Study Of The Effect Of Arsenic Trioxide On Lung Cancer Angiogenesis And The Mechanism

【Background and Objectives】Lung cancer is the leading cause of malignant tumor and cancer related mortality around the world. The overall efficiency of lung cancer drugs and patients’ survival indicators have not been improved for a long time. Looking for new treatment strategies has become an important direction in the field of lung cancer research, and blocking lung cancer angiogenesis is a new breakthrough in recent years. At present, some vascular targeting agents has been used in clinical treatment of lung cancer, such as humanized anti-VEGF monoclonal antibody(bevacizumab) and small molecule inhibitors of VEGFR-2(sorafenib and sunitinib). However, effects of these agents on improving overall survival are not obvious, and their prices are expensive, so their applications are limited in a certain extent.Arsenic trioxide(As2O3) was firstly applied in the treatment of hematological malignancies in our country. It could help patients with acute promyelocytic leukemia(APL) obtain higher complete remission rate, without any side effects such as bone marrow suppression. Due to the high efficiency and low toxicity of its antitumor effect,As2O3 was approved by FDA for the use in the treatment of APL. After that, As2O3 was found to have therapeutic effects on a variety of solid tumors, including breast cancer,renal cell carcinoma, prostate cancer, hepatocellular carcinoma and basal cell carcinoma.China’s Food and Drug Administration approved As2O3 for the use in the treatment of advanced hepatocellular carcinomaasa single drug in 2004. In the field of lung cancer,researchers did some experiments to discuss the inhibitory effect of As2O3 on lung cancer cells, but most of the experiments were in vitro, studies in vivo were rarely reported. Our group firstly carried out a clinical study, in which As2O3 were injected intrapleurally to treat patients with malignant pleural effusion(MPE) caused by advanced lung cancer. We found that As2O3 could significantly reduce the production of MPE, and could change the color of MPE from bloody to light yellow. This suggested the potential application value of As2O3 in the field of lung cancer treatment. However, there is still a lack of studies in vivo to demonstrate the inhibitory effect of systemic application of As2O3 on lung cancer.The mechanism of the inhibitory effect of As2O3 on lung cancer is not clear.According to research results in other types of tumor, possible mechanisms include inhibiting tumor angiogenesis, inhibiting tumor cells proliferation, inducing tumor cells differentiation and apoptosis, reducing telomerase activity and so on. More and more evidences showed that As2O3 has the special effect of inhibiting angiogenesis, which might be one important reason for its anti-tumor effect. Studies in vitro have found that As2O3 could dose-dependently inhibit endothelial cells from forming tube-like formation induced by VEGF on Matrigel. Studies in vivo have found that 2.5 or 5.0 mg/kg of As2O3 intraperitoneal injection could decrease the microvessel density and VEGFR expression in gastric carcinoma xenograft model in a dose-dependent manner. In mice models of fibrosarcoma, 2.0 mg/kg of As2O3 subcutaneous injection could reduce the velocity of blood flow in the irregular microvessels in tumors, inducing the occlusion of tumor central vessels, but no effects on vessels in normal tissues. Our group’s previous research has found that, in mice models of pleura metastasis of lung cancer, As2O3 could reduce the permeability of subpleural capillary and inhibit the formation of MPE through reducing VEGF level, suggesting that angiogenesis inhibition might be a key factor of the therapeutic effect of As2O3 on lung cancer. Currently, there are fewer studies about the regulatory effect of As2O3 on lung cancer angiogenesis, and it is still unclear that whether As2O3 can regulate lung cancer angiogenesis in vivo. Aslo, there is a lack of relevant experimental evidences of the mechanism of the drug’s regulation in tumor angiogenesis.This study will firstly determine the inhibitory effect of As2O3 on the growth of different pathological types of lung cancer xenograft, their angiogenesis, and expression of related factors in VEGF and Dll4-Notch pathway in tumor tissues through in vivo experiments. Then, a series of in vitro experiments will be carried out to observe the effects of As2O3 on multiple stages of lung cancer angiogenesis process including endothelial cell proliferation, migration and tube formation. Finally, the mechanism of the inhibitory effect of As2O3 on tube formation by blocking Dll4-Notch pathway will be demostrated. In additon, the expression of related factors in Dll4-Notch and VEGF pathway in clinical lung cancer tissues will be detected. We hope that our study will provide experimental evidences for the inhibitory effect of As2O3 on lung cancer,demonstrate in-depth mechanism of the regulation of As2O3 in lung cancer angiogenesis and explore the application prospect of As2O3 in lung cancer treatment.