Molecular Mechanism Research Of Quercetin-3-methyl Ether Inhibiting N-nitrosomethylbenzylamine Induced Esophageal Carcinogenesis

Worldwide,esophageal cancer is one of the high incidences and high mortality of cancer.Esophageal squamous cell carcinoma(ESCC)and adenocarcinoma(EAC)are the two major pathological types of esophageal cancer.Currently,the clinical methods of early diagnosis and treatment for esophageal cancer are still very limited,so the five-year survival rate of esophageal cancer is only 15-20%.The occurrence of esophageal epithelial carcinogenesis has a multi-stage development process from hyperplasia,dysplasia to carcinoma in situ and finally esophageal cancer.The remarkable feature of esophageal cancer is its two-way unstable development characteristic.Esophageal cancer has the developmental characteristics of two-way instability: it can rapidly develop into cancer,or remain unchanged for many years at a stage,and even return to normal tissue.Nitrosamines and their precursors are closely related to the occurrence of human esophageal cancer,can also induce the occurrence of esophageal cancer in animals.Therefore,the N-nitrosomethylbenzylamine(NMBA)-induced rat esophageal cancer model has been regarded as a mature animal model for studying the molecular mechanism of esophageal cancer and ESCC chemoprevention.Growth factor and growth factor receptor-mediated signaling pathways play an important role in the cell transformation.The phosphatidylinositol 3-kinase(PI3K)/AKT/mTOR pathway involves in cell differentiation,cellular metabolism and cell proliferation,and when this signaling pathway is aberrantly activated,it promotes tumor growth and cell survival.Activation of ERK1/2 is reported to be involved in cell proliferation,differentiation and migration.Mitogen-activated protein kinase(MAPK)pathway-activated kinases enhance Cyclooxygenase-2(COX-2)inflammation-induced carcinogenesis.Prostaglandin E2(PGE2)produced by COX-2 metabolism promotes cancer progression by regulating signaling pathways that control cell proliferation,migration,and apoptosis.The Pentraxin-related protein(PTX3)is an important component of humoral immunity.PTX3 deficiency promotes the complement-dependent inflammation,which can induce the epithelial carcinogenesis.Chemoprevention has been recognized as one of the effective strategies for preventing esophageal cancer.Quercetin-3-methyl ether(Q3ME),is a quercetin derivative,which found in various medicinal plants.It has been reported that Q3 ME could inhibit skin cancer carcinogenesis by inhibiting ERKs activity.It also has been reported that Q3 ME inhibits growth of lapatinib-sensitive and-resistant breast cancer cells.However,there is no reports about the effects of Q3 ME on esophageal carcinogenesis and its mechanism.In this study,we studied the effect and mechanism of Q3 ME in esophageal cancer.Firstly,the up-regulation of phosphorylation of AKT and ERKs in esophageal cancer was proved by immunohistochemical staining and Western Blot.The targeted inhibitor Q3 ME was found by supercomputer screening,pull-down assay and kinase chip experiment.In vitro,proliferation and clonoy experiments confirmed that Q3 ME could inhibit the proliferation and colony formation of esophageal cancer cells.Western Blot and luciferase assay confirmed that Q3 ME might inhibit the esophageal cancer transformation process by inhibiting AKT/mTOR/p70s6 k and MAPK/ERKs signaling pathways.Further,in vivo experiments verified that Q3 ME could inhibit the occurrence of precancerous lesions in NMBA-induced rats,and immunohistochemistry and ELISA assay confirmed that Q3 ME inhibited the activation of downstream signaling pathways by targeting AKT and ERKs.transcription via Activator protein 1(AP-1)transcription factors.There has more evidence that nearly 20% of human cancers are associated with chronic inflammation.Excessive activation of the COX and Lipoxygenase(LOX)pathways leads to abnormal metabolism of arachidonic acid,which may be one of the causes ofPart ? The expression of p-AKT and p-ERKs in ESCC and found the inhibitor Q3MEChapter ? The phosphorylation levles of AKT and ERKs are increased in human esophageal cancer tissues