拟Smac新型促凋亡多肽分子对凋亡下游蛋白质组的影响及诱导凋亡的研究
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摘要
实验一拟Smac促凋亡多肽的合成及其生物活性的初步研究
目的探讨拟Smac多肽的合成及其对膀胱癌细胞的促凋亡生物活性。方法应用固相多肽合成技术，合成具有细胞膜穿透性SmacN7融合多肽，经RP-HPLC纯化、纯度分析，用质谱仪定性鉴定；通过荧光显微镜观察细胞凋亡形态、细胞增殖抑制率测定及流式细胞仪分析，研究其对低剂量丝裂霉素C诱导的膀胱癌T24细胞的凋亡促进作用。结果可穿透性融合多肽SmacN7产物峰纯度达95％以上，分子量为3278.08，质谱鉴定结果与合成预期结果完全一致；50μg／L～500μg／L SmacN7作用12h～48h，肿瘤细胞出现典型的凋亡形态学改变；随着SmacN7浓度的增加或作用时间的延长，细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后增殖抑制率分别为(9.62±1.07)％～(61.48±1.15)％、(24.17±1.02)％～(72.86±1.68)％、(43.24±1.15)％～(84.91±1.74)％：肿瘤细胞凋亡率也明显增加，分别为(6.12±1.16)％～(49.81±2.11)％、(13.47±1.15)％～(64.54±2.27)％、(28.91±1.08)％～(82.36±2.19)％。结论固相合成的拟Smac融合多肽SmacN7为高纯度的目的肽，能够稳定地转入细胞内且利用率高，并有明显促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡的生物活性，为进一步研究膀胱肿瘤的生物治疗积累了有价值的资料。
实验二人工合成拟Smac融合多肽促低剂量丝裂霉素C诱导膀胱癌细胞凋亡的研究
目的探讨人工合成拟Smac融合多肽(SmacN7)对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞凋亡的促进作用。方法运用固相多肽合成技术人工合成SmacN7细胞可穿透性融合多肽，0.05mg／ml MMC诱导的膀胱癌T24细胞与50μg／L～500μg／L的SmacN7融合多肽共同孵育4h～48h后，采用Annexin—V荧光染色检测肿瘤细胞凋亡；流式细胞术和MTT比色检测诱导后T24细胞凋亡率、增殖抑制率与SmacN7的时间和浓度效应关系。结果50μg／L～500μg／L SmacN7作用4h～48h，肿瘤细胞出现典型的凋亡形态学改变，随着SmacN7浓度的增加或药物作用时间的延长，肿瘤细胞凋亡率也增加，12h为5.67％～56.12％，24h为14.54％～65.24％，48h为31.48％～87.23％，同时细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后增殖抑制率分别为9.58％～63.42％、28.94％～72.3％、44.7％～87.12％。结论SmacN7能够有效地促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡，并具有时间和浓度依赖性，为膀胱肿瘤的生物治疗提供了新思路。
实验三人工合成拟Smac多肽增强膀胱癌细胞化疗敏感性的实验研究
目的：探讨人工合成拟Smac细胞可穿透性促凋亡融合多肽SmacN7对膀胱癌化疗药物敏感性的促进作用。方法：人工合成细胞可穿透性促凋亡融合多肽SmacN7；噻唑蓝(MTT)检测SmacN7融合多肽对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞的相对存活率；Annexin V／PI双标流式细胞术检测T24细胞的凋亡；Western blot检测SmacN7融合多肽与MMC联用后T24细胞内XIAP、Caspase-3蛋白的表达；同时检测Caspase-3活性及SmacN7融合多肽与MMC联用对T24细胞的杀伤作用。结果：SmacN7融合多肽能穿透细胞并与内源性XIAP结合，增加低剂量MMC诱导的T24细胞凋亡并呈时间和浓度依赖性；能显著降低细胞内XIAP的表达水平，增强Caspase-3的表达及活性；在24h和48h，SmacN7+MMC组与单用MMC组相比，T24细胞的存活率分别降低2.22倍和3.61倍。结论：人工合成拟Smac细胞可穿透性促凋亡融合多肽能够促进化疗药物诱导的膀胱癌T24细胞凋亡，抑制细胞增殖，增强膀胱癌细胞对MMC的化疗药物敏感性。
实验四拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及性能研究
目的探讨拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及联合恒定外磁场体外对膀胱癌细胞的凋亡促进作用。方法以羧甲基壳聚糖为骨架与具有超顺磁性的Fe_3O_4纳米粒合成纳米载体粒子，将拟Smac合成肽SmacN7与其结合，制备拟Smac合成肽羧甲基壳聚糖磁性纳米复合物，并通过透射电镜、振动样磁强计等考察其理化性质；Hoechst33258染色观察外加磁场下纳米复合物诱导膀胱癌T24细胞凋亡的形态，MTT比色分析法观察其对肿瘤细胞的生长抑制作用。结果拟Smac合成肽羧甲基壳聚糖磁性纳米粒子的粒径约为46.2nm；磁化曲线提示具有超顺磁性；载药量和包封率分别为(31.8±3.6)％和(65.2±2.4)％并具有良好的药物控释性能；外加磁场下磁性纳米粒子能使肿瘤细胞呈现明显的凋亡形态改变并表现出显著的生长抑制活性。结论拟Smac合成肽羧甲基壳聚糖磁性纳米复合物具有粒径小、较强的磁响应性、载药量和包封率高和良好的药物缓释性能，联合恒定外磁场具有明显的促进肿瘤细胞凋亡的作用，为膀胱肿瘤的生物治疗提供了新的切入点。
