title: Flavonol isolated from ethanolic leaf extract of Thuja occidentalis arrests the cell cycle at G2-M and induces ROS-independent apoptosis in A549 cells, targeting nuclear DNA.
abstract: OBJECTIVE
  The K-ras gene mutation commonly found in lung adenocarcinomas contributes to their non-invasive expansion. Our main objective here was to develop a chemopreventive agent against K-ras-mutated lung adenocarcinoma cell line like-A549.
  
  
  METHODS
  We isolated flavonol from ethanolic leaf extract of Thuja occidentalis, and evaluated its apoptotic potentials on A549 cells. They were treated with 1-10 μg/ml of flavonol and viability was tested retaining normal lung cells L-132 as control. We performed assays such as TUNEL, annexin V, cell-cycle and mitochondrial membrane potentials, by FACS analysis. ROS-mediated oxidative stress and drug-DNA interactions were analysed along with gene expression studies for p53, Bax-Bcl2, cytochrome c, the caspase cascade genes and PARP.
  
  
  RESULTS
  Flavonol reduced A549 cell viability in a dose- and time-dependent manner (IC50 value = 7.6 ± 0.05 μg/ml following 48 h incubation) sparing normal L-132 cells. It effected G2-M phase cell cycle arrest and apoptosis, as indicated by progressive increase in the sub-G1, annexin V and TUNEL-positive cell populations. Apoptotic effects appeared to be mitochondria-dependent, caspase-3-mediated, but ROS-independent. Analysis of circular dichroism data revealed that flavonol intercalated with nuclear DNA. In vivo studies on non small cell lung carcinoma (NSCLC)-induced mice confirmed anti-cancer potential of flavonol.
  
  
  CONCLUSIONS
  Flavonol-induced apoptosis apparently resulted from intercalation of cells' nuclear DNA. Flavonol inhibited growth of induced lung tumours in the mice, indicating its potential as an effective agent against NSCLC.
authors.affiliation: Department of Zoology, Cytogenetics and Molecular Biology Laboratory, University of Kalyani, Kalyani, West Bengal, 741235, India.
authors.forename: A
authors.initials: A
authors.lastname: Mukherjee
authors.forename: S
authors.initials: S
authors.lastname: Sikdar
authors.forename: K
authors.initials: K
authors.lastname: Bishayee
authors.forename: N
authors.initials: N
authors.lastname: Boujedaini
authors.forename: A R
authors.initials: AR
authors.lastname: Khuda-Bukhsh
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000473:pathology; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004273:DNA, Neoplasm / Q000187:drug effects; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D044948:Flavonols / Q000302:isolation & purification / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology; D018515:Plant Leaves / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism; D029783:Thuja / Q000737:chemistry*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004273:DNA, Neoplasm; D044948:Flavonols; D010936:Plant Extracts; D017382:Reactive Oxygen Species