Chloroquine Inhibits Tumor Growth And Angiogenesis In Malignant Pleural Effusion Caused By Lung Cancer

Background Malignant pleural effusion(MPE)is a common complication of lung cancer.Currently,no effective cure exists for MPE.Chloroquine(CQ)has been demonstrated to induce vascular normalization and inhibit tumor growth.The aim of this study was to assess whether CQ affects MPE by impairing angiogenesis.Method1.First of all,we inoculated BALB/c nude mice with a subcutaneous injection of human A549 cells to generate our animal model.Mice were divided into 3 groups matching for tumor volume after 30 days and treated with normal saline(NS)(50μL/3d),CQ(25 mg/kg/3d)or Bevacizumab(BE)(5 mg/kg/3d),respectively.Tumor growth of the mice and micro vascular density(MVD)were monitored.2.And we explored the effect of CQ on the proliferation,survive and proangiogenic signaling of tumor cells in vitro.We further evaluated the effect of CQ on human umbilical vein endothelial cells(HUVECs)viability,migration and tube formation.Chicken chorioallantoic membrane(CAM)Model was carried out to elucidate its effect on angiogenesis.3.By western blot,we analyzed the expression of Akt,p-Akt,Jagged 1 and Ang2 in HUVECs treated by CQ.4.At the end,we established an MPE mice model by injecting Lewis lung carcinoma cells(LLCs)into pleural cavity of BALB/c mice.CQ(25 mg/kg),BE(5 mg/kg)or NS was respectively injected into pleural cavity three times with 3 days interval in each group.The volume of pleural effusion,tumor foci,MVD were evaluated 21 days after the injection of LLCs.Results1.CQ inhibited tumor growth and angiogenesis in vivo and in vitro.In the mouse Xenograft,the CQ group exhibited decreased tumor volume(238.6±74.3 mm3)and tumor weight(0.243±0.1368 g)compared with the NS group(406.1±176.2 mm3 and 0.506±0.2255 g,respectively).The MVD in the CQ group was much lower than that in the NS group.The tumor volume and MVD in the CQ group were not obviously different from those in the BE group.In vitro,CQ also impaired the proliferation of the A549 cells.And CQ enhanced LC3 Ⅰ/Ⅱ in the A549 cells.There was no significant difference in vascular endothelial growth factor(VEGF)expression between NS and CQ groups.Decrease of the amount of MVD in xenograft suggests that CQ impairs vessel development.2.CQ inhibited the proliferation,migration and network formation of HUVECs in vitro and impaired angiogenesis in the CAM model.In order to investigate how CQ impairs vessel development,we examined proliferation,migration and network formation of HUVECs in vitro.The HUVECs treated with CQ exhibited reduced proliferation.The migration ability of the HUVECs treated with CQ was significant reduced compared with the control group at 36 hour.The motion of the HUVECs treated with CQ was weakened in the Transwell assay.The tube formations of the 20 μM and 40 μM CQ-treated groups were reduced compared with the 0 μM CQ group.Additionally,CQ reduced angiogenesis in the CAM model.The quantitative analysis confirmed that the number of blood vessels in the CQ group(28 ± 3)was significantly less than that in the NS group(50±10,P<0.05).3.CQ reduced p-AKT,Jaggedl and Ang2 in the HUVECs.To explore how CQ inhibited the proliferation,migration and network formation of HUVECs in vitro and impaired angiogenesis in the CAM model,some important related proteins were investigated:the Akt,p-Akt,Jagged1 and Ang2.As detected by Western blotting,the levels of p-Akt,Jagged1 and Ang2 were less in CQ group.4.CQ reduced malignant pleural effusion in the nude mice.Male BALB/c nude mice were used as MPE animal models.No obvious pleural effusion was observed in the CQ or BE groups based on CT scanning).The mean pleural effusion volumes in the NS,CQ and BE groups were 0.41 ±0.162 mL,0.13±0.051 mL and 0.18±0.087 mL,respectively.The numbers of pleural nodules in the NS,CQ and BE group were 34.9±3.93,17.8±3.96 and 16.81±3.28,respectively.To further investigate the role of CQ in angiogenesis,we detected the MVD in the pleural nodules of each group.The MVDs in the CQ and BE therapy groups were much lower than that in NS group.There was no significant difference between the CQ and BE groups.The cyologies of the pleural effusions from the three groups revealed cells with large nuclei and visible nucleoli,which indicated that the pleural effusions of all groups were MPEs.The VEGF levels in the MPEs were examined with enzyme-linked immunosorbent assay(ELISA),and the results indicated no significant differences in VEGF expression between the different groups.CQ did not induce mice body weight loss compared with the NS and BE groups.Therefore,CQ could reduce MPE in nude mouse without obvious systemic toxicity.ConclusionOur work demonstrated that CQ played an efficient treatment role in MPE through by antitumor and impairing angiogenesis,which provided a certain theoretical basis for the effectiveness of CQ on the MPE treatment.