| Esophageal cancer is a malignant tumor of the digestive tract derived from epithelium.The incidence of esophageal cancer ranks sixth,and mortality ranks fourth among malignant tumors in China.The morbidity and mortality in females are lower than those in males,and the incidence in urban areas is lower than that in rural areas.Esophageal cancer is divided into esophageal adenocarcinoma(ECA)and esophageal squamous cell carcinoma(ESCC)according to tissue type consist.About 90%of the cases in China are esophageal squamous cell carcinoma.The etiology of esophageal cancer is more complex,although some studies have shown that smoking and heavy drinking are related to esophageal cancer,the specific etiology is still not clear.As the early symptoms of esophageal cancer are not visible,most patients are found in the middle or late stages of the disease,so they have lost the best time for treatment.Despite the improvement in surgical techniques and the emergence of new chemotherapeutic drugs in recent years,the five-year survival rate of esophageal cancer is still at a low level,only about 20-30%.Thus,it is urgent to find active new early diagnosis and treatment to improve the prognosis in future.Substance P(SP)was first found in the brain and intestines of the horse in 1931.It consists of 11 amino acids and is a member of the tachykinin family It is widely distributed in the central and peripheral nervous systems and a variety of tissues and organs.The earliest research on SP is mainly on pain conduction and inflammation.In recent years,this neuropeptide has aroused the interest of many scientists.They found that the expression of SP is significantly increased in a variety of tumor tissues,and is closely related to the occurrence and development of tumors,which provides a new possibility for the treatment of tumors.SP plays its biological role after binding to the receptors.There are mainly three kinds of receptors:NK-1 receptor(NK1R),NK-2 receptor(NK2R),and NK-3 receptor(NK3R).Among them,NK1R has the highest affinity with SP and is overexpressed in tumor tissues.SP plays a role after binding to NK1R.NK1R belongs to G protein-coupled receptor,which has a seven-transmembrane domain and is mainly divided into two subtypes:full-length type(fl-NK1R)and truncated type(tr-NK1R).Fl-NK1R consists of 407 amino acids,while tr-NK1R lacks 96 amino acids at the C-terminal.Therefore,although truncated receptors can also bind to G protein,the efficiency of internalization and desensitization is decreased.NK-1 receptor antagonists are a growing family of compounds to inhibit the binding of SP to NK1R.Nk-1 receptor antagonist can competitively bind NK1R to SP and inhibit the carcinogenic effect of SP.NK-1 receptor antagonists are mainly divided into two categories:peptides and non-peptides.SP and non-peptide NK-1 receptor antagonists bind to different regions of the NK-1 receptor.Both SP and peptide NK-1 receptor antagonists bind to the extracellular loops of the receptor,whereas the non-peptide NK-1 receptor antagonists bind more deeply between the Ⅲ,Ⅳ,Ⅴ,and Ⅵ transmembrane domain.Aprepitant,as a non-peptide NK1R antagonist,is a clinical drug approved by the FDA for the use in intractable nausea and vomiting caused by chemotherapy.Even if given a high dose,the drug has no apparent toxic side effects,so it has an excellent application prospect.However,the role of aprepitant in esophageal cancer remains unclearTo investigate the role of SP/NK-1 receptor system in esophageal cancer,we mainly studied the distribution of SP and the anti-tumor effect of NK-1 receptor antagonist aprepitant by combining tr-NK1R.This study revealed the role and mechanism of the SP/NK1R system in the occurrence and development of esophageal cancer.Part One Expression and significance of SP in esophageal squamous cell carcinomaObjective:Expression of substance P(SP)in esophageal squamous cell carcinoma were investigated the effect of SP on the proliferation,invasion,and migration.Methods:1.Twenty-five patients with esophageal squamous cell carcinoma who did not receive preoperative radiotherapy and chemotherapy in the fourth hospital of Hebei medical university in 2018 were selected as subjects.The expression of SP in esophageal carcinoma and adjacent normal tissues was detected by RT q-PCR method and immunohistochemical staining.2.Immunohistochemical staining was used to detect the expression of immune cells in infiltrating esophageal carcinoma,Double-labeled immunofluorescence was used to detect whether M2 macrophages could secrete SP.3.ELISA method was used to detect the expression of SP in the esophageal cancer cell line and M2 macrophage supernatant.4.CCK-8 method was used to detect the effect of different concentrations of SP on the proliferation of esophageal cancer cells.5.The effect of anti-SP antibody and anti-NK1R on