Abstract: Objective To investigate the effects of andrographolide combined with doxorubicin on the apoptosis of breast cancer cells and its molecular mechanism. Methods The breast cancer cells MDA⁃MB⁃231 and MCF⁃7 were treated with doxorubicin alone, andrographolide alone, or the combination of both. The proliferation and clonogenesis of the cells were detected by MTT assay and clonogenesis assay, respectively. Cell death and ROS level were detected by flow cytometry (PI staining and DCFH⁃DA staining), and the expression of γ⁃H2AX, PARP and α⁃tubulin in breast cancer cells was detected by Western blot. The effect of ROS on DNA was further detected by adding ROS scavenger NAC. A xenograft model of breast cancer in nude mice was established. After treatment with Doxorubicin, andrographolide or the combination of both, the body weight and tumor volume of nude mice were detected, and the cell death and tumor cell proliferation ability were detected by HE pathological staining and Ki⁃67⁃IHC staining, respectively, to verify the therapeutic effect of the combination treatment. Results Compared with the single drug group, the proliferation, survival rate and clonogenesis ability of breast cancer cells in the combined drug group were decreased (P<0.05), the ROS level was increased (P<0.001), the expression of γ⁃H2AX protein was increased and PARP protein cleavage was induced. Compared with the combined drug group, the survival rate of the cells in the NAC+ combined drug group was increased (P<0.05), while the cell ROS level was decreased (P<0.001), and the expression of γ⁃H2AX protein was reduced. In animal experiments, there was no significant difference in the body weight of nude mice between all of the  groups (all P>0.05); compared with the single drug group, the combined drug group had smaller tumor volume, more tumor cell death, and lower Ki⁃67 expression level in nude mice (all P<0.05). Conclusions Andrographolide can enhance the ability of doxorubicin and inhibit the proliferation of breast cancer cells. Andrographolide aggravates cell DNA damage by increasing ROS levels, which may be the mechanism of inducing breast cancer cell death. 

Key words: Breast cancer, Andrographolide, Doxorubicin, ROS, DNA Damage