Metabolic characteristics revealing cell differentiation of nasopharyngeal carcinoma by combining NMR spectroscopy with Raman spectroscopy.

BACKGROUND: The staging system of nasopharyngeal carcinoma (NPC) has close relationship with the degree of cell differentiation, but most NPC patients remain undiagnosed until advanced phases. Novel metabolic markers need to be characterized to support diagnose at an early stage. METHODS: Metabolic characteristics of nasopharyngeal normal cell NP69 and two types of NPC cells, including CNE1 and CNE2 associated with high and low differentiation degrees were studied by combining 1H NMR spectroscopy with Raman spectroscopy. Statistical methods were also utilized to determine potential characteristic metabolites for monitoring differentiation progression. RESULTS: Metabolic profiles of NPC cells were significantly different according to differentiation degrees. Various characteristic metabolites responsible for different differentiated NPC cells were identified, and then disordered metabolic pathways were combed according to these metabolites. We found disordered pathways mainly included amino acids metabolisms like essential amino acids metabolisms, as well as altered lipid metabolism and TCA cycle, and abnormal energy metabolism. Thus our results provide evidence about close relationship between differentiation degrees of NPC cells and the levels of intracellular metabolites. Moreover, Raman spectrum analysis also provided complementary and confirmatory information about intracellular components in single living cells. Eight pathways were verified to that in NMR analysis, including amino acids metabolisms, inositol phosphate metabolism, and purine metabolism. CONCLUSIONS: Methodology of NMR-based metabolomics combining with Raman spectroscopy could be powerful and straightforward to reveal cell differentiation development and meanwhile lay the basis for experimental and clinical practice to monitor disease progression and therapeutic evaluation.

Overexpression of ß-Catenin Decreases the Radiosensitivity of Human Nasopharyngeal Carcinoma CNE-2 Cells.

BACKGROUND/AIMS: Nasopharyngeal carcinoma (NPC) is rare worldwide but remains highly prevalent in endemic regions, notably in southern China. Radiotherapy remains the treatment of choice for NPC, but radioresistance has been identified as a major cause of therapeutic failure. The Wnt/ß-catenin signaling has been found to be involved in NPC radioresistance; however, the effect of ß-catenin overexpression on radioresistance remains unknown in NPC until now. This study aimed to examine the impact of ß-catenin overexpression on the radiosensitivity of human NPC CNE-2 cells. METHODS: Immunohistochemistry was performed to detect the ß-catenin expression in normal nasopharyngeal specimens and NPC specimens. The human NPC CNE-2 cell line overexpressing ß-catenin was modeled by transfection with the pcDNA3.1/Hygro(+)/ß-catenin recombinant vector (transfection group), while cells transfected with the pcDNA3.1/Hygro(+) vector served as negative controls and non-transfected cells served as blank controls. The expression of key molecules of the Wnt/ß-catenin signaling pathway was determined using Western blotting and qPCR assays, and the changes of radiation sensitivity were measured with a colony-formation assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. In addition, the cell cycle and apoptosis was detected using flow cytometry and the TCF/LEF transcriptional activity was measured with a Dual Luciferase Reporter Assay System. RESULTS: Immunohistochemical staining showed high ß-catenin expression in radioresistant NPC specimens, and low expression in radiosensitive NPC specimens and normal nasopharyngeal specimens. Western blotting and qPCR assays detected higher ß-catenin expression in the transfection group than in the negative and blank controls (P < 0.01). Down-regulation of GSK-3ß expression (P < 0.05) and up-regulation of Cyclin D1 expression (P < 0.01) was detected in ß-catenin overexpressing NPC cells exposed to X-ray radiation relative to negative and blank controls. Colony-formation assay revealed higher D0, Dq and SF in the transfection group than in the negative and blank control groups post-radiation, and the SER in the transfection group was 0.75-fold and 0.68-fold greater than that in the blank and negative control groups, respectively. MTT assay revealed that the viability of CNE-2 cells was significantly higher in the transfection group (96% ± 8.72%) than in the negative control group (74.67 ± 7.05%) and the blank control group (75.33% ± 7.02%) 24 h post-exposure to 6 Gy X-ray radiation (P < 0.05). X-ray radiation led to a lower proportion of CNE-2 cells at the G2/M phase and a lower apoptotic rate in the transfection group than in the negative and blank control groups (P < 0.05). In addition, the TCF/LEF transcriptional activity was higher in the transfection group than in the negative and blank control groups (P < 0.01), and 6 Gy X-ray radiation elevated the TCF/LEF transcriptional activity relative to 0 Gy radiation in the transfection group (P < 0.01). CONCLUSION: ß-catenin overexpression may decrease the radiation sensitivity in NPC CNE-2 cells through activating the downstream transcriptional factors of ß-catenin, and reducing G2/M arrest and cell apoptosis.

