Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells

Abstract

The studies on protein–dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA–TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH–HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH–HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH–HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.

Graphical abstract

Keywords:

• human serum albumin
• thionine
• emission spectroscopy
• molecular docking
• MCF-7 breast cancer cells

Acknowledgments

P.M. gratefully acknowledges University Grants Commission for Rajiv Gandhi National Fellowship (UGC-RGNF-F1-17.1/2011-12/RGNF-SC-TAM-4355) and M.I. acknowledges University Grants Commission [UGC-MRP, Project no. 41-309/2012 (SR)] India for the financial support. G. Benelli is supported by PROAPI (PRAF 2015) and University of Pisa, Department of Agriculture, Food and Environment [Grant ID: COFIN2015_22]. Funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Prof. A. Ramu (Madurai Kamaraj University) is gratefully acknowledged for allowing us to record the CD spectra.