The combined use of Camellia sinensis and metronomic zoledronic acid in a breast cancer-induced osteolysis mouse model

Abstract

Purpose: In previous studies, we demonstrated that green tea (Camellia sinensis, CS) water extract had potent anti-tumor and anti-metastasis effects in the 4T1 mouse breast cancer xenograft model, and the metronomic regimen (0.0125 mg/kg twice a week for 4 weeks) of zoledronic acid (ZOL) was also effective in decreasing tumor burden and metastasis when compared with the conventional regimen. This study aimed to investigate the combined use of CS water extract and metronomic ZOL against tumor metastasis and bone destruction in MDA-MB-231-TXSA human breast cancer.

Methods: Female nude mice were injected with MDA-MB-231-TXSA cells into the marrow space of tibia and were treated with CS water extract and/or metronomic ZOL for 4 weeks. Tumor growth and metastasis to lungs and livers were assessed by in vivo bioluminescence imaging. Abilities of migration and invasion of MDA-MB-231-TXSA cells were also evaluated in vitro.

Results: Our results demonstrated that combination of CS and ZOL had the most potent effects on tumor burden and metastasis to bone, lung and liver, while treatment with CS or ZOL alone significantly protected the bone from cancer-induced osteolysis. In vitro, the combined use of CS + ZOL significantly inhibited MDA-MB-231-TXSA cell migration and invasion. Mechanistic zymography studies showed that the enzyme activities of MMP-9 and MMP-2 were significantly suppressed by CS and CS + ZOL.

Conclusions: The combination of CS plus metronomic ZOL demonstrated potent anti-tumor, anti-metastasis and anti-osteolysis effects against breast cancer, suggesting the potential clinical application against breast cancer patients.

Fig. 1

Tumor burden change during CS and/or metronomic ZOL treatment in intratibial breast cancer-induced osteolysis model. a Representative images of tumor burden obtained from each group at different time points as assessed by IVIS system. b Graph showed the bioluminescence measurements according to the average radiance. Data were expressed as mean ± SEM, n = 15. *p < 0.05, as compared with control. “Ctl” means control group; “CS” means CS water extract treatment group in which mice were treated with 0.6 g/kg CS extract, orally fed daily; “ZOL” means metronomic ZOL treatment group in which mice were treated with 0.0125 mg/kg ZOL, i.p. injected twice a week for 4 weeks; “CS + ZOL” means the combination treatment group in which mice were treated with CS plus ZOL, and the dose was the same as the individual treatment of CS or ZOL

Fig. 2

Qualitative and quantitative assessment of bone structure in both tibias after administration of CS and/or metronomic ZOL. a, b Representative X-ray, μ-CT 3D images, section cut and growth plate of a non-tumor-bearing and b tumor-bearing tibias obtained from different groups. c, d Graphs showed the percentage of bone volume to tissue volume (% BV/TV) of c non-tumor-bearing and d tumor-bearing tibias. Data were expressed as mean + SEM, n = 10. **p < 0.01 and ***p < 0.001, as compared with control; ^### p < 0.001, as compared between groups indicated

Fig. 3

Effects of CS and/or metronomic ZOL treatment on lung metastasis in intratibial breast cancer-induced osteolysis model. a Representative images of lungs obtained from different groups at end point of IVIS scan, and the fractional number revealed the frequency of lung metastasis. b Graph showed the bioluminescence measurements (expressed as average radiance) in lungs. c Photographs were the representative H&E-stained sections of lungs from different groups with arrows showing the MDA-MB-231-TXSA tumor nodules. d Graph showed the tumor burden in lungs as assessed by histological analysis and expressed as an average percentage of tumor area to lung area per group. Data were expressed as mean + SEM, n = 15; *p < 0.05, **p < 0.01 and ***p < 0.001, as compared with control. “Ctl” means control group; “Naive” means normal mice without tumor and treatment

Fig. 4

Effects of CS and/or ZOL treatment on liver metastasis in intratibial breast cancer-induced osteolysis model. a Representative images of livers obtained from different groups at end point of IVIS scan, and the fractional number in each group revealed the frequency of metastasis. b Graph represented the bioluminescence measurements (expressed as average radiance) in livers. c Photographs were the representative H&E-stained sections of mouse livers with arrows showing the tumor nodules. d Graph represented the tumor burden in livers as assessed by histological analysis. Data were expressed as mean + SEM, n = 15. *p < 0.05 and **p < 0.01, as compared with control. “Ctl” means control group; “Naive” means normal mice without tumor and treatment

Fig. 5

The effects of ZOL in combination of various doses of CS on MDA-MB-231-TXSA cells viability. Cells were incubated with ZOL (0, 20, 40 and 60 μM) in the presence of various concentrations of CS after 48 h treatment. Data were expressed as mean ± SD (n = 3)

Fig. 6

Effect of CS or CS + ZOL on the MDA-MB-231-TXSA cell migration and invasion. a Representative images of the wounded cell monolayers of MDA-MB-231-TXSA cells after incubated with various concentrations of CS and fixed amount of ZOL (40 μM). b Quantitative analysis of the migration activity of cells after 9 h treatment of CS or CS + ZOL. c Representative images of the stained MDA-MB-231-TXSA cells. d Quantitative analysis of the invasion activity of cells after treated with CS or CS + ZOL. Data were expressed as mean + SD (n = 3). *p < 0.05 and ***p < 0.001, as compared with corresponding untreated control

Fig. 7

Effect of CS and/or ZOL on MMP-9 and MMP-2 activities. Representative zymograms were shown on MDA-MB-231-TXSA cells after treated with CS and/or ZOL for 24 h (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001, as compared with control