Mechanism Studies Of Caudatin Against Human Glioma Growth By Inducing Apoptosis And Antiangiogenesis

Glioma is a type of tumor originating in the brain and arising from glial cells.It is the most common type of primary tumor in the central nervous system.Gliomas are classified by the World Health Organization(WHO)as grade ???,to determine the tumor malignant degree,Grade ? and ? as low grade glioma,grade ? and ? as high grade glioma.According to Central Brain Tumor Registry of the United States,gliomas make up about 1/4 of all central nervous system tumors and 3/4 of all malignant brain tumors.The incidence rate of Glioblastoma(GBM,WHO IV)was the highest among the primary tumors of malignant central nervous system,about 3/100,000,accounting for nearly 1/2.Diffuse astrocytoma had the second highest incidence,about 0.5/100,000.Glioma is highly aggressive and associated with a very poor prognosis and a median survival rate of 12-15 months.At present,the treatment of glioma is mainly surgical excision,in vitro radiotherapy and chemotherapy.The 1-year survival rate of adult gliomas was about 30%,and the 5-year survival rate was only 13%.The median survival time was about 2 to 3 years for anaplastic glioma and only about 1 year for glioblastoma.The role of adjuvant chemotherapy in the treatment of high-grade glioma has been recognized,which can significantly prolong the survival of patients.In addition,with the development of research,the role of adjuvant chemotherapy in the treatment of low-grade glioma was gradually recognized.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.Caudatin is a C-21 steroid isolated from Radix Paeoniae Alba.It has many pharmacological functions such as anti-cancer,immune regulation,liver-protection,anti-aging,anti-oxidation,anti-viral and neuroprotection.The chemical structure of the court is clear.It has been found that Caudatin can induce apoptosis in many types of tumor cell lines.Caudatin can induce apoptosis of HepG2 human hepatoma cells and participate in changes in the Bcl-2 family and activation of ERK and JNK.Apoptosis disorder is a mechanism of many diseases.Accelerated apoptosis was found in acute and chronic degenerative diseases,immune deficiency,while delayed or inhibition of apoptosis was found in tumor and autoimmune diseases.The characteristics of tumor cells exist in almost all types of tumors,including uncontrolled growth,angiogenesis and escape from apoptosis.One of the main functions of apoptosis is to prevent tumorigenesis and induce tumor death.In general,it is an intrinsic pathway of inhibition in cancer,and there are extensive means to inhibite apoptosis.Loss of apoptotic control allows cancer cells to survive longer,and gives more time to accumulate mutations to increase the invasiveness in tumor progression,and to stimulate angiogenesis,regulate cell proliferation and interfere with differentiation.There are many ways for cancer cells to escape apoptosis:the function of caspase is inhibited or the function of triggering apoptosis is disabled.Upregulation of antiapoptotic bcl-2 and loss of BAX and/or BAK are the main escape methods.Although bcl-2 is not an oncogene,its mutation enhances the incidence of tumors.Overexpression of bcl-2 protein is present in more than half of cancers.Therefore,it is of great significance to search for drugs which can inhibit tumor escape from apoptosis and induce tumor cell apoptosis.Our previous study found that Caudatin inhibited human glioma cell growth by inducing cell cycle arrest within 48 hours.However,the underlying mechanism by which Caudatin induce apoptosis is still unclear,especially with regard to the molecular mechanisms that inhibit the growth of human brain tumor cells.Angiogenesis,the formation of new blood vessels initiated by endothelial cell proliferation and migration,is necessary for tumors to provide nutrients and remove metabolic wastes continuously.Tumors cannot grow beyond a certain size without concomitant vascular bed expansion.Therefore,angiogenesis is a key step in tumor growth,and anti-angiogenesis has become an attractive therapeutic strategy in cancer treatment.Angiogenesis is regulated by multiple growth factors and cytokines.Among these factors,vascular endothelial growth factor(VEGF),generated by most cancer cells and endothelial cells as well,is one of the most potent angiogenic factors involved in tumor growth.VEGF stimulates endothelial cell proliferation,migration and tube formation by binding its receptor tyrosine kinases expressed on endothelial cells.In these receptor tyrosine kinases,VEGF receptor 2(VEGFR2)mediates the major function.Activation of VEGFR2 leads to activation of various downstream signal transduction proteins,including focal adhesion kinase(FAK),phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT).Inhibition of VEGF activity or/and disabling the function of VEGFR2 are both accepted as potential strategies for antiangiogenesis intervention in tumor treatment.AKT plays a role in signal transduction from growth factor receptors and intracellular pathways.AKT activation not only promotes endothelial cell survival,but also regulates the vasoconstriction response.PI3K/AKT overexpression can induce angiogenesis and regulate tumor growth.