Mechanism Studies Of Salinomycin Against Human Glioma Growth Through Induction Of Cell Cycle Arrest And Antiangiogenesis

A glioma is a type of tumor originating in the brain that arises from glial cells.Gliomas are a common type of primary brain tumor and include astrocytomas,oligodendrogliomas,and ependymomas.According to Central Brain Tumor Registry of the United States,gliomas make up about 27%of all brain and central nervous system tumors and 80%of all malignant brain tumors.Gliomas are classifiedby the World HealthOrganization(WHO)as grade ???.Glioma is highly aggressive and associated with a very poor prognosis and a median survival rate of 12-15 months.Even though the current clinical treatments for glioma have failed in achieving satisfactory results,the adjuvant chemotherapy after surgical resection and radiotherapy is still irreplaceable.Salinomycin(SAL)as a novel anticancer agent has attracted much attentions since 2009,and its pharmacological activities has been explored widely.Growing evidenceshave revealed that SAL could inhibit kinds ofhuman cancers in vitro and in vivo,such as human nasopharyngeal carcinoma,osteosarcoma,hepatocellular carcinoma,cholangiocarcinoma,gastric cancer and some kind of tumor stem cells.Several possible mechanisms contributed to SAL-induced anticancer potential,such as activation of unconventional pathways of cell death,induction of deoxyribonucleic acid(DNA)damage,inhibition of Wnt signaling pathway,angiogenesis,apoptosis or/andautophagy.Several researchers have reported that SALcould hinder glioma cells proliferation through induction of apoptosis or/and necrosis with involvement of reactive oxygen species(ROS)generation,DNA damage and p53 activation.Increased evidences indicated that ROS may act as a potential important mechanism by which SAL-induced anti-tumor effects.However,SAL-induced cell cycle arrest through triggering oxidative stress response has not been reported.Hence,in the present study,we evaluated the role of ROS,DNA damage and AKT inactivation in SAL-induced cell cycle arrest in U87 human glioma cells.Angiogenesis is well recognized as an essential mechanism for tumor growth and development of metastasis.With the exception of avascular leptomeningealtumor spread,tumors cannot grow beyond a certain size without concurrent vascular bed extension.The angiogenic process is far fromsimple as there are a multitude of players involved,and the final result is determined by thelocal equilibrium of pro-and antiangiogenic factors produced by the tumor cells,infiltratingleukocytes,the stroma and the endothelium itself.lt is now widely accepted that the 'angiogenicswitch' is' off' when the effects of pro angiogenic molecules are balanced by that ofantiangiogenic molecules,and it is 'on' when the net balance is tipped in favor of angiogenesis.The formation of new blood vessels during development called vasculogenesis is distinct fromangiogenesis.Sprouting and intussusceptive angiogenesis are two methods by which new blood vessel formationcan occur.Tumor angiogenesis has prominent features,such as the high degree of proliferation of endothelial cells,andrelatively high vascular permeability,so targeting tumor angiogenesis is a new strategy for the treatment of tumor.Angiogenic growth factors produced by tumor cells induceendothelial cell proliferation.Along with the properties of the surrounding matrix,growth factorsdetermine the success of tumor-related angiogenesisand endothelial cell recruitment.Many of the currentlyinvestigational anti-angiogenic approaches aim at theblockade of angiogenic growth factors and the suppression of endothelial cell recruitment through smallmolecule receptor blockers,specific antibodies,or theuse of endogenous inhibitors.A series of experimental studies have shown that makingvascular endothelial growth factor(VEGF)as a therapeutic target can effectively inhibit angiogenesis.VEGF is an important regulator of blood vessel formation,which can effectively promote the migration of endothelial cells.VEGF is a member of the cytokines family related to the structure and function,which is very important in angiogenesis,promoting cell survival and growth,and regulates the proliferation of endothelial cells via binding specific VEGF receptor.Three different kinds of vascular endothelial growth factor receptors(VEGFR)have been identified,such as VEGFR-1(Flt-1),VEGFR-2(KDR or Flk-1)and VEGFR-3.VEGF can promote the growth of endothelial cells in the arteries,veins and lymphatic vessels after bindingto its receptors.The combination of VEGFR-2 and VEGF plays a important role in the regulation of mitosis and vascular permeability,and in the early phase of angiogenesis,promotes the endothelial cells producing platelet activating factor,stimulates the migration of endothelial cells and mitosis,and increases the permeability of blood vessels.After VEGF and VEGFR-2 bingding together,phosphorylated inositol 3 kinase are activated,which makes the concentration of intracellular inositol three phosphoric acid increased and makes protein kinase B and endothelial nitric oxide synthase activated.Activation of protein kinase B can promote cell migration and increase vascular permeability through inhibiting caspase-9 to promote cell survival.When VEGF and VEGFR-2 are combined,thereafterseveral signal transduction pathways downstream wrer activated,including Ras/MEK/ERK and PI3K/Akt.FAK,a cytoplasmic protein tyrosine kinase,plays a vital role in cell proliferation,survival and migration,which associated with integrin-mediated signal transduction.Integrin clustering-mediated the activation of FAK results in the phosphorylation of Tyr397,which is a binding site for PI3K.Previous work have shown that the activation of FAK by integrin engagement or growth factor stimulation promote the activation of downstream PI3K/Akt signaling pathways,leading to the activation or overexpression of pro-metastatic proteins,such as matrix metalloproteinases,urokinase-type plasminogen activator and VEGF.Studies on the underlying mechanism of action have shown that SAL suppress cancer cells growth via the induction of DNA damage,mitochondrial dysfunction,ROSaccumulationwith involvement of the regulation of FOX03a,Wnt/?