实验五拟Smac合成肽磁性纳米颗粒诱导膀胱癌细胞凋亡的体外实验研究
目的研究拟Smac合成肽磁性纳米颗粒(SmacN7-O-CMC-MNP_S)的制备及其协同恒定外磁场体外对人膀胱癌T24细胞的生长抑制作用并探讨其作用机制。方法氧化还原法制备SmacN7-O-CMC-MNP_S并检测其理化性质，倒置显微镜和电镜观察细胞形态，TUNEL法检测细胞凋亡，MTT、流式细胞术分别观察SmacN7-O-CMC-MNP_S联合化疗药物及恒定外磁场体外对人膀胱癌T24细胞的生长抑制作用，SABC法观察Bcl-2／Bax蛋白的表达，Western blot检测XIAP蛋白的表达。结果SmacN7-O-CMC-MNP_s磁性纳米颗粒直径46.2nm，呈球形，载药量(31.8±3.6)％，磁响应性良好。含相同浓度SmacN7的单纯SmacN7-O-CMC-MNP_S对膀胱肿瘤T24细胞的生长抑制率和凋亡率无明显影响，但协同外磁场及在化疗药物的诱导下其对肿瘤细胞的生长抑制作用明显增强；Bcl-2蛋白表达水平下调同时Bax蛋白的表达增强，外磁场组与化疗药物组差异有统计学意义(P＜0.05)，协同外磁场时Western blot检测不同时间XIAP蛋白的表达显著降低。结论SmacN7-O-CMC-MNP_S的制备不影响SmacN7的生物活性；恒定外磁场下其对肿瘤细胞的促凋亡作用明显增强，为膀胱肿瘤的生物靶向治疗奠定了实验基础。
实验六拟Smac合成肽磁性纳米颗粒在膀胱癌裸鼠移植瘤靶向治疗的体内实验和机制研究
目的研究拟Smac合成肽磁性纳米颗粒对膀胱癌裸鼠移植瘤的体内靶向治疗作用并探讨其机制。方法40只荷瘤裸鼠随机分成5组，每组8只，按不同组别(A、B、C、D、E组)给于不同治疗，各组每天治疗1次，连续1wk，磁场组在肿瘤部位施加0.8T外磁场30min。观察裸鼠的进食、活动及生长情况、测瘤体积并计算抑瘤率；治疗后32d处死动物，HE染色和电镜观察细胞结构，采用SABC法观察Bcl-2／Bax蛋白的表达，采用RT—PCR法检测各组肿瘤组织XIAPmRNA和Caspase-3mRNA的表达，Western blot法检测各组肿瘤组织XIAP和Caspase-3蛋白表达。结果治疗期间及治疗后各组裸鼠的进食、活动及生长情况未见明显异常，体重差异无统计学意义(p＞0.05)，SmacN7-O-CMC-MNP_S磁性纳米颗粒加MMC联合外加磁场组(E组)移植瘤体积增长最为缓慢且对膀胱癌移植瘤有明显抑制作用，抑瘤率为58.4％，高于SmacN7-O-CMC-MNP_S磁性纳米颗粒加MMC组(C组)(24.6％)和SmacN7-O-CMC-MNP_S磁性纳米颗粒加外加磁场组(D组)(32.2％)；免疫组化SABC法检测E组Bcl-2蛋白表达水平下调同时Bax蛋白的表达增强，与C、D组比较差异有统计学意义(p＜0.05)；RT—PCR结果示E组能显著下调XIAPmRNA的表达，同时上调Caspase-3mRNA的表达；Western blot显示E组能下调XIAP蛋白的表达同时上调Caspase-3蛋白的表达。结论在外加磁场和小剂量丝裂霉素C的共同作用下，SmacN7-O-CMC-MNP_S磁性纳米颗粒在体内具有明显的体内促进肿瘤凋亡、抑制肿瘤生长的作用，通过下调XIAP、上调Caspase-3在mRNA和蛋白方面的表达达到肿瘤靶向治疗效果，为膀胱肿瘤的治疗探索出一条新的途径。
Part 1Synthesis and Biological Activities of Smac-mimic Peptide
Objective 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 95％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)％、(24.17±1.02)％～(72.86±1.68)％、(43.24±1.15)％～(84.91±1.74)％and the percentage of apoptosisincreased by (6.12±1.16)％～(49.81±2.11)％、(13.47±1.15)％～(64.54±2.27)％、(28.91±1.08)％～(82.36±2.15)％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 MMC
Objective 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％～56.12％, 14.54％～65.24％and 31.48％～87.23％when treated for12h、24h、48h respectively. The proliferation inhibitory ratio of the cancer cells increased notably by 9.58％～63.42％,28.94％～72.3％and 44.7％～87.12％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 Peptide
