1,3-Benzodioxole Derivatives Improve the Anti-Tumor Efficiency of Arsenicals

Abstract

Arsenicals have been widely used in the treatment of cancers such as leukemia and other tumors. However, their side effects limit their clinical application. Stiripentol, a second-line adjunctive treatment for epilepsy with a good safety profile, inhibits microsomal cytochrome-P450-family enzymes to extend the retention time of co-administration. Inspired by the metabolism of stiripentol, the 1,3-benzodioxole responsible for the inhibition and its metabolic derivatives were conjugated with arsenical precursors. The fabricated arsenicals were eliminated much slower in mice and maintained an efficient concentration in the blood for a longer time than that of the arsenical precursors. They also performed better in anti-proliferation by inhibiting the thioredoxin system to induce oxidative stress, and concomitantly to initiate apoptosis in vitro and in vivo. The fabricated arsenicals reversed the hemogram of tumor-bearing mice to normal and eliminated the tumor without causing damage to any organs, exhibiting a good design strategy and pre-clinical application for leukemia and other tumors.

Keywords: 4T1 tumor; ROS; TrxR; docking; organic arsenicals; stiripentol.

Scheme 1

The ring-opening scheme of 1,3-benzodioxole, the conjugation of arsenical precursors, and the related 1,3-benzodioxole derivatives.

Scheme 2

The conjugation of the arsenical precursors and the 1,3-benzodioxole derivatives.

Figure 1

The arsenicals inhibited TrxR activity by binding to the C–terminal Sec/Cys pair; (A,B) show the binding site of MAZ2 in TrxR1 (PDB: 3EAN) and Trx1 (PDB: 1ERT); (C) is an MS spectrum of a mixture of the peptide AGCUG and MAZ2; (D) is the TrxR activity of the 4T1 cells after a 12 h incubation of compounds (Au: auranofin); (E) is the transcription of the Trx system genes of the 4T1 cells incubated with MAZ2 for 24 h at the dosage of 1IC₅₀ and 1.5IC₅₀; (F) is the protein content of the Trx system of the 4T1 cells; and (G) is an MTT assay of the 4T1 cells with siRNA and MAZ2.

Figure 2

ROS bursting after TrxR inhibition by arsenicals; (A) is the fluorescence intensity of the 4T1 cells stained by DCFH-DA (the fluorescence was detected by flow cytometry); (B) is the 4T1 cells stained by DCFH-DA (the fluorescence was detected by confocal); (C) is the GSH contents; (D) is the cell viability under MAZ2 treatment without GSH or NAC; and (E,F) are the MDA contents of the Molm-13 and 4T1 cells. Significance was set at * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Apoptosis was induced by MAZ2 incubation; (A) is the membrane potential monitored by JC-1 staining; (B) is the ratio of aggregated JC-1 molecules to the monomeric molecules of (A); (C) is the ATP level; (D) is the cytosol’s cytochrome c content; (E) is the caspase 3 activity; (F) is the 4T1 cells’ apoptosis assay by flow cytometry; and (G,H) show the Molm-13 cells’ apoptosis assay by flow cytometry.

Figure 4

The 4T1 tumors were eliminated by two administrations of MAZ2; (A) is a scheme of the mice experiments; (B) is a tumor picture after mice sacrifice; (C) is the tumor volume; (D) is the tumor weight; and (E) is the Trx system protein content determined by WB.

Figure 5

Staining of tumor tissues. ROS: blue-nuclear, red-ROS; TUNEL: blue-nuclear, green-TUNEL; Ki67: blue-nuclear, red-Ki67.

Figure 6

MAZ2 administration had no obvious side effects; (A) is the arsenic content; (B) is the blood arsenic content; (C) is the organ coefficient; (D) is the body weight; (E) is the TrxR and Trx content of the liver and spleen; and (F) is the blood cell counts.

Figure 7

H&E staining of the heart, liver, spleen, lungs, and kidneys from the three groups.

Figure 8

The mechanism of MAZ2.