Moxibustion enhances the antitumor efficacy of Trametinib in breast cancer-bearing mice via MEK/ERK signaling pathway

To explore the synergistic inhibitory effect of moxibustion and mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) pathway inhibitor Trametinib on tumor growth in breast cancer tumor-bearing mice and to analyze its underlying mechanisms.

Fifty female BALB/C mice were randomly divided into blank control, model, inhibitor (Trametinib), direct moxibustion and combination (Trametinib+moxibustion) groups, with 10 mice in each group. Injection of 4T1 cells was used to establish breast cancer tumor-bearing mouse model. Both the blank control and model groups received gavage of 0.1 mL of normal saline once daily. In the inhibitor group, Trametinib solution was administered by gastric gavage at 3 mg/kg, once a day for 21 d. For mice of the direct moxibustion group, moxibustion was applied at bilateral “Zusanli” (ST36), 2 cones per acupoint, once every 2 days for 21 d. The combination group was treated with administration of Trametinib (once daily) by gastric gavage and direct moxibustion (once every 2 d) for 21 d. Body weight and tumor volumes were measured in mice. The tumor weight was quantified and the tumor inhibition rate was calculated. Histopathological alterations in tumor tissues were observed after H.E. staining. The protein expression levels of phosphorylated (p)-MEK, p-ERK, myelocytomatosis viral oncogene homolog (c-Myc), and programmed cell death ligand 1 (PD-L1) in the tumor tissues were assessed using immunohistochemical staining and Western blot, separately. Additionally, the mRNA expression levels of c-Myc and PD-L1 in the tumor tissue were detected using fluorescence quantitative real-time PCR.

After the intervention, compared with the blank control group, the body mass of mice was decreased evidently in both the model and inhibitor groups (P<0.01), rather than in the direct moxibustion and combination groups. Compared with the model group, the body mass of mice was obviously increased (P<0.01), and the tumor volume and weight were obviously decreased in each treatment group (P<0.01, P<0.05). The tumor inhibition rate was 35.19% in the inhibitor group, 30.27% in the direct moxibustion group, and 50.67% in the combination group. The protein expression levels of p-MEK, p-ERK, c-Myc and PD-L1, and the mRNA expression levels of c-Myc and PD-L1 in the tumor tissues were significantly decreased (P<0.01) in each treatment group relatively to the model group. The therapeutic effect of the combination group was significantly superior to that of the inhibitor group in increasing the body mass, and to that of the inhibitor and direct moxibustion groups in reducing the tumor volume, tumor weight, and in down-regulating the immunoactivity and protein and mRNA expressions of c-Myc and PD-L1 (P<0.05, P<0.01). The therapeutic effect of the combination group was also strikingly superior to that of the direct moxibustion group in down-regulating the immunoactivity and expressions of p-MEK and p-ERK (P<0.01, P<0.05). The effect of the direct moxibustion group was superior to that of the inhibitor group in increasing the body mass and up-regulating the immunoactivity and protein expressions of p-MEK and p-ERK (P<0.01, P<0.05). H.E. staining showed that the tumor cells in model group were irregularly arranged and shaped, with obvious cell atypia and enlarged nuclei, but those in the 3 treatment groups displayed obvious cribriform tumor cell degeneration, with more cell debris and smaller density. The degeneration of tumor cells in the combination group was the most obvious.

Moxibustion can enhance the anti-tumor effect of Trametinib by inhibiting the phosphorylation of MEK/ERK pathway and the downstream c-Myc/PD-L1 axis in mice with breast cancer, which provides an experimental basis for the adjuvant targeting therapy of breast cancer with moxibustion.