Study On The Mechanism Of Traditional Chinese Medicine Fuzi In Inhibiting The Growth Of Breast Cancer Cells

Objectives:Breast cancer is one of the most common malignant tumors in women.The incidence and mortality of breast cancer are rising in China,and it has been rankeds first female malignant tumors in China.In traditional Chinese medicine,breast cancer is considered to be yang deficiency and yin accumulation,and Wenyang drugs is recommended for treatment.Fuzi is widely used as a traditional Chinese medicine for breast cancer,as one of Wenyang drugs.However,it is not clear for the molecular mechanism of Fuzi treatment of breast cancer.To further clarify the relationship between Fuzi and breast cancer,we look for evidence from epidemiology and cell level,in order to gain a more comprehensive understanding.Studying the molecular mechanism of Fuzi in inhibiting breast cancer cells through the corresponding signal transduction pathway will increase theoretical support for understanding the role of Fuzi in the treatment of breast cancer,and also contribute to the precise application and development of Fuzi in clinical practice.It will provides the basis for expanding the application of traditional Chinese medicine in cancer treatment.Methods:1.Epidemiological investigation of the relationship between Fuzi planting distribution and breast cancer incidence.At the beginning of 2018,the medical record of breast cancer patients treated in 2017 was collected in the hospital's medical record room of first affiliated hospital,the second affiliated hospital,and the third affiliated hospital(Yunnan Cancer Hospital).Af:ter excluding case of duplicate and extra?provincial,epidemiological investigation was carried out on the relationship between the distribution of Fuzi planting in Yunnan Province and the incidence of breast cancer.2.Cell proliferation assay.Cells were treated Fuzi with concentration gradients of 0.05 mg/mL,0.1 mg/mL,0.2 mg/mL,0.4 mg/mL,and 0.8 mg/mL;at different time points of 2 h,4 h,6 h,12 h,and 24 h,The proliferation were detected in breast cancer cell line MDA-MB-231 and normal breast cell lines MCF10A through MTT assay.3.The effects of Fuzi on the migration were detected in MDA-MB-231 and normal breast cells MCF10A with wound healing assay and transwell assay,respectively.4.The inhibition of Fuzi in breast cancer cell proliferation through apoptotic pathway was texamined by Annexin V-FITC/PI and Hoechst 33342/PI staining.MDA-MB-231 cell was treated with 0.05 mg/mL and 0.1 mg/mL Fuzi,and examined by Annexin V-FITC/PI and Hoechst 33342/PI staining after 24h;MDA-MB-231 cells was treated with DMSO in negative control and treated with 50 ?M H2O2 in positive control.Caspase3 and Caspase9 activities were also detected in the experiment.5.After MDA-MB-231 cells were treated with 0.05 mg/mL and 0.1 mg/mL Fuzi respectively.The fluorescence intensity of intracellular DCFH-DA was measured by flow cytometry and confocal microscopy to analyze the ROS levels of breast cancer cells.6.The effects of Fuzi on proteins expression involved in the endogenous and external apoptotic pathway was detected by Real time PCR to determine whether Fuzi induceds apoptosis of breast cancer cells is mediated by ROS.Results:1.Epidemiological investigations have shown that the incidence of breast cancer in the Fuzi planting area is significantly lower than that in the non-planting area.2.Cellular experiments demonstrated that Fuzi inhibits the malignant phenotype of breast cancer cells but had no effect on normal breast cells.3.Fuzi inhibits migration of breast cancer cells but has no effect on normal breast cells.4.Fuzi can induce apoptosis of breast cancer cells,and the apoptosis rate increases with the increase of Fuzi concentration.5.Moderate Fuzi increases the ROS level of breast cancer cells,and ROS as a second messenger induces apoptosis of breast cancer cells by triggerings the endogenous or external pathway of apoptosis,thus plays a role in the treatment of breast cancer.Conclusions:This study intends to study the molecular mechanism of Fuzi in growth inhibition,apoptosis induction in breast cancer cell and the role of reactive oxygen species in it,the changes of a variety of indicators such as apoptosis and oxidation was detected in breast cancer cell model after Fuzi treatment.The study will reveal the molecular mechanism of Fuzi in inhibiting the malignant phenotype of breast cancer cells by changing the redox state and inducing apoptosis in breast cancer cells,and will help to understand more fully the role of Fuzi in the treatment of breast cancer.It will provide a scientific basis for rational optimal application and potential exploration for Fuzi.The study will also be helpful for finding clues for the determination of active ingredients of Fuzi and for developing "ROS+ROS" strategy in the treatment of tumors by traditional Chinese medicine.