| BackgroundGlioma is the tumor Originated from nerve epithelium,is the most common primary intracranial malignant tumor.[1]Glioma can be divided into astrocytom,medulloblastoma,Glioblastoma multiforme(GBM),ependymoma,oligodendroglioma et.According to the pathological types.Glioma accounts for 2%of all malignant tumors in the adult,and the highest incidence in all types of braintumors.According to foreign literature,glioma accounts for about 50%of the primary central nervous system tumors,80%of all central nervous system malignancies.According to the statistics of our country,glioma is the most of the intracranial tumors,accounting for 35.26%-60.96%(average 44.69%).And more than half of gliomas are highly malignant glioblastoma.The age of onset has two peaks,mainly between 30-40 years old,and at the age of 10-20,there is also a peak.The incidence of gliomas and gender is significant,and the ratio of male to female is about 1.85:1.According to reports,the incidence of brain glioma in China is about 3/100000 to 6/100000,the number of deaths is more than 30 thousands one year.Glioma according to the WHO classification system is divided into WHOI-Ⅳ.The WHOI-Ⅱ grade is low grade glioma,and The WHOⅢ-Ⅳ grade is high grade glioma.At present medical level,the prognosis of the treatment of gliomas is still very poor.In addition to some low-grade gliomas,such as pilocytic astrocytoma,can be cured by surgery.Most of the other gliomas are still prone to relapse after comprehensive treatment,and the prognosis is very poor.According to the literature,the average survival time of glioblastoma(WHOIV)is only 1 year or so,the 5-years survival rate is only 9.8%.[2]Gliomas are malignant,and the growth pattern is mainly infiltration,dispersion,difficult to cure,easy to spread and easy to relapse.Although the world has carried out in all aspects of researches and tries on the treatment of glioma,but the prognosis is still poor.Although there is a great gap between the degree of malignancy and the prognosis of different grates of gliomas,the treatment of gliomas is mainly based on surgical resection,and the combination therapy of postoperative radiotherapy and chemotherapy."The National Comprehensive Cancer Network(NCCN)guidelines","European clinical guidelines"," common CNS tumor diagnosis and treatment program" and "China CNS glioma diagnosis and treatment guidelines"have reached a consensus on the principles of the treatment of gliomas.[3,4,5]For glioblastoma,the internationally recognized standard treatment program is Stupp program(2005).The Stupp program emphasizes that we must resect the tumor to the maximum rangeunder the condition of to protect the normal brain function in the greatest degree first,do the tadiotherapy and temozolomide(TMZ)chemotherapy at the same time after 1 month,and 6 cycles of chemotherapy after radiotherapy.[6]For the anaplastic astrocytoma(WHOⅢ),the guidelines recommend that you can still use the Stupp program,or give radiotherapy alone after surgery and then give chemotherapy.In view of low grade gliomas(WHOⅠ-Ⅱ),surgical treatment is generally accepted as the first choice and We have not reach a consensus about postoperative comprehensive treatment.It is generally believed that patients with total resection of the tumor and no high risk factors can be observed and followed up.The appropriate combined use of radiotherapy and chemotherapy can be given for patients with residual or(and)high risk factors.Stupp program is reported more than 10 years,the treatment of gliomas,especially high-grade gliomas,has been significantly more progress,but the average survival time and prognosis of patients are still not good enough,and the median survival time is only 14.6 months.Surgical is the first choice,the most basic,the most direct treatment of glioma,but also the most important prerequisite for comprehensive treatment.All of the above treatment consensus mention that surgical operation is the first choice for the treatment of glioma.Because the purpose of the operation is to reduce the mortality,protect the function and improve the quality of life,the basic principle of surgical treatment is to remove the tumor on the basis of the function protection.The study of evidence based medicine shows that the degree of surgical resection is closely related to the prognosis of patients.