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.

To observe the effect of wheat-grain moxibustion plus chemotherapy on the activities of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling and expressions of Caspase-3 and Caspase-9 in mice with breast cancer, so as to explore its mechanisms underlying suppression of tumor growth.

Forty female BALB/c mice were randomly divided into blank control, model, chemotherapy, wheat-grain moxibustion (moxibustion) and moxibustion+chemotherapy groups, with 8 mice in each group. The breast cancer model was established by injection of 4T1 cell suspension (0.1 mL) into the subcutaneous fat pad of the mouse’s right fourth nipple. After successful modeling, the mice of the chemotherapy group and moxibustion+chemotherapy group received injection of Adriamycin (2.5 mg/kg) into the mouse’ leg or abdomen in the morning of Monday and Friday every week, 3 weeks altogether. For mice of the moxibustion and moxibustion+chemotherapy groups, wheat-grain moxibustion was applied to “Shenque” (CV8) and bilateral “Zusanli” (ST36) for 2 cones every time, once every other day for 21 days. The body weight, tumor weight and tumor volume were measured. The histopathological changes of the tumor were observed after H. E. staining. The terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) was used to detect the apoptosis of tumor tissues in each group. The expression levels of PI3K, AKT, mTOR, phosphorylated (p)-PI3K, p-AKT, p-mTOR, Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 proteins in the tumor tissues were determined by Western blot.

Compared with the model group, the body weight in the chemotherapy group, the tumor weight, tumor volume, and the expression ratios of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR in the chemotherapy, moxibustion and moxibustion+chemotherapy groups were significantly decreased (P＜0.05), while the apoptosis rate of tumor tissue, the expression levels of Caspase-3, cleaved Caspase-3, Caspase-9 and cleaved Caspase-9 proteins in the chemotherapy, moxibustion and moxibustion+chemotherapy groups were considerably increased (P＜0.05). The therapeutic effect of moxibustion + chemotherapy was significantly superior to that of simple chemotherapy and simple moxibustion in reducing the tumor volume and tumor mass, and in down-regulating the expression ratio of p-AKT/AKT (P＜0.05), and in up-regulating the apoptosis rate and the expression levels of Caspase-3 and cleaved Caspase-3 proteins (P＜0.05), and superior to that of simple moxibustion (not the simple chemotherapy) in up-regulating the expression levels of Caspase-9 and cleaved Caspase 9 protein (P＜0.05). The therapeutic effect of simple moxibustion was evidently inferior to that of simple chemotherapy in reducing tumor volume and tumor mass, in down-regulating the expression ratios of p-PI3K/PI3K and p-mTOR/mTOR (P＜0.05), and in increasing the apoptosis rate, and expression of Caspase-3, Caspase-9 and cleaved Caspase-9 proteins (P＜0.05). H. E. staining showed that the tumor cells of tumor tissue grew diffusely and arranged densely in the model group, while in the chemotherapy, moxibustion and moxibustion+chemotherapy groups, the tumor tissue showed light staining of cytoplasm and nucleus, reduced nuclear division, nuclear debris, smaller density, and different degrees of tumor cell necrosis, and the degree of necrosis was increased in the moxibustion group, chemotherapy group and moxibustion+ chemotherapy group.

Moxibustion plus chemotherapy and simple moxibustion can inhibit tumor growth in breast cancer mice, which may be related to their functions in inhibiting the expression and phosphorylation of PI3K/AKT/mTOR signaling pathway related proteins in the tumor tissue, and then upregulating the expression of apoptosis related proteins Caspase-3 and Caspase-9.

To observe the effects of moxibustion combined with chemotherapy on immune checkpoints including programmed cell death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain (TIM-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in the tumor tissue of breast cancer-bearing mice, so as to explore the effect and mechanisms of moxibustion combined with chemotherapy on breast cancer.

Forty BALB/c female mice were randomly divided into model, chemotherapy, moxibustion and combination groups, with 10 mice in each group. 4T1 tumor cells were inoculated into the fat pad under the left fourth nipple of the mice to establish the breast cancer-bearing mice model. The chemotherapy group was injected intraperitoneally with doxorubicin (DOX) solution at 2.5 mg/kg once every 3 days; the moxibustion group received bilateral “Zusanli” (ST36) moxibustion with 2 moxa cones per point, treated every 2 days; the combination group received both DOX injection and moxibustion intervention. The above interventions lasted for 21 days. The body weight and tumor volume of the mice were recorded daily. HE staining was used to observe tumor tissue pathological morphology. Immunohistochemistry and Western blot were used to detect the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 in the tumor tissue.

Compared with the model group after intervention, the body weight was increased (P<0.01, P<0.05), the tumor volume was decreased (P<0.01), the pathological morphology of tumor tissue showed varying degrees of tumor cells degeneration, and the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 in tumor tissue were decreased (P<0.01, P<0.05) of mice in the chemotherapy, moxibustion and combination groups.Compared with the chemotherapy group, the body weight of mice in the moxibustion group was higher (P<0.01); the tumor volume of mice in the combination group was smaller (P<0.01); the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 were further reduced (P<0.01, P<0.05) of mice in the moxibustion and combination groups. Compared with the moxibustion group, the body weight was decreased (P<0.01), the tumor volume was smaller (P<0.01), the degree of tumor cell degeneration was higher, and the positive expression and protein expression levels of PD-1, TIM-3, and CTLA-4 were decreased (P<0.01) of mice in the combination group.

Moxibustion combined with chemotherapy shows more significant effects in inhibiting tumor growth in breast cancer-bearing mice compared to moxibustion or chemotherapy alone. It reduces the expression of PD-1, TIM-3, and CTLA-4 in tumor tissues, suggesting that lowering immune checkpoint expression levels may be one of the mechanisms by which moxibustion combined with chemotherapy drugs treats tumors.