DNA binding and apoptotic induction ability of harmalol in HepG2: Biophysical and biochemical approaches

doi:10.1016/j.cbi.2016.08.024

Chemico-Biological Interactions

Volume 258, 25 October 2016, Pages 142-152

https://doi.org/10.1016/j.cbi.2016.08.024 Get rights and content

Highlights

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Harmalol shows maximum growth inhibition in HepG₂ with GI₅₀ of 14.2 μM.
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The alkaloid causes conformational changes and stabilization in CT-DNA.
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Induced DNA damage, alterations in cellular morphology and membrane potential.
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Alkaloid shows ROS dependent, p53 and caspase activated cytotoxicity in the cells.
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A significant increase in Sub G_o/G₁ population of HepG₂ cell.

Abstract

Harmalol administration caused remarkable reduction in proliferation of HepG₂ cells with GI₅₀ of 14.2 μM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism (CD) and differential scanning calorimetric (DSC) analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 °C. Binding constant and stoichiometry was calculated using the above biophysical techniques. The Scatchard plot constructed from CD data showed cooperative binding, from which the cooperative binding affinity (K’ω) of 4.65 ± 0.7 × 10⁵ M⁻¹, and n value of 4.16 were deduced. The binding parameter obtained from DSC melting data was in good agreement with the above CD data. Furthermore, dose dependent apoptotic induction ability of harmalol was studied in HepG₂ cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub G_o/G₁ population made the cancer cell, HepG₂, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

Graphical abstract

Introduction

Deoxyribonucleic acid that serves as the repository of genetic information of the cell is the potential therapeutic targets in cancer. Again, plant alkaloids, a natural product as chemotherapeutic agents isolated so far, have been reported to have remarkable anti-cancer applications [1], [2], [3], [4], [5] and consequently there is growing interest in the search for better anti-cancer drugs with high efficacy, low toxicity and minimum side effects. But most of the chemotherapeutic agents due to their rather non-selective nature and dose limiting toxicity, use is often restricted, necessitating search for newer drugs having greater potential and suitability for use. β carbonil is one such large group of natural and synthetic alkaloids (ligand) of indole derivatives. These alkaloids were originally isolated from plants like Peganum harmala L and Banisteriopsis caapi. Our previous investigation showed that beta carboline plant alkaloid, harmalol, is an excellent intercalator preferring hetero G/C sequence [6], [7]. Recently, we further showed its RNA binding ability [8]. Harmalol, has been reported to have several pharmacological, neurophysiological and biochemical activities [9], [10], [11], [12], [13], [14], [15] as well as in vivo and in vitro chemopreventive action against different cancer cell line [16], [17], [18], but its detail apoptotic effect on human cancer cell line has not been studied yet.

Inspired by these results, in the present investigation we elucidate, harmalol inducing apoptotic effects in HepG₂ cells and consequently study its stabilization, binding and conformational changes with CT DNA using different biochemical and biophysical techniques. Further, event like ROS generation and cell cycle arrests were also accompanied in the harmalol induced apoptosis. A complete understanding of these therapeutic aspects of beta carboline alkaloid will enable the researchers for the future drug design for the betterment of mankind.

Section snippets

Biochemicals

Harmalol was obtained from Sigma-Aldrich (St. Louis, MO, USA). The purity of the sample was confirmed as done earlier [6], [7]. Its concentration was determined using molar extinction coefficient value of 19,000 M⁻¹ cm⁻¹ at 371 nm [7]. Double stranded calf thymus DNA (CT DNA) was also obtained from Sigma—Aldrich. Concentration was determined using the molar extinction coefficient (ε) of 6600 M⁻¹ cm⁻¹ at 260 nm. Harmalol and CT DNA was dissolved in 15 mM Citrate–Phosphate (CP) buffer of pH 6.8.

Cell lines and culture conditions

Determination of GI₅₀ by MTT

To evaluate the efficacy of harmalol (Fig. 1A) on different human cancer cell line viz. HeLa, MDA-MB-231, A549, and HepG₂, the alkaloid was tested for its cytotoxicity using the MTT assay. After 48 h treatment, it was found that among the above four cell lines, harmalol showed GI₅₀ values of 42.5 μM in HeLa cells, 23.7 μM in MDA-MB-231 cells, 14.2 μM in HepG₂ cell and 45.2 μM in A549 cells. Hence, the highest degree of concentration-dependent increase in growth inhibition was reported in HepG₂

Acknowledgements

KB and SS are indebted to the Council of Scientific and Industrial Research (CSIR), Government of India for the financial support (Ref. No. 37 (1538)/12/EMR-II). PB is supported by grant from University of Kalyani. Authors are thankful to DST-PURSE and PRG-2015-16, University of Kalyani and also grateful to the Department of Environmental Science, University of Kalyani for providing fluorescence microscope facility.

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¹: Department of Zoology, University of Kalyani, Nadia, Kalyani, 741235.

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DNA binding and apoptotic induction ability of harmalol in HepG2: Biophysical and biochemical approaches

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Biochemicals

Cell lines and culture conditions

Determination of GI50 by MTT

Acknowledgements

J. Photochem. Photobiol. B

J. Pharm. Sci.

Jpn. J. Pharmacol.

Asian Pac. J. Trop. Biomed.

Methods

Biochim. Biophys. Acta

Mutat. Res.

Spectrochim. Acta A

Eur. J. Pharma. Sci.

Biochem. Pharmacol.