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PI3K/AKT pathway-mediated regulation of p27Kip1 is associated with cell cycle arrest and apoptosis in cervical cancer

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Abstract

Background

The cyclin-dependent kinase inhibitor p27Kip1 is known to act as a putative tumor suppressor in several human cancers, including cervical cancer. Down-regulation of p27Kip1 may occur either through transcription inhibition or through phosphorylation-dependent proteolytic degradation. As yet, the mechanism underlying p27Kip1 down-regulation and its putative downstream effects on cervical cancer development are poorly understood. Here we assessed the expression and sub-cellular localization of p27Kip1 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 p27Kip1 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 p27Kip1 localization analyses were carried out using immunofluorescence assays.

Results

We observed p27Kip1 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 p27Kip1, and a cytoplasmic p27Kip1 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.

Conclusion

Our data suggest that p27Kip1 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.

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Acknowledgments

We thank the Department of Biotechnology (DBT) of the Ministry of Science and Technology, India for financial support (BT/PR9246/Med/30/17/2007) to GN, SS and SP, and the Indian Council of Medical Research (ICMR) for providing a Senior Research Fellowship to SBP. The authors are thankful to Dr. V.V.V.S. Murty (Columbia University, New York, USA) for the kind gift of the cervical cancer cell lines.

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The authors declare that they have no competing conflict of interest.

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Correspondence to Gopeshwar Narayan.

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Prasad, S.B., Yadav, S.S., Das, M. et al. PI3K/AKT pathway-mediated regulation of p27Kip1 is associated with cell cycle arrest and apoptosis in cervical cancer. Cell Oncol. 38, 215–225 (2015). https://doi.org/10.1007/s13402-015-0224-x

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