the proliferation of esophageal carcinoma was detected by the CCK-8 method.6.Transwell and Matrigel substrate gel assay were used to detect the effect of SP on the invasion and migration of esophageal cancer cells.Results:1.RT-qPCR analysis showed that the relative expression of SP mRNA in the normal esophageal epithelium tissue and esophageal carcinoma tissue was(1.02±0.21)and(3.50±0.57)respectively.The level of SP mRNA in esophageal carcinoma tissues was higher than that in normal esophageal epithelial tissues,and the difference between two groups was significant(P<0.05).2.Immunohistochemical staining results showed that there was a large number of M2 macrophages infiltrated in esophageal cancer tissues,and co-localization showed that M2 macrophages could secrete SP.3.ELISA assay analysis results showed that both esophageal cancer cells and macrophages could secrete SP.4.CCK-8 assay analysis showed that SP could significantly promote the proliferation of human esophageal cancer cell lines TE1,KYSE150,KYSE170 in vitro(P<0.05).5.CCK-8 assays analysis showed that anti-SP antibody and anti-NK1R antibody could significantly inhibit the proliferation of human esophageal cancer cell line TE1,KYSE150,KYSE170 in vitro(P<0.05).6.Transwell and Matrigel substrate gel experiments showed that SP could promote the invasion and migration of human esophageal cancer cell line TE1,KYSE150,KYSE170 in vitro(P<0.05).Summary:Esophageal cancer cells and macrophages infiltrating in esophageal cancer tissues can promote the proliferation,invasion,and migration of esophageal cancer cells by autocrine and paracrine SP.It is suggested that the neuroendocrine mechanism may involved in the occurrence and development of esophageal cancer,which provides a new theoretical basis for the study of the pathogenesis of esophageal cancer and a new target for the follow-up diagnosis and treatment of esophageal cancer.Part TwoExperimental study on the antitumor effect of the NK-1 receptor antagonist aprepitant by antagonizing the tr-NK1RObjective:The effect and mechanism of NK-1 receptor antagonist aprepitant was investigated on the proliferation,invasion,migration,and apoptosis of esophageal cancer cells.Methods:1.Immunohistochemical staining was used to detect the expression of NK-1 receptor in esophageal carcinoma and adjacent normal esophageal epithelium tissues.2.RT q-PCR method was used to detect the expression of two different subtypes of NK-1 receptors in clinical samples of esophageal cancer.3.Western blotting and RT q-PCR methods were used to detect the expression of full-length NK-1 receptor(fl-NK1R)and truncated NK-1 receptor(tr-NK1R)in esophageal cancer cell lines.4.CCK-8 method was used to investigate the effect of NK-1 receptor antagonist aprepitant on the proliferation of esophageal carcinoma.5.Transwell method and Matrigel matrix gel method were used to detect the effect of aprepitant on the migration and invasion of esophageal cancer cells.6.Annexin V-FITC/PI double staining and TUNEL staining was used to detect the effect of aprepitant on apoptosis of human esophageal cancer cells.7.The changes of apoptosis-related proteins in esophageal cancer cells were detected by Western Blot after treating with aprepitant.8.The changes of PI3K/AKT/mTOR pathway proteins in esophageal cancer cells were detected by the Western Blot method.Results:1.Immunohistochemical staining result showed that the NK1R was almost not expressed in normal tissues adjacent to cancer,but highly expressed in esophageal cancer tissues.2.RT q-PCR analysis showed that the expression of the tr-NK1R receptor was significantly increased in esophageal cancer tissues than normal esophageal epithelium tissues(P<0.05),but the expression of fl-NK1R was no significantly change(P>0.05).3.RT q-PCR analysis showed that the expression of tr-NKIR in human esophageal cancer cell lines TE1,KYSE150,KYSE170 was significantly higher than that in fibroblasts(P<0.05).The results of the tr-NKIR expression in Western Blot assay were consistent with those of RT q-PCR assay.4.CCK-8 assay result showed that esophageal cancer cells with high expression of tr-NK1R were more sensitive to the treatment of aprepitant,and lower concentration could produce apparent growth inhibition,while fibroblasts with low expression of tr-NK1R showed apparent resistance to the treatment of aprepitant.5.CCK-8 detection result showed that NK1R antagonist aprepitant exerted its anti-proliferation effect by competing with SP combined with NKIR.Re-administration of a particular concentration of SP could reverse the anti-proliferation effect of aprepitant.6.Transwell and Matrigel matrix gel assay result showed that aprepitant had a significant inhibitory effect on the invasion and migration of human esophageal cancer cell lines TE1,KYSE150,KYSE170 in vitro.7.Scratch healing experiment