【Research Contents and Methods】Part 1 The inhibitory effect of As2O3 on lung cancer xenografts growth and angiogenesis Lung cancer xenograft models were constructed using human lung adenocarcinoma cell line, human large cell lung cancer cell line and human small cell lung cancer cell line.Pathological features of each type of lung cancer were observed under microscope. After animal models were successfully constructed, they were treated with 2.5 mg/kg or 5.0mg/kg of As2O3 or angiogenic inhibitor sorafenib, with normal saline as control. Tumor volume and the tumor growth inhibition(TGI) of each group were compared. Then anti-CD31 fluorescent antibody was used to measure the number and morphology of new blood vessels in each group. Electron microscope was used to observe the ultra structure of endothelial cells in tumor tissues from each group. The expression of VEGF-A,VEGFR-2, HIF-1, Dll4 and Notch-1 at both protein or m RNA level were detected by methods of immunofluorescence, Western Blot and quantitative PCR.In order to observe the morphology of new blood vessels in vivo better, a mixed transplantation model of human lung adenocarcinoma A549 cells and Matrigel was constructed. After treated with different concentrations of As2O3, transplanted tumor tissues were stained by Masson staining, and red blood cells in capillary were observed.Also, anti-CD31 fluorescent antibody was used to measure the number and morphology of new blood vessels in each group.Part 2 As2O3 influenced multiple stages of lung cancer angiogenesis: inhibiting endothelial cell proliferation, migration and tube formation NSCLC cell line A549, SCLC cell line NCI-H446 and human umbilical vein endothelial cells(HUVECs) were conducted with normal saline, various doses of As2O3 and angiogenesis inhibitor sorafenib. Cell proliferation was detected by CCK-8 assay.The migration ability of HUVECs was detected by wound-healing assay. The ability of HUVECs to form vascular network was detected by Matrigel tube formation assay in vitro. Using Western blot and quantitative PCR, the expression of angiogenic factors and their receptors involved in above stages was detected, including MMP-2, MMP-9,PDGF-BB/PDGFR-β, VEGF-A/VEGFR-2, b FGF/ FGFR-1 and Dll4/Notch-1.Part 3 The inhibitory effect of As2O3 on tube formation of endothelial cell by blocking Dll4-Notch and the mechanism Firstly, the Dll4-overexpression lentivirus and the Notch-1-interfering lentivirus were constructed. HUVECs were infected with these two lentivirus and the relevant negative control lentivirus, and then treated with As2O3 or normal saline. The ability of HUVECs in each group to form vascular tubes on Matrigel was observed, and the expression levels of Dll4, Notch-1 and its downstream target gene Hes-1 in cells in each group were detected by Western blot and quantitative PCR. Finally, the expression of related factors involved in Dll4-Notch and VEGF pathway in clinical lung cancer tissues and adjacent tissues was detected by the method of immunohistochemistry.【Results】Part 1 The inhibitory effect of As2O3 on lung cancer xenografts growth and angiogenesis1. As2O3 could inhibit the growth of lung adenocarcinoma, large cell lung cancer and small cell lung cancer xenografts in a dose-dependent manner. At the end of the intervention, the average tumor volumns in As2O3 groups and the sorafenib group were smaller than the normal saline group. TGI of As2O3 2.5 mg/kg group, As2O3 5.0 mg/kg group and sorafenib group in lung adenocarcinoma model were 28.8%, 42.4% and 64.9%respectively. TGI of the three groups in large cell lung cancer model were 71.1%, 84.9% and 87.2% respectively. TGI of the three groups in small cell lung cancer model were27.7%, 56.8% and 49.1% respectively.2. Xenograft tissue sections were stained with CD31 antibody for immunofluorescence assay to show vascular endothelial structures. Results showed that the microvessel density in As2O3 treating groups was significantly reduced and vasculartube structures in As2O3 treating groups were less than the control group. These phenomenons were observed in all three pathological types of lung cancer xenograft models.3. In the mixed transplantation model of Matrigel and lung cancer cells, the control group was rich in blood vessels in general. Blood vessels were gradually reduced as the increase of As2O3 dose. Masson staining was used to show the red blood cells in capillaries, and the results showed that the control group had the most capillaries full of red blood cells, which were reduced as As2O3 dose increased. CD31 fluorescent antibody was used to show vascular endothelial cells. Results showed that the microvessel density in As2O3 treating groups was significantly reduced, and the tube structures in As2O3 treating groups were obviously abnormal, distorted and even shut down.4. Ultrastructures of endothelial cells in lung cancer xenografts were observed under an electron microscope. Endothelial cell membranes in the control group were smooth and intact with clear nuclear structure, while in As2O3 treating groups, membranes were crinkly, and nucleolus accumulated around nuclear membranes, as well as