Sp1 Predicts Radiotherapy Response In Nasopharyngeal Carcinoma And Berberine Increases Radiosensitivity Of Nasopharyngeal Carcinoma By Inhibiting Sp1

CHAPTER 1 Sp1 IS OVER-EXPRESSED IN NASOPHARYNGEAL CANCER AND IS A POOR PROGNOSTIC INDICATOR FOR PATIENTS RECEIVING RADIOTHERAPYBackground Nasopharyngeal cancer(NPC)is a tumor of epithelial origin with complex etiology.Currently the standard treatment of NPC is radiotherapy,but therapy failure is quite common,making radioresistance an important issue.This study explores the association of specificity protein 1(Sp1)protein expression with clinicopathological significance and disease prognosis in NPC patients receiving radiotherapy.Objective To explores the association of specificity protein 1(Sp1)protein expression with clinicopathological significance and disease prognosis in NPC patients receiving radiotherapy.Methods A total of 82 NPC patients(55 males and 27 females,median age:48 years old)were enrolled and received radiotherapy between September 2011 and March 2014.Tumor tissue and grossly adjacent normal mucosa were obtained in each patient.Sp1 expression was detected by western blot and immunohistochemical analysis,and the associations with clinicopathological status and radiotherapy response were analyzed.Our Results showed Sp1 protein expression was higher in CNE-1 and CNE-2 nasopharyngeal cancer cells than in normal nasopharyngeal mucosal NP69 cells.Results(1)All 82 patients' tissue sections were stained positive for the Sp1 protein,and 39(47.6%)patients showed higher level than adjacent normal mucosa.(2)Sp1-overexpression in the tumor tissue was correlated with a higher tumor stage,nodal status,clinical stage and distant metastasis(P < 0.01).(3)Sp1 expression of nasopharyngeal carcinoma CNE-1 and CNE-2cells is highly than normal nasopharyngeal mucosal NP69 cells.(4)Patients with higher Sp1 expression in pretreatment biopsies had a lower radiotherapy response compared to those with lower expression.Conclusion In conclusion,Sp1 may play roles in radioresistance of nasopharyngeal cancer which attributes to tumor invasiveness,and serve as a novel prognostic marker of NPC radiotherapy.However,further studies are required to validate our findings in larger samples and explore more detailed mechanisms underlying radioresistance of Sp1.CHAPTER 2 BERBERINE SENSITIZES NASPHARYNGEAL CARCINOMA CELLS TO RADIATION THROUGH INHIBITION OF SP1Background Nasopharyngeal carcinoma(NPC)is a tumor of epithelial origin with radiotherapy as its standard treatment.However,radioresistance remains an important issue in the treatment of NPC.This study aims to investigate the effect of berberine on the proliferation,cell cycle regulation,apoptosis,radioresistance of NPC cells and whether Sp1 is a functional target of berberine.Methods The CNE-2 cell line was incubated with different concentrations of berberine(25,50,75,100 ?mol/L)and cell viability and proliferation were detected by MTT assay.The NPC cells were exposed to various doses of 60Co-? rays irradiation(1.33 MV)to measure the radiosensitivity of berberine.Cell cycle analysis was performed by propidium iodide(PI)staining in flow cytometry.Cell apoptosis was determined by Annexin V-FITC and PI double staining.Real-time PCR was performed to analyze the m RNA of Sp1 in berberine treated NPC cells.Western blot was performed to determine protein expressions of Sp1 and EMT marker E-cadherin and Vimentin.Boyden chamber invasion assay was performed to measure tumor invasion capability of NPC cells.Results Berberine showed reduced proliferation and viability of CNE-2 cells in a dose-and time-dependent manner.Berberine induced cell cycle arrest in G0/G1 phase and apoptosis.In CNE-2 cells exposed to 60Co-?irradiation,berberine reduced cell viability at various concentrations(25,50,75,100 ?mol/L).Berberine inhibited cell cycle arrest in G0/G1 phase and induced apoptosis.Berberine significantly decreased m RNA and protein expressions of Sp1 in CNE-2 cells.Mithramycin A,a selective Sp1 inhibitor,can enhance radiosensitivity and apoptosis in CNE-2 cells.Berberine inhibited TGF-?1 induced tumor invasion and suppressed epithelial-mesenchymal transition(EMT)process,as evidenced by increased E-cadherin and decreased Vimentin proteins.Sp1 might be required for TGF-?1-induced invasion and EMT.Conclusion Berberine demonstrated an ability to suppress proliferation,induce cell cycle arrest and apoptosis,and enhance radiosensitivity of the CNE-2 nasopharyngeal carcinoma cells.Sp1 might be a target of berberine which was decreased in radiosensitization of berberine.