| Glioblastoma multiforme(GBM)is the most malignant and aggressive primary brain tumor in adults.Approximately 176,0000 cases of brain tumors are diagnosed annually worldwide,with an estimated annual mortality of 128,000.Despite concerted efforts to improve current therapies,the prognosis of glioblastoma remains very poor with a mean survival of 12 months after diagnosis.Plants have a long history of use in the treatment of cancer and remain the most attractive source of therapeutic candidate compounds as a tremendous chemical diversity is found in million of species of plants.At present,over 60%approved anticancer drugs in the world are derived from natural source including plants and marine organisms.The current estimate indicates that there exists about 500,000-700,000 plant species in this planet and less than 10%of all species have been subjected to investigations for their potential value as a source of drugs.Therefore,there is dire need to screen new plant extracts as a source of new drugs.In the present study,a high throughput screening approach was carried out to identify natural compounds with promising antiglioblastoma activity from Chinese herbs.A total of 300 plant extracts were tested for their antiglioblastoma activity.The plant extracts with promising antiglioma activity were further tested against mouse splenocytes.The plant extracts demonstrating potential anti-glioma activity without or with low toxicity against mouse splenocytes were selected and subjected to the further purification process using high performance liquid chromatography(HPLC)and high speed counter-current chromatography(HSCCC).The structure elucidation was carried out using mass spectrometry(MS)and nuclear megnatic resonance(NMR).Further mechanistic studies for anti-glioma activity were performed using purified single bioactive compounds.Apart from the plant extracts,a library of 400 small molecule single compounds was also screened to identify potential anti-glioblastoma compounds.The bioactive compounds showing promising anti-glioblastoma activity were selected for further mechanistic studies.1.The first report represents the identification,extraction,isolation,purification,structure elucidation and antiglioma activity of "Artabotryside A" a flavonol glycoside isolated from the Descurainia Sophia(L.).This research report demonstrated that artabotryside A significantly reduced the proliferation of U87 cells in a dose-and timedependent manner.By flow cytometric analysis,we found that artabotryside A treatment resulted in an increased apoptosis,mitotic arrest,level of ROS and decreased mitochondrial membrane potential in U87 cells in a time-dependent manner.Furthermore,caspase-3 inhibitor,Ac-DEVD-CHO,significantly inhibited the apoptotic effect of artabotryside A,indicating that artabotryside A induced caspase-dependent apoptosis in U87 cells.In addition,artabotryside A is less toxic to normal mouse splenocytes and glial cells.This selective cytotoxicity of artabotryside A against U87 glioblastoma cells as compared to mouse splenocytes and glial cells is advantageous.Thus artabotryside A might be a safe candidate for prevention and treatment of gliomas.2.The second research report represents the antiglioma activity and molecular mechanism of "Jaceosidin" isolated from Artemisia argyi.Jaceosidin inhibited growth of U87 cells in a time-and dose-dependent manner.Further mechanistic study demonstrated that jaceosidin exerts growth inhibitory effect by arresting the cells at G2/M phase and induction of apoptosis.Furthermore,our study revealed that induction of apoptosis was associated with cell cycle arrest at G2/M phase,up-regulation of p53 and Bax,decrease in mitochondrial membrane potential,release of cytochrome c,and activation of caspase 3.The G2/M phase arrest was found to be associated with up-regulation of p53 and p21 and down-regulation of Cyclin B1/CDK1 complex.This mitochondrial-caspase-3-dependent apoptosis pathway was confirmed by pretreatment with caspase 3 inhibitor,Ac-DEVD-CHO.This is the first report indicating the jaceosidin induces apoptosis through cyclic block.3.The third research report demonstrates the antiglioma mechanism of vitexicarpin,a flavonoid compound identified from compound library.We found that vitexicarpin inhibited the growth of U87 glioblastoma cells in a dose-dependent manner with IC50～22 μM.Vitexicarpin-induced growth inhibition was found to be associated with induction of apoptosis and mitotic arrest.During vitexicarpin-induced apoptosis,up-regulation of Bax,down-regulation of Bcl-2 and cleavage of caspase-3 and PARP were observed.We also found that vitexicarpin induced mitotic arrest by inhibiting tubulin polymerization.Furthermore,pretreatment of cells with z-VAD-fmk reversed the apoptotic effect of vitexicarpin but failed to attenuate mitotic arrest.Taken together,the data revealed that vitexicarpin inhibited the growth of U87 cells by induction of apoptosis and mitotic arrest.Thus vitexicarpin may be a promising candidate for the treatment of glioblastoma.4.In this work,we demonstrated the molecular mechanism of apoptosis induction of Pseudolaric acid B(PLAB)in U87 glioblastoma cells and its organ toxicity in animal mouse model.We found that PLAB inhibited growth of U87 glioblastoma cells in a dose-dependent manner with IC50～10 μM.Flow cytometry analysis showed that apoptotic cell death mediated by PLAB was accompanied with cell cycle arrest at G2/M phase.Using Western blot,we found that PLAB induced G2/M phase arrest by inhibiting tubulin polymerization in U87 cells.Apoptotic cell death was only partially inhibited by pancaspase inhibitor,z-VAD-fmk,which suggested that PLAB-induced apoptosis in U87 cells,is partially caspase-independent.Further mechanistic study demonstrated that PLAB induced caspase-dependent apoptosis via up-regulation of p53,increased level of pro-apoptotic protein Bax,decreased level of anti-apoptotic protein Bcl-2,release of cytochrome c from mitochondria,activation of caspase-3 and proteolytic cleavage of poly(ADP-ribose)polymerase(PARP)and caspase-independent apoptosis through apoptosis inducing factor(AIF).Furthermore,in vivo toxicity study demonstrated that PLAB did not induce significant structural and biochemical changes in mouse liver and kidneys at a dose of 25 mg/kg.Therefore PLAB may become a potential lead compound for future development of antiglioma therapy.5.In this study,we found that alantolactone effectively inhibits growth and triggers apoptosis in glioblastoma cells in a time-and dose-dependent manner.The alantolactoneinduced apoptosis was found to be associated with GSH depletion,reactive oxygen species(ROS)generation,mitochondrial transmembrane potential dissipation,cardiolipin oxidation,up-regulation of p53 and Bax,down-regulation of Bcl-2,cytochrome c release,activation of caspases(caspase 9 and 3)and cleavage of poly(ADP ribose)polymerase.This alantolactone-induced apoptosis and GSH depletion were effectively inhibited or abrogated by a thiol antioxidant,N-acetyl-L-cysteine(NAC),whereas other antioxidant(PEG-catalase and PEG-SOD)did not prevent apoptosis and GSH depletion.Alantolactone treatment inhibited the translocation of NF-κB into nucleus however NF-κB inhibitor,SN50 failed to potentiate alantolactone-induced apoptosis indicating that alantolactone induces NF-κB-independent apoptosis in glioblastoma cells.These findings suggest that the sensitivity of tumor cells to alantolactone appears to results from GSH depletion and ROS production.Furthermore,our in vivo toxicity study demonstrated that alantolactone did not induce significant hepatotoxicity and nephrotoxicity in mouse and can cross blood-brain barrier.Therefore alantolactone may become a potential lead compound for future development of antiglioma therapy. |
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