Nasopharyngeal Carcinoma HONE-1 Cells Research Articles

The improvewment of DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing

To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data. Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.

MACC1 facilitates the escape of nasopharyngeal carcinoma cells from killing by natural killer cells

This study examined the regulation of phenotype and function of natural killer (NK) cells by nasopharyngeal carcinoma (NPC) cells with metastasis-associated in colon cancer-1 (MACC1) overexpression. Thirty patients with NPC and 20 healthy subjects were included. Peripheral blood (5 mL) was collected, and NK cells were purified using Ficoll and negative separation. NPC HONE1 cells were transfected with pcDNA3.1-MACC1 or negative control plasmids, and culture supernatant was collected for co-culture with NK cells. Flow cytometry was used to determine the expression of activated and inhibitory receptors on the cells. Western blotting was used to determine the expression of proteins. The results showed that the ratio of NK cells in the peripheral blood of NPC patients was elevated, but the activity and tumor-killing effect of NK cells were reduced. MACC1 promoted the escape of HONE1 cells from killing by NK cells. HONE1 with overexpression of MACC1 down-regulated the expression of NKG2D in NK cells, inhibited the proliferation of NK cells and escaped the immune surveillance by NK cells. MACC1 down-regulated the expression of NKG2D in NK cells by up-regulating the expression of TGF-β1 in HONE1 cells, thereby inhibiting the activity of NK cells. MACC1 inhibited the cell cycle of NK cells via TGF-β1 pathway. The study demonstrated that MACC1 elevated the expression of TGF-β1 by NPC HONE1 cells, thereby inhibiting NKG2D expression and G1/S transition in NK cells. MACC1 helped NPC HONE1 cells avoid killing by NK cells.

Bauhinia variegata var. variegata lectin: isolation, characterization, and comparison.

Bauhinia variegata var. variegata seeds are rich in proteins. Previously, one of the major storage proteins of the seeds was found to be a trypsin inhibitor that possessed various biological activities. By using another purification protocol, a glucoside- and galactoside-binding lectin that demonstrated some differences from the previously reported B. variegata lectin could be isolated from the seeds. It involved affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose and Mono Q, and also size exclusion chromatography on Superdex 75. The lectin was not retained on Affi-gel blue gel but interacted with Q-Sepharose. The lectin was a 64-kDa protein with two 32-kDa subunits. It had low thermostability (stable up to 50 °C) and moderate pH stability (stable in pH 3-10). It exhibited anti-proliferative activity on nasopharyngeal carcinoma HONE1 cells with an IC50 of 12.8 μM after treatment for 48 h. It also slightly inhibited the growth of hepatoma HepG2 cells. The lectin may have potential in aiding cancer treatments.

Sulfated modification of longan polysaccharide and its immunomodulatory and antitumor activity in vitro

A water-soluble polysaccharide fraction (LP1) was prepared from Dimocarpus longan Lour. by hot water extraction, DEAE-cellulose and Sephadex G-100 chromatography. Its sulfated derivative (LP1-S) was prepared by the sulfuric acid method. Preliminary tests in vitro showed LP1 and LP1-S could stimulate murine lymphocytes proliferation, increase pinocytic activity of murine macrophages and production of nitric oxide (NO), interleukin 6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) in macrophages. Furthermore, LP1-S exhibited higher antiproliferative activity against human nasopharyngeal carcinoma HONE1 cells in vitro than LP1, which might be caused by the sulfate group in its structures. These results indicated that the LP1-S might be useful for developing safe antitumor drugs or health food.

Purification and Characterization of a Glucosamine-Binding Antifungal Lectin from Phaseolus vulgaris cv. Chinese Pinto Beans with Antiproliferative Activity Towards Nasopharyngeal Carcinoma Cells

A lectin has successfully been isolated from Phaseolus vulgaris cv. Chinese pinto bean using affinity chromatography, ion exchange chromatography, and gel filtration in succession, with a 15.4-fold purification. Investigation of its characteristics revealed that Chinese pinto bean lectin (CPBL) was a 58-kDa dimeric glucosamine-binding protein. Its Mg(2+)-dependent hemagglutinating activity was stable at pH 7-8 and at or below 60°C. When the purified lectin was tested against six fungal species including Phyllosticta citriasiana, Magnaporthe grisea, Bipolans maydis, Valsa mali, Mycosphaerella arachidicola, and Setosphaeria turcica, only the mycelial growth of V. mali was reduced by 30.6% by the lectin at 30μM. The lectin did not exert any discernible antiproliferative effects on breast cancer MCF-7 cells, but was able to suppress proliferation of nasopharyngeal carcinoma HONE-1 cells, with an IC50 of 17.3μM, as revealed by the MTT assay. Since few plant lectins demonstrate antifungal activity against V. mali, and not many others have inhibitory effects on HONE-1 cells, CPBL is a distinctive lectin which may be exploited for development into an agent against V. mali and HONE-1 cells.

Physiological β-catenin signaling controls self-renewal networks and generation of stem-like cells from nasopharyngeal carcinoma

BackgroundA few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.ResultsIntroduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24+ and CD44+ populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-β, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays, a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.ConclusionsWnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks.

