Interstitial gene delivery in human xenograft prostate tumors using titanium metal seeds

Gene therapy is a promising approach for the treatment of cancers. Strategies for gene vector delivery include systemic and local-regional approaches. Intratumoral delivery of vectors has generally employed direct injections into single or multiple locations throughout the tumor volume. However, this approach leads to nonuniform distributions of reagents within tumors and becomes cumbersome as the required number of injections is increased. We have investigated the effectiveness of an interstitial plasmid gene delivery based on using tiny metallic seeds (GeneSeeds) analogous to technology used for brachytherapy. Feasibility for interstitial use of GeneSeeds was demonstrated expressing reporter plasmids (green fluorescence protein or beta-galactosidase) in human xenograft prostate tumors. Immunohistochemical analysis confirmed effective interstitial delivery, vector expression, and distributions of reporter genes within tumors. Applicability of GeneSeeds for delivery of radiosensitizing cytokines was examined by generating a cytokine [tumor necrosis factor-alpha (TNF-alpha)] expressing vector under the cytomegaloviral promoter and interstitially implanting GeneSeeds with this vector into prostate cancer tumors. TNF-alpha protein expression was observed around the ends of seeds and decreasing in an exponential gradient as a function of distance. The expression of TNF-alpha resulted in tumor growth delay of a human prostate cancer xenograft. These results demonstrate the feasibility of applying interstitial delivery of gene expressing vectors for the treatment of human cancers.     

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.     

子宫内膜腺癌的分型与PTEN、p53的关系

子宫内膜腺癌可分为雌激素依赖型和雌激素非依赖型。前者早期病变为子宫内膜不典型增生过长，可以发展为子宫内膜样癌，有50％～83％的患者FFEN表达阳性，有20％的患者p53表达阳性；后者早期病变为萎缩的子宫内膜内发生上皮内癌，可以发展为浆液性癌，有90％的患者p53( )，而PTEN(-)。证明PTEN( )的子宫内膜腺癌，多为雌激素依赖型子宫内膜样癌，预后相对较好；p53( )的子宫内膜腺癌，多为雌激素非依赖型浆液性腺癌，预后较差。因此FFEN、p53检测对诊断子宫内膜腺癌及判断预后很有帮助。     

Transferrin-facilitated lipofection gene delivery strategy: characterization of the transfection complexes and intracellular trafficking

We previously showed that mixing transferrin with a cationic liposome prior to the addition of DNA, greatly enhanced the lipofection efficiency. Here, we report characterization of the transfection complexes in formulations prepared with transferrin, lipofectin, and DNA (pCMVlacZ) in various formulations. DNA in all the formulations that contain lipofectin was resistant to DNase I treatment. Transfection experiments performed in Panc 1 cells showed that the standard formulation, which was prepared by adding DNA to a mixture of transferrin and lipofectin, yielded highest transfection efficiency. There was no apparent difference in zeta potential among these formulations, but the most efficient formulation contained complexes with a mean diameter of three to four times that of liposome and the complexes in other gene delivery formulations. Transmission electron microscopic examination of the standard transfection complexes formulated using gold-labeled transferrin showed extended circular DNA decorated with transferrin as compared to extensively condensed DNA found in lipofectin-DNA complexes and heterogeneous structures in other formulations. By confocal microscopy, DNA and transferrin were found to colocalize at the perinuclear space and in the nucleus, suggesting cotransportation intracellularly, including nuclear transport. We propose that transferrin enhances the transfection efficiency of the standard lipofection formulation by preventing DNA condensation, and facilitating endocytosis and nuclear targeting.     

Eradication of p53-mutated head and neck squamous cell carcinoma xenografts using nonviral p53 gene therapy and photochemical internalization.

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.     

