The cytotoxicity of benzaldehyde nitrogen mustard-2-pyridine carboxylic acid hydrazone being involved in topoisomerase IIα inhibition

Abstract

The antitumor property of iron chelators and aromatic nitrogen mustard derivatives has been well documented. Combination of the two pharmacophores in one molecule in drug designation is worth to be explored. We reported previously the syntheses and preliminary cytotoxicity evaluation of benzaldehyde nitrogen mustard pyridine carboxyl acid hydrazones (BNMPH) as extended study, more tumor cell lines (IC50 for HepG2: 26.1 ± 3.5 μM, HCT-116: 57.5 ± 5.3 μM, K562: 48.2 ± 4.0 μM, and PC-12: 19.4 ± 2.2 μM) were used to investigate its cytotoxicity and potential mechanism. In vitro experimental data showed that the BNMPH chelating Fe(2+) caused a large number of ROS formations which led to DNA cleavage, and this was further supported by comet assay, implying that ROS might be involved in the cytotoxicity of BNMPH. The ROS induced changes of apoptosis related genes, but the TFR1 and NDRG1 metastatic genes were not obviously regulated, prompting that BNMPH might not be able to deprive Fe(2+) of ribonucleotide reductase. The BNMPH induced S phase arrest was different from that of iron chelators (G1) and alkylating agents (G2). BNMPH also exhibited its inhibition of human topoisomerase IIα. Those revealed that the cytotoxic mechanism of the BNMPH could stem from both the topoisomerase II inhibition, ROS generation and DNA alkylation.

Figure 1

The chemical structure of BNMPH.

Figure 2

Cytotoxicities of BNMPH in the indicated cell lines (IC₅₀: 57.5 ± 5.0 μM for HCT-116; 19.4 ± 2.2 μM for PC-12; 26.1 ± 3.5 μM for HepG2; and 48.2 ± 4.0 μM for K562).

Figure 3

BNMPH redox activity and induction of DNA fragmentation. (a) ROS generation by Fenton reaction; the ROS product was measured by DCF fluorescence. (b) The ROS induced DNA fragmentation in the presence or absence of BNMPH; the fragmented DNA was separated by agarose gel electrophoresis and visualized by EB staining, and the concentration is as indicated. (c) DNA fragmentation in vivo was evaluated by comet assay, top: control, and bottom: in the presence of 50 μM BNMPH.

Figure 4

DNA alkylation of BNMPH. (a) BNMPH effect on DNA thermal denaturation. The flatted curve (black) indicated that the dissociation of double stranded DNA was blocked during temperature rising due to cross-linking. (b) BNMPH induced DNA cross-linking. The cross-linked DNA was separated by electrophoresis and visualized by EB staining, and the concentrations used are as indicated.

Figure 5

The effect of BNMPH on gene regulation. 1: 12 μM BNMPH; 2: 6 μM BNMPH; 3: control, the genes in the figure as indicated.

Figure 6

BNMPH induced cell cycle arrest. (a) Control; (b) 6 μM BNMPH; and (c) 12 μM BNMPH, the cell distribution in the figure as indicated.

Figure 7

Topoisomerase II inhibition of BNMPH. (a) The docked BNMPH in human topoisomerase II-DNA complex. (b) Comparison of BNMPH and etoposide in topoisomerase II complex. (c) Human topoisomerase IIα inhibition of BNMPH. The concentration is used as indicated. At 50 μM BNMPH almost blocked the topoisomerase catalyzed DNA relaxation.

Figure 8

BNMPH reactivity with DTT and BSA. (a) The absorbance spectral changes of DTT after addition of 26 μM BNMPH to 500 μM DTT (pH 8.0, 37°C). The spectrum was collected after the mixing, and subsequent spectrum at 5 min intervals was recorded. The insert showed a series of increases during the 25 min period, indicating that BNMPH can alkylate thiol group. (b) Spectrum of BNMPH (13 μM) reacted with thiol of BSA (5 μM) in pH 8.0 buffer at 37°C; the insert showed no obvious changes during the 25 min period, indicating that BNMPH may react with thiol group of BSA at very low rate.