Selaginella tamariscina extract suppresses TPA-induced invasion and metastasis through inhibition of MMP-9 in human nasopharyngeal carcinoma HONE-1 cells

BackgroundNasopharyngeal carcinoma (NPC) is known for its high incidence of neck lymph node metastasis, which represents poor prognosis. The present study aimed to examine the anti-metastatic properties of Selaginella tamariscina extract (STE) in human nasopharyngeal carcinoma HONE-1 cells in vitro.MethodsCell viability was examined by MTT assay, whereas cell motility was measured by invasive, migration and would healing assays. Real-time PCR, and promoter assays confirmed the inhibitory effects of STE on matrix metalloproteinase-9 (MMP-9) mRNA level in HONE-1 cells.ResultsThe STE inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HONE-1 cell migration and invasion in a concentration-dependent manner. By zymographic and Western blot analyses, STE was shown to inhibit the activities and expression of MMP-9. Treatment of STE on TPA-induced HONE-1 cells inhibited MMP-9 expression and ERK1/2 phosphorylation without affecting JNK and p38 phosphorylation.ConclusionsSTE inhibits MMP-9 expression and HONE-1 cell metastasis. Its inhibitory effects may involve the Src/FAK/ERK 1/2 pathway. STE may have the potential of being an anti-metastatic agent against NPC.

Cytokeratin 8 silencing in human nasopharyngeal carcinoma cells leads to cisplatin sensitization

By comparing protein profiles of nasopharyngeal carcinoma HONE1 cells to transformed nasopharyngeal epithelial NP 69 cells, several clusters of differentially expressed proteins were identified. The increased expression of cytokeratin 8 (CK8) and pyruvate kinase M2 was a common feature in four NPC cell lines compared to the two transformed epithelial cell lines. Suppression of CK8 was associated with the sensitivity to cisplatin in HONE1 cells; while overexpression of CK8 provided resistance to cisplatin-mediated apoptosis; and this protection occurred through an enhanced phosphorylation of c-Jun NH 2-terminal kinase (JNK). Our findings implicate an underlying molecular mechanism in which CK8 is required for cisplatin resistance.

Apoptosis and expression of cytokines triggered by pyropheophorbide-a methyl ester-mediated photodynamic therapy in nasopharyngeal carcinoma cells

The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5-2μM, MPPa-mediated a drug dose-dependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and beta-chemokines (MIP-1β, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma.

DNA Repair Enzyme,O6-Methylguanine DNA Methyltransferase, Modulates Cytotoxicity of Camptothecin-Derived Topoisomerase I Inhibitors

Two camptothecin-resistant cell lines, CPT30 and KB100, were established and characterized previously in our laboratory. Because enhanced sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and decreased expression of O(6)-methylguanine-DNA methyltransferase (MGMT) protein were observed in these lines, we hypothesized that MGMT may be a determinant of cytotoxicity associated with camptothecin-derived DNA topoisomerase I inhibitors (CPTs). We used the Tet-On system to induce expression of MGMT in Chinese hamster ovary (CHO) cells and RNA interference to knock down MGMT expression in human nasopharyngeal carcinoma HONE-1 cells in order to identify any correlations between MGMT expression and CPTs cytotoxicity. CHO-derived Tet-On-inducible cells (S12+) showed MGMT overexpression and statistically significant more resistance to BCNU, camptothecin, 7-ethyl-10-hydrocamptothecin (SN38), and topotecan than parental CHO cells (p < 0.05), but there was less resistance to CPTs than to BCNU. Knockdown of MGMT expression with small interfering RNA in HONE-1 cells conferred increased sensitivity to BCNU and CPTs compared with mock control. Furthermore, alteration of MGMT expression coincides with CPT-induced cell death and poly(ADP-ribose) polymerase cleavage. There were no differences in protein levels and catalytic activity of topoisomerase I between MGMT-proficient and MGMT-deficient cells from the Tet-On-inducible and small interfering RNA (siRNA) systems. Resistance to CPTs coincided with decreased amounts of protein-linked DNA breaks generated by CPTs in MGMT-proficient cells and vice versa in MGMT-deficient cells. Our data indicate that MGMT can modulate cytotoxicity of CPT-derived topoisomerase I inhibitors.

Chromosome 13q12 region critical for the viability and growth of nasopharyngeal carcinoma hybrids.

Allelic losses of chromosome 13 are often detected in nasopharyngeal carcinoma (NPC) and other cancers, implicating the presence of possible tumor suppressor genes (TSGs) on this chromosome. To identify candidate regions from larger and multiple lost areas observed from direct tumor studies, the technique of monochromosome transfer was utilized to provide functional evidence to verify and define these deletion findings. An intact chromosome 13 was transferred into the NPC HONE1 cell line. Resultant hybrids were used to map putative TSG activity. A critical region at 13q12 was non-randomly eliminated in all surviving microcell hybrids around the marker D13S893; these hybrids were uniformly tumorigenic. Although a known TSG, BRCA2, is mapped close to this critical region, no aberrant expression of this gene was detected in microcell hybrids and other NPC cell lines. These results suggest that at least one novel growth control gene on chromosome 13q12, which is not the BRCA2 gene, is essential for hybrid selection and may play a critical role in tumorigenicity.