| Gastric cancer is one of the common gastrointestinal malignancies.According to the 2020 global cancer statistics released by the International Agency for Research on Cancer(IARC),gastric cancer ranks fifth in the global cancer incidence spectrum and fourth in the cause of death spectrum.In addition,the crude incidence rate of gastric cancer is the highest in East Asia(39.1/10 million).In China,because the early diagnosis rate of gastric cancer is still low,most patients have advanced gastric cancer at the first time.Although the clinical treatment strategy of surgery combined with radiotherapy and chemotherapy for gastric cancer has been developed to some extent,the long-term prognosis of patients with gastric cancer has not reached the expectation due to the inevitable toxic and side effects of traditional chemotherapy drugs,high probability of chemotherapy resistance and tumor recurrence and metastasis.In recent years,with the in-depth study of tumor stem cells,gastric cancer stem cells have been gradually confirmed.Gastric Cancer Stem Cell(GCSC)is a special group of malignant tumor cells,which shows strong self-renewal ability and multidirectional differentiation potential.It is found that gastric cancer stem cells are closely related to the biological behavior of gastric cancer,such as recurrence,metastasis and chemoresistance.Therefore,targeting gastric cancer stem cells is expected to become an effective strategy to treat gastric cancer from the root.Eukaryotic Initiation Factor 5A2(EIF5A2)is one of the candidate oncogenes found in recent years,which plays an important role in the progression and prognosis of many human tumors.However,whether EIF5A2 is specifically expressed in gastric cancer stem cells and the role of EIF5A2 in the occurrence and development of gastric cancer are not completely clear.Through the study of the function of gastric cancer stem cells and its related molecular mechanisms,looking for new molecular markers of gastric cancer will contribute to the early diagnosis of gastric cancer and bring more hope to improve the prognosis of patients with gastric cancer.Disulfiram(DSF)is a drug that can inhibit the activity of acetaldehyde dehydrogenase(ALDH),so it is used as an anti-alcohol dependence drug.In recent years,researchers have found that DSF also has significant anti-tumor effect,which is copper dependent,and has potential targeting role in melanoma,glioma,breast cancer,pancreatic cancer and lung cancer.Disulfiram is cheap.It has been used in clinic for more than 70 years as an alcohol abstinence drug.There are few adverse reactions and low drug risk in clinical use.Due to the unique advantages of low cost,low toxicity and tumor tissue tendency,the new use of disulfiram as a clinical antitumor drug has broad prospects.At present,the antitumor activity of disulfiram on gastric cancer is only involved in a few literatures,and the relevant molecular mechanism has not been clarified.This topic aims to explore the effect of DSF/Cu on the biological characteristics of gastric cancer cells and its molecular mechanism,so as to provide more alternative strategies and theoretical basis for the treatment of gastric cancer.The main contents of this project include the following three parts:(1)The effect of DSF/Cu on gastric cancer cells in vitro;(2)Effect of DSF/Cu on gastric cancer cells in vivo;(3)The molecular mechanism of DSF/Cu inhibiting the proliferation and migration of gastric cancer cells and the characteristics of gastric cancer stem cells.Firstly,the antitumor activity of disulfiram was preliminarily evaluated by MTT and Colony formation assay.The results showed that DSF/Cu inhibited the proliferation of gastric cancer cells SGC-7901 and MGC-803 in a dose-dependent manner;Flow cytometry was used to detect the apoptosis rate.The results showed that DSF/Cu significantly increased the apoptosis rate of SGC-7901 and MGC-803 gastric cancer cells,and further Western Blot results showed that DSF/Cu decreased the level of Caspase-3 protein and increased the level of Cleaved-caspase-3 protein,which confirmed that DSF/Cu could induce apoptosis of gastric cancer cells;The results of Wound healing assay and Transwell migration test confirmed that DSF/Cu could significantly inhibit the migration of gastric cancer cells SGC-7901 and MGC-803;The results showed that DSF/Cu inhibited the protein expression of stem cell marker ALDH1A1 in gastric cancer cell line SGC-7901 in a dose-dependent manner,and down regulated the protein expression and mRNA level of stem cell transcription factors Oct-4,Nanog and Sox-2 in SGC-7901 and MGC-803 gastric cancer cells;Furthermore,gastric cancer stem cells were screened by Tumorsphere formation assay.The results showed that DSF/Cu could inhibit the ability of gastric cancer stem cells to form tumorspheres,which further confirmed the inhibitory effect of DSF/Cu on the characteristics of gastric cancer stem cells.The in vivo effect of DSF/Cu on gastric cancer cells was explored by subcutaneous tumorigenesis experiment in nude mice(subcutaneous injection of SGC-7901)and tail vein metastasis experiment in nude mice(tail vein injection of MKN-45).The tumor volume was recorded every four days.About one month later,the tumor was taken out.The weight and volume of the tumor in the Normal Control Group(NC)and the Experimental Group(DSF/Cu)were measured respectively,and the tumor tissues were stained with Ki67 immunohistochemistry.The results confirmed that DSF/Cu could inhibit the proliferation of gastric cancer cells and down regulate the protein expression level of Ki67;The lung tissue of nude mice was taken from the tail vein metastasis experiment of nude mice for HE staining.The results showed that the lung metastasis of nude mice was significantly reduced and the lesion volume was significantly reduced after DSF/Cu administration,which confirmed that DSF/Cu could inhibit the metastasis of gastric cancer cells in vivo;Western blot and qRT-PCR were used to detect the protein expression and mRNA level of stem cell transcription factors Oct-4,Nanog and Sox-2 in subcutaneous tumor tissues of nude mice.The results showed that DSF/Cu could significantly inhibit the characteristics of gastric cancer stem cells in vivo.In order to further explore the molecular mechanism of the anti-gastric cancer effect of DSF/Cu,we first evaluated its effect on the expression level of EIF5A2.The results of Tumorsphere formation assay and Western Blot showed that EIF5A2 was highly expressed in gastric cancer stem cells,and DSF/Cu could inhibit the protein expression of EIF5A2 in Monolayers and Spheroids,and down regulate the protein expression and mRNA level of EIF5A2 in vivo;Western Blot was used to detect the basic expression of EIF5A2 in MKN-45,HGC-27,AGS,SGC-7901 and MGC-803 gastric cancer cells.HGC-27 with the highest expression level was selected to construct EIF5A2 knockdown cell line.EIF5A2 knockdown cell line was used for Colony formation assay,Trans well migration assay and Tumorsphere formation assay.Finally,it was verified that DSF/Cu inhibited the proliferation and migration of gastric cancer cells and the characteristics of gastric cancer stem cells by down regulating EIF5A2.In conclusion,through the cell function experiment in vitro and the establishment of animal model in vivo,this topic explored the inhibitory effect and molecular mechanism of DSF/Cu on the proliferation and migration of gastric cancer cells and the characteristics of gastric cancer stem cells in vitro and in vivo.The basic expression level of EIF5A2 in five gastric cancer cell lines and its high expression in gastric cancer stem cells were clarified.By constructing EIF5A2 knockdown cell lines,it was finally verified that DSF/Cu inhibited the proliferation and migration of gastric cancer cells and the characteristics of gastric cancer stem cells by down regulating EIF5A2,suggesting that EIF5A2 could be used as a potential specific target of gastric cancer stem cells in the treatment of gastric cancer. |
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