| Objective:Breast cancer has become the most critical threats to women and younger tendency, it has became the focus of society. Neoadjuvant chemotherapy and adjuvant chemotherapy are still predominantly used for the treatment of breast cancer.While advancements in breast cancer treatment and prevention have emerged over the last decade, the mortality rate has been reduced compared to the previous, multidrug resistance (MDR) has been a main cause of breast cancer chemotherapy failure. Psoralen is the main component extracted from traditional Chinese me.dicine. Psoralen is often used to inhibit cell proliferation of skin diseases, and has a strong effect to reverse the ability of multidrug resistance.In 2015, the laboratory has proved of psoralen can reversal the multidrug resistance in breast cancer cell line MCF-7 / ADR cells, but exact mechanism is still not clear. Multidrug resistant breast cancer cell line MCF-7/ADR as a research object,the study to the effect of psoralne on cell cycle and apoptosis provide a theoretical and experimental basis for Multidrug resistant breast cancer cell line .Methods: 1.We measured the MCF-7/ADR cell viability by MTT assay to evaluate cytotoxicity . The cytotoxic effect of psoralen on MCF-7/ADR cells and the IC50 were asssy by the same way. The expriment groups were select by the value of IC5,IC10 and IC20.2.Flow cytometry was used to analyze the cell cycle and apoptosis of MCF-7/ADR cells treated with psoralen.3.Real-time quantitative PCR was conducted to evaluate the expression of p53,Bcl-2 and caspase-3. And further detected by agarose gel electrophoresis experiment.Results: 1. we measured the MCF-7/ADR cell viability by MTT assay to evaluate cytotoxicity of psoralen. Compared to control groups, experiment groups were statistically significant differences (F=140.69, P <0.05). IC50 value of psoralens was 18.785 ± 1.253 μg /ml. To ensure the accuracy of the experiment, 4 μg / ml, 8 μg / ml, 12 μg / ml dose as the experimental group used for the following tests.2. Afte 48 h, flow cytometry showed that the cell ratio in G0/ G1 phase will be increased,blocking entry into S phase, so the decline in the proportion, compared to the control group,experiment groups were significantly different (FGO / G1 = 1272.3 .25, FS = 134.27, p <0.05).These date showed that psoralens can arrest cell cycle in G0/G1 phase, inhibited cell proliferation activity. However, the rate of cell apoptosis was determined by Annexin V-FITC/PI staining. Compared with the negative control group (0.23 士 0.12%),Even at the lowest concentration ( 4μg / ml), psoralen treatment of the MCF-7/ADR cells resulted in increased number of double-stained AnnexinV-FITC-positive and PI-negative cells, treated with 8μg / ml and 12 μg / ml of psoralen don’t exhibited significantly higher rate of apoptosis apoptosis (7.49 ± 0.89%) and (7.56 ± 0.78%) as compared with the negative control group.but no significant difference in each group (p> 0.05).3. Compared to the control group (Oug / ml psoralen group) of MCF-7 / ADR cells After a 48-h exposure to psoralen at non-toxic concentration (8 μg/ml),p53 gene up-regulated about 2.15 土 0.23-fold (P <0.05),apoptosis gene Bcl-2 down-regulate 7.47 ± 1.23-fold (P<0.05),and the execution of apoptosis gene caspase-3 1.35 ± 0.39-fold increase (P <0.05),the differences were statistically significant.Conclusion: 1. By MTT assay, we have a preliminary understanding of the drug resistance of MCF-7/ADR cells, MCF-7/ADR and MCF-7 have a certain resistance to adriamycin, but the effect of MCF-7/ADR is stronger. Further experiments found that psoralen can inhibit the cell proliferation activity, which was dose-dependent. It can be concluded that the MCF-7/ADR has resistance, and psoralen does have drug-resistant cell effect.2.By flow cytometry, we found that the psoralen at different concentrations could arrest cell cycle in GO / G1 phase and reduce the ratio of S phaseand with psoralen concentration changes more obvious. Compared to negative group, the differences is significant(FG0/G1=1272.3.25, FS=134.27, p<0.05). Therefore, we speculate that psoralen can arrest the cell cycle in a dose dependent manner3. To detect apoptosis of MCF-7/ADR cells by flow cytometry, we found that psoralen can induce apoptosis, and the apoptosis ratio was different in different concentration. Compared with the control group, the difference was significant (p<0.05), but there was no significant difference (p>0.05) between different concentration groups. Therefore, we speculate that psoralen can induce cell apoptosis, but no concentration dependence4. By RT-PCR experiments and agarose gel experiment, we found that nontoxic concentrations of psoralen could impact the cell cycle and apoptosis of multidrug resistant breast cancer, such as upregulate the expression of p53 and Caspase-3 expression and inhibiting the expression of anti apoptosis gene bcl-2.. |
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