Effect Of A Novel Smac-mimc Peptide On The Apoptotic Downstream Proteome And Its Induction Of Apoptosis

Part 1Synthesis and Biological Activities of Smac-mimic PeptideObjective A novel Smac-mimic polypeptide (SmacN7) was designed and synthesized tostudy its biological activity that promotes apoptosis of bladder cancer cells. MethodsSmacN7 was synthesized with aid of polypeptide solid phase synthesis technique, purifiedwith reverse-phase HPLC and identified with mass spectrometry. Using fluorescentmicroscopy, MTT assay and flow cytometry, the apoptosis effect on bladder cancer T24cell line with low-dosage of MMC was evaluated. Results The peptide was more than 95ï¼…in purity and the measured value of molecular weight was conformed to its theoreticalvalue. Typical apoptosis morphological changes of the cells were detected after beingincubated by 50μg/L～500μg/L SmacN7 for 12～48h. With the increase of concentrationof SmacN7 or the prolongation of the treating time, the proliferation inhibitory ratio of thecancer ceils increased by (9.62±1.07)％～(61.48±1.15)ï¼…,(24.17±1.02)％～(72.86±1.68)ï¼…,(43.24±1.15)％～(84.91±1.74)ï¼…and the percentage of apoptosisincreased by (6.12±1.16)％～(49.81±2.11)ï¼…,(13.47±1.15)％～(64.54±2.27)ï¼…,(28.91±1.08)％～(82.36±2.15)ï¼…when treated for 12,24,48h respectively. Conclusion The collected fusion peptide SmacN7 is the target peptide with high purity andcan be stably transferred into T24 cells and effectively utilized. It clearly has the biologicalactivity in promoting apoptosis of bladder cancer T24 cell lines with the induction oflow-dosage of MMC. These results accumulate valuable data for the biological therapy ofbladder cancer.Part 2Apoptosis Promoting Effect of a Synthetic Smac-mimc Peptide onBladder Cancer Cell line T24 with the Inductionof Low-dosage of MMCObjective To study the apoptosis improvement effect of a synthetic Smac-mimic peptide(SmacN7) on bladder cancer cell line T24 with the induction of low-dosage of MMC.Methods Penetrating SmacN7 was synthesized with aid of peptide solid phase synthesistechnique. Bladder cancer cell line T24 were incubated with different concentration ofSmacN7 (50μg/L～500μg/L) for 4h～48h. The apoptosis was detected by Annexin-Vfluorescence staining .The relationship between the percentage of apoptosis and the timeand concentration of SmacN7 was assayed by flow cytometry. The inhibitory ratio ofcellular proliferation was evaluated by MTT assay. Results Typical apoptosismorphological changes of the cells were observed after being treated by 50μg/L～500μg/LSmacN7 for 4h～48h. With the increase of concentration of SmacN7 or the prolongation ofthe treating time, the percentage of apoptosis was increased by 5.67％～56.12ï¼…, 14.54％～65.24ï¼…and 31.48％～87.23ï¼…when treated for12h,24h,48h respectively. The proliferation inhibitory ratio of the cancer cells increased notably by 9.58％～63.42ï¼…,28.94％～72.3ï¼…and 44.7％～87.12ï¼…when treated for 12h,24h,48h respectively.Conclusion Apoptosis can be effectively improved by SmacN7 on bladder cancer cellline T24 with the induction of low-dosage of MMC and there are time-dependent anddose-dependent relationships between SmacN7 and apoptosis of cancer cells. SyntheticSmac-mimic peptide may be a new strategy of biological therapy for bladder cancer.Part 3Experiment of Promoting Chemosensitivity of Bladder Cancer Cellby Synthetic Smac-mimic PeptideObjective: To investigate the effect of synthetic Smac peptide (SmacN7) on chemotherapysensitivity of bladder cancer cells. Methods: SmacN7 penetratin peptide was synthesizedand delivered into T24 cells. MTT assay was adopted to evaluate the viability of T24 cellsinduced by low-dosage of MMC; Flow cytometry was applied to analyze the proportions ofapoptosis and Western blot was used to detect the expression ofâ…ªAP and caspase-3; Theactivity of caspase-3 was measured and the effect of SmacN7 combined with MMC on T24cell lines was also determined. Results: SmacN7 penetratin peptide could successfullyinteract with endogenousâ…ªAP and increase the proportions of apoptosis of T24 cell linesinduced by low-dosage of MMC in a dose- and time-dependent manner. An obviousdown-regulation ofâ…ªAP expression and up-regulation of caspase-3 was identified byWestern blot. The activity of caspase-3 in experimental group was significantly increasedas compared with that in the control group; Combining treated with SmacN7 penetratinpeptide, the viability of T24 cells decreased to 2.22 and 3.61 fold in 24h and 48h respectively. Conclusion: SmacN7 penetratin peptide could act as a cell-permeable IAPinhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity ofbladder cancer cells to MMC. When combined with chemotherapy it may be a verypromising strategy for bladder cancer therapy.Part 4Study on Preparation and Biological Activity of Smac-mimicPeptide-loaded O-carboxylmethylated ChitosanMagnetic NanoparticlesObjective To prepare Smac-mimic Peptide-loaded O-carboxylmethylated ChitosanMagnetic Nanoparticles(SmacN7-O-CMC-MNP_S), investigate its physico-chemicalcharacteristics and discuss its apoptotic promotion combined with constant externalmagnetic fields on bladder carcinoma T24 cells in vitro. Methods The magneticSmacN7-O-CMC-MNP_S was composed by O-carboxylmethylated Chitosan (O-CMC),SmacN7 and Fe₃O₄ nanoparticles. Its physicochemical characteristics were confirmed byTEM