[7]Because the glioma is located in the brain parenchyma,the tumor grows infiltrating and lack of visible and clear demarcation region,in clinical it is difficult to complete the full biological cut in the premise of protecting the brain function.The development of the operation mode of gliomas has gradually experienced total tumor resection under the naked eye,total tumor resection under the microscope and imaging total tumor resection.At present the development of surgery assistive technology including:(1)the application of positioning in preoperative functional imaging;(2)the application of neuronavigation in the surgery;(3)the application of intraoperative wake-up technology in operation;(4)ultrasound and MRI imaging technology;(5)the application of electric physiological detection in operation;(6)the application of fluorescent microscope and fluorescein angiography in operation.Alone or in combination with the application of above techniques,improve the accuracy of preoperative localization,realize to avoid the important functional areas in the operation,correct intraoperative brain shift,show residual tumor tissue,improve surgical safety and expand resection.However,the characteristics of the infiltration and diffuse growth of gliomas decide that the simple operation could not complete all cut in Biology,postoperative tumor residual and recurrence is inevitable.Because of this limitation,on the basis of surgical treatment,the adjuvant therapy such as radiotherapy,chemotherapy,immunotherapy,targeted therapy,gene therapy has been developed.Radiotherapy is an important adjuvant therapy for the treatment of gliomas,and its therapeutic effect has long been recognized and affirmed,and mamy studies have indicated that postoperative radiotherapy can improve the prognosis.[8,9,10]At first the radiotherapy for primary gliomas is the whole brain radiation therapy,and the side effects are brain radiation injury.In recent years,with the advancement of radiation technology,the whole brain radiotherapy gradually shifts to local radiotherapy.The aim is to reduce the short-term and long-term normal brain tissue damage,improve the prognosis,and improve the quality of life while as far as possible to inhibit tumor cells.At present,there are Stereotactic radiotherapy,3D-conformal radiation therapy(3D-CRT),Intensity modulated radiation therapy(IMRT)and so on.Chan and other’s research shows that the new radiotherapy technology does not improve the treatment effect of tumor.[11,12]At present,the main function of precise radiotherapy is to reduce the radiation damage of normal brain tissue.Because of the difference of the sensitivity of glioma cells to radiotherapy,the effectiveness of radiotherapy is not guaranteed.The improvement of the efficacy of early conventional chemotherapy drugs in the treatment of glioma is very limited.In 2005,the new England Journal of Medicine published the results of the study of the concurrent radiotherapy and chemotherapy of temozolomide(TMZ)in the treatment of gliomas,which has an epoch making significance in the history of chemotherapy and radiotherapy for gliomas.[6]On the basis of this,the Stupp scheme has become the standard treatment for the treatment of glioblastoma.Although the emergence of TMZ has made a great progress in the treatment of glioma,but its inherent toxic side effects,as well as not through the blood brain barrier,drug resistance and other defects,limiting its application and efficacy.Currently,targeted therapy is mainly targeted at cell proliferation,cell surface receptors related to angiogenesis and signal transduction factors.Mainly include:growth factor and receptor inhibitors,cell signal transduction inhibitors,anti angiogenic drugs,etc..But at present,there are no significant effects on the prognosis of gliomas,and the combined application of various programs is needed.The incidence rate of glioma is high,the degree of malignancy is high,the prognosis is poor,the treatment effect is not satisfied.Human malignant glioma is a serious challenge for all treatments.Therefore,the research and development of new promising antiglioma drugs become urgent needs.[13]Flavonoids are the most important and largest class of plant phytochemicals present in a variety of fruits,vegetables and beverages of plant origin.Flavonoids have been reported to exhibit antioxidant,anticancer and antimutagenic activities.