Effect of Shengmai Yin on Epithelial-Mesenchymal Transition of Nasopharyngeal Carcinoma Radioresistant Cells.

OBJECTIVE: To investigate the mechanism by which Chinese medicine Shengmai Yin (SMY) reverses epithelial-mesenchymal transition (EMT) through lipocalin-2 (LCN2) in nasopharyngeal carcinoma (NPC) cells CNE-2R. METHODS: Morphological changes in EMT in CNE-2R cells were observed under a microscope, and the expressions of EMT markers were detected using quantitative real-time PCR (RT-qPCR) and Western blot assays. Through the Gene Expression Omnibus dataset and text mining, LCN2 was found to be highly related to radiation resistance and EMT in NPC. The expressions of LCN2 and EMT markers following SMY treatment (50 and 100 µ g/mL) were detected by RT-qPCR and Western blot assays in vitro. Cell proliferation, migration, and invasion abilities were measured using colony formation, wound healing, and transwell invasion assays, respectively. The inhibitory effect of SMY in vivo was determined by observing a zebrafish xenograft model with a fluorescent label. RESULTS: The CNE-2R cells showed EMT transition and high expression of LCN2, and the use of SMY (5, 10 and 20 µ g/mL) reduced the expression of LCN2 and reversed the EMT in the CNE-2R cells. Compared to that of the CNE-2R group, the proliferation, migration, and invasion abilities of SMY high-concentration group were weakened (P<0.05). Moreover, SMY mediated tumor growth and metastasis in a dose-dependent manner in a zebrafish xenograft model, which was consistent with the in vitro results. CONCLUSIONS: SMY can reverse the EMT process of CNE-2R cells, which may be related to its inhibition of LCN2 expression. Therefore, LCN2 may be a potential diagnostic marker and therapeutic target in patients with NPC.

Effect of GADD45G on the radioresistance of nasopharyngeal carcinoma cells.

The development of radioresistance by nasopharyngeal carcinoma (NPC) cells almost always results in tumor recurrence and metastasis, making clinical treatment of the disease difficult. In this study, the mechanism of radioresistance in NPC cells was investigated. First, a gene array and quantitative reverse-transcription-PCR assays were used to screen for genes exhibiting significantly altered expression in the DNA damage signaling pathway. Based on those results, GADD45G was further studied in the context of radioresistance. A GADD45G-knockout NPC cell line (CNE-2R-KO) was constructed using CRISPR-Cas9 technology and used for a comparison of differences in radioresistance with other radiosensitive and radioresistant NPC cells, as evaluated using colony formation assays. Cell cycle changes were observed using flow cytometry. Cell proliferation and migration were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and wound healing assays, respectively. The sequencing results revealed the successful construction of the CNE-2R-KO cell line, the radiosensitivity of which was higher than that of its parent radioresistant cell line owing to the GADD45G knockout. This was likely related to the increase in the number of cells in the G1 phase and decrease in those in the S1 phase as well as the increased cell proliferation rate and decreased migratory ability. GADD45G is associated with radioresistance in NPC cells and likely has a role in the occurrence and metastasis of NPC.

Effect of Shengmai Yin on the DNA methylation status of nasopharyngeal carcinoma cell and its radioresistant strains.

Shengmai Yin (SMY) is a Chinese herbal decoction that effectively alleviates the side effects of radiotherapy in various cancers and helps achieve radiotherapy's clinical efficacy. In this study, we explored the interaction mechanism among SMY, DNA methylation, and nasopharyngeal carcinoma (NPC). We identified differences in DNA methylation levels in NPC CNE-2 cells and its radioresistant cells (CNE-2R) using the methylated DNA immunoprecipitation array and found that CNE-2R cells showed genome-wide changes in methylation status towards a state of hypomethylation. SMY may restore its original DNA methylation status, and thus, enhance radiosensitivity. Furthermore, we confirmed that the differential gene Tenascin-C (TNC) was overexpressed in CNE-2R cells and that SMY downregulated TNC expression. This downregulation of TNC inhibited NPC cell radiation resistance, migration, and invasion. Furthermore, we found that TNC was hypomethylated in CNE-2R cells and partially restored to a hypermethylated state after SMY intervention. DNA methyltransferases 3a may be the key protein in DNA methylation of TNC.