| Backgrounds:Cancer remains a serious threat of human health and life in the world. Chemotherapy is one of the most effective methods for cancer treatment. Currently, anti-metabolites account for a large proportion of chemotherapy drugs. Gemcitabine is a cell cycle specific anticancer agent that are effective for therapy of non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer and pancreatic carcinoma and other solid cancers. The primary limitation of gemcitabine is its rapid inactivation by the deoxycytidine deaminase enzyme following its in vivo administration. SL-01is a4-amino derivative of gemcitabine that synthesized by our group. In our previous studies, SL-01was found to be more stable in chemical and enzymatic hydrolysis and have a longer half-life in mice than gemcitabine. In this study, we evaluated the efficacy of SL-01on the growth of human breast cancer by in vitro and in vivo studies and its mechanism of action was also explored.Methods:The experiments were performed on human breast cancer cell line MCF-7. The inhibitory effects of SL-01on MCF-7cells was estimated by the3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) method. Flow cytometry assays were performed to determine the apoptosis cells and cell cycle arrest induced by SL-01. Western blotting assay was carried out to analyze the cell cycle and the related proteins. The efficacy of SL-01in vivo was evaluated in MCF-7xenografts mouse model. Mice were sacrificed and cancer tissues were removed for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and western blotting for apoptosis-related proteins. Samples of liver and stomach were prepared for microscopic examination.Results:MTT assay showed that SL-01(0.125μ M-2.0μ M) significantly inhibited the proliferation of MCF-7cells after24h,48h and72h treatment. Statistical analysis indicated that the IC50value that based on the inhibition rates of72h exposure was0.64μM. Using1μM for72h exposure, the inhibition rate was62.9%for SL-01and58.3%for gemcitabine. Hoechst33258staining results showed that2u M SL-01induced the nuclear chromatin, condensation, or gathering around to the nuclear membrane, or in blocks fragmentation change role in MCF-7cells after24h treatment. PI staining showed that SL-01arrested cell cycle in G1phase. SL-01at0.5,1.0, and2.0μM for24h, the percentages of cell population in G1phase were significantly increased to59.3%,74.6%and77.8%, respectively. Western blotting analysis showed that the levels of p53, p-ATM and p21Waf1/Cip1were elevated and cyclin D1was reduced by SL-01. The activity of SL-01was confirmed in mice bearing MCF-7xenografts. The grafted MCF-7tissue was strongly inhibited by SL-01after3weeks oral administration. SL-01at12.5.25and50μmol/kg, the growth of MCF-7xenografts was reduced by10.1%,34.5%and66.5%, respectively. TUNEL staining indicated that SL-01at12.5,25and50μmol/kg significantly increased the percentages of positive staining cells by19.4%,23.5%and33.8%, respectively. Western blotting suggested the activation of caspase-9, caspase-3, and cleaved poly ADP-ribose polymerase (PARP) and increase of Bax/Bcl-2ratio in the SL-01treated MCF-7xenografts. Furthermore, the levels of PCNA and NF-κB were reduced by SL-01. I listopathologic examination showed that no abnormalities in liver and stomach were found in the SL-01-treated mice during the course of study.Conclusion:SL-01exhibited strong activity against human breast cancer growth with lower toxicity to gemcitabine. The effect of SL-01might arise from its roles in cell cycle G1arrest and apoptosis. We suggested that SL-01might be a potent oral anticancer agent that may supplant the use of gemcitabine. |
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