Inhibitory Effect Of Huaier On Triple Negative Breast Cancer And Its Mechanism Research

ObjectiveBreast cancer is one of the most common malignant cancers in women.Although the current treatment has significantly improved the clinical efficacy of breast cancer,but for the triple negative breast cancer(TNBC)which is estrogen receptor negative,progesterone receptor negative,and human epidermal growth factor receptor-2 negative,because this kind of breast cancer is not sensitive to both endocrine treatment and molecular targeted therapy,it is often lack of effective drugs when occurred recurrence or metastasis.Most patients of TNBC are easily developed to recurrence and metastasis after 1 to 3 years,and appeared rapid progress and even death,which is great challenge for the clinical treatment of TNBC.Related study showed that breast cancer stem cells play an important role in the development,recurrence,metastases and treatment resistance in breast cancer.Therefore,the search for new treatment to suppress the characteristics of triple negative breast cancer stem cells is the key to the treatment of TNBC.In recent years,the role of Huaier in the prevention and treatment of cancer has aroused more and more attention.Studies have shown that Huaier has a significant inhibitory effect on liver cancer,cholangiocarcinoma,ovarian cancer,melanoma and other tumors.Previous studies have shown that Huaier can inhibit breast cancer cell proliferation,invasion and metastasis.However,the inhibition mechanism of Huaier for TNBC has not been fully understood.Therefore,this study proposes from the primary TNBC cell proliferation activity,cell-cycle,apoptosis and tumor stem cell characteristics and other aspects,use molecular biological technology,to make a comprehensive description of Huaier on TNBC inhibition,and to provide pharmacological basis for the comprehensive treatment of TNBC for the clinical application of Huaier.Methods and resultsHuaier Qing cream(Huaier extraction),an independent development of Chinese medicine preparation,has been used for clinical adjuvant treatment of cancer.The main pharmacological components of Huaier extraction are Huaier polysaccharide.This paper carries out a preliminary investigation of Huaier on the TNBC inhibition and its mechanism by using Huaier Qing cream as a research drug.The specific content includes the following two parts:1.The inhibition effect of Huaier on the primary triple negativebreast cancer MethodsFirst,MTT assay was used to detect the effect of Huaier Qing cream on the activity of MDA-MB-231 cell line,and to detemine the optimal concentration and time of action of Huaier Qing cream.The effect of Huaier Qing cream on the invasion and migration of MDA-MB-231 cells was determined by Transwell assay.In addition,flow cytometry was applied to detect the effect of Huaier Qing cream on the cycle and apoptosis of MDA-MB-231 cells.Finally,the effect of Huaier Qing cream on the expression of PI3 K / AKT / m TOR pathway in MDA-MB-231 cells was analyzed by Western blot.ResultsMTT assay results showed that the activity of MDA-MB-231 cells was significantly inhibited in a dose-dependent manner,and it could effectively inhibit the activity of MDA-MB-231 cells at a concentration of 10 mg/ml at 24 hours.The half maximal inhibitory concentrations of Huaier on MDA-MB-231 cells at 24 h,48h and 72 h were 6.508 ± 0.648mg/ml,4.207±0.453mg/ml and 2.281±0.164mg/ml respectively.At the same time,the results of Transwell showed that Huaier Qing cream could inhibit the migration of MDA-MB-231 cells in vitro.Compared with untreated cells,the percentage of cells in G2/M phase of MDA-MB-231 cells exposed to Huaier Qing cream was increased by flow cytometry,and the proportion of G0/G1 phase and S phase cells was decreased compared with untreated cells.The results showed that the cell-cycle of MDA-MB-231 could be induced in G2/M phase.In addition,the late apoptotic rate and early apoptotic rate of MDA-MB-231 cells treated by Huaier Qing cream paste were increased by Annexin V-FITC/PI assay,which indicated that Huaier Qing cream could promote triple negative breast cancer MDA-MB-231 cells,thereby inhibiting tumor proliferation.The mechanism research showed that Huaier Qing cream could decease the proteins related to PI3K/AKT/m TOR pathway,including PI3 K,AKT,p-AKT and m TOR,thus to inhibit the proliferation of MDA-MB-231 cells in triple negative breast cancer.2.The effect of Huaier on the triple negative breast cancer stem cells MethodsMTT assay was used to detect the effect of Huaier Qing cream on cell viability in suspension cells of TNBC,and the volume and number of suspended cells were observed.The effect of Huaier Qing cream on the clonal formation ability of breast cancer was detected by cloning assay.In addition,the effect of Huaier Qing cream on CD44+/CD24-cells was detected by flow cytometry,and the biological marker of cancer stem cells,such as Oct-4,Nestin and Nanog,was analyzed by quantitative polymerase chain reaction.ResultsThe results of MTT showed that Huaier Qing cream could inhibit the cell viability of MDA-MB-231 suspension cells.At the same time,Huaier Qing cream can reduce the number and size of suspended cells.In the clonal formation experiment,we found that Huaier Qing cream can significantly reduce the ability of breast cancer cloning.In addition,flow cytometry showed that the percentage of CD44+/CD24-cells was significantly reduced by 49.20%(2 mg/ml)compared with the control group.In addition,the m RNA levels of tumor stem cell markers such as Oct-4,Nestin and Nanog were also significantly decreased in the Huaier group MDA-MB-231 cells.Conclusions1.Huaier Qing cream can inhibit the activity,invasion and migration of MDA-MB-231 cells.2.The proliferation of MDA-MB-231 cells was induced through PI3K/AKT/m TOR pathway in G2/M phase.3.Huaier Qing cream significantly inhibited the growth of MDA-MB-231 suspension cell spheres.4.Huaier Qing cream significantly inhibited the clonal formation ability of triple negative breast cancer stem cells and reduced the number of CD44+/CD24-cells.