Apoptin induces apoptosis in human bladder cancer EJ and BIU-87 cells

Objective:To investigate whether apoptin is a apoptosis-inducing protein with a potential for bladder cancertherapy. Methods: We constructed a PCDNA3/Apoptin eukaryotic expression vector, and transfected this vectorinto bladder cancer cell lines BIU-87 and EJ, then observed the results by RT-PCR, transmission electronmicroscopy, MTT assay and the flow cytometry (TUNEL method). Results: PCDNA3/Apoptin successfullyinduced a high level apoptosis in both bladder cancer cell lines, compared with the controls (p<0.05). Conclusions:Apoptin can induce high level apoptosis in human bladder cancer EJ and BIU-87 cells, which suggests a potentialfor human bladder cancer therapy.     

Recombinant Apoptin gene retrovirus induces apoptosis in human breast cancer cells 435

Objective  To construct recombinant Apoptin gene (vp3) retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods   vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 (pLVP3) at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry (FCM) was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS) was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results  Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10⁸ pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control (P<0.05) and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1+H and M_3712.4+H, were statistically different between infection group and control group (P<0.05). The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group (t=4.06, P<0.01). TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference (t1=1.05, t2=0.84, P>0.05). Conclusion  The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy. Contributed equally to this work.     

Apoptin induces apoptosis in an oral cancer mouse model

Apoptin, a chicken anemia virus-derived protein, induces apoptosis in various tumor cell lines and xenografted tumors. Its apoptotic activity is not hampered by tumor-suppressor p53 mutations or overexpression of anti-apoptosis proteins Bcl-2 or Bcl-x(L). We report for the first time the effects of apoptin expression in primary oral tumors, induced by the carcinogen 4-Nitroquinoline- 1-oxide in immunocompetent mice. In vivo a significant amount of primary oral tumor cells expressing apoptin cells underwent apoptosis, whereas synthesis of the LacZ control product did not. Ectopical expression of apoptin in passage 1 cell cultures derived from these oral tumors also resulted in apoptin-induced. Both in-vivo and in-vitro treated cells underwent apoptosis via the activation of caspase-3. The fact that apoptin induces apoptosis in primary squamous cell carcinoma cells indicates that apoptin is a potential therapeutic agent for treatment of head and neck squamous cell carcinoma.     

Gelsolin gene therapy by retrovirus producer cells for human bladder cancer in nude mice.

Gelsolin, a regulator of the actin cytoskeleton, has been shown previously to act as a tumor suppressor in vitro and in vivo when introduced into certain cancer cell lines. To investigate the in vivo efficacy of gene therapy with the gelsolin gene, we inoculated nude mice with human urinary bladder cancer cells (UMUC-2 or DAB-1) and tested the effects of adding either retroviral DNA constructs containing gelsolin cDNA or retrovirus producer cells that produce the same retroviral constructs at high levels. The addition of retroviral gelsolin cDNA constructs did not inhibit tumor growth; however, this form of gene therapy, in which retrovirus producer cells were introduced, resulted in marked and reproducible tumor growth inhibition and prolonged survival time in the majority of animals tested. These findings demonstrate the potential for treating human urinary bladder carcinomas with the gelsolin gene.     

Differential expression of tumor-associated antigens in human colon carcinomas xenografted into nude mice

The tumor-associated antigen profile of a number of different human colon carcinomas xenografted into inbred Swiss nude mice was examined to determine whether the tumors could serve as useful models for antigen purification, radioimmunodetection, and immunotherapy. Extreme heterogeneity was observed both by radioimmunoassay and immunohistochemical procedures for the expression of carcinoembryonic antigen (CEA), colon-specific antigens (CSAp), and colonic mucin antigen (CMA) within the tumors. Four of 10 tumors (DLD-2, DLD-3, DLD-5, and HCT-10) were high producers of CEA (greater than 75 micrograms/g wet tissue wt). Two of these (DLD-2 and HCT-10) correlated with a high production of CSAp and CMA, whereas the other 2 produced low quantities of these antigens. Another tumor, HCT-14-OM1, produced large amounts of CMA yet produced moderate amounts of CEA and low quantities of CSAp. Immunohistochemical analyses of CEA gave a mostly diffuse cellular and cell surface staining for all of the tumors. Staining for CSAp was very focal, as in DLD-5 where only a few of the tumor cells were stained. Staining of CMA was limited; however, DLD-2, HCT-10, and HCT-14-OM1 showed intense cystoplasmic and intraluminal staining. A determination of the tumor antigen profile may be useful in characterization and classification of the tumor as well as enabling the selection of the proper antibody or antibodies for immunodetection and immunotherapy.     

