The Inhibitory Effect On Human Breast Cells And Novel Dosage Forms Of Artemisinin

AIMS:Increasing adhesion and migration abilities of tumor cells are the key factor for metastais of cancer.The identifing natural components which have with anti-tumor activations from plants are the focus of researching in the area of anti-tumor drug discovery.Artemisinin,a traditional antimalarial drug,has been reported to have cytotoxicity to many kinds of tumor cells.Vasodilator stimulated phosphoprotein(vasodilator-stimulated phosphoprotein,VASP)is a kind of actin related cytoskeletal proteins that participated in the regulation of adhesion and migration abilities of tumor cells.Here,human breast cancer cells MDA-MB-231 and MCF-7 were employed as an experimental model,and the influence of artemisinin on proliferation,adhesion,migration and invasion of the cells were investigated;the transcription and protein expression level,the structure of VASP were evaluated.It is difficult to make appropriate dosage forms for physical and chemical properties of artemisinin.In order to prepare long-term,targeting artemisinin nanoparticles,the artemisinin mPEG-PLGA nanoparticles were prepared by solvent diffusion technique and the preparation process were optimized by single factor and Box-Behnken design methodology.Methods:1.Breast cancer cells MDA-MB-231(highly invasive)and MCF-7(low invasive)were cultured in vitro and pretreated with artemisinin at different concentration and time.Proliferation of the cells was investigated by MTT assay and plate colony formation assay.Migrartion ability of the cells was detected by Transwell and wound closure assay.The ability of adhesion and invasion of the cells were examed with MTT assay and Transwell assay.2.Subcellular distribution of VASP and F-actin insided in cells were observed under confocal microscope.The mRNA and protein expression of VASP were determined by RT-PCR and Western-Blot.The interaction between artemisinin and VASP was analysised by fluorescence spectroscopy and far-UV CD spectra.And the possible binding sites of artemisinin and VASP were analyzed by Autodock Vina software.Finally,actin polymerization mediated by VASP was tested by actin aggregation assay.3.A method for the determination of artemisinin was established.And Artemisinin nanoparticles were prepared by solvent evaporation method.On the basis of single factor investigation,prescription design and preparation process were optimized by Box-Behnken design methodology.The pharmaceutics characteristics for nanoparticle was evaluated.Results:1.Proliferation of breast cancer cells MDA-MB-231 and MCF-7 could be suppressed by artemisinin in time dependent and concentration dependent mannal.The results of Transwell and wound healing assay showed that the migration ability of the two kinds of cells could be inhibited by artemisinin(concentration>10?M)in concentration dependent mannal.Similarly,the adhesion of the two kinds of cells could also be inhibited by artemisinin.And the invasive ability of the breast cancer cells MDA-MB-231 could be significantely inhibited by artemisinin.2.The polymerization of actin filaments in the cells could be inhibited by artemisinin.But the mRNA and protein expression level of VASP in the cells were not influeced by the artemisinin.Further more,the fluorescence spectroscopy and far-UV CD spectra experiments results indicated that artemisinin could directly inhibit the polymerization of F-actin via changing the space conformation of VASP by directing combine with VASP.3.The detection method of artemisinin concentration by spectrophotometry was established.The prescription prepared technology of artemisinin mPEG-PLGA nanoparticles was determined.The size of the nanoparticles was 265.2±43nm,the entrapment efficiency was 61.2±3.2%,the drug loading was 5.8±0.6%.The drug release was sustained for 72h.Conclusions:Artemisinin could suppress the proliferation,adhesion,migration and invasion ability of breast cancer cells MDA-MB-231 and MCF-7 through directly inhibiting the polymerization of F-actin via changing the space conformation of VASP by directing combine with VASP.The preparation technology of artemisinin nanoparticles was feasible with good reproducibility,and the characteristics of artemisinin mPEG-PLGA nanoparticle in vivo and in vitro need further research.