Proposed new pro-apoptotic Smac peptide molecules downstream of apoptosis proteins and apoptosis effects of group research

Title: Proposed new pro-apoptotic Smac peptide molecules downstream of apoptosis proteins and apoptosis effects of group researchAuthor: Wang JingDegree-granting units: Huazhong University of Science and TechnologyKeywords: Smac;; solid-phase synthesis;; peptides;; biological activity; Smac;; synthetic peptide;; bladder cancer;; Apoptosis; Smac / DIABLO;; synthetic peptide;; bladder cancer;; chemotherapy sensitivity; magnetic nanoparticles;; Smac, synthetic peptides;; O-carboxymethyl chitosan;; bladder cancer;; apoptosis;; Smac;; synthetic peptide;; nanoparticles;; constant magnetic field;; bladder cancer;; apoptosis; Smac;; synthetic peptide;; nanoparticles;; model Neodymium Magnets animals;; bladder cancer;; targeted therapySummary:
Experiment I be pro-apoptotic Smac peptide synthesis and biological activity of the preliminary study
Objective To investigate the proposed Smac peptide synthesis and apoptosis of bladder cancer biological activity. Methods The solid-phase peptide synthesis, synthesis of membrane fusion peptides penetrating SmacN7 by RP-HPLC purification, purity analysis, qualitative identification with mass spectrometry; by fluorescence microscopy morphology of apoptosis, cell proliferation and flow rate measurement cytometry analysis, the study of low-dose mitomycin C-induced apoptosis in T24 cells to promote the role. Results can be penetrating fusion peptide SmacN7 product peaks more than 95% purity, molecular weight of 3278.08, the results of mass spectrometry results were in agreement with the expected synthesis; 50μg / L ~ 500μg / L SmacN7 role 12h ~ 48h, typical apoptotic tumor cells morphological changes; with SmacN7 concentration increases or the role of time, the cell http://www.everbeenmagnet.com/en/products/110-sintered-neodymium-magnets proliferation rate has increased significantly, drug 12h, 24h, 48h after the proliferation inhibition rate was (9.62 ± 1.07)% ~ (61.48 ± 1.15)% , (24.17 ± 1.02)% ~ (72.86 ± 1.68)%, (43.24 ± 1.15)% ~ (84.91 ± 1.74)%: the rate of tumor cell apoptosis was significantly increased, respectively (6.12 ± 1.16)% ~ (49.81 ± 2.11)%, (13.47 ± 1.15)% ~ (64.54 ± 2.27)%, (28.91 ± 1.08)% ~ (82.36 ± 2.19)%. Conclusion The proposed solid-phase synthesis of Smac fusion peptide SmacN7 the purpose of high-purity peptides that can stably into cells and the high utilization rate, and significantly promote the low-dose mitomycin C-induced apoptosis in T24 cells biological activity, in order to further study the biological treatment of bladder cancer has accumulated valuable information.
Experiment two synthetic Smac fusion polypeptide to be promoting low-dose mitomycin C-induced apoptosis of bladder cancer
Objective To explore the synthetic peptide to be Smac fusion (SmacN7) to low doses of mitomycin C (MMC)-induced apoptosis in T24 cells facilitating role. Method using solid phase peptide synthesis technique synthetic SmacN7 cells penetrating fusion peptide, 0.05mg/ml MMC-induced bladder cancer T24 cells and 50μg / L ~ 500μg / L of SmacN7 fusion peptides were incubated 4h ~ 48h after using Annexin -V fluorescence staining of tumor cell apoptosis; flow cytometry and MTT colorimetric detection of T24 cells after induction of apoptosis, proliferation inhibition rate and SmacN7 time and concentration-effect relationship. Results of 50μg / L ~ 500μg / L SmacN7 role 4h ~ 48h, apoptotic tumor cells typical morphological changes, with increasing concentration SmacN7 or drug treatment time, increase the rate of tumor cell apoptosis, 12h to 5.67% ~ 56.12%, 24h to 14.54% ~ 65.24%, 48h to 31.48% ~ 87.23%, while inhibition of cell proliferation rate has increased significantly, drug 12h, 24h, 48h after the growth inhibition rates were 9.58% ~ 63.42%, 28.94% ~ 72.3%, 44.7% ~ 87.12%. Conclusion SmacN7 can effectively promote the low-dose mitomycin C-induced apoptosis in T24 cells, and a time-and concentration-dependent manner, the biological treatment of bladder cancer provides a new idea.
