title: PI3K/AKT pathway-mediated regulation of p27(Kip1) is associated with cell cycle arrest and apoptosis in cervical cancer.
abstract: BACKGROUND
  The cyclin-dependent kinase inhibitor p27(Kip1) is known to act as a putative tumor suppressor in several human cancers, including cervical cancer. Down-regulation of p27(Kip1) may occur either through transcription inhibition or through phosphorylation-dependent proteolytic degradation. As yet, the mechanism underlying p27(Kip1) down-regulation and its putative downstream effects on cervical cancer development are poorly understood. Here we assessed the expression and sub-cellular localization of p27(Kip1) and its effects on proliferation, cell cycle progression and (inhibition of) apoptosis in cervical cancer cells.
  
  
  METHODS
  Primary cervical cancer samples (n = 70), normal cervical tissue samples (n = 30) and cervical cancer-derived cell lines (n = 8) were used to assess the expression of p27(Kip1) and AKT1 by RT-PCR, Western blotting and immunohistochemistry, respectively. The effects of the PI3K inhibitor LY294004 and the proteasome inhibitor MG132 on cervical cancer cell proliferation were investigated using a MTT assay. Apoptosis and cell cycle analyses were carried out using flow cytometry, and sub-cellular p27(Kip1) localization analyses were carried out using immunofluorescence assays.
  
  
  RESULTS
  We observed p27(Kip1) down-regulation (p = 0.045) and AKT1 up-regulation (p = 0.046) in both the primary cervical cancer samples and the cervical cancer-derived cell lines, compared to the normal cervical tissue samples tested. Treatment of cervical cancer-derived cell lines with the PI3K inhibitor LY294002 resulted in a reduced AKT1 activity. We also observed a dose-dependent inhibition of cell viability after treatment of these cell lines with the proteasome inhibitor MG132. Treatment of the cells with LY294002 resulted in a G1 cell cycle arrest, a nuclear expression of p27(Kip1), and a cytoplasmic p27(Kip1) accumulation after subsequent treatment with MG132. Additionally, we found that the synergistic effect of MG132 and LY294002 resulted in a sub-G1 cell cycle arrest and apoptosis induction through poly (ADP-ribose) polymerase (PARP) cleavage.
  
  
  CONCLUSIONS
  Our data suggest that p27(Kip1) down-regulation in cervical cancer cells is primarily regulated through PI3K/AKT-mediated proteasomal degradation. The observed synergistic effect of the MG132 and LY294002 inhibitors may form a basis for the design of novel cervical cancer therapies.
authors.affiliation: Cancer Genetics Laboratory, Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi, 221005, India.
authors.forename: Shyam Babu
authors.initials: SB
authors.lastname: Prasad
authors.forename: Suresh Singh
authors.initials: SS
authors.lastname: Yadav
authors.forename: Mitali
authors.initials: M
authors.lastname: Das
authors.forename: Arusha
authors.initials: A
authors.lastname: Modi
authors.forename: Soni
authors.initials: S
authors.lastname: Kumari
authors.forename: Lakshmi Kant
authors.initials: LK
authors.lastname: Pandey
authors.forename: Sunita
authors.initials: S
authors.lastname: Singh
authors.forename: Satyajit
authors.initials: S
authors.lastname: Pradhan
authors.forename: Gopeshwar
authors.initials: G
authors.lastname: Narayan
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D059447:Cell Cycle Checkpoints / Q000502:physiology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000473:pathology*
chemical_list: C436291:CDKN1B protein, human; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt
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