Metabolic profiling of normal hepatocyte and hepatocellular carcinoma cells via 1 H nuclear magnetic resonance spectroscopy.

Hepatocellular carcinoma (HCC) causes death mainly by disseminated metastasis progression from the organ being confined. Different metastatic stages are closely related to cellular metabolic profiles. Normal hepatocyte and HepG2 cell line from low metastatic HCC were studied by NMR-based metabolomic techniques. Multivariate and univariate statistical analyses were utilized to identify characteristic metabolites from cells and cultured media. Elevated levels of acetate, creatine, isoleucine, leucine, and phenylalanine were observed in HepG2 cells, suggesting more active in gathering nutrient components along with altered amino acid metabolisms and enhanced lipid metabolism. High glucose consumption was significantly different in low metastatic cells. A series of characteristic metabolites were identified and served as biomarkers. Relative metabolic pathway analysis shows that low metastatic HepG2 cell line exhibits active behaviors in metabolisms and biosynthesis of specific amino acids and energy metabolism. Moreover, characteristic metabolites-based classification models executed by support vector machines algorithm perform robustly to classify normal hepatocyte and HepG2 cell line. It is concluded that NMR-based metabolomic analyses of cell lines can provide a powerful approach to understand metastasis-related biological alterations. The present study also provides a basis for metabolic markers determination of hepatic carcinoma in the future clinical study.

Raman spectral analysis of nasopharyngeal carcinoma cell line CNE2 after microwave radiation.

This study aimed to study the effects of microwave radiation on the nasopharyngeal carcinoma cell line CNE2 by Raman spectroscopy. The cells were separated into a control group and radiated groups with radiation times of 2, 5, 10, and 25 min, respectively. Both principal components analysis and support vector machine were employed for statistical analysis of Raman spectra. The results show that the relative content of C-H deformation and amide I begin to change when the radiation time is over 10 min, and principal components analysis further confirms there are significant differences after 10 min of radiation. Moreover, support vector machine is simultaneously used to classify radiated samples from control samples. The classification accuracy is low until the radiation time reaches over 10 min. In conclusion, this study reveals the Raman spectral characteristics of CNE2 under different microwave radiation exposure timesand demonstrates Raman spectroscopy can be a potential method to explore cellular characterization after radiation. The final results may help in elucidating the mechanism by which microwave radiation interacts with tumor cells.

Raman microspectroscopy as a diagnostic tool to study single living nasopharyngeal carcinoma cell lines.

Raman spectroscopy can provide molecular-level fingerprint information about the biochemical composition and structure of cells and tissues with excellent spatial resolution. In this study, Raman spectroscopy of 3 different nasopharyngeal carcinoma cell lines C666-1, CNE1, and CNE2 and 1 nasopharyngeal normal cell line NP69 acquired on a piece of silica glass slide are presented to investigate the differences among them. The results show the ratio of I1657/I1449 (= 0.7) could provide good distinction between tumor and normal cell lines very easily, which coincides with existing reports about the study of different cell lines and bronchial tissue. In addition, several statistical analytical methods were used to classify these 4 different cell lines and then achieved an exciting result with great sensitivity and specificity of >90%, respectively. The findings of this work further support former work where cells' Raman spectra were acquired on a different substrate. All of these results indicate Raman spectroscopy has the potential to discriminate between normal and tumor cells and have potential use in early diagnosis of nasopharyngeal carcinoma.

