Casticin Inhibits Nasopharyngeal Carcinoma Cell Proliferation And Drug Resistance Through Targetting PI3K

Background and ObjectivesNasopharyngeal carcinoma(NPC)is a malignant tumors that occurs in the nasopharyngeal epithelium.In China this disease often occurs in southern area,especially in Guangdong Province,also named "Guangdong cancer".Nasopharyngeal cancer is tumor with high degree of malignancy in which cervical lymph node metastasis and distant metastasis occurs early,and is closely associated with Epstein Barr virus,most patients missed the best treatment time when they make definite diagnosis,this phenomenon increased economic burden of patients,and affecting the quality of life.Therefore,study on the traditional chinese medicine monomer taget the primary genes and signaling pathways involved of NPC may contribute to the exploration of pathogenesis.Phosphatidylinositol 3-kinases(PI3Ks)are a family of lipid kinases that integrate signals from cytokines,growth factors,and components of the cell environment,and translate those into intracellular signals that regulate multiple signaling pathways.These pathways control many cellular processes and physiological functions,including cell survival,growth,motility,proliferation,and metabolism.In recent years,to research the relationship between PI3K/AKT signaling pathway and drug resistance is more and more important to the clinical research,PIK3CA is considered as a new target for involed in chemotherapy and drug resistance.PIK3CA paly key role in the upstream of the PI3K/AKT pathway and it involved in the expression and activation of multiple tumors.Abnormal PI3Kactivating alterations are frequent in a variety of cancers,making this class of enzymes important drug targets.Tremendous efforts have been devoted to developing effective PI3K inhibitors for cancer therapy.However,clinical trials of PI3K-directed drugs,consisting largely of non-isoform-selective pan-PI3K inhibitors,have not yielded exciting results.With the continuous progress of modern drug research,tumors drug targeted therapy has made rapid development.Traditional Chinese medicine is an important part of medicine.In recent years,a large number of basic and clinical research found that a variety of chinese herbal medicine has anti-tumors effect,the main pathways include inhibition of tumors cell proliferation and metastasis,which induced tumors cell autophagy,apoptosis,necrosis,promote cancer stem cell differentiation and regulation of tumors cell signaling to the occurrence and development of anti-tumors.As a traditional Chinese medicine,the study of its anti-tumors effect has provided great support for the development of cancer therapy.Traditional Chinese medicine targeting tumors abnormal expression of the kinase and abnormal activation of the signal pathway has become more and more important and the drug research can bring new hope for the treatment of cancer in the future.Casticin(3,5-dihydroxy-3,4’,6,7-tetramethoxyflavone),which also known as vitexicarpin,is one of the main active constituents from Chinese traditional herb Fructus Viticis.It is used as an anti-inflammatory agent[5].Many studies reported that Casticin significantly inhibits proliferation and migration of many type ofcancer cells,including leukemia,ovary,stomach,liver,lung,and colon cancer cells.However,the role of casticin in inhibiting nasopharyngeal carcinoma has not been investigated.More importantly,the molecular target and mechanisms are still unknown.The study be based on large number of literature and explore the drug target and more pharmacological action Casticin on nasopharyngeal carcinoma and also its molecular mechanism in in vivo and in vitro.Purpose1.To study the effects of Casticin on the growth,proliferation,invasion and migration of nasopharyngeal carcinoma cell lines;2.To study the effects of Casticin on the nasopharyngeal carcinoma stem cells;3.To study the effect of Casticin on the apoptosis and cell cycle of nasopharyngeal carcinoma;4.Reverse virtual screening analysis screening of drug targets for Casticin and verify it;5.To investigate whether Casticin affects the biological behavior of NPC cells by PI3K/mTOR signaling pathway;6.To study whether Casticin affects the proliferation of nasopharyngeal carcinoma cells in vivo.Contents and method1.Effects of Casticin on the biological function of nasopharyngeal carcinoma cells1.1 The effect of Casticin on the cell viability of 12 nasopharyngeal carcinoma cell lines including normal nasopharyngeal epithelium named NP69 was detected by CCK8 assay,and the IC50 value of drug action was clarified;1.2 Wound Healing experiment,plate clone formation experiment to observe the effect of Casticin on cell growth and proliferation ability,to determine the inhibition and killing effect of Casticin on nasopharyngeal carcinoma cells;1.3 Tumors sphere culture was