Inhibitory Effect And Mechanism Of Niclosamide On Adrenocortical Carcinoma SW-13 Cells

BackgroundAdrenocortical carcinoma(ACC)is a kind of epithelial malignant tumor that comes from adrenal cortex.It is rare at clinical.The annual incidence rate is about 1-2/1000 million people,and the peak age of onset are more common in two segments,<5 year old children and adults around the age of 50,and the average onset age were 45 years,the incidence of women slightly higher than the male.The pathophysiology of ACC is still not clear.It is believed that ACC pathology is a complicated process with many factors.Chromosomal aberrations,gene mutation,methylation of DNA,and dysfunction of insulin-like growth factor(IGF)and Wnt/?-catenin signaling pathway were all play an important role on the occurrence and development of ACC.ACC has high degree of malignancy.The diameter of most tumors was>5cm,and the average diameter was 10cm.Most tumor bodies were accompanied by hemorrhage and necrosis,and the progression is rapid and occult,so most patients were diagnosed with advanced cancer when they come to the clinic,and were accompanied by lymph node or distant metastasis,the most common metastasis were in the lymph nodes of lung,liver and peritoneum and in bones.The clinical manifestations of ACC are decided by tumor function and volume.The majority of adult ACC have endocrine function.Secretion of cortisol and sex hormone in Cushing syndrome(Cushing syndrome,CS)and masculinized are the most common syndrom in clinic,accounting for nearly 40%of ACC,pure CS and masculine expression accounting for 30%and 20%respectively,feminization accounting for about 10%,secreting aldosterone was rare in clinic,accounting for about 2%.Over 90%ACC in Children has endocrine function,manifesting as masculinization or pseudopuberty,among which androgen secreting tumors were the most common,accounting for more than 55%of children with ACC,mixed secretion of cortisol and androgen accounted for 30%,cortisol secreting tumors are rare,accounted for less than 5%.Due to the occult onset of non-functional ACC,most of the general clinical manifestations were abdominal symptoms caused by tumor progression.Imaging and endocrine examination are the main methods for clinical diagnosis of ACC.Due to the lack of specific chemotherapy and target drug treatment of ACC,surgery is still the main treatment,but 30%-85%patients have tumors in progress when they see a doctor,accompanied by lymph or other organ metastasis,so they cannot receive effective or radical surgical resection.The average survival time are only 3 to 9 months,most of the survival time was less than 1 year,5 years survival rate is less than 15%,and 5 years survival rate is less than 30%even after surgical treatment.So drug treatment should be considered in patients with inoperable or unable to receiving radical surgery,or for those who have received surgery to prolong long-term survival.Mitotane was recommended for ACC drug treatment at abroad,but its effectiveness and safety remains controversial.EDPM(cisplatin,etoposide,doxorubicin,mitotane)and Sz/m(streptomycin,mitotane)is the first-line treatment of mitotane plus cytotoxic drugs for the treatment of progression ACC,but the total effect is very limited.Radiotherapy,radiofrequency ablation,interventional embolization can only benefit a small number of patients,so in order to effectively improve the survival rate of patients with ACC,surgery combined with drug therapy is still an important research topic.Because ACC is not sensitive to traditional chemotherapy drugs,it is the focus of current research on ACC to find effective,safe and highly selective drugs.Niclosamide also known as yomesan,its chemical name is N-(2'-chloro-4'-nitrophenyl)-5-chloro salicylic amide,and molecular formula is C13H8C12N2O4.It was clinically used in the treatment of human intestinal parasitic diseases,and was recommended to treat worm by the U.S.Food and Drug Administration(FDA).The mechanism is inhibiting parasite intracellular oxidative phosphorylation and mitochondrial function,which is blocking NADH conversion to NADP+,inhibiting the production of ATP,and change of cell energy metabolism to play a therapeutic role.In recent years,in the process of research and development of antitumor drugs,through a large number of drugs screening experiments,niclosamide was also found has potential antitumor effects.Compared with the new synthetic anticancer drugs,niclosamide has advantages of strong cytotoxicity,and small side effects on human body.In 2009,Wang reported that niclosamide could inhibit the expression of leukemia cell K562 target genes.Then other scholars found that niclosamide can inhibit the proliferation of tumor cells and induce apoptosis by influencing the signaling pathways of Wnt/?