| Colorectal cancer is a malignant tumor originating from the mucosal epithelium of the colorectal.Every year,there are more than 250,000 new cases of colorectal cancer,and about 140,000 deaths,showing an upward trend.In recent years,due to the improvement of people’s living standards and unhealthy diets,the proportion of people with colorectal cancer in the total cancer patients has gradually increased.The treatment of colorectal cancer is often determined according to the cancer stage of the patient.For patients with early colorectal cancer,minimally invasive surgery is the most commonly used treatment,and for patients with middle and advanced stage,in addition to surgical resection,chemotherapy,molecular targeted therapy or immunotherapy should be combined.However,the toxic side effects of chemotherapy,radiotherapy and other therapies as well as the emergence of acquired drug resistance often seriously affect the prognosis of patients,resulting in tumor recurrence,metastasis and other phenomena.Disulfiram(DSF)is irreversible inhibitor of acetaldehyde dehydrogenase,which leads to acetaldehyde accumulation in the body and causes severe headache,hot,anxiety,tachycardia and other physical discomfort phenomena in drinkers,so that alcoholics have aversion and fear to drinking to achieve the purpose of alcohol withdrawal.With the deepening of the clinical application of disulfiram,it has been found that disulfiram has a broad spectrum anti-tumor effect when it is used with copper.Disulfiram combined with copper(DSF/Cu)can play a curative effect by inhibiting tumor stem cells,tumor cell proliferation and migration,and inducing tumor cell apoptosis.Guanine nucleotide binding protein like 3(GNL3),as a GTP-binding protein,is highly expressed in a variety of stem cells and cancer cells.It is involved in cell self-renewal,cell cycle regulation,apoptosis and cell proliferation by binding to a variety of proteins,such as p53,mouse dual microsomal 2(MDM2),and ribosomal L1 domain containing 1(RSL1D1).Combined with the high expression of GNL3 in colorectal cancer cells and its effects on various cellular biological processes,GNL3 can be used as an effective prognostic indicator to evaluate the treatment of patients with colorectal cancer.At present,there are few clinical studies on the application of DSF in the treatment of colorectal cancer.Therefore,this study aimed to explore the effects of disulfiram combined with copper on the treatment of colorectal cancer cells and to understand the molecular mechanism involved.The research contents mainly include the following three sections:(1)in vitro pharmacological study of inhibiting effects of DSF/Cu on the proliferation and migration of colorectal cancer cells;(2)in vivo pharmacological evaluation of inhibiting effects of DSF/Cu on the growth and metastasis of colorectal cancer;(3)exploration of molecular mechanism of DSF/Cu for the treatment of colorectal cancer.(1)In vitro pharmacological study of inhibiting effects of DSF/Cu on the proliferation and migration of colorectal cancer cells:The cytotoxic effects of Cu,DSF and DSF/Cu on colorectal cancer cells HCT116,LOVO and SW480 were evaluated.Then the antitumor activities of disulfiram on HCT116 cells were determined by using colony formation,woundhealing and Transwell assays.The MTT assay results showed that disulfiram exhibited copperdependent inhibition of the proliferation of colorectal cancer cells and the impact on cell survival became stronger with increasing drug concentration as well as intervention time.The IC50 values of DSF/Cu on colorectal cancer cells HCT116,LOVO and SW480 at 24 h/48 h/72 h were 482.6 nM/323.6 nM/316.0 nM、384.5 nM/317.1 nM/292.3 nM、381.1 nM/314.6 nM/249.9 nM,respectively.The colony formation assay results showed that the number of colonies formed in DSF/Cu was less than that in Cu and DSF groups,which further indicated that DSF/Cu could inhibit the proliferation of HCT116 cells.The wound-healing assay and Trans well assay results demonstrated that DSF/Cu significantly inhibited the migration ability of HCT116 cells.(2)In vivo pharmacological evaluation of inhibiting effects of DSF/Cu on the growth and metastasis of colorectal cancer:Both the subcutaneous tumor and tail vein metastasis models with HCT116 cells were established in nude mice.The control and DSF/Cu treatment groups(50 mg/kg)were set up in both models.In the subcutaneous tumor model group,the tumor tissues were stripped after treatment and the Ki67 immunohistochemical assay was performed.The experimental results showed that the subcutaneous tumor volumes and Ki67 protein positive expressions of nude mice in the DSF/Cu treatment group were significantly lower than those in the control group.These results indicated that DSF/Cu inhibited the growth of colorectal cancer cells in vivo,and the tumor inhibition rate was 44.03%.After the treatment of the tail vein metastasis group for 30 days,fresh lung tissues of nude mice were taken for HE staining.Multiple lung metastases with obvious tumor atypia were observed in the lung tissues of the control group while the number and size of lung metastases were significantly higher than that of the DSF/Cu treatment group,indicating that DSF/Cu inhibited the metastasis of colorectal cancer cells in vivo.(3)Exploration of molecular mechanism of DSF/Cu for the treatment of colorectal cancer:In order to further explore the molecular mechanism of DSF/Cu against colorectal cancer,studies on the influence of GNL3 expression in vitro and in vivo confirmed that DSF/Cu inhibited the GNL3 expression,and GNL3 had a certain co-expression correlation with the multi-drug resistant protein MRP1.Western Blot and RT-PCR were used to detect the GNL3 positive expression in colorectal cancer cell lines Caco-2,HCT116,HT-29,LOVO and SW480.The HCT116 cells with the highest expression were selected to establish GNL3 knockdown and overexpression models.It was verified that the GNL3 expression showed the same trend in the proliferation and migration abilities of colorectal cancer cells and the expression of MRP 1 by using various pharmacological techniques such as colony formation,wound-healing,Transwell,Western Blot and RT-PCR assays.DSF/Cu intervention in HCT116-GNL3-OV cells antagonized the cell proliferation and migration caused by the overexpression of GNL3.Finally,HCT116-PLVX cells and HCT116-GNL3-OV cells were inoculated into the subcutaneous tissues of nude mice to establish the transplanted tumor model.Four experimental groups were set up as follows:HCT116-PLVX control,HCT116-PLVX+DSF/Cu,HCT116-GNL3-OV,and HCT116-GNL3-OV+DSF/Cu.In vivo experimental results showed that DSF/Cu treatment inhibited tumor growth in nude mice promoted by GNL3 overexpression.The Western Blot analyses of GNL3 and MRP 1 proteins in the tumor tissues of nude mice showed the same results as those of the in vitro cell experiments.In conclusion,this study demonstrated that DSF/Cu inhibited the proliferation and metastasis of colorectal cancer cells in vitro and in vivo based upon the cell function experiments and animal models.The molecular mechanism of anti-tumor activities of DSF/Cu was explored by constructing HCT116-GNL3 knockdown and overexpression cell lines.The results showed that DSF/Cu inhibited the proliferation,migration and drug resistance of colorectal cancer cells by regulating the expression of GNL3.This work established the research basis for the subsequent clinical treatment of colorectal cancer with disulfiram-related drugs and for the development of molecular targeted therapeutic drugs for colorectal cancer with GNL3 as a specific target. |
