The Anti-angiogenesis Effects Of Arsenic Trioxide On The Metastasis Of Small Cell Lung Cancer

【Background and Objective】Lung cancer is one of the most serious threats to human health and life.In the world,there are more than 1 million 200 thousand new found lung cancer patients,and about 1 million 100 thousand related death every year.Small cell lung cancer（SCLC）is one of the most malignant types of lung cancer,accounting for about 15% to 20% of lung cancer.SCLC is a kind of undifferentiated carcinoma with rapid growth,early lymphatic metastasis and invasion of blood vessels to distant organs.In all kinds of lung cancer,it has the worst prognosis,its 2-year survival rate of patients with less than 5%.The invasion and metastasis is the most important and most characteristic biological marker of malignant tumor.In fact,most patients with malignant tumors eventually died of tumor invasion and metastasis.Therefore,if we can inhibit tumor invasion and metastasis,we will reduce the mortality of patients with cancer and prolong the survival time.In the current medical conditions,the early diagnosis rate of SCLC is low,many patients in the newly diagnosed had already metastasized,lost operation chance.The efficacy of chemotherapy and radiotherapy is not ideal,and SCLC is still lack of effective targeted therapy drugs for a long time,the survival rate of patients with SCLC has no obvious increase.In this case,we need to find a new effective method for the treatment of SCLC,and the control of early metastasis of SCLC is a breakthrough..Arsenic trioxide（As₂O₃）is used in the treatment of acute promyelocytic leukemia（APL）at first.It brings a high complete remission rate in APL patients,showing relatively low toxicity and high efficiency in antitumor effect.In solid tumors,there are a number of clinical and basic reports to prove that it has therapeutic effect.Wang et al found As₂O₃ could inhibit the growth of HCC in vivo and in vitro.So far,As₂O₃ has been approved for the use in the treatment of acute promyelocytic leukemia and advanced liver cancer by FDA.The previous research of our research group has confirmed that As₂O₃ has an inhibitory effect on lung cancer.At the cellular level,it can make the cell cycle of lung cancer cell arrest and apoptosis of lung cancer cells,the proliferation was inhibited;in vivo,we found that As₂O₃ can inhibit NSCLC and SCLC in nude mice.And the tumor growth inhibitory effect on SCLC is particularly significant.It is probable that through the inhibition of VEGF and Dll4-Notch pathways,As₂O₃ could block the blood supply of lung cancer.In clinical studies,we found that As₂O₃ has a good therapeutic effect on malignant pleural effusion caused by pleural metastasis of lung cancer.However,we still lack relevant evidence to prove that As₂O₃ can inhibit the metastasis of lung cancer,the VEGF pathway downstream in internal endothelial cell signaling mechanism is not clear,the purpose of this study is to investigate As₂O₃ on the role of SCLC in metastasis in vivo and its possible mechanism.【Methods】Part1 Effects of arsenic trioxide on the proliferation and migration of vascular endothelial cells 1、Effects of arsenic trioxide on the proliferation of vascular endothelial cellsAfter mix the logarithmic phase of vein endothelial cells,we put them into 96 hole plate.then 0,0.5,1,2,4 and 8μmol/L As₂O₃ were put into the plate for 24 h,48 h,72 h,cell proliferation activity was detected by CCK8 kit.2.Effects of arsenic trioxide on the migration of vascular endothelial cellsAfter culture vein endothelial cells to the logarithmic growth phase,we use trypsin digest it,and then culture in serum-free medium and mix it,put 50 thousand cells every hole into the upper chamber,add medium containing 20% fetal bovine serum to the lower chamber,after 24 hours’ culture,terminate and observe after staining.3.Effects of arsenic trioxide on the expression of endothelial cell migration related moleculeVein endothelial cells were treated with 0,2,4μmol/L As₂O₃ for 48 h,then qPCR and Western blot were used to detect the expression of DSCR1、NFAT2、calcineurin A、 CXCR7、RND1 in nucleic acid and protein levels.Part2 Inhibitory effect of arsenic trioxide on small cell lung cancer metastasis in animal models 1.Construction of human small cell lung cancer metastasis model in nude miceThe human small cell lung cancer H446 were cultured to the logarithmic growth phase,after