Anti-metastatic and pro-apoptotic effects elicited by combination photodynamic therapy with sonodynamic therapy on breast cancer both in vitro and in vivo

Abstract

Sono-Photodynamic therapy (SPDT), a new modality for cancer treatment, is aimed at enhancing anticancer effects by the combination of sonodynamic therapy (SDT) and photodynamic therapy (PDT). In this study, we investigated the antitumor effect and possible mechanisms of Chlorin e6 (Ce6) mediated SPDT (Ce6-SPDT) on breast cancer both in vitro and in vivo. MTT assay revealed that the combined therapy markedly enhanced cell viability loss of breast cancer cell lines (MDA-MB-231, MCF-7 and 4T1) compared with SDT and PDT alone. Propidium iodide/hoechst33342 double staining reflected that 4T1 cells with apoptotic morphological characteristics were significantly increased in groups given combined therapy. Besides, the combined therapy caused obvious mitochondrial membrane potential (MMP) loss at early 1h post SPDT treatment. The generation of intracellular reactive oxygen species (ROS) detected by flow cytometry was greatly increased in 4T1 cells treated with the combination therapy, and the loss of cell viability and MMP could be effectively rescued by pre-treatment with the ROS scavenger N-acetylcysteine (NAC). Further, Ce6-SPDT markedly inhibited the tumor growth (volume and weight) and lung metastasis in 4T1 tumor-bearing mice, but had no effect on the body weight. Hematoxylin and eosin staining revealed obvious tissue destruction with large spaces in the Ce6-SPDT groups, and TUNEL staining indicated tumor cell apoptosis after treatment. Immunohistochemistry analysis showed that the expression level of VEGF and MMP were significantly decreased in the combined groups. These results indicated that Ce6-mediated SPDT enhanced the antitumor efficacy on 4T1 cells compared with SDT and PDT alone, loss of MMP and generation of ROS might be involved. In addition, Ce6-mediated SPDT significantly inhibited tumor growth and metastasis in mouse breast cancer 4T1 xenograft model, in which MMP-9 and VEGF may play a crucial role.