal carcinoma].

OBJECTIVE: To quantitatively measure plasma level of human telomerase reverse transcriptase (hTERT) mRNA in patients with nasopharyngeal carcinoma (NPC) and explore its implications for NPC diagnosis and treatment. METHODS: With 24 healthy volunteers serving as controls, the plasma level of hTERT mRNA was detected in 33 NPC patients by real-time PCR before and after treatments with chemotherapy or radiotherapy, and its association with the clinicopathological parameters of the patients were analyzed. RESULTS: The NPC patients showed a significantly higher mean plasma level of hTERT mRNA than the healthy volunteers (10.75 ± 4.29 vs 0.95 ± 0.37, P<0.05). The plasma hTERT mRNA level in the NPC patients was significantly correlated with clinical staging, tumor size, and degree of nodal metastasis (P<0.05) but with gender or age (P>0.05). In patients with stage I and II NPC, the plasma hTERT mRNA level decreased significantly after radiotherapy (5.60 ± 2.33 vs 3.43 ± 1.42); in patients in advanced stages (III and IV), plasma hTERT mRNA level decreased significantly from 12.68 ± 3.08 to 10.68 ± 2.48 (P<0.05) after chemotherapy and to 3.13 ± 1.69 (P<0.05) after radiotherapy. CONCLUSION: Radiotherapy and chemotherapy can effectively suppress elevated plasma hTERT mRNA levels in NPC patients. Plasma hTERT mRNA level is closely related to the clinicopathological factors and provides important information for early diagnosis and therapeutic effect evaluation of NPC.

[The role of Nods like receptors in the patients with allergic rhinitis].

OBJECTIVE: To explore role of Nods (nucleotide-binding oligomerization domain Nod Like receptors) kind of pattern recognition receptors (PRR) in patients with allergic rhinitis. METHOD: The mRNA and protein of Nod1, Nod2 of Nalp3 were analyzed in the turbinate mucosa of patients with allergic rhinitis, nasal septum deviation (NSD) nasal mucosa of patients and nasal polyp mucosa with Real-Time RT-PCR, Western blot and immunohistochemistry respectively, and Nod1 expression changes was explored in PBMC with wad explored Western-blot and then the level of IL-4, IL-6, IL-10, IFN-γ were detected in serum of AR after desensitization treatment. RESULT: These Nods like receptors, mainly found in nasal mucosa epithelial cells, glandular epithelium and inflammatory cells (e. g. plasma cells, eosinophils), were expressed in the nasal mucosa tissues. In AR group, Nod1 (mRNA and protein) expression were lower than NSD group (P<0.05), Nalp3 expression were higher than (P<0.05), while, there was no significant difference of Nod2 (mRNA and protein) between groups. After 6 months desensitization therapy, the change of Nod1 in PBMC was negatively correlated with the change of IL-10 in the peripheral blood, r=-0.88, P<0.05; while, change of Nod1 was positively correlated, with the change of IL-6, r=0.57, P>0.05. CONCLUSION: Nod1, Nod2 and Nalp3 expression were seen in the two groups,and the Nod1 expression in allergic rhinitis group was lower than other two groups, while, the Nalp3 was higher than other two groups. It showed Nod1, Nalp3 may be involved in the pathogenesis of allergic rhinitis. Expression of Nod1 in PBMC reduced after sublingual desensitization treatment. Besides, the change of Nod1 was negatively correlated with the change of IL-10 in PBMC. So,it seemed that Nod1 may regulate IL-10 changes and be involved in sublingual desensitization therapy.

Individualized treatment for allergic rhinitis based on key nasal clinical manifestations combined with histamine and leukotriene D4 levels / Tratamento individualizado para a rinite alérgica baseado nas principais manifestações clínicas nasais e dos níveis de histamina e leucotrieno D4