Objective: 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 ofⅪAP 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 endogenousⅪAP 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 ofⅪAP 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 Nanoparticles
Objective 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 Fe_3O_4 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 )％and (65.2±2.4)％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 Cells
Objective 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 ofⅪAP 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 )％. 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 ofⅪAP 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 vivo
Objective 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 ofⅪAPmRNA and Caspase-3mRNA was measured by RT-PCR;Protein expression level ofⅪAP 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 58.4％, which was remarkably higherthan those of the C group(24.6％)and D group(32.2％); 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 ofⅪAP 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 theⅪAP 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 expressionofⅪAP and up-regulating the mRNA and protein expression of Caspase-3.
目的探讨拟Smac多肽的合成及其对膀胱癌细胞的促凋亡生物活性。方法应用固相多肽合成技术，合成具有细胞膜穿透性SmacN7融合多肽，经RP-HPLC纯化、纯度分析，用质谱仪定性鉴定；通过荧光显微镜观察细胞凋亡形态、细胞增殖抑制率测定及流式细胞仪分析，研究其对低剂量丝裂霉素C诱导的膀胱癌T24细胞的凋亡促进作用。结果可穿透性融合多肽SmacN7产物峰纯度达95％以上，分子量为3278.08，质谱鉴定结果与合成预期结果完全一致；50μg／L～500μg／L SmacN7作用12h～48h，肿瘤细胞出现典型的凋亡形态学改变；随着SmacN7浓度的增加或作用时间的延长，细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后增殖抑制率分别为(9.62±1.07)％～(61.48±1.15)％、(24.17±1.02)％～(72.86±1.68)％、(43.24±1.15)％～(84.91±1.74)％：肿瘤细胞凋亡率也明显增加，分别为(6.12±1.16)％～(49.81±2.11)％、(13.47±1.15)％～(64.54±2.27)％、(28.91±1.08)％～(82.36±2.19)％。结论固相合成的拟Smac融合多肽SmacN7为高纯度的目的肽，能够稳定地转入细胞内且利用率高，并有明显促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡的生物活性，为进一步研究膀胱肿瘤的生物治疗积累了有价值的资料。
实验二人工合成拟Smac融合多肽促低剂量丝裂霉素C诱导膀胱癌细胞凋亡的研究
目的探讨人工合成拟Smac融合多肽(SmacN7)对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞凋亡的促进作用。方法运用固相多肽合成技术人工合成SmacN7细胞可穿透性融合多肽，0.05mg／ml MMC诱导的膀胱癌T24细胞与50μg／L～500μg／L的SmacN7融合多肽共同孵育4h～48h后，采用Annexin—V荧光染色检测肿瘤细胞凋亡；流式细胞术和MTT比色检测诱导后T24细胞凋亡率、增殖抑制率与SmacN7的时间和浓度效应关系。结果50μg／L～500μg／L SmacN7作用4h～48h，肿瘤细胞出现典型的凋亡形态学改变，随着SmacN7浓度的增加或药物作用时间的延长，肿瘤细胞凋亡率也增加，12h为5.67％～56.12％，24h为14.54％～65.24％，48h为31.48％～87.23％，同时细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后增殖抑制率分别为9.58％～63.42％、28.94％～72.3％、44.7％～87.12％。结论SmacN7能够有效地促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡，并具有时间和浓度依赖性，为膀胱肿瘤的生物治疗提供了新思路。