PTEN、p53和Survivin表达在胃癌组织中的意义

目的探讨PTEN、p53和Survivin表达在胃癌组织中的临床意义。方法选择2010~2013年陕西省中医医院收治的60例胃癌患者的胃癌标本作为A组,选择同期于陕西省中医医院手术切除吻合口切缘处的正常胃黏膜60例作为B组,另取不典型增生胃黏膜石蜡标本65例作为C组,采用免疫组织化学链霉亲和素方法检测Survivin、PTEN和p53基因在3组中的表达情况。结果胃癌组织中的Survivin、p53基因阳性率高于其他两组,差异具有统计学意义(P0.05);Survivin基因的阳性表达和p53阳性呈正相关(P0.05),和PTEN阴性率呈负相关(P0.05);统计结果显示,3种基因在胃癌不同病理指标中的阳性表达差异有统计学意义(P0.05)。结论 Survivin、PTEN和p53基因异常表达和胃癌发生有密切的关系,其中PTEN与p53基因是判断胃癌生物学行为的重要指标,Survivin、PTEN、p53基因在胃癌发生及发展中有着相互协同的作用。     

腺病毒介导的PTEN和p16基因对前列腺癌细胞系的作用

[目的]探讨腺病毒介导的PTEN和p16基因蛋白对抑制前列腺癌细胞增殖和调控其凋亡的作用.[方法]构建携带人PTEN和p16基因的腺病毒载体,转染体外培养的前列腺癌细胞系PC-3,采用RTPCR、Westem b1ot检测目的基因的表达.通过细胞生长试验、流式细胞仪检测PC-3转染前后细胞增殖和凋亡的变化.[结果]病毒滴度 Ad-PTEN为1.8×107 pfu/ml、Ad-p16为1.2×1010 pfu/ml,RT-PCR检测可见PTEN-mRNA(462bp)和p16-mRNA(520bp)表达,Western blot检测有PTEN蛋白(60KD)和p16蛋白(16KD)特异表达,并可明显抑制PC-3细胞的增殖,诱导其凋亡,联合基因治疗组与单基因组相比差异显著.[结论]PTEN和p16可明显的抑制前列腺癌细胞株PC 3的增殖,增加细胞的凋亡,转染携带PTEN和p16的重组腺病毒载体有望成为治疗前列腺癌的有效方法.     

姜黄素对前列腺癌PC-3M细胞抗侵袭作用的研究

目的 探讨姜黄素对前列腺癌PC-3M细胞抗侵袭作用。方法 0—40μmol／L姜黄素分别处理PC-3M细胞24h后，Western blot法检测MMP-2和TIMP-2蛋白表达，免疫组化sp法检测细胞内NF-kB蛋白的表达水平。结果 姜黄素可下调MMP-2和上调TIMP-2的表达，抑制NF-kB的表达。结论 姜黄素通过下调MMP-2和上调TIMP-2的蛋白表达发挥抗侵袭作用，抑制NF-kB的表达可能是抗侵袭作用的机制之一。     

Combination of PTEN gene therapy and radiation inhibits the growth of human prostate cancer xenografts

The resistance of prostate cancers to radiation therapy has been linked to abnormalities in overexpression of Bcl-2, an oncogene associated with inhibition of apoptosis. In this study, we evaluated whether the combination of the overexpression of phosphatase and tensin homolog (PTEN), a protein known to inhibit Bcl-2 expression, and radiation therapy would inhibit proliferation of Bcl-2-expressing human prostate cancer cells inoculated into the subcutis of athymic mice. Compared with either treatment alone, the combination of adenoviral vector-expressed PTEN (AdPTEN) and radiation (5 Gy) significantly inhibited xenograft tumor growth. Median tumor size on day 48 was 1030 mm3 in untreated controls, 656 mm3 in mice treated with radiation (5 Gy) alone, 640 mm3 in mice treated with AdPTEN alone, and 253 mm3 in mice treated with the combination (p<0.001). Treatment was well tolerated in all cases. Combination treatment also enhanced apoptosis (p=0.048), inhibited cellular proliferation (p=0.005), and inhibited tumor-induced neovascularity (p=0.030). Interestingly, this treatment increased apoptosis not only in tumor cells but also in tumor-associated endothelial cells. Together, these findings indicate that AdPTEN strongly inhibits the growth of human prostate tumors, especially when combined with radiation therapy, and that this effect is mediated by the induction of apoptosis and by the inhibition of angiogenesis and cellular proliferation.     

Failure of wild-type p53 gene therapy in human cancer cells expressing a mutant p53 protein.