digestion with trypsin,the cells were resuspended in physiological saline,then 2*106 H446 cell were injected into 5 weeks old nude mice through caudal vein.The mice were observed on the 21 th and 28 th day by fluorescence imaging technique.On the 28 th day,the mice were dissected to observe the tumor growth in lung and liver 2.The inhibitory effect of arsenic trioxide on the metastasis of small cell lung cancer in nude miceLung cancer metastasis in mice model was constructed,24 of them were divided into 4 groups,The first group was control group and saline group.The second and third group were intraperitoneal injection of 2.5mg/kg/d and 5mg/kg/d arsenic trioxide for 10 days,the fourth groups received intraperitoneal injection of cyclosporine A 20mg/kg/d for 10 days.10 days later,vivisection,HE staining were used to observe the metastasis of lung and liver.【Results】 Part 1 Effects of arsenic trioxide on proliferation and migration of vascular endothelial cells 1.Effects of arsenic trioxide on the proliferation of vascular endothelial cellsAfter 24 hours of treatment,the inhibition of As₂O₃ on human umbilical vein endothelial cells was not significantly different from that of the control group（p>0.05）.After 48 hours of treatment,each group of As₂O₃ can inhibit cell proliferation.And group 0.5μM,4μM,8μM were statistically significant（P<0.05）,with the increase of drug concentration,the inhibition trend is more obvious.After 72 hours of treatment,each group of As₂O₃ can inhibit cell proliferation.And group 2μM,4μM,8μM were statistically significant（P<0.05）,with the increase of drug concentration,the inhibition trend is more obvious.2.The effect of arsenic trioxide on the migration of vascular endothelial cellsAs₂O₃ has an obvious inhibitory effect on human umbilical vein endothelial cell migration.In group 2μM and 4μM,the migrated number of vein endothelial cells were significantly lower than the control group(p<0.05.Compared to the low concentration group（2μM）,the high concentration group（4μM group）decreased more obviously（p<0.05）.Otherwise,the cyclosporine A group was lower than the control group（p<0.05）.The As₂O₃ 4μM group was lower than the cyclosporine A group（p<0.01）3.The effect of arsenic trioxide on the expression of endothelial cell migration related molecule DSCR1 Using qPCR and Western blot,respectively,in the level of nucleic acid and protein,arsenic trioxide can up regulate the expression of DSCR1,down regulate the expression of NFAT2,Calcineurin A,CXCR7,and RND1.Part 2 The inhibitory effect of arsenic trioxide on small cell lung cancer metastasis in animal models 1.Construction of human small cell lung cancer metastasis model in nude miceWe construct the nude mouse model of lung cancer metastasis successfully,28 days after injection of the tumor cells into the tail vein,we found lung tumor nodules in nude mice lung and liver through vivisection,we can also see tumor in lung and liver under the microscope through He staining,in vivo imaging showed pulmonary metastases.2.The inhibitory effect of arsenic trioxide on the metastasis of small cell lung cancer in nude miceAs₂O₃ can inhibit the metastasis of human small cell lung cancer cells in nude mice,and the inhibitory effect is more obvious with the increase of arsenic trioxide concentration..After compared the number of metastatic tumor nodules in the liver,we found that group 2.5mg As₂O₃,group 5mg As₂O₃ and group cyclosporine A were lower than the control group.（P<0.01,P<0.01,P<0.05）However,there was no significant difference between the groups of 2.5mg As₂O₃ and 5mg As₂O₃（P>0.05）.After compared the area of metastatic tumor,we found that the liver metastasis of As₂O₃ 2.5mg group was significantly lower than the control group（P<0.05）,As₂O₃ 5mg group and cyclosporine A group had no significant difference compared with saline control group statistically.（p>0.05）【Conclusion】 1.Arsenic trioxide could inhibit the proliferation and migration of vascular endothelial cells 2.As₂O₃ can regulate the signaling pathway of VEGF pathway in vascular endothelial cells,including up regulation of DSCR1,down regulation of NFAT2,Calcineurin A and its downstream factors CXCR7,RND1 and other factors 3.Arsenic trioxide could inhibit the metastasis of small cell lung cancer in nude mice 4.It is possible that arsenic trioxide inhibit the metastasis of small cell lung cancer through inhibiting angiogenesis.