实验三人工合成拟Smac多肽增强膀胱癌细胞化疗敏感性的实验研究
目的：探讨人工合成拟Smac细胞可穿透性促凋亡融合多肽SmacN7对膀胱癌化疗药物敏感性的促进作用。方法：人工合成细胞可穿透性促凋亡融合多肽SmacN7；噻唑蓝(MTT)检测SmacN7融合多肽对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞的相对存活率；Annexin V／PI双标流式细胞术检测T24细胞的凋亡；Western blot检测SmacN7融合多肽与MMC联用后T24细胞内XIAP、Caspase-3蛋白的表达；同时检测Caspase-3活性及SmacN7融合多肽与MMC联用对T24细胞的杀伤作用。结果：SmacN7融合多肽能穿透细胞并与内源性XIAP结合，增加低剂量MMC诱导的T24细胞凋亡并呈时间和浓度依赖性；能显著降低细胞内XIAP的表达水平，增强Caspase-3的表达及活性；在24h和48h，SmacN7+MMC组与单用MMC组相比，T24细胞的存活率分别降低2.22倍和3.61倍。结论：人工合成拟Smac细胞可穿透性促凋亡融合多肽能够促进化疗药物诱导的膀胱癌T24细胞凋亡，抑制细胞增殖，增强膀胱癌细胞对MMC的化疗药物敏感性。
实验四拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及性能研究
目的探讨拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及联合恒定外磁场体外对膀胱癌细胞的凋亡促进作用。方法以羧甲基壳聚糖为骨架与具有超顺磁性的Fe_3O_4纳米粒合成纳米载体粒子，将拟Smac合成肽SmacN7与其结合，制备拟Smac合成肽羧甲基壳聚糖磁性纳米复合物，并通过透射电镜、振动样磁强计等考察其理化性质；Hoechst33258染色观察外加磁场下纳米复合物诱导膀胱癌T24细胞凋亡的形态，MTT比色分析法观察其对肿瘤细胞的生长抑制作用。结果拟Smac合成肽羧甲基壳聚糖磁性纳米粒子的粒径约为46.2nm；磁化曲线提示具有超顺磁性；载药量和包封率分别为(31.8±3.6)％和(65.2±2.4)％并具有良好的药物控释性能；外加磁场下磁性纳米粒子能使肿瘤细胞呈现明显的凋亡形态改变并表现出显著的生长抑制活性。结论拟Smac合成肽羧甲基壳聚糖磁性纳米复合物具有粒径小、较强的磁响应性、载药量和包封率高和良好的药物缓释性能，联合恒定外磁场具有明显的促进肿瘤细胞凋亡的作用，为膀胱肿瘤的生物治疗提供了新的切入点。
实验五拟Smac合成肽磁性纳米颗粒诱导膀胱癌细胞凋亡的体外实验研究
目的研究拟Smac合成肽磁性纳米颗粒(SmacN7-O-CMC-MNP_S)的制备及其协同恒定外磁场体外对人膀胱癌T24细胞的生长抑制作用并探讨其作用机制。方法氧化还原法制备SmacN7-O-CMC-MNP_S并检测其理化性质，倒置显微镜和电镜观察细胞形态，TUNEL法检测细胞凋亡，MTT、流式细胞术分别观察SmacN7-O-CMC-MNP_S联合化疗药物及恒定外磁场体外对人膀胱癌T24细胞的生长抑制作用，SABC法观察Bcl-2／Bax蛋白的表达，Western blot检测XIAP蛋白的表达。结果SmacN7-O-CMC-MNP_s磁性纳米颗粒直径46.2nm，呈球形，载药量(31.8±3.6)％，磁响应性良好。含相同浓度SmacN7的单纯SmacN7-O-CMC-MNP_S对膀胱肿瘤T24细胞的生长抑制率和凋亡率无明显影响，但协同外磁场及在化疗药物的诱导下其对肿瘤细胞的生长抑制作用明显增强；Bcl-2蛋白表达水平下调同时Bax蛋白的表达增强，外磁场组与化疗药物组差异有统计学意义(P＜0.05)，协同外磁场时Western blot检测不同时间XIAP蛋白的表达显著降低。结论SmacN7-O-CMC-MNP_S的制备不影响SmacN7的生物活性；恒定外磁场下其对肿瘤细胞的促凋亡作用明显增强，为膀胱肿瘤的生物靶向治疗奠定了实验基础。
实验六拟Smac合成肽磁性纳米颗粒在膀胱癌裸鼠移植瘤靶向治疗的体内实验和机制研究
目的研究拟Smac合成肽磁性纳米颗粒对膀胱癌裸鼠移植瘤的体内靶向治疗作用并探讨其机制。方法40只荷瘤裸鼠随机分成5组，每组8只，按不同组别(A、B、C、D、E组)给于不同治疗，各组每天治疗1次，连续1wk，磁场组在肿瘤部位施加0.8T外磁场30min。观察裸鼠的进食、活动及生长情况、测瘤体积并计算抑瘤率；治疗后32d处死动物，HE染色和电镜观察细胞结构，采用SABC法观察Bcl-2／Bax蛋白的表达，采用RT—PCR法检测各组肿瘤组织XIAPmRNA和Caspase-3mRNA的表达，Western blot法检测各组肿瘤组织XIAP和Caspase-3蛋白表达。