In Experiment 3, the proposed synthetic Smac peptide enhanced the chemosensitivity of bladder cancer experimental study
Objective: To investigate the synthetic penetrating cells can be pro-apoptotic Smac fusion peptide SmacN7 chemosensitivity of bladder cancer promoting effect. Methods: The synthetic pro-apoptotic cells penetrating fusion peptide SmacN7; thiazolyl blue (MTT) test SmacN7 fusion peptide of low-dose mitomycin C (MMC)-induced bladder cancer T24 cells, the relative survival rate; Annexin V / PI double-labeled T24 cells analyzed by flow cytometry of apoptosis; Western blot detection SmacN7 fusion peptides associated with the MMC after T24 cells with XIAP, Caspase-3 protein expression; simultaneous detection of Caspase-3 activity and SmacN7 fusion peptides associated with MMC use of T24 cells in vitro. Results: SmacN7 fusion peptides can penetrate the cell and combine with endogenous XIAP, increased low-dose MMC-induced apoptosis in T24 cells and was time-and concentration-dependent manner; can significantly reduce the level of intracellular expression of XIAP, Caspase-3 in enhancing expression and activity; at 24h and 48h, SmacN7 + MMC group compared with MMC alone, T24 cell viability decreased by 2.22 times and 3.61 times. Conclusion: Synthetic Smac cells can be penetrating fusion peptide can promote apoptosis induced by chemotherapeutic drugs in bladder cancer T24 cell apoptosis and inhibit cell proliferation, enhanced bladder cancer chemosensitivity of the MMC.
Experiment 4 to be Smac synthetic peptide of carboxymethyl chitosan nano-composite magnetic Preparation and Properties
Objective To explore the synthetic peptide to be Smac carboxymethyl chitosan preparation of magnetic nanocomposites and joint constant external magnetic field of bladder cancer cells in vitro apoptosis-promoting effect. Methods and carboxymethyl chitosan as the backbone of Fe_3O_4 with superparamagnetic nanoparticles synthesis of nano-carrier particles, will be combined with SmacN7 Smac peptide synthesis, synthetic Smac peptides to be prepared carboxymethyl chitosan nano-composite magnetic material, and by transmission electron microscopy, vibrating sample magnetometer and other investigation of their physical and chemical properties; Hoechst33258 staining nanocomposites under applied magnetic field induced apoptosis in T24 cells form, MTT colorimetric assay to observe the inhibition of tumor cell growth. The results of the proposed synthetic Smac peptide carboxymethyl chitosan magnetic nanoparticles of diameter about 46.2nm; magnetization curve tips superparamagnetism; drug loading and encapsulation efficiency was (31.8 ± 3.6)% and (65.2 ± 2.4 )% and has a good drug delivery performance; external magnetic field of magnetic nanoparticles allows the tumor cells showed obvious morphological changes of apoptosis and showed significant growth inhibitory activity. Conclusions to be Smac synthetic peptide of carboxymethyl chitosan magnetic nanocomposites with small particle size, a strong magnetic response, drug loading and encapsulation of the drug release rate and good performance, combined with the obvious constant external magnetic field promote tumor cell apoptosis, the biological treatment of bladder cancer provides a new starting point.