载双歧杆菌胞外多糖纳米粒子诱导人胃癌裸鼠移植瘤细胞的凋亡

目的: 制备载双歧杆菌胞外多糖纳米粒子（Bifidobacterium exopolysaccharideloaded nanoparticles，B.EPSNPs)，探讨B.EPSNPs对人胃癌细胞裸鼠移植瘤增殖和凋亡的影响。方法：取Fe3O4纳米粒子和B.EPS，采用乳化高温固化法制备B.EPSNPs。建立人源胃癌细胞(BGC823)裸鼠移植瘤模型，接种6 d后随机分为5组，分别以生理盐水、NPs、环磷酰胺（cytoxan,CTX）、游离B.EPS及B.EPSNPs进行灌胃治疗。观察各组瘤体的生长情况，TUNEL法检测细胞凋亡，透射电镜观察细胞超微结构，免疫组化法检测增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)及凋亡调控基因bcl2、bax蛋白表达水平。结果：空载NPs和B.EPSNPs平均粒径分别为（560±21）nm、（960±32）nm，B.EPSNPs载药率为30％。B.EPSNPs对移植瘤的抑瘤率为（46.4±2.94）%，高于游离B.EPS组的(32.0±1.62)％和NPs组的(4.9±0.98)％。B.EPSNPs组移植瘤细胞凋亡指数明显高于B.EPS组\[（66.8±5.58）％ vs （41.3±4.26）％\]（P<0 .01)。透射电镜观察到B.EPSNPs治疗后的移植瘤细胞核染色质凝集成块状，出现了典型的凋亡特征。与B.EPS相比，B.EPSNPs组PCNA及bcl2的表达水平有所降低（P<0.01），bax的表达水平有所提高（P<0 .01）。结论：纳米粒子运载B.EPS增强了后者对胃癌细胞(BGC823)移植瘤的增殖抑制作用和促凋亡作用，提高了B.EPS的抗肿瘤活性。      

Induction of apoptosis in tumor-associated endothelial cells and therapy of orthotopic human pancreatic carcinoma in nude mice

Although gemcitabine has been accepted as the first-line chemotherapeutic reagent for advanced pancreatic cancer, improvement of response rate and survival is not sufficient and patients often develop resistance. We hypothesized that the inhibition of phosphorylation of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) on tumor cells and tumor-associated endothelial cells, combined with gemcitabine, would overcome the resistance to gemcitabine in orthotopic pancreatic tumor animal model. L3.6pl, human pancreatic cancer cells growing in the pancreas, and tumor-associated endothelial cells in microorgan environment highly expressed phosphorylated EGFR, VEGFR, and Akt, which regulates antiapoptotic mechanism. Oral administration of AEE788 (dual tyrosine kinase inhibitor against EGFR and VEGFR) inhibited the phosphorylation of EGFR, VEGFR, and Akt on tumor-associated endothelial cells as well as tumor cells. Although intraperitoneal (i.p.) injection of gemcitabine showed limited inhibitory effect on tumor growth, combination with AEE788 and gemcitabine produced nearly 95% inhibition of tumor growth in parallel with a high level of apoptosis on tumor cells and tumor-associated endothelial cells, and decreased microvascular density and proliferation rate. Collectively, these data indicate that dual inhibition of phosphorylation of EGFR and VEGFR, in combination with gemcitabine, produces apoptosis of tumor-associated endothelial cells and significantly suppresses human pancreatic cancer in nude mice.     