Recombinant adenovirus-p53 (Gendicine) sensitizes a pancreatic carcinoma cell line to radiation.

OBJECTIVE: In this study, we examine the effects of recombinant adenovirus-p53 (rAd-p53) on the pancreatic carcinoma cell line SW1990. Specifically, we determine if expression of rAd-p53 sensitizes these cells to radiation. METHODS: Following transfection of SW1990 cells with rAd-p53, we measured expression of P53, P21 and Bax by immunocytochemistry. Both transfected and control cell lines were irradiated with a range of doses, and the survival fractions (SF) were calculated. Dose survival curves were constructed and modeled for comparison. RESULTS: Transfection of SW1990 cells with rAd-p53 resulted in increased expression of P53, P21 and Bax in a time-dependent manner. At 96 h after transfection, 89.92% of cells expressed P53, 56.8% expressed P21, and 76.50% expressed Bax. The SF following radiation was lower in the rAd-p53 transfected cells compared to the control cells, suggesting that rAd-p53 sensitizes SW1990 cells to radiation (D0 for the experimental and control groups was 2.199 and 2.462, respectively). CONCLUSIONS: Use of the adenoviral vector is an effective means of transfecting SW1990 cells with wild-type P53, and this sensitizes the cell line to irradiation. This work suggests that combining rAd-p53 with radiation therapy in pancreatic cancer may be therapeutically beneficial.

RNU12 inhibits gastric cancer progression via sponging miR-575 and targeting BLID.

Gastric cancer (GC) is one of the major causes of cancer deaths with 5-year survival ratio of 20%. RNU12 is one of long noncoding RNAs (lncRNAs) regulating the tumor progression. However, how RNU12 affecting GC is not clear. qRT-PCR was utilized for determining the RNU12 expression in cell lines, 113 cases of paired gastric cancer (GC) and their adjacent normal gastric tissues. The biofunction alterations of RNU12 were assessed by its overexpression or knockdown in GC cells. MTT and cloning assay were assayed for the cell proliferation, the flow cytometry for the detection of cell cycle and the wound healing assay (WHA) and transwell invasion assay (TIA) for examining the migration and invasion of cells. The expressions of a set of genes related proliferation and migration were investigated with the Western Blotting (WB). RNA immunoprecipitation (RIP), biotinylated RNA pull-down and dual luciferase reporter tests were used to detect the interactions of RNU12 with miR-575/BLID. The in vivo proliferation and migration ability of RNU12 infected cells were determined in zebrafish system. This study revealed that RNU12 inhibited proliferation, invasion and metastasis by sponging of miR-575 and regulating the downstream BLID and modulated EMT of GC cells. The RNU12/miR-575/BLID axis is likely to be the prognosis biomarkers and drug targets of GC.

Knockdown of Annexin A2 Enhances Radiosensitivity by Increasing G2/M-Phase Arrest, Apoptosis and Activating the p38 MAPK-HSP27 Pathway in Nasopharyngeal Carcinoma.

Annexin A2 (ANXA2) has been found to be involved in cancer proliferation, metastasis and prognosis; however, its exact role in nasopharyngeal carcinoma (NPC) radioresistance remains unknown. We found that ANXA2 expression was correlated with prognosis in NPC patients, and longer overall survival in NPC patients with low ANXA2 expression than those with high ANXA2 expression. ANXA2 knockdown increased the radiosensitivity in radioresistant NPC cells, and ANXA2 overexpression decreased the radiosensitivity in NPC cells. Knocking-down ANXA2 expression increased the irradiation-induced apoptosis of radioresistant NPC cells, and ANXA2 overexpression decreased the irradiation-induced apoptosis of NPC cells. ANXA2 knockdown induced G2/M phase arrest in NPC cells post-irradiation, and ANXA2 overexpression abrogated G2/M phase arrest in NPC cells post-irradiation. ANXA2 overexpression resulted in inhibition of the p38 MAPK-HSP27 pathway, while ANXA2 knockdown resulted in activation of the p38 MAPK-HSP27 pathway. In addition, ANXA2 knockdown increased the radiosensitivity of the xenografted tumors in nude mice. Our data demonstrate that knockdown of Annexin A2 enhanced radiosensitivity in NPC by increasing G2/M-phase arrest, apoptosis and activating the p38 MAPK-HSP27 pathway. ANXA2 may be a promising target used to overcome radioresistance in NPC.

