Study Of Resveratrol-mediated Radiosensitization Of Nasopharyngeal Carcinoma Cells

ObjectivesThe study aims to eludidate the molecular mechanism by which resveratrol(RSV)enhances the radiosensitivity of nasopharyngeal carcinoma(NPC)cells.The research data lays foundation for the potential clinical application of resveratrol.Method1.Effect of resveratrol on viability and proliferation of NPC cells.1.1 The viability of NPC cells after treatment of resveratrol was examined by MTT method.1.2 Colony Formation Assay was used to determine the proliferation of on drug-treated or untreated NPC cells.·1.3 NPC cells were exposed to different concentrations of resveratrol and the cell cycle profile was analyzed by flow cytometry.2.Effect of resveratrol on the radiosensitivity of NPC cells.2.1 Colony formation assay used to determine the the survival rate of NPC cells with the combinatory treatment of IR and resveratrol.Cell survival curves conform with the linear-quadratic formula.2.2 The apoptosis of NPC cells treated with IR and resveratrol was analyzed by Flow cytometry with PI/Annexin V stain.3.Effects of resveratrol on the expression of radiosensitization-related proteins.3.1 Western Blotting used to detect the expression of radiosensitivity-related proteins after treatment of resveratrol(50 ?moL/L).3.2 Western Blotting used to detect the expression in NPC cells after treatment of resveratrol with different doses.3.3 Western Blotting was used to detect the expression of related proteins in NPC cells after different time intervals of resveratrol treatment.3.4 The effect of resveratrol on the proliferation of of NPC cells after knockdown of E2F1 by lentivirus system.3.5 The effect of resveratrol on the proliferation of of NPC cells stably overexpressing E2F1.4.In vivo experiment:the effect of resveratrol on radiosensitization of NPC cells exografted to the flank of nude mice.Results1.Effects of resveratrol on the viability and proliferation of NPC cells.1.1 The NPC cells was treated with different concentrations of resveratrol for 24 hours.MTT assay showed 25 ?moL/L or 50 ?moL/L resveratrol has no effect on cell viability of NPC cells.1.2 The NPC cells was treated with 50 ?moL/L of resveratrol for 24 hours.The rate of colony formation of NPC cells was significantly inhibited.1.3 With exposure to different concentrations of resveratrol,the percentage of NPC cells in G0/G1 phase increased from 33.40%by 81.68%,indicating that resveratrol could in dose-dependent manner block NPC cells in G0/G1 phase.1.4 We optimized the treatment dose and time of resveratrol(50 ?moL/L,24 hours)for the following experiment.2.Effect of resveratrol on sensitization of NPC cells in radiotherapy.2.1 Irradiation torture decreased the colony formation rate in dose-dependent manner.2.2 The apoptotic rate of NPC cells was significantly increased after resveratrol combined with radiotherapy(p<0.01).3.Effect of resveratrol on the expression of radiosensitization-related proteins in NPC cells.3.1 Resveratrol(50 ?moL/L)inhibited expression of E2F1,down-regulated the level of p-AKT.By contrast,the level of p-ERK has no change.3.2 Resveratrol downregulated the expression level of E2F1 and p-AKT in dose-dependent manner,but showed no effect on the level of p-ERK and ERK.3.3 Resveratrol time-dependently downregulated the expression level of E2F1 and p-AKT,but showed no effect on the level of p-ERK.3.4 NPC cells with stable knockdown of E2FI(CNE-1-shE21)and its controlCNE-1-NC was constructed using lentivirus system.The depletion of E2F1 significantly surpressed the expression of p-AKT,but had no effect on the level of p-ERK.The colony formation rate of CNE-1-shE2Fl cells was significantly decreased comparing with CNE-1-NC cells.Cell cycle analysis by flow cytometry showed that CNE-1-shE2F1 arrested at GO/G1 phase.3.5 NPC cells with stable overexpressing of E2FI(CNE-1-E2F1)and its control CNE-1-NC was constructed using lentivirus system..After treated with 50 ?moL/L of resveratrol,WB was detected by overexpression of E2F1 in CNE-1,Which could significantly inhibit the expression of p-AKT,but had effect on the level of p-ERK.The clonal formation showed that E2F1 overexpression in CNE-1 could inhibit the colony formation rate induced by resveratrol.Cell cycle analysis by flow cytometry showed that overexpression of E2F1 in CNE-1 inhibits resveratrol-induced arrested at GO/G1 phase.4.In vivo experiments showed that the combination treatment of radiotherapy and resveratrol significantly reduced the tumor volume and weight comparing with resveratrol or IR treatment alone(p<0.01).These result indicated that resveratrol can enhance the NPC cells sensitivity to radiation therapy.ConclusionThis study showes that resveratrol can enhance the sensitivity of NPC cells to radiotherapy,whose mechanism might be down-regulating E2F1 and inhibiting p-AKT after resveratrol treatment on NPC cells.