Carnosic Acid Mediates Production of Reactive Oxygen Species to Regulate Mitogen‐Activated Protein Kinase Pathway Phosphorylation and Induce Apoptosis in Human Breast Cancer Cells

ABSTRACT Background Reactive oxygen species (ROS) can induce cancer cell apoptosis, which plays a crucial role in breast cancer therapy. Carnosic acid (CA) exerts an anti‐tumor effect via generating ROS or activating the mitochondria‐related apoptosis pathway in vitro and in vivo. The deoxy‐glucose derivative CN5DG labeled with 99m Tc can be used for breast cancer imaging and evaluating the therapeutic efficacy of CA. Methods Inhibition of cancer cell proliferation by CA was assessed by MTT and cell colony‐formation assays. The mechanism of CA‐induced cancer cell apoptosis was examined by western blotting and the apoptosis rate was detected by flow cytometry. The in vivo anti‐tumor effect of CA was assessed by immunohistochemistry and 99m Tc‐CN5DG imaging. Its biodistribution was determined to evaluate the accumulation of 99m Tc‐CN5DG in xenografts. Results CA promoted cancer cell apoptosis via ROS generation, which activated c‐Jun N‐terminal kinase (JNK) and p38 phosphorylation. The apoptosis rates of T47D and MCF7 cells treated with CA (IC50 concentration) for 24 h were 44.97% ± 1.56% and 39.74% ± 1.78%, respectively. The antioxidant N‐acetyl‐L‐cysteine (5 μM) abolished CA‐induced apoptosis. MCF‐7 xenografts without CA were visualized in vivo at all time points by 99m Tc‐CN5DG imaging (tumor/muscle ratio: 2.52 ± 0.10 at 4 h), but xenografts treated with CA were not visualized (tumor/muscle ratio: 1.36 ± 0.34 at 4 h). Evaluation of the biodistribution by γ‐counter also demonstrated the efficacy of 99m Tc‐CN5DG. Bcl‐2 and Ki‐67 expression were higher while Bax expression was lower in the control group compared with the CA treatment group. The tumor/muscle radioactivity count ratio was lower following treatment with CA compared with the control group. Conclusion CA can induce breast cancer apoptosis via ROS generation and activation of JNK and p38 phosphorylation. The anti‐tumor effect of CA can be assessed using 99m Tc‐CN5DG SPECT imaging.