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Synthesis, characterization, and evaluation of Cd[L-proline]2, a novel histone deacetylase inhibitor that induces epigenetic modification of histone deacetylase isoforms in A549 cells

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Summary

Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression through their effects on the compact chromatin structure. In clinical studies, several classes of histone deacetylase inhibitors (HDACi) have demonstrated potent anticancer activities with metal complexes. Hence, we synthesized cadmium–proline complexes using both the D- and L-isomers of proline and evaluated their biological activities by observing the efficiency of their inhibition of HDAC activity, ability to reduce the expression of HDAC isoforms in A549 cells and effect on apoptosis. The synthesized compounds were characterized by UV, IR, NMR spectroscopy and elemental analysis. In-vitro cell toxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the 50% inhibitory concentration (IC50; 2 μM) was obtained at 12 h. The morphological study at nuclear levels was performed by acridine orange/ethidium bromide (AO/EB) and Hoechst staining, and the results showed an association with cell cycle arrest at the G2/M phase. Both cadmium–proline complexes intensely inhibited HDAC activity at 2 μM concentration. Interestingly, Cd[L-proline]2 was found to be a potent inhibitor for all HDAC isoforms, whereas Cd[D-proline]2 inhibited only HDAC1 and 2. HDACi are novel chemotherapeutic drugs that induce hyperacetylation of histones H3 and H4, counteracting the aberrant repression of genes, such as insulin-like growth factor-binding protein 3 (IGFBP-3), p53, and p21. ERK/MAPK signaling pathway resulted in the downregulation of the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), contributing to the inhibition of metastasis in A549 cells. Apoptosis induction was accompanied by the activation of death receptors and their ligands which recruit initiator caspase 8, decrease in mitochondrial membrane potential (ΔΨm), as well as increased Bax/Bcl2 ratio, followed by activation of caspases 9 and 3. Our finding suggests that Cd[L-proline]2 complex accelerates epigenetic rearrangement by HDAC inhibition, which may be the key mechanism for its anticancer activity.

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Acknowledgements

The first author is grateful to Bharathidasan University, Tiruchirappalli, India, for providing financial support through their University Research fellowship (URF) scheme (Ref. number 055441/URF/K7/2013). The fourth author thanks the Department of Science and Technology, Science and Engineering Board (DST-SERB, SB/FT/CS-086/2013), India, for providing research fund. We acknowledge Dr. C. Prahalathan and Dr. A. Antony Joseph Velanganni, Department of Biochemistry, Bharathidasan University, Tiruchirappalli, India, for their help with gel documentation and fluorescence microscopic studies. We are grateful to the Department of Science and Technology–Fund for Improvement of S&T Infrastructure in Universities and Higher Educational Institutions (DST-FIST) for their infrastructure support to our department.

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Authors and Affiliations

  1. Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Anusha Chidambaram, Kalaiarasi Arunachalam & Ravikumar Vilwanathan

  2. Department of Biotechnology, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Arunachalam Sekar

  3. Organic Chemistry Laboratory, Department of Chemistry, Veltech University, Avadi, Chennai, 600 062, Tamil Nadu, India

    Kavya S.H. & Ramesh Kumar Chidambaram

  4. Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, 605 006, Tamil Nadu, India

    Senthilkumar G.P.

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  1. Anusha Chidambaram
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Correspondence to Ramesh Kumar Chidambaram or Ravikumar Vilwanathan.

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The study was supported by the Department of Science and Technology, Science and Engineering Board (DST-SERB, SB/FT/CS-086/2013), India.

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Electronic supplementary material

ESM 1

Electronic spectra of Cd[D-proline]2 and Cd[L-proline]2 complexes in H2O (DOCX 47 kb)

ESM 2

FT-IR spectra of Cd[proline]2 complexes (DOCX 60 kb)

ESM 3

The sequence of primers used in RT-PCR (DOCX 18 kb)

ESM 4

FT-IR spectral data of the Cd[proline]2 complexes (DOCX 13 kb)

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Chidambaram, A., Sekar, A., S.H., K. et al. Synthesis, characterization, and evaluation of Cd[L-proline]2, a novel histone deacetylase inhibitor that induces epigenetic modification of histone deacetylase isoforms in A549 cells. Invest New Drugs 35, 691–705 (2017). https://doi.org/10.1007/s10637-017-0489-1

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