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.Tumor angiogenesis can provide essential nutrients and oxygen to the tumor microenvironment,which plays a crucial role in tumor growth,progression and metastasis.Inhibition of tumor angiogenesis is one of the most promising strategies in cancer therapy.Caudatin can effectively inhibit the growth of human glioma cells in vitro and in vivo by triggering cell cycle arrest and apoptosis.However,little information is available about the anti-angiogenic properties of Caudatin in human gliomas.This study was to investigate the anti-angiogenic effects of Caudatin on human gliomas and to explore their underlying molecular mechanisms.Objective1.Investigate the inhibition effect of Caudatin on the growth of human glioma cell lines in vitro,explore the underlying mechanism.2.Construct a tumor-bearing model of glioma in nude mice,and evaluate the molecular mechanism of Caudatin inhibitting tumor growth in vivo.3.Evaluate the effects and molecular mechanisms of Caudatin on tumor angiogenesis in vivo and in vitro.Methods1.Human glioma cell lines U251 and U87 were used as the main experimental materials.Different concentrations of Caudatin was added to the culture medium with diverse time.Cell morphology was observed under the phase contrast microscope.Cell viability was detected by MTT assay,and U251 cell apoptosis induced by Caudatin was analyzed by flow cytometry.Expression of apoptosis indicators such as Cleaved-PARP,active caspase-3,active caspase-7 and active caspase-9 was detected by Western blotting.Fluorescent probe was used to detecte ROS,superoxide and Mitochondrial membrane potential(??m).Z-VAD-fmk(ordinary caspase inhibitor)was used to further investigate whether Caudatin induce apoptosis in glioma cells by caspase-dependent manner.2.Human glioma cells U251(1×107 in 100 ?l serum-free medium)were subcutaneously injected into the right oxter of male nude mice.After 8-day growth (tumor volume about 60 mm3),the mice were treated with Caudatin.Caudatin(0,25 and 50 mg/kg)were injected from the caudal vein every other day from the first day until the sixteenth day(8 times).After experiments,tumors were harvested and measured.Sections of tumors were used for western blotting and immunohistochemistry assay(IHC).3.The human umbilical vein endothelial cells(HUVECs)was used as an experimental material.The cytotoxicity of Caudatin to HUVECs was first tested by MTT assay.Subsequently,the migration,invasion,and angiogenesis ability of HUVEC cells were detected by Transwell and Matrigel culture methods,respectively.The expression of related proteins in the VEGF-VEGFR2-AKT/FAK pathway was detected by Western blot to verify the effect of Caudatin on angiogenesis.Finally,the in vivo transplantation tumor experiment was carried out,and the immunodeficient nude mice of U251 xenograft tumor were used.IHC Immunohistochemistry and Western blot was performed to evaluate the inhibitory effect of Caudatin on angiogenesis in glioma and its potential mechanism in vivo.Results1.The results showed that Caudatin significantly inhibited the growth of U251 and U87 cells in a time-and dose-dependent manner.Flow cytometry analysis showed that Caudatin-induced cell growth inhibition was achieved by inducing apoptosis,and by the increase of Sub-G1 peak,PARP cleavage and activation of caspase-3,caspase-7 and caspase-9.Treatment with Caudatin also causes mitochondrial dysfunction,which is associated with an imbalance in the Bcl-2 family members.Further studies have shown that Caudatin has triggered the apoptosis of U251 cells by disturbing the redox homeostasis to induce the production of reactive oxygen species(ROS).In addition,pretreatment with cysteine protease inhibitors significantly attenuated Caudatin-induced cell apoptosis,PARP cleavage,caspase activation,and ultimately reversed Caudatin-mediated apoptosis.2.Tumor-bearing model of glioma was successfully constructed.The growth of glioma was significantly inhibited by Caudatin treatment in vivo,as convinced by the decrease of tumor volume and tumor weight.The mechanism studies revealed that Caudatin inhibited tumor growth by induction of cell apoptosis,as convinced by the activation of caspase-3.Caudatin treatment obviously inhibited cell proliferation(Ki67 staining)and angiogenesis(CD31 staining)in vivo through evaluation by IHC method.3.Caudatin treatment significantly inhibited the proliferation,and blocked the migration,invasion and capillary-like tube formation of HUVECs.Treatment of HUVECs with Caudatin caused significant inactivation of FAK,and this effect was enhanced at the presence of PF562271(specific FAK inhibitor),which suggests Caudatin inhibited cell migration of HUVECs through FAK dephosphorylation.Caudatin disturbs the VEGF-VEGFR2-AKT/FAK signal axis,which contributed to Caudatin-induced inhibition against proliferation and angiogenesis.Xenograft experiments found that Caudatin significantly inhibited the expression of VEGF,CD31,CD34,and the phosphorylation of FAK and AKT,thereby inhibiting angiogenesis in vivo.Conclusion1.Caudatin inhibits growth of glioma cells by inducing apoptosis through the caspase pathway.2.Caudatin can induce apoptosis through causing mitochondrial dysfunction and ROS-mediated oxidative stress.3.Caudatin can significantly inhibit tumor growth in vivo by induction of cell apoptosis.The mechanism is consistent with that in vitro.4.Caudatin abolish glioma growth by suppression of angiogenesis in vitro and in vivo.