SNHG8 Promotes the Progression of Epstein-Barr Virus-Associated Gastric Cancer via Sponging miR-512-5p and Targeting TRIM28.

SNHG8, a family member of small nucleolar RNA host genes (SNHG), has been reported to act as an oncogene in gastric carcinoma (GC). However, its biological function in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) remains unclear. This study investigated the role of SNHG8 in EBVaGC. Sixty-one cases of EBVaGC, 20 cases of non-EBV-infected gastric cancer (EBVnGC), and relative cell lines were studied for the expression of SNHG8 and BHRF1 (BCL2 homolog reading frame 1) encoded by EBV with Western blot and qRT-PCR assays. The relationship between the expression levels of SNHG8 and the clinical outcome in 61 EBVaGC cases was analyzed. Effects of overexpression or knockdown of BHRF1, SNHG8, or TRIM28 on cell proliferation, migration, invasion, and cell cycle and the related molecules were determined by several assays, including cell proliferation, colony assay, wound healing assay, transwell invasion assay, cell circle with flow cytometry, qRT-PCR, and Western blot for expression levels. The interactions among SNHG8, miR-512-5p, and TRIM28 were determined with Luciferase reporter assay, RNA immunoprecipitation (RIP), pull-down assays, and Western blot assay. The in vivo activity of SNHG8 was assessed with SNHG8 knockdown tumor xenografts in zebrafish. Results demonstrated that the following. (1) BHRF1 and SNHG8 were overexpressed in EBV-encoded RNA 1-positive EBVaGC tissues and cell lines. BHRF1 upregulated the expressions of SNHG8 and TRIM28 in AGS. (2) SNHG8 overexpression had a significant correlation with tumor size and vascular tumor thrombus. Patients with high SNHG8 expression had poorer overall survival (OS) compared to those with low SNHG8 expression. (3) SNHG8 overexpression promoted EBVaGC cell proliferation, migration, and invasion in vitro and in vivo, cell cycle arrested at the G2/M phase via the activation of BCL-2, CCND1, PCNA, PARP1, CDH1, CDH2 VIM, and Snail. (4) Results of dual-luciferase reporter assay, RNA immunoprecipitation, and pull-down assays indicated that SNHG8 sponged miR-512-5p, which targeted on TRIM28 and promoted cancer malignant behaviors of EBVaGC cells. Our data suggest that BHRF1 triggered the expression of SNHG8, which sponged miR-512-5p and upregulated TRIM28 and a set of effectors (such as BCL-2, CCND1, CDH1, CDH2 Snail, and VIM) to promote EBVaGC tumorigenesis and invasion. SNHG8 could be an independent prognostic factor for EBVaGC and sever as target for EBVaGC therapy.

The Role of ARL4C in Erlotinib Resistance: Activation of the Jak2/Stat 5/ß-Catenin Signaling Pathway.