Abstract Introduction The types of allergic rhinitis are roughly classified based on the causative antigens, disease types, predilection time, and symptom severity. Objective To examine the clinical typing and individualized treatment approach for allergic rhinitis and to determine the optimal treatment method for this disease using various drug combination therapies. Methods A total of 108 participants with allergic rhinitis were divided into three groups based on symptoms. Subsequently, each group was further categorized into four subgroups based on the medications received. The efficacy of the treatments was evaluated using the visual analog scale VAS scores of the total and individual nasal symptoms, decline index of the symptom score, histamine and leukotriene levels, and mRNA and protein expression levels of histamine 1 and cysteinyl leukotriene 1 receptors. Results Loratadine + mometasone furoate and loratadine + mometasone furoate + montelukast significantly improved the sneezing symptom and reduced the histamine levels compared with the other combination therapies (p < 0.05). Meanwhile, montelukast + mometasone furoate and montelukast + mometasone furoate + loratadine considerably improved the nasal obstruction symptom and decreased the leukotriene D4 levels compared with the other combination therapies (p < 0.05). Conclusion Clinical symptom evaluation combined with experimental detection of histamine and leukotriene levels can be an objective and accurate method to clinically classify the allergic rhinitis types. Furthermore, individualized treatment based on allergic rhinitis classification can result in a good treatment efficacy.

Resumo Introdução A rinite alérgica é basicamente classificada de acordo com os antígenos causadores, tipos de doença, peridiocidade e gravidade dos sintomas. Objetivo Avaliar os tipos clínicos e a abordagem terapêutica individualizada para cada tipo de rinite alérgica e determinar o método de tratamento ideal utilizando várias terapias de combinação de fármacos. Método Um total de 108 participantes com rinite alérgica foram divididos em três grupos com base nos sintomas. Posteriormente, cada grupo foi subsequentemente categorizado em quatro subgrupos com base nos medicamentos recebidos. A eficácia dos tratamentos foi avaliada utilizando os escores da escala visual analógica EVA dos sintomas nasais totais e individualmente, índice de declínio do escore de sintomas, níveis de histamina e leucotrienos e níveis de expressão de mRNA e proteína dos receptores de histamina 1 e cisteinil-leucotrieno 1. Resultados As associações entre loratadina + furoato de mometasona, assim como a de loratadina + furoato de mometasona + montelucaste melhoraram significativamente o sintoma de espirros e reduziram os níveis de histamina em comparação às outras terapias combinadas (p < 0,05). Por outro lado, a associação montelucaste + furoato de mometasona, assim como a associação montelucaste + furoato de mometasone + loratadina melhoraram consideravelmente o sintoma de obstrução nasal e diminuíram os níveis de leucotrieno D4 em comparação com as outras combinações (p < 0,05). Conclusão A avaliação clínica dos sintomas combinada com a detecção experimental dos níveis de histamina e leucotrieno pode ser um método objetivo e preciso para classificar clinicamente os tipos de rinite alérgica. Além disso, o tratamento individualizado baseado na classificação da rinite alérgica pode resultar no aumento da eficácia do tratamento.

[Role of Nods like receptors in the patients with nasal polyps].

OBJECTIVE: To investigate the expression and role of a new pattern-recognition receptors (PRR), nucleotide binding oligomerization domain (Nod) like receptors (NLRs), in the patients with nasal polyps and nasal septum normal control group. METHOD: The expressions of Nod1, Nod2 and Nalp3 mRNA and protein were explored with real-time RT-PCR, Western-Blot and immunohistochemistry respectively. RESULT: The protein levels of Nod1, Nod2 and Nalp3 were expressed in nasal polyp and the control, but the expression of Nod1 and Nalp3 in nasal polyps were higher than those in control. No significant difference of Nod2 was seen between the two groups. And then, there was no significant difference of Nod1, Nod2, Nalp3 mRNA between two groups with Real-time RT-PCR. CONCLUSION: The expression of Nod1 and Nalp3 are increased in nasal polyp tissues and maybe a etiological factors in the formation of nasal polyps.

[Effect of nasal epithelial lining prognosis and turnover changed by vesicles molecular immuno-pathology after nasal endoscopic surgery].