结果治疗期间及治疗后各组裸鼠的进食、活动及生长情况未见明显异常，体重差异无统计学意义(p＞0.05)，SmacN7-O-CMC-MNP_S磁性纳米颗粒加MMC联合外加磁场组(E组)移植瘤体积增长最为缓慢且对膀胱癌移植瘤有明显抑制作用，抑瘤率为58.4％，高于SmacN7-O-CMC-MNP_S磁性纳米颗粒加MMC组(C组)(24.6％)和SmacN7-O-CMC-MNP_S磁性纳米颗粒加外加磁场组(D组)(32.2％)；免疫组化SABC法检测E组Bcl-2蛋白表达水平下调同时Bax蛋白的表达增强，与C、D组比较差异有统计学意义(p＜0.05)；RT—PCR结果示E组能显著下调XIAPmRNA的表达，同时上调Caspase-3mRNA的表达；Western blot显示E组能下调XIAP蛋白的表达同时上调Caspase-3蛋白的表达。结论在外加磁场和小剂量丝裂霉素C的共同作用下，SmacN7-O-CMC-MNP_S磁性纳米颗粒在体内具有明显的体内促进肿瘤凋亡、抑制肿瘤生长的作用，通过下调XIAP、上调Caspase-3在mRNA和蛋白方面的表达达到肿瘤靶向治疗效果，为膀胱肿瘤的治疗探索出一条新的途径。
Part 1Synthesis and Biological Activities of Smac-mimic Peptide
Objective 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 95％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)％、(24.17±1.02)％～(72.86±1.68)％、(43.24±1.15)％～(84.91±1.74)％and the percentage of apoptosisincreased by (6.12±1.16)％～(49.81±2.11)％、(13.47±1.15)％～(64.54±2.27)％、(28.91±1.08)％～(82.36±2.15)％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 MMC
Objective 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％～56.12％, 14.54％～65.24％and 31.48％～87.23％when treated for12h、24h、48h respectively. The proliferation inhibitory ratio of the cancer cells increased notably by 9.58％～63.42％,28.94％～72.3％and 44.7％～87.12％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 Peptide
Objective: 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 ofⅪAP 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 endogenousⅪAP 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 ofⅪAP 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 Nanoparticles
Objective 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 Fe_3O_4 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 )％and (65.2±2.4)％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 Cells
Objective 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 ofⅪAP 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 )％. 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 ofⅪAP 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 vivo
Objective 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 ofⅪAPmRNA and Caspase-3mRNA was measured by RT-PCR;Protein expression level ofⅪAP 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 58.4％, which was remarkably higherthan those of the C group(24.6％)and D group(32.2％); 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 ofⅪAP 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 theⅪAP 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 expressionofⅪAP and up-regulating the mRNA and protein expression of Caspase-3.