[14]These phytochemicals are believed to have a significant protective role in several chronic diseases including malignant tumors.This also explains the well-known relations between decreased risk of numerous cancers and high consumption of fruits and vegetables containing flavonoids.[15,16,17,18,19]Chalcone structure comprises of an open-chain flavonoid with two aromatic rings connected by a 3-carbon fragment bearing a,3-unsaturated carbonyl motif.They are isolated from the plants and are believed to be the synthons of natural products like flavonoids and isoflavonoids.[20]Tomato is a popular plant,is planted almost all over the world.Tomato contains a variety of pophenols,such as chlorogenic acid and rutin.Naringenin chalcone also belong to the flavonoid phytochemicals found in red tomato peel.[21]To the best of our knowledge,there are no such reports on the antiglioma activity of naringenin chalcone reported in the published literature.So the aim of the present investigation was to study the antitumor potential of naringenin chalcone in U87MG human glioblastoma cells and in xenograft mice model along with evaluating its effects on apoptosis,autophagy and PI3K/Akt pathway.ObjectiveTo investigate the in vitro and in vivo anti-glioma effects of naringenin chalcone in U87MG glioma cells and xenograft model.MethodsThe first test is to investigate the vitro antitumor effects of naringenin chalcone on U87MG glioma cells.U87MG glioma cells with different concentrations(0,2.5,5,10,50,75 and 100μM)naringenin chalcone,different culture time(24,48,72h),respectively by MTT cell proliferation assay.U87MG glioma cells with different concentrations(0,10,50 and 100μM)naringenin chalcone after cultured for 48 hours,respectively by acridine orange/ethidium bromide and Hoechst33342 staining under a fluorescence microscope to assess cell apoptosis.U87MG glioma cells with different concentrations(0,10,50 and 100μM)naringenin chalcone after cultured for 48 hours,observe the number and size of autophagic vacuoles in cells under transmission electron microscope.U87MG glioma cells with different concentrations(0,10,75 and 100μpM)naringenin chalcone after cultured for 48 hours,Annexin V-FITC and PI stain after treatment,use flow cytometry to detect U87MG human glioma cell early apoptosis and late apoptosis.U87MG glioma cells are starved for 4 hours in serumfree medium,and then add different concentrations(0,10,50 and 100μM)naringenin chalcone and culture for 40 minutes and 48 hours,use the Western blot to evaluate the effect of naringenin chalcone on PI3K/Akt signal transduction protein.Use a mouse xenograft model to evaluate the in vivo anti-tumor effect of naringenin chalcone on U87MG glioma cells.The U87MG human glioblastoma cells are subcutaneously injected into the right rear flank of each mouse to produce tumors in mice.After tumor development,the mice are divided into 5 groups and are given different doses of naringenin chalcone injection(5,20,40 and 80 mg/kg).24 days later,the mice are killed,the weight,length and width of the tumor are measured,and the tumor volume is calculated.ResultsNaringenin chalcone has inhibitory effect on proliferation of U87MG cells,the cytotoxicity increase with concentration and time.That is to say,the cytotoxic effect of naringenin chalcone on U87MG cells is with dose and time dependent.The naringenin chalcone treated U87MG cells are stained by acridine orange/ethidium bromide under fluorescence microscopy,are observed the formation of cell shrinkage and apoptosis.After Hoechst 33342 staining,chromatin condensation,DNA fragmentation,cytoplasmic vacuoles are observed under the fluorescence microscope.These are the morphological characteristics of apoptosis.Under transmission electron microscopy it shows that the naringenin chalcone can induce the formation of autophagic vacuoles,and the number and volume of autophagic vacuoles increases with naringenin chalcone dose increases.Detected by flow cytometry,naringenin chalcone can induce U87MG glioma cell early apoptosis and late apoptosis.Naringenin chalcone can also inhibite phosphorylated and non phosphorylated PI3K and Akt protein,inhibite NF-κB、Bcl-2,activite Caspase-3.In vivo test,the results show that the tumor volume and weight treated with different doses of naringenin chalcone are less than the control group.Conclusionnaringenin chalcone exhibits antitumor activity in U87MG human glioblastoma cells and in xenograft mice model by inducing apoptosis,autophagy and PI3K/Akt pathway. |
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