The Inhibit Effects And Mechanisms Of Caffeic Acid Phenethyl Ester In Nasopharyngeal Carcinoma

Background Nasopharyngeal carcinoma(NPC),derived frequently from the epithelium of Rosenmüller fossa.,is a special subtype of head and neck malignancy,The racial/ethnic and geographic distribution of NPC is distinctive,with high incidence in some countries including Southeast Asia,southern China,especially in Guangdong and Guangxi province.The annual incidence of southern China is about 30 cases per 100000 persons.NPC poses a serious health problem in southern China and is one of the most comman cancers in china.In contrast to fewer than 1 case per 100000 persons in the Western country.The pathogenesis and development of NPC is a multistep process.Previous epidemiological studies suggest three major aetiological factors for NPC: genetic susceptibility,environmental factor and latent EBV infection.With the development of radiotherapy technology,NPC patients in early-stage(stage I),treated with radiotherapy alone has good efficacy,and leads to a 5-year overall survival rate of over 90%.However,up to 70% of patients present with locoregionally advanced(stage III-IV)disease at diagnosis.Although locoregionally advanced patients can be treated with chemoradiotherapy(CRT),there still were approximately 30% of patients failed in CRT because of distant metastasis..So it is positived for improving over surviral rate of patients with advanced nasopharyngeal carcinoma and important to identify some new drugs sensitived to radiotherapy or chemotherapy.Epstein-Barr virus(EBV)infection play an important role in carcinogenesis.However,most NPC cell lines may not express EBV genome in long-term culture,so the preclinical study that focus on drug and EBV targeted should be more reliable by choosing EBV-carrying NPC cell lines.Caffeic acid phenethyl ester(CAPE)is the main polyphenol extracted from honeybee propolis and polyphenol.It has anti-bacterial infection,anti-oxidant,anti-inflammatory,and anti-tumor properties.CAPE can inhibit NF-?B pathway by supressing the expression TNF,inhibiting phosphorylation and degradation of I?Ba,impeding nuclear translocation of p65 in cancer cells.The sustained activation of nuclear factor-?B(NF-?B)signaling is a key event in the regulation of the initiation and progression of human cancer.NF-?B signaling play an important role during NPC carcinogenesis and NF-?B is continuously activated in NPC.The EBV-encoded LMP1 is one of the major oncoproteins in NPC and most of downstream effects of LMP1 overexpression are mainly dependent on it's activation on NF-k B signaling.Constitutive activation of NF-k B signaling contribute to persistence of EBV and immune escape of EBV infected cells.NF-k B is the main carcinogenic pathway and negative regulators of the NF-k B pathway has high prevalence of genetic variants in NPC.Reprogramming of tumor cell energy metabolism is one of characteristics of cancer.In addition to glucose metabolism,lipid metabolism is also a very important metabolic pathway in the body.Its storage and utilization in cells is critical to maintaining normal energy metabolism of cells,and participates in the maintenance of intracellular homeostasis.Fatty acid metabolism is an important component of the body's lipid metabolism.More and more studies have shown that fatty acid metabolism is closely related to the occurrence and development of tumors,and participates in the occurrence and development of various malignant tumors.Purpose 1.This study aimed to assess the inhibitory effect of CAPE in nasopharyngeal carcinoma(NPC),and further elucidate the possible molecular mechanism.2.This study aimed to explor the synergistic action of CAPE in radiotherapy sensitivity of NPC cell lines.3.To evaluate the effect of CAPE on NPC lipid energy metabolism and dysregulation of key gene in the metabolic pathway.Methods and Result 1.CAPE inhibited the malignant biological behavior of NPC cells 1)CAPE suppressed the proliferation and colony-formation ability in NPC cells CCK-8 assay was used to analyze cell proliferation ability.The data shown that CAPE can inhibit NPC cell proliferation.With increasing CAPE concentration and time of treatment,the viability of these cells remarkably decreased.But the non-malignant nasopharyngeal epithelial cell lines NP69 and NP460 were more resistant to CAPE treatment.Colony formation assay was used to evaluate the clonogenic ability and the result indicated that CAPE suppressed the colony-formation ability of NPC cells.2)CAPE treatment retarded the growth of NPC cells xenograft in nude mice Administration of CAPE by gavage resulted in reduction of tumor volume compared with DMSO control group,suggesting that CAPE treatment inhibited NPC tumor growth both in vivo and in vitro.3)CAPE attenuate migration and invasion of NPC cells To further determine the effect of CAPE on NPC cell migration and invasive abilities,we used transwell and wound-healing assay.CAPE treatment markedly attenuated the migratory ability in NPC cells.Similarly,on wound-healing assay,cellular motility was significantly slower with CAPE than DMSO treatment.Thus,CAPE could inhibit NPC cell migration and invasion in vitro.2.Possible anti-cancer mechanism of CAPE 1)CAPE promoted apoptosis in NPC cells Cell apoptosis was detected by flow cytometry,As compared with the DMSO control,after treatment with CAPE for 24 hr,the proportion of total apoptotic cells