The Exploration Of Growth Inhibition Of BD-15 In A549 And H1299 Cells

In recent years,the tumor incidence rise persistently.According to statistic data,there are 4.2916 million morbidity and 2.8142 million dead about cancer at China in 2005.The highest morbidity of which is lung cancer.The number of the morbidity is about seven hundred thousand.Classified by pathology,the lung cancer is divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).The 80% of patient belongs to NSCLC.There are operative treatment and chemo radiotherapy and targeted therapies about the therapies.Protein kinase C(PKC)is the effector of the family of G-protein-coupled receptors.The system which belongs to the kinase of serine and threonine includes Conventional、Novel and Atypical families.Protein kinase C depends on liquid,It can be activated by the presence of diacylglycerol and calcium ion and then the signaling pathways downstream of PKC are activated to regulate the gene expression.In recent years,the intimate connection between PKC and the occurrence and development of tumors are founded.It will be of important significance to look into the relationship between PKC and tumors.Bisindolylmaleimides is the inhibitor of PKC.Manny of Effectors compounded from it have the growth inhibition effect.There are one drug library about the ramification of bisindolylmaleimides.We expected that a new medicine which can inhibit tumors growth is screened out and then the mechanism of the new medicine can be exploration.Alkaloid is a kind of alkaline organic compounds containing nitrogen It mainly exists in nature(mostly in plants,and some others in animals),so it used to be called Constraint alkali.Most of alkaloids have complex ring structure which more nitrogen containing in the ring.According to scientist,Most of them have significant biological activity,which is one of important effective components of Chinese herbal medicine.There are Bisindolymaleimide and Indole carbazole alkaloids which has diverse structure and good biological activity and thus attracted much attention.They can be applied to a wide variety of tumor related targets,such as protein kinase C and DNA topoisomerase and so on.Three point ester alkali is one alkaloids which can inhibit the tumor growth.It is separated from the three plants of the genus pointed Chinese fir in our country and has the specific influence in cell cycle where targets the G1 and G2 phase but not in S phase.It can affect DNA compound enzyme in the early stages of polymer formation and Transcription interference ribosomal protein for normal function,beside that,The synthesis of DNA also has inhibitory effect.Objective: A new medicine which named BD-15 screened out from the Bisindolylmaleimides drug library has the effect of tumor growth inhibition.There are many experiments used to study the mechanism of BD-15’s growth inhibition effects in A549 and H1299 cells,such as MTT、Immunofluorescence 、Western blotting and flow cytometry and so on.Methods:(1)The model of A549 and H1299 cells which is NSCLC is used,then the MTT experiment will be used to verify the growth inhibition and detect the multiplication capacity when the cells are added BD-15 that the concentration is 1mM for 24 h.(2)The forms of A549 and H1299 cells are screened by inverted microscope and inverted fluorescence microscope.(3)The change of cell cycle has a detection by flow cytometry when the cells were added BD-15 which the concentration is 1mM for 24h(4)After the Western blotting is used,there are some proteins which the expression are detected when the cells dosed for 24 h.for example,the p-PKC substrate、p-ERK、P53 and P21 in A549 cells and the p-PKC substrate、p-ERK.(5)The exploration whether the BD-15 can inhibit the growth of A549 and H1299 tumor in nude mice is used.Results:(1)By MTT experiment,the conclusions showed that the proliferation of A549 and H1299 cells which added BD-15 decrease(2)The phenomenon observing from inverted microscope and inverted fluorescence microscope shows that: BD-15 has the effects of stuck in the middle mitosis and change the form in the two cells.(3)The result from flow cytometry shows that BD-15 can retardant the cell cycle which reach the G2/M period.(4)The Western-blotting shows the consequence that as times by,BD-15 has the capacity that not only the protein expression of p-PKC substrate、P53、P21 increase in A549 and H1299 cells,but also the phosphorylation of P21 elevated.Conclusions :(1)BD-15 can inhibit the growth of A549 and H1299 cells.(2)BD-15 doesn’t have only the effect of stuck in the middle mitosis,but also arrested in G2/M period.(3)When added BD-15.The PKC pathway and cell apoptosis can be activated and the phosphorylation of P21 can increase.(4)The growth rate of A549 and H1299 tumors in nude mice can be slow down by BD-15.