1-Amino-4-hydroxy-9,10-anthraquinone - An analogue of anthracycline anticancer drugs, interacts with DNA and induces apoptosis in human MDA-MB-231 breast adinocarcinoma cells: Evaluation of structure-activity relationship using computational, spectroscopic and biochemical studies

Abstract

The X-ray diffraction and spectroscopic properties of 1-amino-4-hydroxy-9,10-anthraquinone (1-AHAQ), a simple analogue of anthracycline chemotherapeutic drugs were studied by adopting experimental and computational methods. The optimized geometrical parameters obtained from computational methods were compared with the results of X-ray diffraction analysis and the two were found to be in reasonably good agreement. X-ray diffraction study, Density Functional Theory (DFT) and natural bond orbital (NBO) analysis indicated two types of hydrogen bonds in the molecule. The IR spectra of 1-AHAQ were studied by Vibrational Energy Distribution Analysis (VEDA) using potential energy distribution (PED) analysis. The electronic spectra were studied by TDDFT computation and compared with the experimental results. Experimental and theoretical results corroborated each other to a fair extent. To understand the biological efficacy of 1-AHAQ, it was allowed to interact with calf thymus DNA and human breast adino-carcinoma cell MDA-MB-231. It was found that the molecule induces apoptosis in this adinocarcinoma cell, with little, if any, cytotoxic effect in HBL-100 normal breast epithelial cell.

Keywords: 1-AHAQ; 1-AHAQ, 1-amino-4-hydroxy-9,10-anthraquinone; Apoptosis; Calf thymus DNA; DFT and spectroscopy; DFT, Density Functional Theory; NBO, natural bond orbital; PED, potential energy distribution; VEDA, Vibrational Energy Distribution Analysis; X-ray diffraction; ct DNA, calf thymus DNA.

Graphical abstract

Scheme 1

1-Amino-4-hydroxy-9,10-anthraquinone.

Fig. 1

X-ray crystallographic structure of 1-AHAQ (per unit cell two identical molecules are present).

Fig. 2

Optimized molecular structure of 1-amino-4-hydroxy-9,10-anthraquinone using B3LYP/6-31+G(d,p) protocol.

Fig. 3

The isodensity plot of HOMO, LUMO, HOMO-1 and LUMO+1 for 1-amino-4-hydroxy-9,10-anthraquinone.

Fig. 4

Important molecular orbitals involved in electronic transitions of 1-amino-4-hydroxy-9,10-anthraquinone.

Fig. 5

(a). Absorption spectra of 1-AHAQ (20 µM) in the absence (Curve-1) and in the presence of different calf thymus DNA concentrations (Curve- 2–7). pH=7.4, [NaCl]=120 mM, 25 °C. (b). Double reciprocal plot, (A₀/A–A₀) vs. 1/[DNA], of 1-AHAQ–calf thymus DNA interaction; [1-AHAQ]₀ = 20 µM, pH=7.4, [NaCl]=120 mM, 25 °C.

Fig. 6

(a) Double reciprocal plot of 1-AHAQ – calf thymus DNA interaction using UV–vis spectroscopy; [1-AHAQ]₀ = 20 µM, pH=7.4, [NaCl]=120 mM, 25 °C. (b). Binding isotherm of 1-AHAQ and calf thymus DNA and corresponding non-linear fit using UV–vis spectroscopy; [1-AHAQ]₀=20 µM, pH=7.4, [NaCl]=120 M, 25^○C.

Fig. 7

Absorption spectra of 1-AHAQ in the absence (solid line) and presence (dotted line) of single stranded ct DNA in phosphate buffer at pH 7.4. [1-AHAQ] = 30 µM, [Phosphate Buffer]=50 µM, [NaCl]=120 mM, [ss DNA]=80 µM, 298.15 K.

Fig. 8

(a) MTT assay for 1-AHAQ for 24 h and 48 h in MDAMB 231 cells. (b) MTT assay for 1-AHAQ for 24 h and 4 8 h in HBL-100 cells.

Fig. 9

The effect of 1-AHAQ on MDAMB 231 cells with Acridine orange and ethidium bromide staining. (a) Representative morphological changes observed against MDAMB 231 cells after 24 h incubation with 1-AHAQ. (b) Relative percentage of morphological changes was determined and classified into three categories: viable, apoptosis and necrosis as compared with the control cells after 24 h incubation.

Fig. 10

The effect of 1-AHAQ on MDAMB 231 cells with Hoechst staining. (a) Representative morphological changes observed against MDAMB 231 cells after 24 h incubation with 1-AHAQ. (b) Relative percentage of morphological changes was determined and classified into two categories: Normal and abnormal nuclei as compared with the control cells after 24h incubation.