Retinoid receptor mRNA expression profiles in human bladder cancer specimens

Retinoids, which include vitamin A (retinol) and its derivatives, have previously been investigated as potential chemopreventive and chemotherapeutic agents in bladder cancer. We examined mRNA expression of the retinoid receptors RARalpha, RARbeta2, RARgamma and RXRalpha, as well as two putative RARbeta2 target genes, DAB2 and Midkine, in normal and malignant bladder tissue specimens from human patients. We evaluated 24 normal and malignant bladder specimens for retinoid receptor, DAB2 and Midkine mRNA expression using RT-PCR. We also examined the effects of retinoic acid and retinol on the expression of these genes in five human bladder cancer cell lines. Expression of RARalpha, RARbeta2, RARgamma and RXRalpha mRNA was detected in all of the non-neoplastic patient bladder specimens. RARbeta2 mRNA expression was undetectable in 7/13 tumors, RARalpha in 3/13, RARgamma in 1/13 and RXRalpha in 2/13. DAB2 mRNA was expressed in all non-neoplastic and all tumor specimens, while Midkine mRNA was detected in 8/11 non-neoplastic specimens versus 11/13 tumors. Two of the five bladder cancer cell lines expressed RARbeta2 independent of retinoid exposure; in three cell lines RARbeta2 expression was induced by retinoids. RARalpha, RARgamma and RXRalpha mRNA expression was detected in 5/5 cell lines, independent of retinoid exposure. We found a reduction in retinoid receptor mRNA expression, particularly for RARbeta2, in human bladder cancer specimens. We also demonstrated induction of RARbeta2 mRNA expression in some of the retinoid-treated bladder cancer cell lines. We suggest that restoration of RARbeta2 expression may be a reasonable biomarker for developing bladder cancer preventive and/or therapeutic drugs.     

转染外源p53基因在荷瘤裸鼠人肠癌细胞中表达的检测

采用直接注射外源ＤＮＡ－脂质体复合物的方法分别在荷瘤裸鼠人肠癌组织中转染外源正义ｐ５３ｃＤＮＡ和反义ｐ５３ｃＤＮＡ的重组表达质粒及非整合型哺乳动物表达载体ｐＲＥＰ９，并以注射等量生理盐水为对照，然后应用链菌素亲生物蛋白－过氧化酶免疫组化法检测肿瘤组织中突变型ｐ５３蛋白的表达。结果表明：体外培养ＳＷ１１１６细胞中约有５０％为突变型ｐ５３蛋白阳性；转染反义ｐ５３ｃＤＮＡ表达质粒组的裸鼠移植瘤细胞ｐ５３蛋白阳性率为２５％，而注射生理盐水、转染ｐＲＥＰ９及正义ｐ５３ｃＤＮＡ表达质粒组阳性率分别为６６．７％，７７．８％，６６．７％；前者与后三者之间有显著住差异（Ｐ＜０．０１，Ｘ［２］检验），但各组间肿瘤大小并无显著性差异。据此认为，肿瘤体内直接转染外源反义ｐ５３基因能封闭肠癌ＳＷ１１１６细胞中突变型ｐ５３基因的表达。     

Apoptin gene transfer via modified wheat histone H4 facilitates apoptosis of human ovarian cancer cells

Nonviral approaches have been used extensively for intracellular gene transfer and gene therapy. A modified wheat histone H4 protein, H4TL (H4-TAT-LHRH), as a protein-based gene delivery vector that was able to form stable complexes with plasmid DNA and increase gene delivery efficiency has been described previously. In this study, H4TL has been used to deliver apoptin gene into a human ovarian carcinoma cell line HO8910. After transfection, increased expression of apoptin at both mRNA and protein levels was detected in HO8910 cells, accompanied by reduced rate of growth of HO8910 cells in vitro and the loss of mitochondrial membrane potential in these cells. These data demonstrate that H4TL-mediated transfer of apoptin initiates mitochondrial death pathway in ovarian cancer cells and suggest a novel therapeutic strategy for cancer.     

Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells

Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms – a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte–macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12. © 1999 Cancer Research Campaign     

MIA PaCa-Ⅱ胰腺癌细胞系神经转移动物模型建立的实验研究

目的建立MIA PaCa-Ⅱ人胰腺癌细胞系裸鼠胰腺原位移植瘤模型并观察神经侵袭情况。方法将MIA PaCa-Ⅱ细胞行裸鼠背部皮下接种,建立高转移特性胰腺癌裸鼠皮下移植瘤模型,然后将高转移性移植瘤组织接种于裸鼠胰腺被膜下,分别于4周、6周和8周处死裸鼠行移植瘤组织解剖和病理学检查,测量移植瘤大小和重量,并用HE和银染方法观察神经侵袭情况。结果MIA PaCa-Ⅱ细胞裸鼠皮下接种成瘤率为100.0%（10/10）,皮下移植瘤呈局限性生长,无脏器和神经转移。原位移植4周、6周和8周后,胰腺癌原位移植瘤成瘤率均为100.0%（10/10）,神经转移率分别为50.0%（5/10）、80.0%（8/10）和60.0%（6/10）,并可见多个脏器转移,以肝脏、肝门淋巴结、胃窦转移和腹膜播散多见。结论人胰腺癌细胞系MIA PaCa-Ⅱ裸鼠胰腺原位移植瘤模型为一较理想的＂拟人＂神经浸润转移模型,可用于胰腺癌体内嗜神经性机制的研究。     