OBJECTIVE: To observe Effect of nasal epithelial lining prognosis and turnover changed by vesicles molecular immuno-pathology after nasal endoscopic surgery. METHOD: Forty patients (80 sides) with chronic nasal polyps in accordance with the EPOS standard after endoscopic sinus surgery were randomly divided into treatment group and control group according to weather scraping the vesicles. Compared the speed of epithelialization of nasal cavity mucosa in the two groups. Observed the pathological features of vesicles through HE staining and transmission electron microscopy. Divided the treatment group into 1-3 weeks group, 6-8 weeks group and 11-14 weeks group. And make the inferior turbinate mucosa and nasal polyps during surgery the control group. Compared the level of vascular endothelial growth factor(VEGF) and transforming growth factor beta1 (TGF beta1) between the groups. RESULT: Vesicles were divided into mild groups and moderate to severe groups according to the sizes and amonts. Vesicles in moderate to severe groups were faster epithelialization than the mild groups, 1.5 weeks shortened on average (P < 0.05). There wes no significant difference between the two groups in mild group. The level of TGF beta1 in the nasal polyps, 1-3 weeks group and 6-8 weeks group were significantly higher than the inferior turbinate mucosa and 11-14 weeks group (P < 0.05). The level of VEGF in the nasal polyps, 6-8 weeks group were significantly higher than the inferior turbinate mucosa, 1-3 weeks group and 11-14 weeks group (P < 0.05). Vesicles were displayed discontinuous basal membranes, interstitial edama, infiltration of inflammatory cells, increased pathological glands, abnormal microtubule structure, reduction of mitochondrials. CONCLUSION: Vesicles is a dynamic process, which may predict the increased levels of inflammatory factors (VEGF and TGF beta1) and contribute to some of the patheologic changes observed in nasal polyps. The level of VEGF and TGF beta1 in vesicles after endoscopic sinus surgery can be used as indicators of prognosis. Treating the moderate to severe vesicles after surgery is benefit to the epithelium of the nasal mucosa.

PinX1 regulation of telomerase activity and apoptosis in nasopharyngeal carcinoma cells.

BACKGROUND: Human interacting protein X1 (PinX1) has been identified as a critical telomerase inhibitor and proposed to be a putative tumor suppressor gene. Loss of PinX1 has been found in a large variety of malignancies, however, its function in inhibiting telomerase activity of tumor cells is not well documented. Here we show that PinX1 is essential for down-regulation telomerase activity of nasopharyngeal carcinoma. METHODS: Expression vectors of human PinX1 (pEGFP-C3-PinX1) and its small interfering RNA (PinX1-FAM-siRNA) were constructed and transfected into NPC. Their effects on mRNA of telomerase catalytic subunit (hTERT), telomerase activity, cell proliferation, cell migration, wound healing, cell cycles and apoptosis were examined using semi-quantitative RT-PCR, stretch PCR, MTT assay, Transwell, scratch assay and flow cytometry, respectively. RESULTS: Transfection of pEGFP-C3-PinX1 and PinX1-FAM-siRNA increased and reduced PinX1 mRNA by 1.6-fold and 70%, respectively. Over-expression of PinX1 decreased hTERT mRNA by 21%, reduced telomerase activity, inhibited cell growth, migration and wound healing ability, arrested cells in G0/G1 phase, and increased apoptotic index. In contrast, down-regulation of PinX1 did not alter the above characteristics. CONCLUSIONS: PinX1 may play important roles in NPC proliferation, migration and apoptosis and has application potential in tumor-targeted gene therapy.

[Study on the relationship between TK gene regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells].

OBJECTIVE: To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells. METHOD: The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines, telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in malignant tumour cells pre- and post-transfected by enhanced vector. Meanwhile the relationship between TK and telomerase was analyzed. RESULT: (1) A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group, pGL3-basic-EGFP-TK-hTRETp, and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. (2) Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV. (3) After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp, pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector. CONCLUSION: TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed. But it is unclear how the telomerase are down-regulated by TK gene.

[Enhanced thymidine kinase gene vector and its killing effect on nasopharyngeal carcinoma in vitro and in vivo].