-catenin,STAT3/Skp2 and Akt/NF-KB/mTOR signaling pathways.However,few attentionswere paid on the antiangiogenic potential of SAL.In this study,SAL was evaluated as a potential angiogenic agent in vitro and in vivo.The underlying molecular mechanisms by which SAL caused angiogenesis inhibition and cell death in human glioma were also elucidated.Objective1.Investigate the inhibition effect of SAL on the growth of U87 and U251 human glioma cell lines in vitroand U87 tumor xenograftsin vivo,explorethe underlying mechanism of cell cycle arrest.2.Investigate the inhibition effect of SAL on the biological behavior of HUVECsin vitro and U251 tumor xenografts in vivo,and explorethe underlying mechanism of angiogenesis.Methods1.U87 and U251 cells were amplified and randomly divided into normal control group and SAL-treated group.After treated with diverseconcentration(0,1,2,4,8 uM)for 48 h,the phase contrast microscope was used to observe cell morphology;and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)analysis was used to determine the cell viability.Flow cytometrywas applied to analyize the influence of SAL on U87 and U251 cell cycle distribution;the DCFH-DA probe and ultra oxygen anion kits were applied to check ROS and superoxide anion generation;Western blotting was used to detect the influence of SAL on expression of cell cycle regulatory protein cyclin A,CDK-2,Cyclin B1 and Cyclin D1,DNA injury markers of H2A and p53,and cell signal pathway proteins AKT in U87.At the same time,SAL was combined with multiple pathway protein inhibitors to study its effects on cell cycle distribution.Human glioma U87 cells(1×107)were injected into the right lower hind flank of each six-week male nude mice to form U87 tumor xenografts in vivo.Tumors from each group were used for western blotting and immunohistochemical(IHC)assay.2.U251 cells and HUVECs were cultured and amplified.After treated with diverseconcentration(0,0.1,0.5,1,2,4,8 uM)for 48 h,the phase contrast microscope was used to observe cell morphology;and MTT analysis was used to determine the cell viability.Afyer treated with SAL,the scratch motility(wound-healing)assay,transwell invasion assay and tube formation assay are used to evaluatemigration,invasion and capillary-like tube formation of HUVECs,employing VEGF and cisplatin as the positive and negative control respectively.Western blotting was used to detect the influence of SAL on expression of VEGF,VEGF receptor,and cell signal pathway proteins p-FAK,FAK,AKT and p-AKT in HUVECs.At the same time,SAL was combined with multiple pathway protein inhibitors to study its effects onangiogenesis.Human glioma U251 cells(1×107)were injected into the right lower hind flank of each six-week male nude mice to form U251 tumor xenografts in vivo.Tumors from each group were used for western blotting and IHC assay.Results1.MTT results showed a time/dose dependent inhibition of SAL on U87 and U251 cell growth,and the cells were observed rounding,synaptic reduction,and the cell number was significantly reduced;flow cytometry showed that SAL induced S-phase arrest significantly in U87 and G0/G1-phase arrest in U251,which is consistent with the reduction of cyclin A,CDK-2,Cyclin B1 and Cyclin D1 expression;ROS test showed that SAL treatment induced DNA damage by ROS increasing in cells;Western blotting showed that SAL treatment significantly increased DNA damage marker H2A and p53 expressions,with decreasing expressions ofp-AKT.In vivo,SAL treatment effectively suppressed the tumor volume and weight.IHC results suggested that SAL treatment effectively inhibited the U87 cells cell proliferation(Ki-67 staining)and angiogenesis(CD-31 staining)in vivo.The western blotting studies in vivo revealed that SAL activated Serl5-p53 and Serl39-H2A,decreased cyclin A,and induced AKT inactivation,which were consistent with the mechanism in vitro.2.MTT analysis showed a dose dependent inhibition of SAL on HUVECsgrowth,and the cells were observed rounding,synaptic reduction,and the cell number was significantly reduced.The results of scratch motility assay,transwell invasion assay and tube formation assay indicated that the migration,invasion and capillary-like tube formation of HUVECs were blocked by SAL treatment.Western blotting showed that SAL treatment significantly decreased the expressions of p-FAK,VEGF,p-VEGFR2 and p-AKT.In vivo,The average tumor weights and tumor volumes were significantly suppressed by SAL treatment.IHC staining showed that the expression of Ki-67,a biomarker of proliferation,and CD34,an important mark of hematopoietic progenitor cells and the small vessel endothelium,were significantly inhibited by SAL treatment.The datas of Western blotting indicated that SAL treatment moderatelydepressed the expressions of p-FAK and p-FAK.Conclusion1.Salinomycin inhibits U87 human glioma cells growth by induction of S-phase cell cycle arrest and U251 human glioma cells growth by induction of G0/G1-phase cell cyclethrough triggering ROS-mediated DNA damage and AKT inactivation.2.SAL can block the angiogenesis and inhibit human glioma growththrough suppression of VEGF-VEGFR2-AKT/FAK signal axis,which validated the potential application of SAL in treatment of human glioma.