and esophageal cancer cellsMethods 1?Esophageal cancer adjacent normal tissue as a control,the expression of p-AKT(Ser473)and p-ERKs(Thr202/Tyr204)in esophageal cancer tissue microarray was evaluated by immunohistochemistry(IHC).2?To detect the expression of p-AKT(Ser473)and p-ERKs(Thr202/Tyr204)in human esophageal squamous carcinoma cells compared with SHEE cell by Western blot.Results 1?The phosphorylation levles of AKT(Ser473)and ERKs(Thr202/Tyr204)are increased in human esophageal cancer tissues compared with normal tissues.IHC result showed the phosphorylation levles of AKT(Ser473)and ERKs(Thr202/Tyr204)increased in esophageal cancer tissues compared with adjacent normal tissues.And the different expression of p-AKT(Ser 473)and p-ERKs(Thr202/Tyr204)was statistically significant(*p < 0.05,**p < 0.01).This suggests that p-AKT(Ser473)and p-ERKs(Thr202/Tyr204)may play a role in the development of esophageal cancer.2?The phosphorylation levles of AKT(Ser473)and ERKs(Thr202/Tyr204)are increased in human esophageal cancers.The results of Western blot showed that the phosphorylation levles of AKT(Ser473)and ERKs(Thr202/Tyr204)were increased in different esophageal squamous cell carcinoma cells compared with SHEE cells,while there was no significant difference of AKT and ERKs total proteins expression in esophageal squamous carcinoma cells compared with SHEE cells.Chapter ? Q3 ME binds with AKT and ERKs and inhibits the related downstream pathways.Methods 1?To screen and mimic the interaction between Q3 ME and AKT or ERKs,we conducted the silico docking study.2?To confirm the binding of Q3 ME with AKT and ERKs,we performed binding assay between Q3ME-conjuated SepharoseTM 4B beads and esophageal cancer cell lysate.3?Human Phospho-Kinase array was performed to detect the changes of signaling pathways after Q3 ME treated.Results 1?Q3ME binds with AKT and ERKs at the ATP-binding pocket.The results from computational binding models showed that interaction bonds were formed between Q3 ME and AKT1/2 or ERK1/2.2? Q3 ME binds with AKT and ERKs ex vivo.Pull-dowm assay results showed Q3ME-conjuated SepharoseTM 4B beads could bind with AKT and ERKs in KYES450 and KYSE510 cells.3? Q3 ME inhibits some kinases activity.The results of the kinase microarray showed that compared with the control group,the NMBA treated group could induce the phosphorylation of c-Jun,p70S6 k,RSK2 and other proteins,while Q3 ME could inhibit the phosphorylation of c-Jun,p70S6 k and RSK2.This result indicates that Q3 ME can bind with AKT and ERKs and inhibit their activity,thereby inhibiting the activation of downstream signaling pathways.Part ? Q3 ME inhibits the proliferation of ESCC cells and the study of molecular mechanismChapter ? Q3 ME can inhibit anchorage-independent growth and proliferation in esophageal cancer cellsMethods 1?To screen and synthesize small molecule inhibitor Q3 ME against AKT and ERKs by using computational methods.2?To determine the cytotoxic effect of Q3 ME by MTS assay.3?To examine the inhibitory effect of Q3 ME on esophageal cancer cells colony formation,we performed soft agar assay.4?To detect the effect of Q3 ME on esophageal cancer cells proliferarion,we performed MTS assay.Results 1?Synthesis the specific compound,Q3 ME,which binds to the ATP domain of AKT and ERKs and inhibits the kinase activity.2?Q3ME has no effect on SHEE cells vitality.The MTS results showed that the human SHEE cells were treated with different concentrations of Q3 ME for 24 h or 48 h.The cells treated with the concentration below 10 ?M had a cell survival rate of more than 80% compared with the DMSO-treated group,suggesting that the compound below 10 ?M are no effect on SHEE cells vitality and can be used in subsequent experiments.3?Q3ME can inhibit the anchorage-independent cell growth in esophageal cancer cells.The colony formation experiment showed that the SHEE cells had malignant transformation ability after EGF induced,while Q3 ME inhibited the transformed ability of SHEE cells which induced by EGF in a dose-dependent manner.At the same time,Q3 ME inhibited the colony formtion of esophageal cancer cells KYSE450 and KYSE510 dose-dependently.4? Q3 ME inhibits