The Inhibitory Effect And Mechanism Of Ginkgolic Acid In Growth And Development Of Nasopharyngeal Carcinoma

Introduction:Nasopharyngeal carcinoma(NPC)is a malignant tumor originating from the nasopharyngeal epithelium.The overall incidence of NPC is not high in the world.But most of the patients are in East and Southeast Asia.Radiotherapy combined with chemotherapy was the main measure for the treatment of locally advanced NPC.However,the radiotherapy and chemotherapy resistance often leads to the occurrence of distant metastasis,which also causes poor prognosis for patients with NPC.In order to improve the treating dilemma,it is imperative to develop new anti-tumor drugs.Ginkgolic acid(GA),as a small molecule compound with potential anti-tumor activity,has received increasing attention in recent years.But its effect on nasopharyngeal carcinoma and the related mechanisms are still unclear.This study aimed to assess the inhibitory effect of GA in NPC,evaluate the synergistic action of GA in chemotherapy sensitivity of NPC cell lines and further elucidate the possible molecular mechanism involved.Methods:This was investigated in the present study using 5-8F and CNE2 NPC cells and NP69 human immortalized nasopharyngeal epithelial cells treated with GA.We analyzed cell proliferation with the Cell Counting Kit-8 assay,cell migration and invasion with the wound healing and transwell invasion assays and apoptosis with flow cytometry respectively.And the expression of proteins related to these cellular processes were analyzed by western blotting,which including the cycle-related proteins Cyclin D3,Cyclin D2,cyclin-dependent kinase 6(CDK6)and apoptosis-related protein B-cell leukcmia/lymphoma 2(Bcl-2),Bcl2-associated X protein(Bax),c-casp-9,c-casp-3,poly-ADP ribose polymerase(PARP)cleavage fragment and protein kinase B(Akt)/nuclear factor kappa-B(NF-κB)signaling pathway related proteins NF-κBp65,NF-κBp-p65,Akt,p-Akt,IκBα,and p-IκBα,respectively.Finally,the inhibitory effect of the combined GA and 5-fluorouracil(5-FU)treatment for NPC cells was verified in vitro by flow cytometry and in vivo through xenograft model.Results:We found that GA treatment decreased the survival and invasive capacity of NPC cells and GA also induced cell apoptosis in a dose-dependent manner;this was accompanied by down-regulation of Bcl-2,up-regulation of Bax,and activation of caspase-9/-3 and PARP.Moreover,GA induced G0/G1 phase arrest in NPC cells with the low expression of CDK6 and its regulators cyclin D2 and cyclin D3.The expressions of NF-κBp-p65,Akt,p-Akt,IκBα,and p-IκBα were significantly reduced by the treatment of GA(0μM,50μM and 100μM)and there was no significant difference in the expression level of NF-κBp65.Besides,GA inhibited the activation of Akt/NF-κB signaling to NPC cells.This effect was potentiated by combined treatment of GA and 5-FU,which also enhanced 5-FU-induced apoptosis.In the nude mouse xenograft models,GA significantly inhibited the growth of 5-8F transplanted tumors by comparing the GA group and the solvent group.At the same time,comparing the 5-FU group and the GA and 5-FU combined group,we found that the combined application of GA and 5-FU had a synergistic tumor suppressive effect.Conclusions:Ginkgolic acid inhibits the proliferation,migration and invasion of 5-8F and CNE2.Moreover it induces the G0/G1 phase arrest of nasopharyngeal carcinoma cells by inhibiting the expression of CDK6,Cyclin D3 and Cyclin D2.In addition,ginkgolic acid induces 5-8F and CNE2 apoptosis by inhibiting the caspase pathway,and mediates anti-tumor effects by down-regulating the Akt/NF-κB signaling pathway.Our findings also reveal that GA and 5-FU not only induce the apoptosis of NPC cells synergistically in vitro,but also inhibit the growth of NPC xenografts synergistically in vivo.And their anti-tumor effects may be mediated by down-regulation of Akt/NF-κB signaling pathway.These findings indicate that GA may be effective as an adjuvant to conventional chemotherapy drugs in preventing the progression of NPC.