title: Molecular targeting of Akt by thymoquinone promotes G(1) arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells.
abstract: OBJECTIVE
  Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells.
  
  
  METHODS
  MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies.
  
  
  RESULTS
  Studies revealed G1 phase arrest till 24h incubation with TQ while extended exposure showed phase shift to subG1 indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K.
  
  
  CONCLUSIONS
  Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.
authors.affiliation: School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
authors.forename: Shashi
authors.initials: S
authors.lastname: Rajput
authors.forename: B N Prashanth
authors.initials: BN
authors.lastname: Kumar
authors.forename: Kaushik Kumar
authors.initials: KK
authors.lastname: Dey
authors.forename: Ipsita
authors.initials: I
authors.lastname: Pal
authors.forename: Aditya
authors.initials: A
authors.lastname: Parekh
authors.forename: Mahitosh
authors.initials: M
authors.lastname: Mandal
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000187:drug effects / Q000378:metabolism; D016227:Benzoquinones / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019938:Cyclin D1 / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D005260:Female; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D061986:MCF-7 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D058990:Molecular Targeted Therapy; D014176:Protein Biosynthesis / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D051017:Apoptosis Regulatory Proteins; D016227:Benzoquinones; D019938:Cyclin D1; D051057:Proto-Oncogene Proteins c-akt; C003466:thymoquinone
keywords: Cyclin D1; G(1) phase arrest; PI3K/Akt signaling pathway; Thymoquinone; Thymoquinone (PubChem CID: 10281); Translational proteins