The introduction of exogenous wild-type p53 into human cancer cells bearing p53 mutation does not necessarily result in inhibition of tumor growth. We have demonstrated this in MDA-MB468 breast cancer cells which are hemizygous for p53 mutation and also in KM12SM colorectal carcinoma cells which are heterozygous for p53 mutation. The wtp53 transfectants decreased three- to four-fold the number of colonies compared with controls. Most wtp53-expressing cells died by apoptosis at early passages, but some cells were able to form colonies and their proliferation rate was similar to control transfectants. This reversion was observed in three of the six MDA-MB-468 clones selected. When MDA-wtp53 transfectants were implanted orthotopically in nude mice only one clone showed prolonged tumor latency. No differences were found in either tumor proliferation or apoptosis in tumors. Integration and expression of exogenous wtp53 was assessed in early and late passages in vitro, and in tumors growing in vivo. Consistently, we found mutations in the exogenous wtp53 gene of MDA-MB468 transfectants. Excision of the exogenous gene was an alternative to abrogate the wtp53 function that was extremely efficient in KM12 cells, although they maintained resistance to geneticin. These results were corroborated by the functional assay in yeast. In conclusion, wtp53 is inactivated in these cancer cells by different mechanisms. The presence of mutated p53 may confer genome instability and mutator ability, which allows cells to escape the effects of the exogenous wtp53 and contributes to the failure of wtp53 gene therapy.     

基底细胞样型浸润性乳腺癌中PTEN、p53、Ki-67的表达及意义

目的探讨基底细胞样型浸润性乳腺癌中PTEN、p53、Ki-67的表达情况,并分析其意义。方法采用免疫组织化学方法检测57例基底细胞样型浸润性乳腺癌中PTEN、p53、Ki-67的表达水平。结果基底细胞样型浸润性乳腺癌中PTEN、p53、Ki-67的阳性表达率分别是38．6％（22／57）、56．1％（32／57）、82．5％（47／57）,与对照组非特殊类型浸润性导管癌中PTEN、p53、Ki-67的表达水平相比,差异有统计学意义（P〈0．05）。PTEN阳性标本中Ki-67的阳性率（68．2％,15／22）与PTEN阴性标本中Ki-67的阳性率（91．4％,32／35）相比差异显著,统计学分析显示PTEN表达与Ki-67表达呈负相关（P〈0．05）。结论抑癌基因PTEN、p53的异常表达与基底细胞样型浸润性乳腺癌的发生和发展有关,PTEN在一定程度上可以抑制肿瘤细胞的增殖。     

三氧化二砷对前列腺癌PC-3细胞周期的阻滞作用

目的:研究三氧化二砷As2O诱导前列腺癌PC-3细胞生长抑制、周期()3阻滞的作用。方法:实验于2003-10/2004-05在解放军第四军医大学唐都医院中心实验室完成。应用体外细胞生长抑制试验(MTT比色法)研究As2O3对细胞生长的影响;流式细胞仪检测细胞周期分布的情况;Westernblotting检测细胞周期调节分子周期素依赖性激酶CDKs)、周期素依赖(性激酶抑制素(CDKI)和周期素的变化。结果:As2O抑制人前列腺癌细胞系PC-3生长具有时间、剂量依赖性,3MTT比色试验显示1,2,5μmol/L的三氧化二砷处理1～6d,各组之间吸光值差异有显著性意义(F=22.220,P<0.001);流式细胞术检测细胞周期分布见As2O(1,2,5μmol/L)处理72h后,随着浓度的增加,3聚集G1期的细胞增加犤(51.8±2.4)%～(58.8±2.3)%犦,各组之间比较,差异有显著性意义(χ2=15.846,P=0.01),说明可诱导PC-3细胞G1期阻滞;免疫印迹试验提示As2O可诱导PC-3细胞Cip1/p21和3Kip1/p27呈计量依赖性增加,而CDK2,6和周期素E,A下调。结论:As2O通过调节细胞周期调节素的表达来阻滞前列腺癌PC-3细3胞周期进程、抑制细胞生长,有必要进一步研究,为其在临床上用于治疗前列腺癌提供依据。     