-catenin,mTORC1,STAT3,NF-?B,Notch,and Hedgehog,and by influencing mitochondria and lysosomes in tumor cells.Further studies found that niclosamide not only can be applied to tumor cells,but also to tumor stem cells,which have the ability to self renew and can produce heterogeneity cellular.In addition,in other studies,it has been found that niclosamide can increase the sensitivity of tumor cells to chemotherapy and radiotherapy,and even reverse the tolerance of tumor cells to chemotherapy and radiotherapy.Niclosamide has little effect on the energy metabolism and signal transduction of normal cells.As a potential antitumor drug,it has a good application prospect.Objective:1.To study the effects of niclosamide on cell proliferation,cell cycle,cell apoptosis,cell migration and invasion of human adrenocortical carcinoma cell line SW-13 and its mechanism.2.To study the inhibitory effect and mechanism of niclosamide on the growth of adrenocortical carcinoma SW-13 cells subcutaneous tumor in nude mice.Methods:1.SW-13 cells were cultured in vitro,and the effects of different concentrations of niclosamide on cell proliferation were detected by CCK8 and iCELLigence real-time label free cell proliferation monitor.2.Flow cytometry was used to detect the effect of niclosamide on the cell cycle distribution of SW-13 cells.3.AO/EB staining and Annexin V/PI staining flow cytometry were used to detect the effect of niclosamide on SW-13 cell apoptosis after SW-13 cells were treated with niclosamide.4.Transwellexperiment and cell scratch test were used to detect the effect of niclosamide on SW-13 cell invasion and migration ability after SW-13 cells were treated with niclosamide.5.Western Blotting was used to detect the effect of niclosamide on the expression of?-catenin,LRP6 vimnetin,and E-cadherin after SW-13 cells were treated with niclosamide.6.Established a xenograft model of SW-13 cells in nude mice,and divided them into the control group,DMSO group,low concentration niclosamide group,and high concentration niclosamide group randomly.After intraperitoneal injection,the tumor diameter and tumor weight were dynamically monitored.The effects of niclosamide on tumor volume and tumor weight were observed,and growth inhibition rate was caculated?Part of the tumor tissue was observed by HE staining under microscope.7.Cisplatin group was used as the positive control,blood samples were collected from the inner canthus vein before the death of the mice in each group,and were analyzed by blood cells and biochemical analysis8.TUNEL method was used to detect the apoptosis rate of transplanted tumor cells in nude mice after it was treated with niclosamide.9.Immunohistochemical staining was used to detect the expression of ?-catenin?LRP6?vimentin?E-cadherin?Bcl-2 and Caspase-3 in transplanted tumor tissues in nude mice after it was treated with niclosamide.Results:1.After treated with different concentrations of niclosamide 24,48,72,96,and 120 hours,the proliferation of SW-13 cells showed a time and concentration dependence inhabitation(F=157.93,P<0.001).2.Flow cytometry cell cycle test showed that after 24,48,and 72 hours,compared with the control group,the SW-13 cell cycle was blocked in the G0/G1 phase(P=0.001,0.005,0.008).3.The results of AO/EB staining and Annexin V/PI staining showed that the apoptosis rate of niclosamide group was higher than that of the control group(all P<0.001).4.The results of Transwell experiment showed that the invasion ability of SW-13 cells was significantly lower than that of the control group(P<0.001).The results of cell scratch test showed that the migration ability of SW-13 cells was significantly lower than that of the control group(P=0.002).5.Western Blotting results showed that the expression of ?-catenin?LRP6?vimentin in the SW-13 cells of the niclosamide group was lower than that of the control group(all P<0.001),the expression of E-cadherin higher than that of the control group(P=0.004).6.HE staining has confirmed the successful establishment of xenograft model of SW-13 cells in nude mice,the terminal volume of the tumor and tumor weight in low concentration niclosamide group and high concentration niclosamide group were lower than that of the control group(all P<0.001),there is no significant difference between the control group and DMSO group(all P>0.05).7.White blood cell in cisplatin group was lower than that in niclosamide group and control group(all P<0.001),and there was no significant difference between the niclosamide group and the control group(all P>0.05).There were no significant differences in erythrocytes and platelets between the niclosamide group and the cisplatin group as compared with the control group(all P>0.05).The urea nitrogen,creatinine,total bilirubin and alanine aminotransferase in the cisplatin group were higher than those in the niclosamide group and the control group(all P<0.001),and there was no significant difference between the niclosamide group and the control group(all P>0.05).8.TUNEL assay of transplanted tumor tissue showed that the cell apoptosis of low concentration niclosamide group and high concentration niclosamide group were higher than that of the control group(P=0.004,P=0.005).9.Immunohistochemistry of tumor tissues showed that the expression of p-catenin.LRP6?vimentin and Bel-2 of low concentration niclosamide group and high concentration niclosamide group were lower than that of the control group(all P<0.001),and the expressions of E-cadherin and caspase-3 were higher than that of control group(all P<0.001).Conclusions1.Niclosamide inhibited the cell proliferation,block of the cell cycle,inhibit the invasion and migration ability and promote cell apoptosis of human adrenocortical carcinoma SW-13 cells in a time/dose dependent manner.It inhibited the growth of SW-13 cells subcutaneous tumor in nude mice.This may be involved in the inhibition of Wnt/?-catenin signal pathway and epithelial mesenchymal transition(EMT),as well as the influence of the expression of apoptosis related proteins.2.The effect of niclosamide on blood cell,liver and kidney function is less than cisplatin.