Molecular Mechanism Of Selenocysteine Against Malignant Osteosarcoma Growth

Osteosarcoma is a common cancer in younger and children,which does harm to people's health and human family.Patients usually face to death because of its high malignant degree and poor prognosis.Patients with osteosarcoma often suffer from pain in the early stage,and gradually becomes persistent pain,which severe affected the basic life of the patients.The patient with osteosarcoma is prone to pathological fracture and deformity,and difficult to heal,which affects the normal physiological function and healthy life.Surgery and neoadjuvant chemotherapy represent the main treatment of osteosarcoma in clinic.Surgery is to have the tumor resection,and even amputation.Chemotherapy is used to prevent postoperative recurrence and metastasis.However,surgery can affect the normal physiological function,the patient's body,and reduce the survival of patients,which brings great economic and ideological burden to the patients and society.Chemotherapies often have a large of toxic side effect,which can damage the patient's immune system,and may even accelerate the death of the patient.Therefore,searching a safe,painless,efficient treatment for osteosarcoma is urgently needed.Selenium acts as an indispensable trace element for human body.There are two forms of selenium,including organic selenium and inorganic selenium.Selenium as a multifunctional nutrient is often used for the prevention or treatment of more than 40 kinds of diseases.Such as bone disease,cardiovascular disease,diabetes,liver disease,prostate disease and cancer.Recent years,selenium and selenium-containing compounds have shown more advantages than traditional chemotherapeutic drugs in treatment of tumors.More and more people are paying attention to its biological property.The inhibitory effect of selenium and selenium-containing against human tumors is popularly studied.Selenium and selenium-containing have the potential to inhibit the proliferation,migration of tumor cells,promote the cancer cells apoptosis through various ways,inhibit angiogenic factors,and abolish neovascularization through inhibiting nutrient and eventually reach the"starving" status.Selenium and selenium compounds can inhibit tumor angiogenesis,prevent tumor growth and metastasis.Tumor metastasis and growth often depend on the tumor itself to establish a neovascular network.Selenium and selenium-containing compounds can inhibit the establishment of this neovascular network,cut off the supply of nutrients to tumor cells.The tumor can not grow certain size if it can not get the nutrition source.Tumor tissue can not be discharged and the tumor is gradually degenerated and necrotic if cutting off the metabolism of the tumor.Antiangiogenesis by selenium and selenium-containing compounds is a new strategy that affects tumor angiogenesis.Studies have shown that selenium and selenium-containing compounds can inhibit the expression of MMP-2 and VEGF(an angiogenic factor)in endothelial cells in a variety of tumor cell lines.It is also reported that selenium and selenium-containing compounds can act as inhibitors of early tumors angiogenesis.Accumulated evidences indicated that selenium treatment can effectively down-regulate the osteopontin gene expression.Selenocystine(SeC)is a natural available selenium-containing amino acid displayed novel anticancer activity against human cancers.SeC can effectively inhibited human cancers growth through induction of cell cycle arrest and cell apoptosis by inducing ROS-mediated oxidative damage.It is of great significance to evaluate its anticancer effect and mechanism.However,the anticancer effect and mechanism against human osteosarcoma in vitro and in vivo have not been insvestigated.The mechanism also remains elusive.This study is aim to culture MG-63 human osteosarcoma cells.The growth inhibition effect and mechanism of SeC against MG-63 cells were evaluated.The tumor model in nude mice was established and the anticancer mechanism in vivo was examined.Moreover,the effect of SeC on HUVECs migration,invasion and tube formation was also explores.The in vivo anti angiogenesis of SeC was laso examined.This study will provided evidence that selenium-containing will be potential chemotherapy agents.Purpose1.To explore the effect and mechanism of SeC on