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Expression/localization patterns of sirtuins (SIRT1, SIRT2, and SIRT7) during progression of cervical cancer and effects of sirtuin inhibitors on growth of cervical cancer cells

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Tumor Biology

Abstract

Sirtuins belong to the family of class III histone deacetylases; its role in neoplasia is controversial as both tumor-suppressive and promoting functions have been reported. There are very few reports available, where expressions of sirtuin isoforms are comprehensively analyzed during neoplasia. Therefore, in the present study, the expression of SIRT1, SIRT2, and SIRT7 during different stages of cervical cancer progression was analyzed. The normal cervical epithelium showed feeble expression of sirtuin isoforms, SIRT1, SIRT2, and SIRT7. A significant increase in SIRT1 expression was noted in the cytoplasm as well as in the nucleus of proliferative layers of cervical epithelium in squamous intraepithelial lesions (SIL); however, in the squamous cell carcinomas (SCC), a heterogeneous pattern of SIRT1 expression varying from low to high was noted. A progressive increase in the expression of both SIRT2 and SIRT7 was noted during cancer progression in the following order: normal < preneoplasia < cancer. Cervical cancer cell lines, HeLa and SiHa, showed higher levels of SIRT1 and SIRT2 in comparison to the immortalized cell counterpart, HaCaT. Specific inhibitors of SIRT1 (Ex527) and SIRT2 (AGK2) impaired the growth of the cervical cancer cells, SiHa, but not of the HaCaT cells. SIRT1 inhibition caused cell death, while SIRT2 inhibition resulted in cell cycle arrest. In conclusion, we report the overexpression of SIRT2 and SIRT7 proteins in cervical cancer and suggest probable application of sirtuin inhibitors as therapeutic targets. Further, a specific increase in the levels of SIRT1 in intraepithelial lesion makes it a promising candidate for identification of preneoplastic changes.

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Abbreviations

SIRT:

Sirtuin

IHC:

Immunohistochemistry

SIL:

Squamous intraepithelial lesion

SCC:

Squamous cell carcinoma

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Acknowledgments

We thank Dr. Lucy M. Anderson for the valuable suggestions, critical reading, and editing. SS is supported by a fellowship from the Department of Biotechnology. Laboratory of GR is supported by the funding from the Department of Biotechnology, New Delhi, India. We thank Mr. Bala for the help in flow cytometry. We thank Dr Nirupama Chatterjee for the help with statistical analysis. The critical comments and suggestions from the anonymous reviewers have helped immensely to improve the manuscript.

Conflicts of interest

None

Author contribution

SS performed all the experiments with the help from ST and SK. PAR helped in immunohistochemistry; BS and SS helped in immunoblotting. VVR performed the statistical analysis. The pathological evaluations were by UK. GR designed the study and wrote the manuscript with inputs from SS.

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Author notes

  1. Gayatri Ramakrishna

    Present address: Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences (ILBS), D1 Block, Vasant Kunj, Delhi, 110070, India

Authors and Affiliations

  1. Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

    Sapna Singh & Shashi Kiran

  2. National Institute of Nutrition, Hyderabad, India

    Sapna Singh, P. Uday Kumar & Vishnu Vardhan Rao

  3. BioGenex Lab Inc, Hyderabad, India

    Suresh Thakur & A. R. Poongothai

  4. Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences (ILBS), D1 Block, Vasant Kunj, Delhi, 110070, India

    Bijoya Sen & Shreya Sharma

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Electronic supplementary material

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Supplementary Data 1

(SD1) Details of the antibodies used for immunohistochemistry and the standardized conditions. (DOCX 14 kb)

Supplementary Data 2

(SD2) SIRT1 immunohistochemistry in cervical epithelium. A. Immunohistchemistry for SIRT1 expression in normal, SIL and SCC cases. Note the intense cytoplasmic and nuclear expression of SIRT1 in SIL cases compared to normal. B. Pie chart indicating heterogeneous expression pattern of SIRT1 in squamous cell carcinoma (SCC) as noted in immunohistochemistry. (GIF 253 kb)

High Resolution Image (TIFF 20154 kb)

Supplementary Data 3

(SD3) 1A Chi-Square Values of immunohistochemical expression profile of SIRT1, SIRT2 and SIRT7 in Normal, SIL and SCC cases as shown in Table 2 of the main text. 1B Chi-Square Values of Co-expression/association of sirtuin isoforms and p27 as shown in Table 3. of the main text. 1C: Chi-Square Values of Co-expression of sirtuin isoforms and p27 in various stages of cancer. (DOC 53 kb)

Supplementary Data 4

(SD4) Expression analysis of sirtuin proteins (SIRT1, SIRT2 and SIRT7) by immunoblot as seen in different replicate experiments. (GIF 6 kb)

High Resolution Image (TIFF 1502 kb)

Supplementary Data 5

(SD5) Effect of Sirtinol on growth of SiHa cells. A. Bar diagram indicating viability of SiHa cells treated with either solvent alone or with different doses of Sirtinol. Superscripts a and b indicate significant difference with control and treatment with 50 μM dose of Sirtinol respectively (P < 0.05). B. Phase contrast images of cells treated with 100 μM of Sirtinol (20× magnification). C and D. Cell cycle profile and distribution of SiHa cells in various cell cycle phases following treatment with Sirtinol (100 μM) and evaluated by flow cytometry. (GIF 44 kb)

High Resolution Image (TIFF 17956 kb)

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Singh, S., Kumar, P.U., Thakur, S. et al. Expression/localization patterns of sirtuins (SIRT1, SIRT2, and SIRT7) during progression of cervical cancer and effects of sirtuin inhibitors on growth of cervical cancer cells. Tumor Biol. 36, 6159–6171 (2015). https://doi.org/10.1007/s13277-015-3300-y

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  • DOI: https://doi.org/10.1007/s13277-015-3300-y

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