title: Induction of apoptosis and inhibition of PI3K/Akt pathway in PC-3 and LNCaP prostate cancer cells by ethanolic neem leaf extract.
abstract: OBJECTIVE
  The present study is aimed to delineate the effect of ethanolic neem leaf extract on PI3K/Akt and apoptotic pathway in prostate cancer cell lines (PC-3 and LNCaP).
  
  
  METHODS
  To test the hypothesis, two different prostate cancer cell lines LNCaP (androgen dependent) and PC-3 (androgen independent) were taken. Cells were exposed to various concentrations of ethanolic neem leaf extract (ENLE) (25-125 μg/ml). The doses were fixed by cell viability (MTT) assay. For apoptotic detection in situ apoptosis assay, caspase-3 activity and protein expression of cytochrome c and Poly-ADP Ribose Polymerase (PARP) were analysed as well as mRNA expression of Bcl-2 family proteins was studied by RT-PCR. The phosphoinositide 3-kinase (PI3K) and p-Akt were analysed by western blotting and mRNA expression of Akt 1 and 2, PTEN was performed by RT-PCR. Immunoblotting of cyclin D1 and p21 was done to access the inhibition of cell proliferation.
  
  
  RESULTS
  ENLE gives 50% inhibition at a dose of 100 μg/ml in both PC-3 and LNCaP cells and considered as effective dose. ENLE decreased the protein expression of PI3K as well as p-Akt and the mRNA expression of Akt 1and 2 in both the cells. There was a significant decrease in mRNA expression of PTEN in LNCaP cells. ENLE induced apoptosis and inhibited cell proliferation by inhibiting PI3K/Akt pathway. Decrease in p-Akt leads to increase in the protein level of Bad, p21 and decrease in the cyclin D1, respectively. ENLE treatment increased the cytochrome c expression and caspase-3 activity as well as regulated the mRNA expression of Bcl-2 family proteins thereby inducing apoptosis to both the cell lines. In situ apoptosis assay showed increased red fluorescence in 100 μg/ml of ENLE in both PC-3 and LNCaP cell lines.
  
  
  CONCLUSIONS
  The results suggested that ENLE induces apoptosis and inhibits cell proliferation through inhibiting PI3K/Akt pathway in both PC-3 and LNCaP cells.
authors.affiliation: Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, Tamilnadu, India.
authors.forename: Dharmalingam N
authors.initials: DN
authors.lastname: Gunadharini
authors.forename: Perumal
authors.initials: P
authors.lastname: Elumalai
authors.forename: Ramachandran
authors.initials: R
authors.lastname: Arunkumar
authors.forename: Kalimuthu
authors.initials: K
authors.lastname: Senthilkumar
authors.forename: Jagadeesan
authors.initials: J
authors.lastname: Arunakaran
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D031601:Azadirachta / Q000737:chemistry*; D001483:Base Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D017931:DNA Primers; D000431:Ethanol / Q000737:chemistry; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry*; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000473:pathology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018797:Cell Cycle Proteins; D017931:DNA Primers; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010936:Plant Extracts; D047428:Protein Kinase Inhibitors; D000431:Ethanol; D051057:Proto-Oncogene Proteins c-akt