Effect of osthole on proliferation and radiosensitivity of poorly differentiated CNE2 stem cells from nasopharyngeal carcinoma / 中草药

Objective To investigate the effect and the underlying mechanism of osthole on the proliferation and radiosensitivity of CNE2 stem cells, one of the poorly differentiated cell lines from nasopharyngeal carcinoma. Methods Poorly differentiated CNE2 stem cells were isolated and cultured in serum-free medium (SFM). Flow cytometry was used to detect biomarkers (CD44+/CD24-low) of stem cells and the activity of ALDH. CNE2 stem cells was treated with different concentrations of osthole (0, 20, 40, 80 μg/mL), and then subject to MTT assay. Additionally, CNE2 stem cell was treated with 40 μg/mL osthole plus different dose of radiation (0, 2, 5 Gy) followed by colony formation assay. Consequently, Western blotting was used to detect the difference of pGSK-3β, β-catenin, and Cyclin D1 protein expression in CNE2 stem cells after the treatment of osthole with or without radiation. Results Poorly differentiated CNE2 stem cells isolated and cultured in serum-free medium (SFM) could be passaged stably. The ratio of CD44+/CD24-low and the activity of ALDH were significantly higher in CNE2 stem cells than that in the parental cells (P < 0.05). Compared to the control group, osthole could obviously suppress the proliferation of CNE2 stem cells in a dose and time dependent manner (P < 0.05). Moreover, colony formation assay revealed that inhibition rate of CNE2 stem cell colony formation was highest after the treatment of osthole plus radiation, followed by the treatment of osthole or radiation alone (P < 0.05). Western blotting indicated that in contrast to the controls, the expression of pGSK-3β, β-catenin, and Cyclin D1 protein was mostly down-regulated in the CNE2 stem cells treated with osthole plus radiation, followed by that treated with osthole or radiation alone (P < 0.05). Conclusion Osthole effectively inhibited the proliferation and increased the radiosensitivity of CNE2 stem cells, probably due to the down-regulation of pGSK-3, β-catenin, and Cyclin D1 in tumor stem cells by osthole.

Study on promoter methylation status of E-cadherin gene in nasopharyngeal carcinoma cell lines / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the role of methylation on E-cadherin inactivation in nasopharyngeal carcinoma (NPC) cell line HNE1 and CNE2, as well as evaluate the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-dC) on cell abilities of proliferation and invasion.</p><p><b>METHODS</b>The expression level of E-cadherin was measured by RT-PCR, Western blot and immunohistochemistry (polymer method), the methyaltion status was analyzed by methylation-specific PCR (MSP), and cell proliferation and invasion were examined by MTT and invasion assay, separately before and after treatment with demethylating agent 5-Aza-dC.</p><p><b>RESULTS</b>The expression level of E-cadherin was down-regulated compared with the normal tissue, simultaneously partially methylated in gene promoter. Treatment with 20 µmol/L 5-Aza-dC increased the expression of E-cadherin and reduced the methylation degree. Moreover, it also significantly suppressed cell growth (27.6% for HNE1 cells and 34.3% for CNE2 cells, P < 0.05) and invasiveness (37.2% for HNE1 cells and 29.7% for CNE2 cells, P < 0.05).</p><p><b>CONCLUSIONS</b>Aberrant methylation around gene promoter region may play an important part in down regulation of E-cadherin in NPC, suggesting a potential therapeutic strategy for demethylating agents such as 5-Aza-dC.</p>

Inhibitory effect of ginsenoside Rg3 on the tube-like structure formation in human nasopharyngeal carcinoma HNE-1 cell line in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the anti-angiogenic effect of ginsenoside Rg3 (Rg3 in abbreviation) in human nasopharyngeal carcinoma HNE-1 cells in vitro.</p><p><b>METHODS</b>The tube-like structure (TLS) formation of HNE-1 cells, cultured in medium with different concentrations of Rg3, was determined by in vitro anti-angiogenic test based on preliminary experiment observing the TLSs formed by HNE-1 cells on Matrigel and their structural characteristics. The VEGF expression level in HNE-1 cells was determined by immunohistochemistry (IHC) and Western-blot test after 48-hour cultured in medium with different concentrations of Rg3.</p><p><b>RESULTS</b>HNE-1 cells could form TLSs and mosaic vessels when mix-cultured with CRL-2480 on Matrigel. Rg3 could inhibit the TLS formation of HNE-1 cells. After 24-hour culture in medium with Rg3 at concentrations of 0, 50, 100 and 200 µg/ml, the number of TLSs were 75.50 ± 6.86, 55.00 ± 11.92, 39.75 ± 7.93 and 24.50 ± 6.25, respectively, which were negatively correlated with the concentrations of Rg3 (r = -0.928; P < 0.01). After 48 hours of culture, the expressions of VEGF significantly declined by IHC test with results as 0.19 ± 0.03, 0.13 ± 0.02, 0.11 ± 0.01, and 0.08 ± 0.01, respectively, which were negatively correlated with the concentrations of Rg3 (r = -0.911; P < 0.01). The expressions of VEGF also gradually decreased as revealed by Western blot test, with corresponding results as 119.49, 111.51, 86.45, and 38.29. All of the tests showed significantly declined results in the group at the concentration of 200 µg/ml Rg3.</p><p><b>CONCLUSION</b>Rg3 can inhibit the vasculogenic mimicry of HNE-1 cells, and the possible mechanism might be associated with the down-regulation of VEGF protein expression in HNE-1 cells.</p>

The promotive effects of N-nitrosopiperidine on the malignant transformation of the immortalized esophageal epithelium induced by human papillomavirus / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Study on the promotive effects of N-nitrosopiperidine on carcinogenesis process was performed, based on the immortalization of human fetal esophageal epithelium induced by human papillomavirus (HPV) 18E6E7 genes.</p><p><b>METHODS</b>The immortalized esophageal epithelium SHEE was induced by HPV18E6E7. The cells at 17th passages were cultured in 50 ml flasks. The N-nitrosopiperidine (NPIP) 0, 2, 4, 8 mmol/L added to the cultured medium of SHEE cells for 3 weeks. The morphology, proliferation and apoptosis of the cells were studied by phase contrast microscopy and flow cytometry. Modal number of chromosomes was analyzed by standard method. Tumorigenicity of the cells was assessed by soft agar colony formation and by transplantation of cells into nude mice. Expression of HPV was detected by Western blot.</p><p><b>RESULTS</b>When cells were exposed to high concentration (8 mmol/L) of NPIP, cell death was increased, leaving a few live cells. In normal cultural medium instead of NPIP proliferative status of the cells restored after 4 weeks and the cells progressed to the proliferation stage with continuous replication and atypical hyperplasia. At the end of the 8th week, the cells appeared with large colonies in soft-agar and tumor formation in transplanted nude mice. When the cells were cultured in 2, 4 mmol/L NPIP the doubling passage was delayed and without tumor formation in transplanted nude mice. Modal number of chromosomes was 61-65, in 8 mmol/L NPIP group and control group, 56-61. Expression of HPV18 appeared in experimental and control groups.</p><p><b>CONCLUSION</b>NPIP promotes malignant change of the immortalized esophageal epithelial cells induced by HPV18E6E7. HPV18E6E7 synergy with NPIP will accelerate malignant transformation in esophageal epithelium.</p>