Effects Of Jiuxiangworm Decoction On The Growth Of Breast Cancer Cells In Vitro And In Vivo

Cancer threatens human health,breast cancer is one of the most common cancers in women,which has the highest morbidity and mortality.It is a serious threat to women's lives and health,and its prevention,control and treatment situation is grim.Aspongopus chinensis Dallas is used in the treatment of cancer in combination with other drugs clinically and used to treat cancer as decoction commonly.In this study,the effect of A.chinensis decoction(ACD)on the growth of breast cancer in vivo and in vitro were investigated,and the safety of ACD was preliminarily clarified,so as to provide experimental basis for clinical research and application of A.chinensis and provide references for traditional Chinese medicine in the treatment of breast cancer.The main experimental methods and results are as follows:1.CCK-8 method was used to detect the effects of different concentrations of ACD on the proliferation of human breast cancer cells MDA-MB-453 and HCC1937 and mice breast cancer cells 4T1 after 24 h.The results showed that ACD could significantly inhibit the proliferation of these three breast cancer cells(P<0.001)in a dose-dependent manner,the half maximal inhibitory concentration(IC₅₀)of MDA-MB-453,HCC1937 and 4T1 were 0.036,0.024 and 0.101 g/m L,respectively.2.Cell wound healing assay was used to investigate the effect of ACD on the migration ability of MDA-MB-453 and HCC1937.The results showed that the migration rates of MDA-MB-453 cells treated with 0.02,0.04 and 0.06 g/m L ACD for24 h and 48 h were significantly lower than those of the control group(P<0.01).The migration rate of MDA-MB-453 cells in the experimental group was significantly lower than control group(P<0.01).After treated with 0.02 and 0.04 g/m L ACD for 8 h and24 h,the cell migration rates of HCC1937 cells were significantly lower than control group(P<0.05).In conclusion,the ACD can inhibit the migration of MDA-MB-453and HCC1937 of human breast cancer cells,and the migration rate in dose-and time-dependent.3.Annexin V-FITC/PI staining was used to detect the apoptotic effect of ACD on breast cancer cells by flow cytometry.After treatment with 0.02,0.06 and 0.10 g/m L ACD,the apoptosis rates of MDA-MB-453 and HCC1937 cells were significantly higher than those of the control group(P<0.0001);the early apoptosis rate was significantly higher than that of control group(P<0.0001),the late apoptosis rate of cells treated with 0.10 g/m L ACD was significantly higher than control group(P<0.0001).In a word,the MDA-MB-453 and HCC1937 cells could be induced apoptosis in a dose-dependent manner after being treated with the ACD for 24 h,mainly in early apoptosis.4.Flow cytometry was used to detect the effect of the ACD on the cell cycle of breast cancer.The results showed that after 24 h treatment with the ACD on MDA-MB-453 cells,the proportion of cells in G1 phase was significantly increased(P<0.001),and the proportion of cells in S phase was significantly decreased(P<0.001),while there was no significant difference in G2 phase.After being treated with ACD of HCC1937 for 24 h,the proportion of cells in the G1 phase and S phase was significantly decreased(P<0.05),while the proportion of cells in the G2 phase was significantly increased(P<0.001).The cell cycle of MDA-MB-453 was arrested in G1 phase,while HCC1937 was arrested in G2 phase.5.Fluorescence quantitative PCR was used to detect the expression of apoptosis-related m RNA(caspase-3,caspase-8,Bax,Bcl-2),migration-related m RNA(MMP-2,MMP-9)and cell cycle related m RNA(CDK-1,CDK-2,cyclin A1 and cyclin B1)in MDA-MB-453 and HCC1937 cells treated with 0.06 g/m L ACD for 24 h.The pro-apoptotic related m RNA caspase-3,caspase-8 and Bax were significantly up-regulated(P<0.001),the anti-apoptosis related m RNA Bcl-2 down-regulated(P<0.0001),suggesting that the ACD may induce apoptosis by up-regulating caspase-3,caspase-8and Bax and down-regulating the Bcl-2 pathway.MMP-2 and MMP-9 were significantly down-regulated in both cells(P<0.0001),suggesting that ACD may inhibit cell migration by down-regulating MMP-2 and MMP-9.The cell cycle related m RNA CDK-1 was significantly down-regulated in both cells(P<0.05),CDK-2 was down-regulated,but there was no statistical difference(P>0.05),cyclin A1 and cyclin B1 were significantly down-regulated(P<0.05).The ACD can block the cell cycle,possibly by down-regulating CDK-1,cyclin A1 and cyclin B1.6.To investigate the effect of ACD on the proliferation of breast cancer in mice,the breast cancer xenograft in Balb/c mice was established.The volume and weight of tumor were compared after autopsy.Intragastric administration ACD had inhibitory effect on tumor growth of breast cancer mice,but there was no statistical difference compared with the NS group.The results of H&E staining of tumor tissue showed that the tumor cell density of mice treated with the ACD was lower,and there were fewer sinusoid capillaries in the tumor tissues.No cancer cell metastasis was found in the liver,spleen,lung,kidney and stomach.7.To investigate the effect of the ACD on tumor growth in breast cancer mice by intraperitoneal injection.Low and middle-dose of ACD had no inhibitory effect on the growth of mice breast cancer,but the high-dose group promoted the growth of tumor,and the combination of high dose ACD decoction and DDP reduced the tumor inhibition effect of DDP alone.By calculating the organ coefficient of mouse organs and H&E staining,compared with the NS group,the organ coefficient of lung in low dose and medium dose groups was significantly higher(P<0.05;P<0.0001),organ coefficient of liver and spleen in high-dose was significantly larger(P<0.001).There were a few neutrophils and lymphocytes infiltrating in liver cells and around the central vein treated by high dose group,and in all three ACD groups,the alveolar space and alveolar interval became wider and could find a large number of neutrophils and monocytes infiltration,which showed the pulmonary congestion and inflammation.With the increase of intraperitoneal injection of ACD,the side effect of would increase.8.The acute toxicity of ACD on Kunming mice was observed by the maximum tolerance dose method.Comparing with the control group,there was no toxic symptoms and abnormal activity reactions in administration group mice.Blood biochemical indexes of the mice were measured.The results showed that GLU was lower in male mice of administration group and other indexes were normal.TP,ALB,GLB,GLU and UA were lower in female mice,while T-Bil and D-Bil were higher.There was no significant difference in the organ coefficients of male and female mice in the administration group compared with the control group.H&E staining sections of liver,spleen,kidney,lung,stomach,ovary and testis were analyzed.Lymphocyte infiltration was found in the liver of female mice in the administration group,and no abnormality was observed in the rest.There was no lethal effect at 20 g/kg ACD,the MTD(maximum tolerated dose)>20 g/kg.In conclusion,ACD can inhibit the proliferation of both human and mice breast cancer cells in vitro,and inhibit the cell migration,regulate of cell cycle and induce apoptosis.In vivo,ACD significantly inhibit the growth of breast cancer,maximum tolerated dose of ACD in Kunming mice was more than 20 g/kg.