cell proliferation in esophageal cancer cells.MTS results showed that different doses of Q3 ME significantly inhibited the proliferation of esophageal cancer cells KYSE450 and KYSE510.The results at 48 h and 72 h showed statistical differences.Chapter ? Mechanism of inhibition of cell proliferation and colony formation by Q3MEMethods 1?To examine the effect of Q3 ME on signaling pathway by using western blot.2?Detection the effect of Q3 ME on AP-1 transcriptional activity by using luciferase assay.Results 1? Q3 ME inhibits activation of AKT and ERKs downstream signaling pathways in esophageal cancer cell lines.Western Blot results showed that Q3 ME inhibits phosphorylation level of m TOR,p70S6 k and c-Jun in esophageal cancer cells KYSE450 and KYSE510.2?Q3ME inhibits AP-1 transcriptional activity in esophageal cancer cells.The results of luciferase assay showed that the AP-1 transcriptional activities of esophageal cancer cells KYSE450 and KYSE510 were enhanced,while Q3 ME inhibited the transcriptional activity of AP-1 in esophageal cancer cells,and the inhibitory effect was positively correlated with the dose of Q3 ME.Part ? The inhibitory effect of Q3 ME in rat esophageal carcinogenesis and the mechanism studyChapter ? Construction of NMBA induced esophageal precancerous lesions rats animal model and detection the effect of Q3MEMethods 1?To induce esophageal precancerous lesions in rats by using NMBA 2?To classify the precancerous lesions of rat and determine the effect of Q3 ME by H&E staining.Results 1?The model of esophageal precancerous lesions was constructed in rat.Animal experiment results showed that: after the induction of NMBA for 35 weeks,F344 rat esophageal epithelium showed hyperplasia,mild dysplasia,moderate dysplasia and severe dysplasia precancerous lesions.2?Q3ME inhibits the formation of esophageal precancerous lesions in rats.The NMBA-induced group caused different types of precancerous lesions in rats.Compared with the NMBA group,Q3 ME treated with low dose and high dose significantly inhibited esophageal epithelial hyperplasia,mild dysplasia,moderate dysplasia and severe dysplasia lesions.And the effect of high-dose Q3 ME was better than that of the positive control treated group.Chapter ? Mechanism study of Q3 ME on inhibiting esophageal carcinogenesis in rat.Methods 1?IHC method was used to detect the expression levels of proliferation related proteins and inflammation related proteins in rat precancerous lesions.2?ELISA assay was used to detect the expression of COX-2 and PTX3 in rat serum.Results 1?Q3ME inhibits the expression of Ki-67,c-Jun,p70S6 k and the expression of NF-?B,COX-2 and CD11 B.The IHC results showed that Ki-67,c-Jun and p70S6 k expression in the esophageal epithelial tissues of the NMBA group was significantly higher than that of the normal rat esophageal epithelium,and Q3 ME significantly inhibited the expression of Ki-67,c-Jun,and p70S6 k.NMBA induction increased the expression of NF-?B,COX-2 and CD11 B in rat esophageal epithelium,and Q3 ME also significantly inhibited the expression of inflammatory markers NF-?B,COX-2 and CD11 B.This result further demonstrates that the inhibitory effect of Q3 ME on esophageal precancerous lesions is associated with the inhibition of proliferation and inflammation.2?Q3ME inhibits the expression of COX-2 in rat serum and up-regulates the expression of PTX3 The results of ELISA showed that high dose of Q3 ME significantly inhibited the expression of COX-2 in rat serum and increased the concentration of PTX3 in rat serum compared with NMBA group.The ELISA results further confirmed that Q3 ME can act by regulating the homeostasis between inflammation and the body.Conclusions 1?Q3ME targets AKT and ERKs and inhibits the phosphorylation and activation of downstream signaling pathway-related proteins,ultimately inhibiting the proliferation and transformation of esophageal cancer cells.2?Q3ME inhibits NMBA-induced esophageal carcinogenesis in rats,which may be mediated by AKT and ERKs signaling pathways,thus inhibiting cell proliferation and regulating inflammation.Q3 ME is expected to become a promising drug for prevention and treatment of esophageal cancer.