引文
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8. Guo F, Nimmanapali R, Paranawthana S, et al. Ectopic overexpression of second mitochondria- derived activator of caspase (Smac/DIABLO) or cotreatment with N-terminals of Smac/DIABLO peptide potentiates epothilone B derivative (BMS 247550) and Apo-2L/TRAIL-induced apoptosiss[J]. Blood, 2002,99(9):3419-3426.
9. Nikolovska-Coleska Z,Wang RX,Fang XL, et al.Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization [J]. Analytical Biochemistry, 2004, 332:261-273.
10. Kipp RA, Case MA, Wist AD, et al. Molecular targeting of inhibitor of apoptosis proteins based on small molecule mimics of natural binding partners [J]. Biochemistry,2002, 41: 7344-7349.
11. Console S, Marty C, Garci'a-Echevern'a C et al. Antennapedia and HIV Transactivator of Transcription (TAT) "Protein Transduction Domains" Promote Endocytosis of High Molecular Weight Cargo upon Binding to Cell Surface Glycosaminoglycans [J]. Biol Chem, 2003, 278(37):35109-35114.
12. Thoren P E, Persson D, Karlsson M, et al. The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J]. FEBS Lett, 2000,482(3):265-268.
13. Lubbe AS, Alexin C, Bergemann C. Clinical application of magnetic drug targeting [J].J Surg Res,2001, 95: 200-206.
[1] Shi Y.A conserved tetrapeptide motif: potentiating apoptosis through IAP - binding [J].Cell Death and Differ, 2002, 9(2):93-95.
[2] Hasenjager A, Gillisen B, Muller A, et al.Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xl in a strictly caspase-3-dependent manner in human carcinoma cells [J].Oncogene, 2004, 23(26):4523-4535.
[3] Thoren PE, Persson D, Karlsson M, et al.The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J].FEBS Lett, 2000, 482(3):265-268.
[4] Liu Z, Sun C, Olejniczak ET ,et al.Structure basis for binding of Smac/DIABLO to XIAP BIR3 domain[J].Nature, 2000, 408(6835): 1004-1008.
[5] Shi Y.Mechanisms of caspase activation and inhibition during apoptosis [J].Mol Cell, 2002, 9(3):459-470.
[6] Chai J, Du C, Wu J, et al.Structure and biochemical basis of apoptotic activation by Smac/DIABLO [J].Nature, 2000, 406(6798):855-862.
[7] Vucic D, Deshayes K, Ackerly H, et al.Smac Negatively Regulates the Anti-Apoptotic Activity of Melanoma Inhibitor of Apoptosis (ML-IAP)[J].J Biol Chem, 2002,277(14): 12275-12279.
[8] Mizukawa K, Kawamura A, Sasayama T, et al.Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines [J].J Neuro Oncol, 2006, 77(3): 247-255.
[9] Yang L, Mashima T, Sato S, et al.Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide [J]. Cancer Res, 2003,63(4):831-837.
[10] Arnt CR, Chiorean MV, Heldebrant MP, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP_1 in situ [J].J Biol Chem, 2002,277(46):44236-44243.