was increased.Compared with increasing the activity of caspase3/7,upregulating Bax expression and downregulating Bcl-xl expression in protein level.2)CAPE induced G1 cell cycle arrest in NPC cells Through flow cytometry,we further found reduce number of cells in the G2/M phase and increased number in the G1 or S phase in NPC cells under CAPE treatment.Further western blot indicated that the protein level of Rb,p-Rb,CDK4,and CDK6(some cell cycle relative protein)was downregulated.3)CAPE show the effect of reversing the epithelial-mesenchymal transition(EMT)pathway in NPC cells.Western blot analysis revealed that the expression of E-cadherin(a marker of epithelium cell)was elevated and the expression of vimentin(a marker of mesenchymal cell)was remarkably downregulated after CAPE treatment,while MMP9(an important effector for cell invasion ability)was decreased upon the treatment of CAPE.4)CAPE impeded nuclear translocation of p65 to inhibit NF-?B signaling pathway in NPC cells We found a marked reduction of p65 level in the nuclear fraction but not cytoplasm,so CAPE mainly hindered the nuclear translocation of p65,thereby suppressing the NF-?B signaling pathway CAPE specifically inhibited nuclear factor ?B(NF-?B)signaling pathway by suppressing p65 subunit translocation from cytoplasm to nucleus and then cause inhibition of NF-?F-regulated downstream molecules and transcriptional activity signaling pathways 3.CAPE enhanced the sensitivity of radiation in NPC cell lines and combined with cisplatin has the effect of inhibiting the proliferation of NPC cells,rendering NPC cells more sensitive to cisplatin Through the colony formation experiment,we found that the colony forming ability of NPC cells treated with CAPE was significantly decreased after exposure to radiation,suggesting that CAPE can increase the radiosensitivity of NPC cells.In addition,through the CCK8 experiment,we found that CAPE and cisplatin have synergistic effects in inhibiting the proliferation of NPC cells.These results suggest that CAPE may be used as an adjunct to chemotherapeutic and radiationtherapy sensitization for nasopharyngeal carcinoma therapy.4.CAPE reduced accumulation of lipid droplets in NPC cell cytoplasm and regulated the content of fatty acid in NPC cells by regulating key genes of fatty acid metabolism,including FASN,ACACA,ATGL,HSL Through the Bodipy-stained lipid droplets experiment,we found that the accumulation of lipid droplets in the cytoplasm of NPC cells after CAPE treatment was reduced.Analysis of micro-array indicated that,CAPE alter the expression of lipid metabolism related genes.And the key molecule ATGL,HSL were upregulated both in the protein level and the m RNA level.It was further proved by experiments of Quantitative Real-time PCR(Q-PCR)andImmunofluorescence.Q-PCR also show that the expression of FASN,ACACA was downregulated by CAPE.Meanwhile,gas chromatography-mass spectrometry(GC-MS)indicated that CAPE caused change of fatty acid content.5.Different expression profile of CAPE-regulated fatty acid metabolism genes in nasopharyngeal carcinoma and the correlation between transcription level of these four genes and clinical stage or progression-free survival of NPC patients.Analysis of expression profiling by array from GEO platform indicated that,FASN,ACACA appeared higher expression in tumor tissue and ATGL,HSL appeared lower expression compared with non-tumor tissue.Using clinical imformations for analysis,the expression of FASN,ACACA was relative high while ATGL,HSL was relative low in late stage NPC patients.Survival analysis indicated that,high expression of ATGL,HSL are associated with better progression-free survival and low expression of FASN,ACACA are associated with better progression-free survival.Conclusion Our results indicate that CAPE inhibits the malignant biological behavior of NPC cells,including proliferation,colony formation,invasion and migration.In addition,CAPE increased apoptosis and caused cell cycle arrest in NPC cells.The inhibitory mechanisms of CAPE included promotion of apoptosis by increasing caspase3/7 activity and dysregulation of anti-apotosis or pro-apoptosis protein,induction of cell cycle arrest by dysregulation of cell cycle relative protein,inhibition of NF-?B signaling pathway by inhibiting the entry of the P65 subunit into the nucleus and upregulating the expression of the NF-?B negative regulatory gene and reversal of EMT.The combination of CAPE enhances the radiosensitivity of NPC cells and synergistically inhibits the proliferation of NPC cells with the chemotherapy basic drug cisplatin.CAPE regulated the fatty acid metabolism in NPC cells by regulating the key genes of fatty acid metabolism(including down-regulating FASN,ACACA,up-regulating ATGL,HSL).CAPE may play a tumor suppressing role by altering the fatty acid metabolism of NPC cells.It is suggested that CAPE is a potential dual-targeted NPC drug that targets NF-?B and energy metabolism.Our study further demonstrated the differential expression profiles of four key lipid metabolism genes between nasopharyngeal carcinoma and normal nasopharyngeal tissues.It was found that FASN and ACACA were up-regulated while ATGL and HSL were down-regulated in nasopharyngeal carcinoma.These expression is associated with clinical stage and disease progression-free survival prognosis in NPC patients.So these gene may be a new therapeutic target for NPC or a prognosis-related gene.