the increased vacuoloid changes in the cytoplasm.5. The results of quantitative PCR, Western blot and immunofluorescence assay showed that the expression levels of VEGF-A, VEGFR-2, HIF-1α,Dll4 and Notch-1 in As2O3 treating groups were lower than those in the control group.Part 2 As2O3 influenced multiple stages of lung cancer angiogenesis: inhibiting endothelial cell proliferation, migration and tube formation1. Cell proliferation assay showed that, at 24 h after drug treatment, there were no no significant difference of NSCLC and SCLC cell proliferation between As2O3 treating groups and the control group. At 48 h after treatment, NSCLC and SCLC cell proliferation in As2O3 treating groups was slightly lower than in the control group. On the other hand, As2O3 could significantly inhibit the proliferation of HUVECs at 24 h after treatment, and the inhibitory effect got more obvious at 48 h after treatment. 4 μM As2O3 showed comparative inhibitory effect with angiogenesis inhibitor sorafenib on HUVECs proliferation.2. HUVUCs wound-healing assay showed that, at 24 h after scratch, the wound repair ability in As2O3 treating groups was weaker than other groups. At 48 h after scratch, cell migration distancesin As2O3 treating groups were significantly shorter than the normal saline control group, and the phenomenon was more obvious in As2O3 4 μM group than in 2 μM group. There were no significant difference of cell migration distance between the sorafenib group and the normal saline control group.3. Matrigel tube formation assay showed that As2O3 could significantly reduce the number of HUVECs cell cords, intervene the normal tube formation and break the cell connections that form network structures. Sorafenib could also reduce the number of cell cords, but each tube structure remained intact. The lumen morphology in the sorafenib group was similar to that in the control group.4. As2O3 dose-dependently down-regulated the protein expression level of VEGF-A,b FGF, MMP-2, MMP-9 and PDGF-BB in lung cancer cells, and the of protein expression level of VEGFR-2, FGFR-1, MMP-2, MMP-9 and PDGFR-β in HUVECs. Aslo, As2O3dose-dependently down-regulatedthe m RNA expression level of HIF-1α, an upstream factor of VEGF-A, in lung cancer cells. As2O3 could inhibit the expression of Dll4 in lung cancer cells and Notch-1 in HUVECs at the protein level.Part 3 The inhibitory effect of As2O3 on tube formation of endothelial cell by blocking Dll4-Notch and the mechanism1. The Dll4-overexpression lentivirus and the Notch-1-interfering lentivirus were successfully constructed, and HUVECs were infected with them. The Dll4-overexpression lentivirus could increase the expression of Dll4, and the Notch-1-interfering lentivirus could reduce the expression of Notch-1.2. Matrigel tube formation assay showed that HUVECs infected by negative control lentivirus could form cross-linked vascular network in the absence of As2O3, but could not in the presence of As2O3. When Dll4 was overexpressed, HUVECs could form cross-linked vascular network in the absence of As2O3, but could not in the presence of As2O3 except for a few of cord-like structures. However, when Notch-1 was inhibited,HUVECs could not form cross-linked vascular network even in the absence of As2O3,and there were no cord-like structures at all in the presence of As2O3.3. Dll4-Notch pathway and its downstream factorwere detected by Western blot. In HUVECs infected by negative control lentivirus, the effect of As2O3 on the expression of Dll4 was not obvious, but As2O3 could significantly inhibit the expression of Notch-1 and its downstream target gene Hes-1. In HUVECs with Dll4 overexpression, As2O3 could still down-regulate the expression of Notch-1 and Hes-1. In HUVECs with Notch-1m RNA interference, Notch-1 and Hes-1 expression was reduced, and As2O3 made the down-regulation of Notch-1 and Hes-1 to a greater degree.4. Clinical samples of lung cancer tissues were detected by immunohistochemistry.The results showed that the expression of Dll4, Notch-1, Hes-1 and VEGFR-2 in lung cancer tissue was higher than that in adjacent tissues.【Conclusion】1. As2O3 could inhibit the growth of lung cancer xenografts by anti-angiogenesis.As2O3 not only reduced the number of new vessels in lung cancer, but also prfevented the formation of normal tube structures and destructed the ultrastructure of vascular endothelial cells in lung cancer, which might be related to the down-regulation of VEGF and Dll4-Notch pathway.2. As2O3 could inhibit multiple stages in the process of lung cancer angiogenesis,including extracellular matrix degradation, endothelial cell migration, proliferation and tube formation. As2O3 could also inhibits the expression of multiple angiogenic factors involved in the stages above mentioned.3. As2O3 blocked the Dll4-Notch signaling pathway and its downstream factors by targeting Notch-1, thereby inhibited the ability of endothelial cells to form vascular tubes.4. Dll4, Notch-1, Hes-1 and VEGFR-2 were highly expressed in lung cancer tissues,suggesting that Dll4-Notch and VEGF pathway might be the target of As2O3 in lung cancer treatment.