and vibrating sample magnetometer. The apoptotic morphology was observed byHoechst33258 staining and the inhibition rates (IR) was assayed by MTT when SmacN7-O-CMC-MNP_S was combined with constant external magnetic fields. Results The meandiameter of SmacN7-O-CMC-MNP_S was 46.2nm with round shape and the its loading doseand trapping efficiency was (31.8±3.6 )ï¼…and (65.2±2.4)ï¼…respectively. Magnetizationcurve showed superpara-magnetism. SmacN7-O-CMC-MNP_S nanoparticles couldapparently induce apoptotic morphology and inhibit the growth of the bladder cancer cellswhen combined with constant external magnetic fields. Conclusions Smac-mimic Peptide-loaded O-carboxylmethylated Chitosan Magnetic Nanoparticles (SmacN7-O-CMC-MNP_S)has small particle size, good magnetic responsiveness, high loading rate andembedding ratio. It can sharply promote apoptosis of bladder cancer cells when combinedwith constant external magnetic fields and implies a promising future for the biologicaltherapy of bladder cancer.Part 5Magnetic Nanoparticles of Synthetic Smac-mimic Peptide InducedApoptosis—A Study in vitro on Bladder Cancer CellsObjective To prepare SmacN7-O-carboxymethyl chitosan magnetic nanoparticles(SmacN7-O-CMC-MNP_S), investigate its effective inhibition combined with constantexternal magnetic fields on human bladder cancer cells and then explore its mechanism ofaction in vitro. Methods SmacN7-O-CMC-MNP_S were prepared by the periodate oxidationborohydride reduction technique and their physico-chemical property detected. Cellmorphometry were observed by means of optical microscopy and electron microscopy;Combined with chemotherapeutic drug and constant external magnetic fields ofSmacN7-O-CMC-MNP_S, the cell apoptosis index(AI) and the inhibition rates(IR) wereassayed by using TUNEL and MTT assay, apoptosis of cells was observed by Annxin-V/PIdouble labeled flow cytometry; The expressions of Bcl-2/Bax protein were observed bymeans of SABC assay and the expression ofâ…ªAP was measured by Western blot in vitro.Results The mean diameter of SmacN7-O-CMC-MNP_S was about 46.2nm with round shape, good magnetic response and SmacN7 loading (31.8±3.6 )ï¼…. The differences of thecell apoptotic index (AI) and the inhibition rates (IR) were not significant between thecontrol group and SmacN7-O-CMC-MNP_S group. At the same drug concentration thegrowth inhibition and the expression of Bcl-2/Bax proteins induced by chemotherapeuticdrug and constant external magnetic fields showed sharply significant. Compared with theSmacN7-O- CMC-MNP_S+MMC group, the differences of SmacN7-O-CMC-MNP_S +Mgroup were more obvious. At different time the expression ofâ…ªAP decreased notablywhen combined with constant external magnetic fields of SmacN7-O-CMC-MNP_S.Conclusions The preparation method exert no action on bio-activity of SmacN7, combinedwith constant external magnetic fields SmacN7-O-CMC-MNP_S can effectively improvedthe apoptosis of cancer cells and can build up a solid experimental basis for the magnetictargeting therapy of the bladder cancer.Part 6Effect of Smac-mimic Magnetic Nanoparticles on the Targeting Therapyof the Bladder Carcinoma Xenografts in Nude Mice in vivoObjective To study the effect of SmacN7-O-CMC-MNP_S magnetic nanoparticles on thetargeting therapy of the bladder carcinoma xenografts in nude mice and explore itsmechanism in vivo. Methods Forty nude mices were divided into 5 groups of 8 mice anddifferent treatments were given according to different groups. This therapy was done onetime per day and continued one week and magnetic field with the strength of 0.8T was added to the tumor surface about 30 min in the meantime. The diet, activity and growth ofthe nude mice were observed and the volume of tumors was measured. After 32 days, themice were killed and the tumors were taken out and the cells structure of the tumor wereobserved; The expressions of Bcl-2/Bax protein were detected by means of SABC assayand the expression ofâ…ªAPmRNA and Caspase-3mRNA was measured by RT-PCR;Protein expression level ofâ…ªAP and Caspase-3 was detected by western blot between theexperimental group and control group in vitro. Results Targeting therapy study displayedthat the differences of diet, activity, growth and weight body in nude mice showed notsignificant; The growth speed of tumor in the E group significantly slowed down than othergroups; The tumor inhibition rate of E group was 58.4ï¼…, which was remarkably higherthan those of the C group(24.6ï¼…)and D group(32.2ï¼…); The expression of Bcl-2 proteindecreased and the expression Bax protein increased in E group by SABC assay andcompared to the group C and D the differences showed significant; RT-PCR showed thatthe mRNA expression ofâ…ªAP of E group is the lowest and the mRNA expression ofCaspase-3 of E group is the highest of all groups(P＜0. 01); Western blot revealed that in Egroup theâ…ªAP protein expression is the lowest and the Caspase-3 protein expression isthe highest of all groups (p＜0.01) . Conclusions SmacN7-O-CMC-MNP_S has obviouseffect of promoting apoptosis and inhibiting growth of the bladder cancer when combinedwith constant external magnetic fields and low dosage of MMC in vivo and can obtain thetargeting therapeutic achievements by down-regulating the mRNA and protein expressionofâ…ªAP and up-regulating the mRNA and protein expression of Caspase-3.