1.Liu Z, Sun C, Olejniczak ET, et al.Structural basis for binding ofSmac / DIABLO to the XIAPBIR3 domain[J].Nature, 2000, 408(6815): 1004 -1008.
2.Hasenjager A, Gillisen B, Muller A, et al.Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xl in a strictly caspase-3-dependent manner in human carcinoma cells [J].Oncogene, 2004, 23(26):4523-4535.
3.Thoren P E, Persson D, Karlsson M, et al.The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J].FEBS Lett, 2000, 482(3):265-268.
4.Arnt C R, Chiorean M V, Heldebrant M P, et al.Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP_1 in situ [J].J Biol Chem, 2002, 277(46):44236-44243.
5.Li L, Thomas RM, Suzuki H, et al.A small molecular Smac mimic potentiates TRAIL-and TNFa-mediated cell death [J].Science, 2004, 305(5689): 1471-1474.
6．符伟军，刘佳，郭丽，等．低剂量丝裂霉素C短时加药诱导膀胱癌细胞凋亡的研究[J]．中华实验外科杂志，1999，16(1)：16-17．
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2. Hickman JA. Apoptosis induced by anticancer drugs [J]. Cancer Metestasis Rev, 1992,11:121-139.
3. Shi Y. Mechanisms of caspase activation and inhibition during apoptosis [J]. Mol Cell,2002, 9:459-470.
4. Vucic D, Deshayes K, Ackerly H, et al. SMAC negatively regulates the anti-apoptic activity of melanoma of apoptosis (ML-IAP) [J]. J Biol Chem, 2002,277:12275-12279.
5. Liu Z, Sun C, Olejniczak ET, et al.Structural basis for binding of Smac / DIABLO to the XIAP BIR3 domain [J]. Nature, 2000, 408: 1004-1008.
6. Korga A, Korobowicz E, Dudka J. Role of mitochondrial protein Smac/Diablo in regulation of apoptotic pathways [J]. Pol Merkur Lekarski, 2006,20: 573-576.
7. Chauhan D, Neri P, Velankar M, et al. Targeting mitochondrial factor Smac/DIABLO as therapy for multiple myeloma (MM) [J].Blood, 2007, 109: 1220-1227.
8. Glover CJ, Hite K,DeLosh R. A high-throughout screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay [J]. Analytic Biochemistry, 2003, 320:157-169.
9. Fulda S, Wick W, Weller M, et al. Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo [J]. Nat Med, 2002, 8:808-815.
10. Yang L, Mashima T, Sato S, et al. Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide [J]. Cancer Res, 2003, 63:831-837.
11. Thoren PE, Persson D, Karlsson M, et al. The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J]. FEBS Lett, 2000,482:265-268.
12. Mizukawa K, Kawamura A, Sasayama T, et al. Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines [J]. J Neurooncol, 2006, 77: 247-55.
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6. Gao ZH, Tian Y, Wang JR.,et al. A dimeric Smac/Diablo peptide directly relieves Caspase-3 inhibition by XIAP[J]. Biol Chem, 2007, 282 (42): 30718-30727.
7. Wang XD. The expanding role of mitochondria in apoptosis [J]. Genes & Dev, 2001,15: 2922-2933.
8. Guo F, Nimmanapali R, Paranawthana S, et al. Ectopic overexpression of second mitochondria- derived activator of caspase (Smac/DIABLO) or cotreatment with N-terminals of Smac/DIABLO peptide potentiates epothilone B derivative (BMS 247550) and Apo-2L/TRAIL-induced apoptosiss[J]. Blood, 2002,99(9):3419-3426.
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[1] Shi Y.A conserved tetrapeptide motif: potentiating apoptosis through IAP - binding [J].Cell Death and Differ, 2002, 9(2):93-95.
[2] Hasenjager A, Gillisen B, Muller A, et al.Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xl in a strictly caspase-3-dependent manner in human carcinoma cells [J].Oncogene, 2004, 23(26):4523-4535.
[3] Thoren PE, Persson D, Karlsson M, et al.The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J].FEBS Lett, 2000, 482(3):265-268.
[4] Liu Z, Sun C, Olejniczak ET ,et al.Structure basis for binding of Smac/DIABLO to XIAP BIR3 domain[J].Nature, 2000, 408(6835): 1004-1008.