used to observe the effect of Casticin on the size and ball formation rate of nasopharyngeal neoplasms.Western Blotting technique was used to detect the expression of Nanog,SOX2 and Oct-4 in nasopharyngeal carcinoma and to study the effect of Casticin on cancer stem gene of NPC;1.4 Flow cytometry for apoptosis experiments;1.5 Flow cytometry was used to detect the cell cycle of nasopharyngeal carcinoma cell line and observe the effect of Casticin on the apoptosis and cell cycle of nasopharyngeal carcinoma cells;1.6 The effect of Casticin on nasopharyngeal carcinoma cells was observed by subcutaneous tumors formation in nude mice.2.The mechanism of Casticin on nasopharyngeal carcinoma inhibition2.1 Drug reverse virtual screening screening Casticin potential drug target,molecular docking software were used to simulate the interaction model between Casticin and the targetting protein;2.2 Kinase spectrum test to verify the virtual screening of drug targets;2.3 Westernblot was used to detect the effect of Casticin on the phosphorylation of the downstream substrate protein of the candidate protein kinase and compared with the known kinase inhibitor.3.SPSS 20.0 statistical software is used for data analysis.Scaling experiments,Transwell experiments,tumor sphere formation test results uesd two independent samples t test(Independent-Samples T,Test);two or more samples of the average number of single factor analysis(One-way ANOVA),such as variance Homogeneous,using Levene’s Test method,such as variance,using Welch method of approximate variance analysis,multiple comparison using Dunnett T3 method.In this paper,the inhibitory rate of drug growth on cells was analyzed by factorial design variance.For nude mice in vivo,the tumor volume was compared using the repeated data to measure the variance analysis.The tumor weight and organ weight ratio were compared with the independent t test.The statistical results in this paper are expressed as mean±standard deviation(x±s).P<0.05 was considered statistically significant.Results1.Casticin inhibited the proliferation of nasopharyngeal carcinoma cells1.1 CCK8 experiments showed that Casticin can inhibit the cell viability of different nasopharyngeal carcinoma cell lines in concentration and time dependent manner.However,the normal nasopharyngeal mucosal epithelial cells which name is NP69 was insensitive to Casticin;1.2 Wound Healing test showed that the cell lateral migration was decreased after Casticin treatment;and the results of transwell and Boyden test show that Casticin can inhibit longitudinal migration capability of NPC cells;1.3 Tumor sphere formation experiments showed that the number of tumor sphere was less than the control group after been treated with Casticin,and the sphere diameter of tumor sphere was significantly smaller than the untreated group.Western blot results showed that Casticin can reduce the expression of cancer stem protein in NPC.;1.4 FITC/PI apoptosis test showed that Casticin induce the early and also late apoptosis rate of nasopharyngeal carcinoma cells in a dose-dependent and timedependent manner.Western blot results showed that Casticin could increase the expression level of apoptosis-related proteins Cleaved Caspase3 and PARP,increased Bax/Bcl2 expression ratio.2.In vivo experiments and mechanism validation2.1 Nude mice subcutaneous tumor formation test proved that Casticin can significantly reduced the tumorigenic ability of nasopharyngeal carcinoma cells in vivo;2.2 Drug reverse virtual screening gives a series of potential target proteins of Casticin,in which the PI3K family and PDPK1 get high socre.And the results of Autodock molecular docking show that the molecular structure of the inhibitor of PI3K110a is similar with Casticin,and both of them take bond by hydrogen and hydrophobic bond between small molecule compounds and the target protein,suggesting that PI3K110a may be one of potential targets for Casticin;2.3 The results of Kinase spectrum showed that Casticin had strong sensitivity to PI3K family and especially the mutant type;2.4 Westerntblot results show that Casticin inhibits PI3K/AKT pathway.Conclusions1.Casticin inhibits the migration,proliferation and invasion of nasopharyngeal carcinoma cells.2.Casticin inhibits the stemness of nasopharyngeal carcinoma cells in tumor sphere formation and also in protein level.3.Casticin affecting the cycle of nasopharyngeal carcinoma cells,and make induction of apoptosis through regulation of expression of apoptosis-related proteins.4.Casticin inhibits the proliferation of nasopharyngeal carcinoma cells through PI3K/AKT/Mtor signaling pathway.5.PI3K is medicine target of Casticin,and cansticin is a pan-inhibitors of the PI3K protein family incloud its mutant type.6.Casticin plays a combination effect of PI3K inhibitors and PDPK1 inhibitors,so can reverse the drug resistance of BYL179.