Antitumor effect of low-frequency ultrasound combined with autophagy blockade in the mouse model of 4T1 breast cancer / 中华超声影像学杂志

ABSTRACT

Objective:To investigate the effect of low-frequency ultrasound combined with hydroxychloroquine (HCQ) on the growth and invasion of 4T1 breast cancer in mice.Methods:4T1 cells in logarithmic growth phase were divided into 4 groups control group, ultrasound group, HCQ group and ultrasound combined with HCQ group. Western blot was performed to detect the effects of ultrasound combined with HCQ on the protein expression levels of autophagy-related proteins LC3, p62 and matrix metalloproteinase 9 (MMP-9). Transmission electron microscopy was used to observe the formation of autophagic vesicles. The cell proliferation and cell viability were determined by EdU and CCK-8. Transwell assay was performed to detect the effect of ultrasound combined with HCQ on the invasive ability of 4T1 cells. Flow cytometry was performed to detect the effect of ultrasound combined with HCQ on the apoptosis of 4T1 cells. The transplantation tumor model of 4T1 breast cancer in BALB/c mice was constructed.The mice were randomly divided into 4 groups ( n=5 for each group), including control group, ultrasound group, HCQ group, ultrasound combined with HCQ group. The tumor volume and mice body weight were evaluated and measured in each group every 2 days. Results:The expression of LC3-Ⅱ and p62 protein levels increased in the ultrasound combined with HCQ group, and intracellular autophagosome accumulation was evident by transmission electron microscopy. In cellular experiments, compared with the other groups, the ultrasound combined with HCQ group showed stronger growth inhibition, significantly decreased cell proliferation rate, decreased expression of MMP-9, and significantly inhibited invasion (all P<0.050). In the in vivo experiments, compared with the control group, the tumor growth rate of all 3 inter vention groups of mice decreased (all P<0.050), and the ultrasound combined with HCQ group had better therapeutic effects than the ultrasound and HCQ groups. The treatment effect of ultrasound combined with HCQ group was better than that of ultrasound and HCQ groups (all P<0.001). Conclusions:The combination of low-frequency ultrasound and hydroxychloroquine could synergistically inhibit tumor cell proliferation, promote apoptosis, and significantly inhibit tumor growth in 4T1 tumor-bearing mice.