OBJECTIVE: To construct a modified and enhanced thymidine kinase (TK) vector regulated by human telomerase catalytic subunit promoter (hTERT) promoter and cytomegalovirus (CMV) enhancer and its killing effect on nasopharyngeal carcinoma in vitro and in vivo and its safety in vivo. METHODS: The pGL3-basic, as basic vector template, was linked and constructed into TK vector regulated by hTERT promoter and CMV enhancer with mono-promoter vector as control. Enhanced TK expression was confirmed by fluorescent microscopy and real time fluorescent quantitative PCR. Telomerase activity was measured by stretch PCR. Tumour killing effects were examined by MTT and Boyden areola. The effects of enhanced TK on the invasiveness of tumor cell NPC 5-8F and the growth of xenograft implanted in nude mice were investigated. RESULTS: Compared with non-enhanced vector, TK expressed by the enhanced vector significantly increased in NPC 5-8F and MCF-7 cells, telomerase activity was positive in human in NPC 5-8F cells and breast cancer MCF-7 cells and negative in control human blood vessel endothelium ECV-304 cells. After ganciclovir(GCV) treatment, NPC 5-8F cell survival rate and invasiveness decreased and tumor progress of NPC xenograft implanted in nude mice was inhibited, without obvious toxicity effects on mouse liver and kidney. CONCLUSIONS: The enhanced TK vector regulated by hTERT promoter and CMV enhancer can obviously and specifically inhibit and kill nasopharyngeal carcinoma cells in culture and nasopharyngeal carcinoma xenograft in nude mice in vivo, without obviously toxic side effects on nude mice. The targeted and enhanced TK gene vector with high performance may be a new tumour targeted gene therapy strategy clinically to aim directly at most malignant tumours including nasopharyngeal carcinoma, with more extensive anti-cancer spectrum.

Induction of programmed cell death in Arabidopsis and rice by single-wall carbon nanotubes.

PREMISE OF THE STUDY: Single-walled carbon nanotubes (SWCNTs) have many unique structural and mechanical properties. Their potential applications, especially in biomedical engineering and medical chemistry, have been increasing in recent years, but the toxicological impact of nanoparticles has rarely been studied in plants. • METHODS: We exposed Arabidopsis and rice leaf protoplasts to SWCNTs and examined cell viability, DNA damage, reactive oxygen species generation, and related gene expression. We also tested the effects of nanoparticles on Arabidopsis leaves after injecting a SWCNT solution. EM-TUNEL (electron-microscopic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and a cerium chloride staining method were used. • KEY RESULTS: SWCNTs caused adverse cellular responses including cell aggregation, chromatin condensation along with a TUNEL-positive reaction, plasma membrane deposition, and H(2)O(2) accumulation. The effect of SWCNTs on the survival of cells was dose dependent, with 25 µg/mL inducing 25% cell death in 6 h. In contrast, activated carbon, which is not a nano-sized carbon particle, did not induce cell death even 24 h after treatments. The data indicated that the nano-size of the particle is a critical factor for toxicity. Moreover, endocytosis-like structures with cerium chloride deposits formed after SWCNT treatment, suggesting a possible pathway for nanoparticles to traverse the cell membrane. • CONCLUSIONS: Consequently, SWCNTs have an adverse effect on protoplasts and leaves through oxidative stress, leading to a certain amount of programmed cell death. Although nanomaterials have great advantages in many respects, the benefits and side effects still need to be assessed carefully.

Targeted gene therapy of nasopharyngeal cancer in vitro and in vivo by enhanced thymidine kinase expression driven by human TERT promoter and CMV enhancer.

BACKGROUND/AIM: To explore the therapeutic effects of thymidine kinase (TK) expressed by enhanced vector pGL3-basic- hTERTp-TK-EGFP-CMV driven by human telomerase reverse transcriptase promoter (hTERTp) as well as cytomegalovirus immediate early promoter enhancer (CMV). MATERIALS/METHODS: Enhanced TK-EGFP expression was confirmed by fluorescent microscopy, real time PCR and telomerase activity. Its effects were examined by survival of tumor cells NPC 5-8F and MCF-7, index of xenograft implanted in nude mice and histology. RESULTS: Compared with non-enhanced vector pGL3-basic-TK-hTERTp-EGFP, TK expressed by the enhanced vector significantly decreased NPC 5-8F and MCF-7 cell survival rates after ganciclovir (GCV) treatment (p < 0.001) and tumor progress in nude mice with NPC xenograft and treated with GCV, without obvious toxicity to mouse liver and kidney. CONCLUSION: The enhanced TK expression vector driven by hTERTp with CMV enhancer has brighter clinical potentials in nasopharyngeal carcinoma therapy than the non-enhanced vector.