| Nereid belong Annelida,Polychaeta,wandering eyes,Nereis families,invertebrates,widely distributed in the Sea of Japan and the Pacific Rim,is a species endemic to Japan and China.In recent years,studies in vivo biological active substances Nereis abroad gradually increased,Japanese scholars separated from Grube in a polypeptide having the biological activity of these peptides on annelid esophagus with a strong contraction,Jilin University Bethune Medical College,the body was first isolated and purified from the Nereid protease which has a strong fibrin and fibrinogen degradation effect(Nereis active protease,NAP),and the study found a variety of in vitro protease Nereis leukemia cells significantly inhibited.But has yet to see Grube active protease reported on a variety of cancer cells.We obtained to cultured cancer cell lines,the optimal screening tumor cell lines were determined by MTT assay,The typical morphologic changes were observed in the SPC-A-1cells and H1299 cells with inverted microscope、HE、transmission electron microscopy,and AO/EB staining.The mechanism that NAP induced cancer cells apoptosis were studied in vitro experiments by Annexin V-FITC/PI,mitochondrial membrane potential method,real-time quantitative PCR,Western blotting Western blotting and in vivo experimental with establishing cancer tumor-bearing nude mice.which can lay the foundation for future research.ContentsThe cultured cancer cell was treated with NAP which was madethe different concentrations of drugs.The following some experiments were conducted to explore the mechanisms underlying the cancer cells.1、 Concentration-dependent responses of the effect of NAP on PC-3、DU-145、H1299、SPC-A-1、95C、A549、SK-BR-3、Hep G2 cell proliferation were examined using the method of MTT assay.To elect the most advantageous effect cell lines.2、 The morphological changes of SPC-A-1 and H1299 cells were observed by inverted microscope、HE staining,AO/EB staining methods during the cell apoptosis with treatment of NAP.3、 Cell ultrastructural changes were observed by transmission electronmicroscopy after the NAP act on SPC-A-1 and H1299 cells for 24 h.4、 The early apoptosis rate and The mitochondrial membrane potential changes were observed by flow cytometry after the SPC-A-1 and H1299 cells treated with different concentrations of NAP for 24 hours.5、 The expression of apoptosis-related proteins(Bax,Bcl-2,caspase-3,caspase-9,poly(ADP-ribose)polymerase(PARP)were detected by Western Blot analysis after the SPC-A-1 and H1299 cells treated with different concentrations of NAP for 24 hours.6、 The expression of apoptosis-related m RNA(Bax,Bcl-2)were detected by QC-PCR analysis after the SPC-A-1 and H1299 cells treated with different concentrations of NAP for 24 hours.7、 In vivo tumor inhibition rate and mechanism were detected through establishing Cancer nude mouse model after the H1299 cells treated with different concentrations of NAPResults1、 MTT assay showed that NAP has a certain inhibition rate on different tumor cell among the best inhibitory effect on lung cancer cells.MTT assay showed that different concentrations of the NAP have significantly inhibitory effect on human prostate cancer cell line the SPC-A-1 and H1299 cells,which showed thesignificant time-and dose-effect relationship.2、 The results of inverted microscope 、 HE staining showed that the morphology of SPC-A-1 cells and H1299 cells treated with different concentrations of NAP after 24 h changed a lot.The results showed cellular morphological changes: volume reduced,cellular shape irregular,empty bubble in cytoplasm,Numbers of nucleolus reduced,chromatin condensed and so on.3、 The results of AO/EB staining exhibited that there were more apoptotic cells in NAP-treated groups than control groups.early apoptosis morphological cellular features were foud in Low concentrations of NAP,such as cavitation bubble,budding in cytoplasm.With the increase of concentration of NAP,late apoptotic cells and high concentrations of early apoptotic cells increased significantly,showing orange fluorescence.4、 The results of transmission electron microscope observed the changes of ultrastructure of SPC-A-1 cells and H1299 cells with different concentrations of NAP for 24 hours,In the control group,the microvilli in SPC-A-1 cells and H1299 cells surface are obvious,and the plasma membrane and nuclear membrane both keep integrity.However,in the SPC-A-1cells and H1299 cells incubated by NAP,the microvilli of the cell surface disappear,5、 heterochromatin side dimerization,.and the endoplasmic reticulum becomes vacuolization.At high concentrations of NAP,the chromatin gathers vary significantly,and the smooth endoplasmic reticulum expands significantly.We can see that there are significant apoptosis after using the drug through thechanges in organelle morphology.6、 FCM disclosed that there were apoptosis cells in control group of SPC-A-1 and H1299 cells.The early apoptosis rate of SPC-A-1 cells increased from 13.50% to 22.98%,when the NAP concentration ranged from 30 μg/m L to 50 μg/m L.The early apoptosis rate of H1299 cells increased from 12.95% to 25.28%,when the NAP concentration ranged from30μg /m L to 50μg /m L.7、 FCM disclosed that there were the degree of decline in mitochondrial membrane of SPC-A-1 and H1299 cells with NAP.The percentage decrease in mitochondrial membrane potential of SPC-A-1 cells increased from 12.95% to 25.38%,when the NAP concentration ranged from 30 μg/m L to 50 μg/m L.The percentage decrease in mitochondrial membrane potential of H1299 cells increased from 11.01% to 27.78%,when the NAP concentration ranged from 30 μg/m L to 50 μg/m L.8、 The results of Western Blotting showed that the expression of proteins had change a lot.After SPC-A-1 and H1299 cells with treatment of NAP,the expression of Bcl-2 protein decreases,but the expression of Bax protein enhances.The ratio of Bax / Bcl-2 also increases significantly,the expression of cytochrome C 、 Cleaved-Caspase 9 、Cleaved-Caspase 3、Cleaved-PARP protion decreases.9、 In vivo results show that,NAP for H1299 xenografts inhibited,The results of Western Blotting showed that the expression of Bax protein enhances,but the expression of Bcl-2protein decreases.which initiates intrinsic apoptosis pathway,activation of the protein of caspase 9,The activated caspase 9 activation of downstream effector caspase 3,causing apoptosis,resulting in smaller xenografts.Conclusion1、 NAP has a broad-spectrum anti-tumor effect.2、 NAP had an obvious inhibition on the growth of SPC-A-1 cells and H1299 cells in concentration-and time-dependent manner.3、 The morphological changes of SPC-A-1 and H1299 cells were observed by inverted microscope、Fluorescence microscopy,transmission electron microscopy during the cell apoptosis with treatment of NAP.4、 FCM disclosed that there were apoptosis cells and the degree of decline in mitochondrial membrane of SPC-A-1 and H1299 cells with NAP5、 In vitro experiments showed the mechanism of apoptosis of the NAP-induced SPC-A-1cells and H1299 cells is that the NAP induces lower mitochondrial membrane potentialby down-regulating the expression of the protein Bcl-2 and up-regulating the expression of the protein Bax protein.Then it cause cytochrome C transfer and Caspase family activated and eventually stimulate apoptosis,which might be mitochondrial apoptotic pathway.6、 Tn vivo mechanism of action is similar to in vitro,the experiments showed the mechanism of apoptosis of the NAP-induced SPC-A-1 cells and H1299 cells is that the NAP induces lower mitochondrial membrane potentialby down-regulating the expression of the protein Bcl-2 and up-regulating the expression of the protein Bax protein.Occur the mitochondrial apoptotic pathway,Then it induces up-regulating the expression of the protein Cleaved-caspase 9,then it cause Cleaved-caspase 3 activated and eventually stimulate apoptosis.Resulting in smaller xenografts. |
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