锌对前列腺癌PC-3M细胞侵袭能力的影响

目的探讨锌对人前列腺癌PC-3M细胞侵袭能力的影响。方法用MIT比色法检测不同剂量锌对PC-3M细胞增殖的影响;Tramwell小室检测锌对PC-3M细胞侵袭能力的影响;RT-PCR检测锌作用24h后MMP-2、MMP-9mRNA的表达;酶谱分析检测锌作用后PC-3M细胞MMP-2、MMP-9的活性变化。结果MTT检测结果表明一定剂量的锌可抑制PC-3M细胞的生长;Transwell小室实验结果显示,锌作用下PC-3M细胞穿透Matrigel膜的细胞数减少;RT-PCR结果显示锌抑制了PC-3M细胞bIMP-2和MMP-9的表达;酶谱分析表明锌作用后PC-3M细胞MMP-2、MMP-9的活性下降。结论一定剂量的锌可降低PC-3M细胞的侵袭能力,其机制可能与抑制MMP-2、MMP-9的合成和激活有关。     

结直肠癌p53基因点突变和蛋白表达的研究

目的了解中国人结直肠癌ｐ５３基因的突变谱，探讨聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术用于研究结直肠癌中ｐ５３基因突变的可行性。方法应用ＰＣＲ－ＳＳＣＰ银染技术检测４１例结直肠癌ｐ５３基因突变，以ＡＢＣ免疫组织化学染色检测结直肠癌Ｐ５３蛋白的表达。结果３４％（１４／４１）的病例显示有ｐ５３基因突变，其中外显子５，６，７和８各有４，１，５和４例突变。２０例有Ｐ５３蛋白异常表达，阳性率为４９％。结论导致Ｐ５３蛋白异常堆积的ｐ５３基因突变是结直肠癌的一种常见的分子结构改变，可能在结直肠癌的发生和发展中起着重要的作用。ＰＣＲ－ＳＳＣＰ银染技术是一简便、快速、有效的检测基因点突变的方法     

Cox-2和PTEN/MMAC1在非小细胞肺癌中的相关性表达及其意义

目的探讨环氧化物酶-2(Cox-2)和PTEN/MMAC1在非小细胞肺癌中表达的相关性及其意义。方法应用免疫组化EnVision二步法检测50例非小细胞肺癌、30例癌旁正常肺组织中Cox-2和PTEN/MMAC1的表达。结果 50例非小细胞肺癌中Cox-2和PTEN/MMAC1阳性率分别为78%(39/50)和36%(18/50),30例癌旁正常肺组织Cox-2和PTEN/MMAC1阳性率分别为20%(6/30)和83.3%(25/30)。非小细胞肺癌组织中Cox-2阳性表达率显著高于癌旁正常肺组织,PTEN/MMAC1阳性率显著低于癌旁正常肺组织,Cox-2和PTEN/MMAC1蛋白表达呈负相关(P<0.05);Cox-2高表达和PTEN/MMAC1低表达与非小细胞肺癌临床分期、脉管浸润、淋巴结转移及患者生存期呈显著相关(P<0.05),与患者性别、年龄、肿瘤大小、组织学类型无关(P>0.05),PTEN/MMAC1与肿瘤组织分化相关(P<0.05),Cox-2与肿瘤组织分化无关(P>0.05)。结论 Cox-2和PTEN/MMAC1与非小细胞肺癌浸润转移,推测Cox-2和PTEN/MMAC1蛋白表达可共同作为非小细胞肺癌浸润、转移、预后的评估指标。     

超声造影剂介导PTEN基因影响卵巢癌侵袭的实验研究

目的 研究超声介导造影剂增效PTEN基因在卵巢癌细胞中的转染，探讨该基因抑制肿瘤细胞侵袭的机制。方法分别于接种人卵巢黏液性囊腺癌细胞（SKOV3）的6孔板中每孔加入200μl造影剂和5μl脂质体，以诊断超声剂量辐照60s，细胞转化48h后，采用重组基底膜侵袭模型，观察转染前后SKOV3细胞侵袭力的变化。结果转染PTEN基因可以明显抑制肿瘤细胞的侵袭力。结论PTEN基因可以通过抑制肿瘤细胞侵袭力而抑制卵巢肿瘤的生长；超声介导造影剂增效基因转染，可望成为临床基因治疗的新手段。