Cancer patients who initially benefit from Erlotinib, a drug targeting EGFR path, eventually develop resistance to the drug. The underlying mechanism is largely unknown. This study investigated the role of ARL4C in Erlotinib resistance development of NSCLC. qRT-PCR and Western blotting were performed to analyze the expression of mRNA and protein of ARL4C in two NSCLC cell lines (HCC827 and PC-9). Several assays (MTS, colony formation, transwell migration, luciferase reporter, and chromatin-immunoprecipitation) were used to explore the role of ARL4C in biofunctional changes of Erlotinib-resistant cells and their associations with Jak2/Stat 5/ß-catenin signaling. Results demonstrated that (1) long-term use of Erlotinib resulted in downregulation of ARL4C; (2) overexpression of ARL4C could regain the sensitivity to Erlotinib in the drug-resistant HCC827/ER cells, while downregulation of ARL4C increased HCC827, and PC-9 cells' resistance to the drug; (3) Erlotinib-induced downregulation of ARL4C resulted in phosphorylation of Jak2/Stat5 and upregulation of ß-catenin and their related molecules Axin2, CD44, Ccnd1, Lgr-5, and MMP7, which promoted the malignant behaviors of Erlotinib-resistant cells; (4) chromatin immunoprecipitation and luciferase reporter assay revealed that Stat5 could bind to ß-catenin promoter to upregulate molecules to maintain the malignant behaviors, which might count for how Erlotinib-resistant cell survived while EGFR path was blocked; (5) the expression of ARL4C was not associated with known EGFR gene mutations in both Erlotinib-resistant cells and NSCLC tissues. Our data suggest that Erlotinib resistance of NSCLCs is associated with downregulation of ARL4C via affecting Jak/Stat/ß-catenin signaling. ARL4C could serve as a biomarker to predict the effectiveness of TKI targeting therapy and a potential therapeutic target for overcoming Erlotinib resistance in NSCLC.

Stanniocalcin 2 (STC2) expression promotes post-radiation survival, migration and invasion of nasopharyngeal carcinoma cells.

Background: Stanniocalcin 2 (STC2) expression is upregulated under multiple stress conditions including hypoxia, nutrient starvation and radiation. Overexpression of STC2 correlates with tumor progression and poor prognosis. Purpose: We previously demonstrated that overexpression of STC2 in nasopharyngeal carcinomas (NPC) positively correlates with radiation resistance and tumor metastasis, two major clinical obstacles to the improvement of NPC management. However, it remains elusive whether STC2 expression is a critical contributing factor for post-radiation survival and metastasis of NPC cells. Materials and methods: Using the radiation resistant CNE2 cell line as a model, we examined the importance of STC2 expression for post-radiation survival, migration and invasion. Here, we report the establishment of STC2 knockout lines (CNE2-STC2-KO) using the CRISPR/Cas9-based genome editing technique. Results: Compared with the parental line, STC2-KO cells showed similar proliferation and morphology in normal culture conditions, and loss of STC2 did not compromise the cell tumorigenicity in nude mice model. However, STC2-KO lines demonstrated increased sensitivity to X-radiation under either normoxic or hypoxic conditions. Particularly, upon X-radiation, parental CNE2 cells only slightly whereas STC2-KO cells remarkably decreased the migration and invasion ability. Cell cycle analysis revealed that loss of STC2 accumulated cells in G1 and G2/M phases but decreased S-population. Conclusion: These data indicate that the expression of STC2, which can be stimulated by metabolic or therapeutic stresses, is one important factor to promote survival and metastasis of post-radiation NPC cells. Therefore, targeting STC2 or relative downstream pathways may provide novel strategies to overcome radiation resistance and metastasis of NPC.

Down-regulation of miR-214 reverses erlotinib resistance in non-small-cell lung cancer through up-regulating LHX6 expression.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are standard treatments for advanced non-small-cell lung cancer (NSCLC) patients. However, acquired resistance to EGFR-TKIs is widely detected across the world, and the exact mechanisms have not been fully demonstrated until now. This study aimed to examine the role of miR-214 in the acquired resistance to erlotinib in NSCLC, and elucidate the underlying mechanisms. qRT-PCR assay detected higher miR-214 expression in the plasma of NSCLC patients with acquired EGFR-TKI resistance than prior to EGFR-TKI therapy, and in the generated erlotinib-resistant HCC827 (HCC827/ER) cells than in HCC827 cells. Bioinformatics analysis and dual-luciferase reporter assay indentified LHX6 as a direct target gene of miR-214, and LHX6 expression was detected to be down-regulated in erlotinib-resistant HCC827 cells. Transwell invasion assay revealed that overexpressing LHX6 reversed the increase in the invasive ability of HCC827 cells induced by miR-214 overexpression, and the CRISPR-Cas9 system-mediated LHX6 knockdown reversed the reduction in the invasion of erlotinib-resistant HCC827 cells caused by miR-214 down-regulation. The results of the present study demonstrate that down-regulation of miR-214 may reverse acquired resistance to erlotinib in NSCLC through mediating its direct target gene LHX6 expression.

