| Objective:In this study, based on the previous work, the effects of TET, ATO, and their combination on the proliferation inhibition and apoptosis promotion of MDA-MB-435S cells and HCC1937 cells and the mechanism involved were analyzed in vitro. For the in vivo experiments, using MDA-MB-435S cells-inoculated nude mice as lung metastasis model, the anti-tumor effects TET, ATO, their combination, and chemotherapy drug cisplatin and the corresponding expressions of tumor angiogenesis-related signals were observed, and simultaneously the liver and kidney function of nude mice in each group was detected to determine the safety of these toxic traditional Chinese medicines, providing experimental basis for the combination therapy of breast cancer and its lung metastasis by compatibility of Chinese medicine under the guidance of "combat poison with poison."Methods:The inhibitory effects of TET, ATO, and TET combined with ATO at various concentrations on MDA-MB-435S and HCC1937 cell lines at different time points (24,48, 72, and 96 hours) as well as the relationship between drug concentration and time were detected in vitro by using MTT assay, the optimal drug ratio for the combination treatment was determined by using the median-effect principle (Chou-Talalay combination index) to guide the subsequent experiments, and the morphological changes of MDA-MB-435S and hcc1937 breast cancer cells 48 hours after intervention with TET, ATO, and TET combined with ATO at various concentrations were observed using by using inverted phase contrast fluorescent microscope, to explore the effect of these drugs.Annexinv FITC and PI double staining flow cytometry were used to determine the changes of the proportion of early apoptosis and late apoptosis of MDA-MB-435S and HCC1937 breast cancer cells 48 hours after treatment with TET, ATO, and their combination at different concentrations to study the effects of two drugs on tumor cell apoptosis.The tumor invasion assay was used to determine the inhibitory effects of TET, ATO, and their combination at various concentrations on the invasion abilities of MDA-MB-435S and HCC1937 breast cancer cells 48 hours after treatment as well as the degree of the inhibitions.The protein expressions of breast cancer cell apoptosis-related genes Caspase-3, PARP, Bid, Bax, Bcl-2, survivin, AKT and the related mechanism were analyzed in MDA-MB-435S and HCC1937 cells 48 hours after treatment with TET, ATO, and their combination by using RT-PCR and Western blot methods.For the in vivo tests, a model for breast cancer with lung metastasis was established in BALB/c nude mice inoculated with high pulmonary metastasis human breast carcinoma cell line MDA-MB-435S under breast pad. After the success of modeling, tetrandrine hydrochloride injection, arsenious acid injection, cisplatin injection, and the combined injection were injected to the model animals to observe the general living conditions, tumor inhibition rates, lung metastasis rates, and indexes of liver and kidney function. HE staining, immunohistochemistry, RT-PCR, and Western blot were performed for both tumor and lung tissues to observe the gene and protein expressions of vascular angiogenesis-related genes, such as VEGF and MMPs, and therefore to elucidate the signal pathways.Results:It was found in in vitro experiments that TET, ATO, and TET combined.with ATO had significant inhibitory effects on the in vitro growth of MDA-MB-435S and HCC1937 breast cancer cells, with a significant relationship with dose and action time, respectively. The inhibition rates increased with prolonged time and increased drug concentrations, and an interaction effect between time and the drug dosage was also observed. Based on the median-effect principle (Chou-Talalay combination index) and calculation by calcusyn2.0 software, TET (1g/ml) combined with ATO (1.5μg/ml) was found to have the best synergistic action on the MDA-MB-435S and HCC1937 cells, and thereafter, this combined dosage was chosen for subsequent tests.Morphological studies showed that cell morphology was changed 48 hours after treatment with TET, ATO, and TET combined with ATO in a dose-dependent way, and combination treatment was better than the single drug alone. A large number of cells died and disintegrated, and the number of adherent cells was significantly decreased.Cell apoptosis detected by Flow cytometry double staining method revealed that the early apoptosis and late apoptosis rates of MDA-MB-435S and HCC1937 cells increased 48 hours after the treatment with TET, ATO, and TET combined with ATO, and the apoptosis rate increased by combined drugs was higher than that of single drug alone.Tumor invasion assay results showed that there were differences in the cell numbers that penetrated into lower chamber 48 hours after MDA-MB-435S and HCC1937 cells were treated with TET, ATO, and TET combined with ATO, and the number of membrane-penetrating cells decreased with the increasing of drug dosage. The combined effects of the two drugs were not significantly different from that of the single drug at the same concentration.The analysis of apoptosis-related gene expression profile by RT-PCR shows that the expression levels of related genes such as Caspase-3, bax, and bid in MDA-MB-435S and HCC1937 cells were increased along with the increase doses of drugs 48 hours after treatment with TET, ATO, and TET combined with ATO, while those of regulatory genes such as Bcl-2 and Suivivin were decreased. And the combined effects of the two drugs were superior to the single treatment, respectively.Western blot results showed that the expressions of pro-Caspase 3 in MDA-MB-435S and HCC1937 cells were reduced by drug treatments, while that of the cleaved Caspase 3 was increased. The enzymatic digestion bands of the downstream PRAP suggested the occurrence of apoptosis. Bcl-2 expression decreased, while the expressions of Bax and Bid increased, and the drug combination was superior to the single drug treatment alone at the same concentration. The expression of Survivin protein was not significantly different between the control group and the single drug alone treatment groups, respectively, while that of the combined treatment group was significantly decreased.The in vivo experiments demonstrated that tetrandrine hydrochloride injection, arsenious acid injection, and the combined injection could slow down the growth of the tumor and inhibit lung metastasis, with slightly poor effects when compared with cisplatin. There were no significant damages on the liver and kidney function in nude mice either single or in combination. The RT-PCR and Western blot analysis of tumor and lung tissue in nude mice showed that tetrandrine hydrochloride injection, arsenious acid injection, and the combined injection could reduce the expression of VEGF, and the expression level in the combined treatment group was lower than that of each single treatment group.Conclusions:TET, ATO, and TET combined with ATO significantly inhibit the cell proliferation of MDA-MB-435S and hcc1937 cells in vitro, with a strong correlation with the drug dose and treatment duration, and optimal synergistic action may be achieved when TET (1g/ml) is combined with ATO (1.5g/ml).TET, ATO, and Y+TET combined with ATO at different concentrations may increase the early apoptosis and late apoptosis rates and reduce the invasion function of MDA-MB-435S and HCC1937 cells 48 hours after each treatment. The apoptosis of breast cancer cells was induced in vitro by the decreased expression of Bcl-2, the increased expressions of Bid and Bax, and the consequently effected Caspase-3.In vivo tests indicate that breast cancer model with a high pulmonary metastasis rate is successfully established by inoculation of MDA-MB-435S cells into nude mice. Tetrandrine hydrochloride injection, arsenious acid injection, and the combined injection can inhibit the tumor growth and lung metastasis. The mechanism involved may be related to the decrease of VEGF expression and the effect on tumor angiogenesis.
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