Experiment 5 Smac synthetic peptide to be induced by magnetic nanoparticles apoptosis in bladder cancer cells in vitro
Purpose of the proposed synthetic Smac peptide magnetic nanoparticles (SmacN7-O-CMC-MNP_S) Preparation and coordination constant external magnetic field in vitro on human bladder cancer T24 cell growth inhibition and to explore its mechanism. Prepared by redox method SmacN7-O-CMC-MNP_S and detect physical and chemical properties, inverted microscope and electron microscopy of cell morphology, TUNEL assay of apoptosis, MTT, flow cytometry were observed SmacN7-O-CMC-MNP_S chemotherapy drugs and constant external magnetic field in vitro on human bladder cancer T24 cell growth inhibition, SABC Bcl-2/Bax protein expression was observed, Western blot detection of XIAP protein expression. The results SmacN7-O-CMC-MNP_s magnetic nanoparticles diameter 46.2nm, spherical, drug loading (31.8 ± 3.6)%, the magnetic response was good. Containing the same concentration SmacN7 the simple SmacN7-O-CMC-MNP_S T24 bladder cancer cell growth inhibition and apoptosis was no significant effect, but together and in the external magnetic field induced by the chemotherapeutic drugs on tumor cell growth inhibition was enhanced; Bcl-2 protein levels down while Bax protein expression increased, the magnetic field outside the group and chemotherapy group difference was statistically significant (P <0.05), coordinated by Western blot when the external magnetic field at different times of XIAP protein expression was significantly reduced. Conclusion SmacN7-O-CMC-MNP_S SmacN7 preparation does not affect the biological activity; constant external magnetic field under its pro-apoptotic effect of tumor cells significantly enhanced for targeted therapy of bladder cancer biological experimental basis.
Experiment six synthetic peptides to be Smac magnetic nanoparticles in bladder cancer xenografts in nude mice in vivo targeted therapy and mechanism of
Purpose of the proposed synthetic Smac peptide magnetic nanoparticles for bladder cancer xenografts in nude mice in vivo role of targeted therapy and its mechanism. Methods 40 tumor-bearing nude mice were randomly divided into five groups of eight, according to the different groups (A, B, C, D, E group) to the different treatment groups 1 day treatment, continuous 1wk, the magnetic field group at the tumor site to exert 0.8T magnetic field 30min. Observed in nude mice eating, activity and growth, the measured tumor volume and calculate the inhibition rate; 32d after treatment the animals were killed, HE staining and electron microscopy cellular structures, using the SABC method Bcl-2/Bax protein expression observed by RT- PCR method to detect the tumor tissue XIAPmRNA and Caspase-3mRNA expression, Western blot method to detect the tumor XIAP and Caspase-3 protein expression. Results during treatment and after treatment of nude mice in each group to eat, activities, and no obvious abnormalities in growth, body weight difference was not statistically significant (p> 0.05), SmacN7-O-CMC-MNP_S magnetic nanoparticles combined magnetic field plus MMC group (E) tumor volume and the slowest growth of bladder cancer xenografts significantly inhibited tumor inhibition rate was 58.4%, higher than SmacN7-O-CMC-MNP_S magnetic nanoparticles plus MMC group (C) (24.6% ) and SmacN7-O-CMC-MNP_S magnetic nanoparticles plus an external magnetic field group (D) (32.2%); immunohistochemical SABC Group E was detected expression levels of Bcl-2 down while Bax protein expression increased, and C, D group, the difference was statistically significant (p <0.05); RT-PCR results showed E group significantly reduced XIAPmRNA expression, while increased expression of Caspase-3mRNA; Western blot shows Group E can down-regulate the expression of XIAP protein also increased Caspase -3 protein expression. Conclusion Low-dose external magnetic field and the joint action of mitomycin C, SmacN7-O-CMC-MNP_S magnetic nanoparticles in the body has a significant body of tumor apoptosis and inhibit tumor growth, by down-regulating XIAP, Caspase up 3 in the expression of mRNA and protein in tumor-targeting treatment to achieve for the treatment of bladder cancer to explore a new way.Degree Year: 2009