Detection of Genomic DNA Damage from Radiated Nasopharyngeal Carcinoma Cells Using Surface-Enhanced Raman Spectroscopy (SERS).

Structural changes and chemical modifications in DNA during interactions with X-ray radiation are still not clear within 48 h of incubation. We investigate genomic DNA from the radiated CNE2 cell line within 48 h of incubation using surface-enhanced Raman spectroscopy (SERS). Multivariate methods including principal component analysis (PCA) and random forest are proposed to explore the statistical significance before and after radiation. Our results show that intensities of several bands change after radiation, which indicates backbone damage and base-unstacking. Biological effects from DNA damage repairing process may be simultaneously stimulated and different from incubation time. Under doses of 10 Gy (with 24 and 48 h of incubation) and 20 Gy (with 48 h of incubation), the relative contents of C against T and A against G deviate obviously from the control level. Statistical results strengthen significantly the idea that modification in DNA bases is associated with the disruption of base-stacking in the DNA duplex. Our findings provide vital information for radiation-induced the DNA damage at the molecular level, which may provide insight into the effect and mechanism of anticarcinogens in tumor therapy.

Early discrimination of nasopharyngeal carcinoma based on tissue deoxyribose nucleic acid surface-enhanced Raman spectroscopy analysis.

Surface-enhanced Raman spectroscopy (SERS) was employed to detect deoxyribose nucleic acid (DNA) variations associated with the development of nasopharyngeal carcinoma (NPC). Significant SERS spectral differences between the DNA extracted from early NPC, advanced NPC, and normal nasopharyngeal tissue specimens were observed at 678, 729, 788, 1337, 1421, 1506, and 1573??cm?1, which reflects the genetic variations in NPC. Principal component analysis combined with discriminant function analysis for early NPC discrimination yielded a diagnostic accuracy of 86.8%, 92.3%, and 87.9% for early NPC, advanced NPC, and normal nasopharyngeal tissue DNA, respectively. In this exploratory study, we demonstrated the potential of SERS for early detection of NPC based on the DNA molecular study of biopsy tissues.

Early changes in apparent diffusion coefficients predict radiosensitivity of human nasopharyngeal carcinoma xenografts.

OBJECTIVES/HYPOTHESIS: Our objective was to predict the radiosensitivity of human nasopharyngeal carcinoma xenografts in nude mice models through an examination of early changes in apparent diffusion coefficient (ADC) values. STUDY DESIGN: Randomized. METHODS: BALB/c-nu nude mice (n = 20) were divided into two groups that were subcutaneously injected with CNE1 or CNE2 cell lines. Xenograft volumes were measured after tumor formation, mice were scanned with a diffusion-weighted imaging sequence, and the mean ADC values were measured (ADC(0) ). Fifteen to 20 hours after tumors received 15 Gy, mice were scanned again and ADC values (ADC(1) ) were measured. RESULTS: ADC(0) and ADC(1) values of the CNE1 group showed no significant difference (P = .692). The difference between the ADC(0) and ADC(1) values of the CNE2 group was statistically significant (P < .001). ADC(0) values of the two groups exhibited no statistically significant difference (P = .204). ADC(1) , ADC(1-0) , and ΔADC of the two groups exhibited statistically significant differences (P < .001; P = .001 and .002, respectively). After irradiation, volume changes ΔV(8) , ΔV(10) , and ΔV(12) of two groups were statistically different (all P < .001). Pearson correlation analysis showed ADC(1-0) and ΔADC were positively correlated with ΔV(8) , ΔV(10) , and ΔV(12) . The cut point was found by means of a receiver operating characteristic curve, and the ΔV(12) of the two redivided groups showed a statistically significant difference (P = .001). CONCLUSIONS: This study found that changes in ADC values correlated with volume changes after irradiation. Therefore, ADC values have the potential to predict the radiosensitivity of nasopharyngeal carcinoma xenografts.