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Aim: The anti-breast cancer effect and mechanism of ethyl acetate extract of persimmon leaves (PLE) were determined. Methods: Persimmon leaves were extracted by reflux at 80°C with 80% ethanol as the solvent. The total extracts of persimmon leaves were extracted with ethyl acetate, and the yield was calculated by weighing. The mouse breast cancer cell line 4T1 was cultured in vitro, and different concentrations of PLE were added. At the same time, the effects of PLE at different concentrations (25, 50, 100 µg/ml) on cell apoptosis ability were detected by Acridine Orange and Ethidium Bromide (AOEB) and flow cytometry experiments. In addition, real-time quantitative PCR (real-time PCR, RT-PCR) was used to test the expression of the Bax, Bcl-2, ERK1/2, MEK1/2 and RAF genes. In vivo tumor-bearing mouse model: A breast cancer transplant tumor model was established with BALB/c mice. The doses of PLE were 30, 60 and 120 mg/kg body weight/d, and the dose of CTX was 20 mg/kg body weight/d. The tumor inhibition rate and the effects of PLE on immune organs in tumor-bearing mice with 4T1 breast cancer were determined. The expression levels of IL-6, TNF-α, TGF-β and VEGFA in the serum of mice were detected by ELISA. The expression of the Bax, Bcl2, ERK1/2, MEK1/2 and RAF genes was determined by RT-PCR. The protein expression levels of Bax, Bcl-2, Caspase-3, p-MEK, p-JNK and p-P38 in tumor tissues were detected by immunohistochemistry. In addition, the protein expression levels of MAPK pathway components were assessed through Western blotting.Results: A total of 119.34 g ethyl acetate extract was obtained from 3 kg persimmon leaves with a yield of 3.98%. In vitro: MTT results indicated a strong antiproliferative effect of PLE on breast cancer cell lines. AOEB and flow cytometry assays showed that PLE promoted the apoptosis of breast cancer cells. PCR results showed that PLE could inhibit Bcl-2, promote Bax expression, and downregulate ERK1/2, MEK1/2, and RAF gene expression. In vivo: PLE had a significant inhibitory effect on breast cancer, and the tumor inhibition rates were 11.65%, 33.71% and 47.24% from low dose to high dose, respectively, showing a concentration dependence. The tumor inhibition rate of CTX was 57.74%. Meanwhile, PLE can increase the spleen and thymus index of 4T1 mice and decrease the liver index of 4T1 mice. Compared with the model group, PLE significantly reduced the expression levels of IL-6, TNF-α, TGF-β and VEGFA in the serum of mice. PCR results showed that PLE could inhibit Bcl-2, promote Bax expression, and downregulate ERK1/2, MEK1/2, and RAF gene expression. Immunohistochemical results showed that the PLE group and CTX group significantly promoted the expression of Bax and Caspase-3 proteins and downregulated the expression of Bcl-2, p-MEK, p-JNK and p-P38 proteins. WB results showed that PLE regulated the expression of proteins in the MAPK pathway.Conclusion: PLE enhances immunity, inhibits angiogenesis, inhibits 4T1 cell proliferation and induces apoptosis. Its apoptosis mechanism is related to the regulation of Bax/Bcl-2/Caspase-3 protein and the phosphorylation of regulatory proteins related to the MAPK signaling pathway.

Metastasis causes approximately 90% of breast cancer-related deaths in women. Previously, we have demonstrated that 2-dodecyl-6-methoxycyclohexa-2,5-diene- 1,4-dione (DMDD) remarkably inhibited the growth of human breast cancer cells with little toxicity. In this study, we investigated the toxicity and efficacy of DMDD to treat metastatic breast tumors using an in vivo mouse model of the 4T1 mammary carcinoma. DMDD caused no observable toxicity and significantly extended the survival of 4T1 tumor-bearing mice. DMDD effectively inhibited the growth of 4T1 cells in vitro, and suppressed the growth and metastasis of mammary tumor in vivo. The levels of inflammatory cytokines in the serum (TNF-α, IL-6, IL-12, TGF-β, and VEGF) were down regulated by DMDD. Immunohistochemical analysis demonstrated that the inhibition of tumor growth and metastasis was associated with activation of Bax, cleaved caspases-3 and -9, and down-regulation of Bcl-2, MMP-2 and -9, NF-κB and IκBα. We speculate that DMDD inhibits cytokine production in the tumor cells in mice, which leads to deactivation of NF-κB pathway, and consequently inhibits the expression of many anti-apoptosis and metastasis-promoting genes, such as Bcl-2 and MMPs. Collectively, our results demonstrate the potential of DMDD as a safe and effective antitumor agent in the treatment of late-stage breast cancer.