Anti-breast cancer sinomenine derivatives via mechanisms of apoptosis induction and metastasis reduction

Abstract

Sinomenine, a morphinane-type isoquinoline-derived alkaloid, was first isolated from stems and roots of Sinomenium diversifolius (Miq.) in 1920. Later discovery by researchers confirmed various essential biological efficacy sinomenine exerted in vitro and in vivo. In this study, a series of 15 sinomenine/furoxan hybrid compounds were designed and synthesised in search of a TNBC drug candidate. Some of the target compounds exhibited strong antiproliferative activities against cancer cell lines, especially for TNBC cells, compared to positive controls. Among them, hybrid 7Cc exerted superior cytotoxic effects on cancer cell lines with exceptionally low IC₅₀ (0.82 μM) against MDA-MB-231 cells with the highest safety index score. Further studies in mechanism displayed that 7Cc could induce an S phase cell cycle arrest, stimulate apoptosis in MDA-MB-231 cells, disrupt mitochondrial membrane potential and exert a genotoxic effect on DNA in cancer cells. In addition, 7Cc also notably inhibited MDA-MB-231 cells in both migration, invasion and adhesion.

Keywords: Sinomenine; apoptosis; breast cancer; furoxan.

Figure 1.

The chemical structures of reported Sinomenine and furoxan derivatives.

Scheme 1.

Synthesis of 1–4, 5A–E, 6A–E(a–c) and 7A–E(a–c). Reagents and conditions: (a) ClCH₂COOH, NaOH (aq), reflux, 2 h; (b) 30% H₂O₂, AcOH, rt, 3 h; (c) fuming HNO₃, 100 °C, 8 h; (d) corresponding diol, THF, 30% NaOH, 0 °C, 1 h; (e) corresponding anhydrides, TEA, DMAP, DCM, rt, 2 h; (f) DMAP, EDCI, rt, 4 h.

Figure 2.

NO-releasing ability of several selected target compounds. The values were expressed as averages of three independent experiments.

Figure 3.

MDA-MB-231 cells were treated with 7Cc and Cells were stained with PI and investigated by flow cytometry. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control group. Statistical analyses were carried out on GraphPad.Prism software under ordinary one-way ANOVA method and compared with the control group.

Figure 4.

Flow cytometry analysis of apoptosis induced by 7Cc in MDA-MB-231 cells. Data are represented as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 vs control group. Statistical analyses were carried out on GraphPad.Prism software under ordinary one-way ANOVA method and compared with the control group.

Figure 5.

7Cc induced mitochondrial depolarisation in MDA-MB-231 cells. Data are represented as mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001 vs control group. Statistical analyses were carried out on GraphPad.Prism software under ordinary one-way ANOVA method and compared with the control group.

Figure 6.

(a) MDA-MB-231 cells were treated with 7Cc, sterile pipette tips were used to scratch evenly, the incubation was continued, and representative images were captured. (b) MDA-MB-231 cells were seeded onto chambers and incubated with 7Cc, stained with crystal violet, and representative images were photographed. (c) MDA-MB-231 cells were incubated with 7Cc, then fixed, washed and photographed with a fluorescence microscope. All data are represented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group. Statistical analyses were carried out on GraphPad.Prism software under ordinary one-way ANOVA method and compared with a control group.

Figure 7.

MDA-MB-231 cells were incubated with 7Cc. Comet assay was used to evaluate DNA damage and photomicrographs were provided. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 vs control group. Statistical analyses were carried out on GraphPad.Prism software under ordinary one-way ANOVA method and compared with a control group.