result showed that the scratch healing ability of esophageal cancer cell lines TE1,KYSE150,KYSE170 was decreased after treatment with aprepitant 12h and 24h.8.Annexin V-FITC/PI double staining and TUNEL staining result showed that the number of apoptotic cells increased significantly with the increase of aprepitant concentration in human esophageal cancer cell lines TE1,KYSE150,KYSE170.9.Western Blot assay result showed that the expression of apoptosis-related proteins cleaved PARP and cleaved Caspase-3 in esophageal cancer cell lines increased gradually with the increase of aprepitant concentration.10.Western blot assay result showed that the expression of PI3K-p110αand phosphorylated AKT,mTOR,4EBP-1,P70S6K protein in human esophageal cancer cell lines TE1,KYSE150,KYSE170 in a concentration-dependent manner of aprepitant.Summary:1.In esophageal cancer tissues and cell lines,the expression of truncated NK-1 receptor is significantly increased,suggesting that the truncated NK1 receptor plays an essential role in the carcinogenesis of esophageal cancer.2.In vitro,the application of NK-1 receptor antagonist aprepitant in esophageal cancer cell line TE1,KYSE150,KYSE170 could inhibit the proliferation,invasion,and migration of esophageal cancer cells.3.SP/NK-1 receptor system may regulate the biological processes such as proliferation,invasion,and apoptosis of esophageal cancer cells by PI3K/AKT/mTOR signal transduction pathway.Part Three Experimental study of the effect of aprepitant on the biological behavior of esophageal cancer in vivoObjective:To study the effect of aprepitant on tumor growth and neovascularization in the mouse model of xenografted tumor of esophageal cancer.Methods:1.To establish a subcutaneous transplanted tumor model in nude mice,we injected the human esophageal cancer cell KYSE170 into the right forepaw.When the tumor volume reached 100mm3,the rats were randomly divided into two groups:the aprepitant treatment group and the control group.2.The growth,size,and weight of tumors in each group were measured,and the general condition of tumor-bearing nude mice was observed at any time.3.Immunohistochemical staining assay was used to detect the expression of SP,NK1R and Ki-67,CD31,MMP-9 protein in the aprepitant treatment group and control group.4.RT q-PCR method was used to detect the expression of tr-NK1R and fl-NK1R at the level of mRNA in the aprepitant treatment group and control group.5.HE staining was used to detect the effect of aprepitant on the morphology of tissues in essential organs of tumor-bearing nude mice.Results:1.In the tumor-bearing nude mice with the esophageal cancer cell,aprepitant could significantly inhibit the growth of tumor in vivo,and the tumor volume and weight were significantly lower than those in the control group(P<0.05).2.Immunohistochemical staining result showed that the positive rate of Ki-67 decreased significantly in tumor-bearing nude mice after treatment with aprepitant(P<0.05).3.Immunohistochemical staining result showed that there was no significant difference in the expression of SP and total NK1R in the transplanted tumor tissue between the treatment group and the control group(P>0.05).4.RT q-PCR analysis showed that the expression of tr-NK1R in tumor-bearing nude mice of aprepitant treatment group was lower than that in the control group at the level of mRNA(P<0.05),but there was no significant change in the expression of fl-NK1R(P>0.05).5.Immunohistochemical staining result showed that the expression of protein CD31 related to neovascularization and matrix metalloproteinase-9(MMP-9)related to the invasion was significantly decreased in tumor-bearing nude mice of aprepitant treatment group(P<0.05).6.HE staining showed that no noticeable structural and morphological changes were found in the critical organs of mice after treatment with aprepitant.Summary:NK-1 receptor antagonist aprepitant decreased the expression of Ki-67,CD31,and MMP-9,and inhibited tumor growth and neovascularization in the esophageal cancer cell xenograft model.Conclusions:1.SP may participate in the occurrence and development of esophageal cancer through autocrine and paracrine,while the NK-1 receptor antagonist plays a role in cancer inhibition through the competitive binding of the NK1R to SP.2.The expression of tr-NK1R was significantly increased in esophageal cancer cells and tissues.The NK-1 receptor antagonist aprepitant mainly binds to the tr-NK1R and can play an anti-tumor role by directly inhibiting the growth of tumor cells and promoting the apoptosis of tumor cells.3.The NK-1 receptor antagonist aprepitant may inhibit the PI3K/AKT/mTOR signaling pathway,reduce the expression of AKT,mTOR,and other proteins,promote the apoptosis of the tumor cells and inhibit the growth of tumor cells. |
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