[5] Shi Y.Mechanisms of caspase activation and inhibition during apoptosis [J].Mol Cell, 2002, 9(3):459-470.
[6] Chai J, Du C, Wu J, et al.Structure and biochemical basis of apoptotic activation by Smac/DIABLO [J].Nature, 2000, 406(6798):855-862.
[7] Vucic D, Deshayes K, Ackerly H, et al.Smac Negatively Regulates the Anti-Apoptotic Activity of Melanoma Inhibitor of Apoptosis (ML-IAP)[J].J Biol Chem, 2002,277(14): 12275-12279.
[8] Mizukawa K, Kawamura A, Sasayama T, et al.Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines [J].J Neuro Oncol, 2006, 77(3): 247-255.
[9] Yang L, Mashima T, Sato S, et al.Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide [J]. Cancer Res, 2003,63(4):831-837.
[10] Arnt CR, Chiorean MV, Heldebrant MP, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP_1 in situ [J].J Biol Chem, 2002,277(46):44236-44243.
1.Liu Z, Sun C, Olejniczak ET, et al.Structural basis for binding ofSmac / DIABLO to the XIAPBIR3 domain[J].Nature, 2000, 408(6815): 1004 -1008.
2.Hasenjager A, Gillisen B, Muller A, et al.Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xl in a strictly caspase-3-dependent manner in human carcinoma cells [J].Oncogene, 2004, 23(26):4523-4535.
3.Thoren P E, Persson D, Karlsson M, et al.The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J].FEBS Lett, 2000, 482(3):265-268.
4.Arnt C R, Chiorean M V, Heldebrant M P, et al.Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP_1 in situ [J].J Biol Chem, 2002, 277(46):44236-44243.
5.Li L, Thomas RM, Suzuki H, et al.A small molecular Smac mimic potentiates TRAIL-and TNFa-mediated cell death [J].Science, 2004, 305(5689): 1471-1474.
6．符伟军，刘佳，郭丽，等．低剂量丝裂霉素C短时加药诱导膀胱癌细胞凋亡的研究[J]．中华实验外科杂志，1999，16(1)：16-17．
1. Hasenjager A, Gillisen B, Muller A, et al. Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xl in a strictly caspase-3-dependent manner in human carcinoma cells [J] . Oncogene, 2004,23:4523-4535.
2. Hickman JA. Apoptosis induced by anticancer drugs [J]. Cancer Metestasis Rev, 1992,11:121-139.
3. Shi Y. Mechanisms of caspase activation and inhibition during apoptosis [J]. Mol Cell,2002, 9:459-470.
4. Vucic D, Deshayes K, Ackerly H, et al. SMAC negatively regulates the anti-apoptic activity of melanoma of apoptosis (ML-IAP) [J]. J Biol Chem, 2002,277:12275-12279.
5. Liu Z, Sun C, Olejniczak ET, et al.Structural basis for binding of Smac / DIABLO to the XIAP BIR3 domain [J]. Nature, 2000, 408: 1004-1008.
6. Korga A, Korobowicz E, Dudka J. Role of mitochondrial protein Smac/Diablo in regulation of apoptotic pathways [J]. Pol Merkur Lekarski, 2006,20: 573-576.
7. Chauhan D, Neri P, Velankar M, et al. Targeting mitochondrial factor Smac/DIABLO as therapy for multiple myeloma (MM) [J].Blood, 2007, 109: 1220-1227.
8. Glover CJ, Hite K,DeLosh R. A high-throughout screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay [J]. Analytic Biochemistry, 2003, 320:157-169.
9. Fulda S, Wick W, Weller M, et al. Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo [J]. Nat Med, 2002, 8:808-815.
10. Yang L, Mashima T, Sato S, et al. Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide [J]. Cancer Res, 2003, 63:831-837.
11. Thoren PE, Persson D, Karlsson M, et al. The antennapedia peptide penetratin translocates across lipid bilayers-the first direct observation [J]. FEBS Lett, 2000,482:265-268.
12. Mizukawa K, Kawamura A, Sasayama T, et al. Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines [J]. J Neurooncol, 2006, 77: 247-55.
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