title: Direct inhibition of retinoblastoma phosphorylation by nimbolide causes cell-cycle arrest and suppresses glioblastoma growth.
abstract: OBJECTIVE
  Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica, controls glioblastoma growth.
  
  
  METHODS
  Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma.
  
  
  RESULTS
  We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastoma multiforme in vitro and in vivo. Azt caused cell-cycle arrest, most prominently at the G1-S stage in glioblastoma multiforme cells expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma protein, cell-cycle arrest at G1-S, and cell death. Independent of retinoblastoma hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastoma multiforme cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor-induced phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific because Azt did not affect mTOR or other cell-cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of glioblastoma multiforme growth.
  
  
  CONCLUSIONS
  Our preclinical findings demonstrate nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.
authors.affiliation: Departments of Oncology, Cincinnati Children's Hospital Medical Center, OH.|James L. Winkle College of Pharmacy, University of Cincinnati, OH.
authors.forename: Swagata
authors.initials: S
authors.lastname: Karkare
authors.affiliation: Departments of Oncology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Rishi Raj
authors.initials: RR
authors.lastname: Chhipa
authors.affiliation: Departments of Oncology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Jane
authors.initials: J
authors.lastname: Anderson
authors.affiliation: Departments of Oncology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Xiaona
authors.initials: X
authors.lastname: Liu
authors.affiliation: Departments of Oncology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Heather
authors.initials: H
authors.lastname: Henry
authors.affiliation: Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Anjelika
authors.initials: A
authors.lastname: Gasilina
authors.affiliation: Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Nassar
authors.affiliation: Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Jayeeta
authors.initials: J
authors.lastname: Ghosh
authors.affiliation: Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Clark
authors.affiliation: Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Ashish
authors.initials: A
authors.lastname: Kumar
authors.affiliation: James L. Winkle College of Pharmacy, University of Cincinnati, OH.
authors.forename: Giovanni M
authors.initials: GM
authors.lastname: Pauletti
authors.affiliation: Maryville University, St. Louis, MO.
authors.forename: Pradip K
authors.initials: PK
authors.lastname: Ghosh
authors.affiliation: Departments of Oncology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Biplab
authors.initials: B
authors.lastname: Dasgupta
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D031601:Azadirachta / Q000737:chemistry; D001932:Brain Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D051358:Cyclin-Dependent Kinase 4 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051361:Cyclin-Dependent Kinase 6 / Q000037:antagonists & inhibitors / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D005909:Glioblastoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D036701:Limonins / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology; D018515:Plant Leaves / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D036701:Limonins; D010936:Plant Extracts; D016160:Retinoblastoma Protein; C042198:nimbolide; C512478:EGFR protein, human; D066246:ErbB Receptors; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6