2-methoxyestradiol Inhibits The Proliferation And Migration And Reduces The Radioresistance Of Nasopharyngeal Carcinoma CNE-2 Stem Cells Via NF-κB/HIF-1 Signaling Pathway Inactivation And EMT Reversion

Objective :Accumulating evidence indicates that cancer stem cells(CSCs) are the source of resistance to radiation therapy(RT); however, the mechanism of this resistance remains unclear. 2-Methoxyestradiol(2-ME2) is a metabolic product of estrogen in the body. Recent studies have found that 2-ME2 regulates the activation of transcription factors, including NF-κB/HIF-1, thus contributing to tumor cell apoptosis and chemosensitivity; therefore, 2-ME2 is being studied as a potential anticancer drug. The purpose of this study is to determine the effect and mechanism of 2-ME2 inhibits nasopharyngeal carcinoma CNE-2 stem-like cell(NPCSC) proliferation and migration and reduces NPCSC radioresistance. This study has important significance for reducing the radioresistance of these cells to improve the cure rate of NPC.Methods : 1 NPCSC formation and identification Enriched nasopharyngeal carcinoma stem cells(NPCSC) from CNE-2 cells with serum-free culture system and identified the stemness by re-differentiation observation, soft AGAR cloning, Transwell assay, flow cytometry(FCM), RT-PCR and clone formation assay. Then, the NPCSC was treated by 2-ME2 with 0/4/8μM. CCK-8 and Transwell assay were used to detect the proliferation and migration of NPCSC. 2 The effect and mechanism of 2-ME2 on NPCSC The NPCSC treated with 2-ME2 with different concentrations were radiated by X ray with 0/2/4/8 GY, clone formation assay was used to detect the radiosensitivity of NPCSC. Meanwhile, flow cytometry(FCM) and western blot were used to detect the proportion of CD133+ cells and the expression of HIF-1α and NF-κB p65 and makers of epithelial-mesenchymal transition(EMT). 3 The effect of NF-κB p65 on the anti-cancer of 2-ME2 to NPCSC Knock down of NF-κB p65 using shRNA. The NPCSC treated with 2-ME2 with different concentrations were radiated by X ray with 2 GY. CCK-8, Transwell assay and colony formation assay was used to detected the effect of NF-κB p65 on the anti-cancer of 2-ME2 to NPCSC. RT-PCR and western blot was used to detect the related mechanism of gene and protein.Results: 1 The NPCSC enriched with this method had unlimited self-renewal and re-differentiation ability, stronger cloning and migration ability, higher percentage of CD133 possitive cells(p <0.01) and stemnessrelevant gene proportion(p <0.05), and resistant to radiation, compared with parental cells. 2 After NPCSC treated with 2-ME2, with the increase of the concentration, the proliferation and migration decreased significantly; the radio-resistance decreased. Meanwhile, the proportion of CD133+ cells decreased; the protein expression of HIF-1α and NF-κB p65 and N-cad and Vimentin decreased(p <0.01), E-cad increased(p <0.01). 3 The viability of CSC-NC significantly decreased in a 2-ME2 concentration-dependent manner; the number of CSC-shR that migrated to the other side of the membrane decreased more significantly with increasing concentrations of 2-ME2 compared to the CSC-NC; 2-ME2 reduced the radioresistance of NPCSCs and that NF-κB p65 further enhanced the anti-radioresistance effects of 2-ME2; after NF-κB p65 knockdown, the mRNA and protein levels of p65 and HIF-1α decreased significantly; The protein levels of the EMT markers N-cadherin and Vimentin were decreased, but the protein expression of E-cadherin increased.Conclusion: 2-ME2 inhibits NPCSC proliferation, migration and radioresistance possibly by inhibiting the NF-κB/HIF-1 signaling pathway and reversing EMT.