cell cycle arrest and cell apoptosis in MG-63 human osteosarcoma.2.To investigate the effect and mechanism of SeC on HUVECs migration,invasion and angiogenesis in vitro and in vivo.MethodsMG-63 and HUVECs cells were cultured in vitro.Groups were randomly divided into control group and drug administration group.The cell morphology and density of each treatment group were observed by contrast microscope.MTT staining method was used to detect cell viability under different treatment conditions.The changes of cell cycle were detected by flow cytometer after PI staining.Mitochondrial dysfunction in cells was examined by JC-1 probe.Mito-traker was used to detect mitochondrial structure.ROS accumulation in cells was detected using DCFH-DA.Mito-SOX was used to detect superoxide.Scratch motility(wound-healing)assay was employed to detect the changes of cell migration.Transwell invasion assay was used to detect cell invasion.The effect of SeC on angiogenesis in osteosarcomas was detected by tube formation assay.The Western blotting method was used to detect the cell cycle and apoptosis-related protein factors.The effect of SeC on osteosarcoma cells in vivo was detected by xenotransplantation of nude mice by immunohistochemistry(IHC).Results1.Compared with the control group,the cells after SeC treatment showed obvious apoptotic changes and reduction of cell number.MTT results showed that compared with normal cells,SeC treatment time-dependently and dose-dependently inhibited MG-63 cells viability(P<0.05).Flow cytometry results showed that SeC treatment caused obvious cells apoptosis and S-phase arrest,as convinced by the increase of Sub-G1 peak and down-regulation of Cyclin A and CDK-2.DCFH-DA staining results showed that SeC induced significant accumulation of ROS.Mito-SOX revealed that SeC induced significant superoxide anions generation in cells.JC-1 probe results found that SeC caused the lost of mitochondrial membrane potential(??m).Mito-tracker staining revealed that SeC lead to mitochondrial change from threadiness to punctiform.MG-63 nude mice xenograft tumor results showed that SeC treatment in vivo effectively inhibited tumor weight and tumor volume,but not affect mice body weight.SeC treatment also induces tumor cell apoptosis and Serl5-p53 phosphorylation.Immunohistochemical(IHC)staining showed that SeC treatment also inhibited the cell proliferation(Ki-67 staining)and inhibited tumor angiogenesis(CD-34).Western blotting results showed that SeC treatment induced dose-dependent PARP cleavage and the activation of caspase-3,caspase-7 and caspase-9.SeC also increased Bad and Bax expression and decreased the Bcl-2 and Bcl-xl expression.Moreover,SeC treatment also triggered p53 phosphorylation at Ser 15,Ser 20 and Ser 392 with a time-dependent and dose-dependent manner.2.Wound healing and transwell invasion experiment found that VEGF addtion significantly promoted the UHVECs migration,invasion and tube formation.However,SeC treatment effectively inhibited the migration,invasion and tube formation of HUVEC.Western blotting found that the SeC treatment can inhibit p-FAK expression time-dependently and dose-dependently.PF562271(FAK inhibitors)addition chanced SeC-induced inhibition against HUVECs migration,indicating that SeC inhibited HUVECs migration with FAK-dependent manner.Mechanism revealed that SeC disrupted VEGF-VEGFR2-AKT signaling axis,as reflected by the decrease of VEGFR2 and AKT expression.LY294002(AKT upstream inhibitor)addition effectively enhanced SeC-induced cells growth inhibition against HUVECs,revealing that SeC inhibited HUVECs growth with AKT-dependent manner.The nude mouse model of osteosarcoma revealed that SeC treatment in vivo effectively inhibited p-FAK and p-AKT expression.The IHC result indicated that SeC treatment blocked vascular endothelial growth factor(VEGF)expression.The HE staining result also revealed that SeC treatment in vivo inhibited the formation of blood vessels,as convinced by the decreased number of erythrocyte-containing blood vessels.Conclusion1.SeC induced ROS generation,mitochondrial dysfunction and caused DNA damage and p53 phosphorylation,and eventually leaded to S-phase arrest and apoptosis in human osteosarcoma.2.SeC blocked osteosarcoma migration,invasion and angiogenesis through dysfunction of VEGF-VEGFR2-AKT/AK signaling axis.