人类胃癌裸鼠淋巴结转移模型的建立

 目的　研究胃癌淋巴结转移动物模型的建立方法。方法　人类胃癌低分化细胞系SGC-7901体外培养、传代并扩增后，收集细胞行皮下种植成瘤，鼠间传代至第6代，以皮下肿瘤组织块原位种植于裸鼠胃壁建立动物模型。种植后第9周处死裸鼠，观察原位种植瘤生长、淋巴结转移及其他脏器转移情况，测定荷瘤裸鼠血清癌胚抗原（CEA）值。结果　原位移植瘤种植成功率100 ％，胃周淋巴结转移率93.3 ％，移植瘤可发生局部浸润及远处脏器转移，荷瘤裸鼠CEA值明显高于正常裸鼠（P＜0.01）。结论　应用SGC-7901细胞系可成功建立胃癌的淋巴结转移动物模型。     

流式细胞术检测肿瘤相关蛋白在人卵巢上皮性肿瘤裸鼠冻融卵巢组织移植中的意义

目的 通过对人卵巢上皮性肿瘤裸鼠原位移植瘤的癌旁组织相关蛋白的检测,探讨癌旁正常卵巢组织筛选的标准及冻融卵巢组织移植的可行性.方法 将人卵巢上皮性肿瘤OVCAR3细胞于裸小鼠颈背部近腋窝处皮下种植获取瘤源,行卵巢原位移植后,取癌组织、癌旁近端组织、癌旁中段组织、癌旁远端组织及正常裸鼠卵巢组织,采用流式细胞术检测各组织中的细胞角蛋白-7（CK-7）、CA125、p53、Survivin、基质金属蛋白酶-2（MMP-2）、金属蛋白酶组织抑制物-2（T1MP-2）的表达量;分别取全部指标阴性组、CK-7（-）CA125（-）Survivin（-）组、CK-7（＋）CA125（＋）Survivin（＋）组的组织、癌组织和冻融正常裸鼠卵巢组织进行移植,分析各组移植后的癌变率及CA125水平变化,同时检测原位移植后不同病变程度的裸鼠血清CA125水平.结果 从52份人卵巢上皮性肿瘤裸鼠原位移植瘤模型中获取46份（88.5％）活组织检查正常的癌旁残余卵巢组织;仅卵巢种植裸鼠血清CA125水平高于正常裸鼠（P＜0.01）,低于卵巢外种植裸鼠（P＜0.05）.原位移植瘤组织中CK-7、CA125、p53、Survivin、MMP-2及TIMP-2的表达率分别为93.3 ％（28/30）、93.3 ％（28/30）、86.7％（26/30）、86.7％（26/30）、83.3％（25/30）、83.3％（25/30）,癌旁近端组织的表达率分别为70.0％（21/30）、70.0％（21/30）、63.3％（19/30）、63.3％（19/30）、60.0％（18/30）、56.7％（17/30）,癌旁中段组织的表达率分别为33.3％（10/30）、30.0％（9/30）、30.0％（9/30）、30.0％（9/30）、26.7％（8/30）、23.3％（7/30）,癌旁远端组织的表达率分别为26.7％（8/30）、23.3％（7/30）、26.7％（8/30）、26.7 ％（8/30）、30.0％（9/30）、30.0％（9/30）;20份原位移植正常卵巢组织上述指标均为阴性.癌旁近端组织中CK-7、CA125、p53、Survivin、MMP-2及TIMP-2的表达率低于癌组织（P＜0.05）,高于癌旁中段组织及癌旁远端组织（P＜0.01）;癌旁中段组织及癌旁远端组织表达率差异无统计学意义（P＞0.05）.癌旁组织CK-7、CA125、Survivin的强阳性表达率明显高于p53、MMP-2、TIMP-2（P＜ 0.05）.全部指标表达阴性组或CK-7（-）CA125（-）Survivin（-）组的组织移植后,未发现癌变,其癌变率及CA125水平低于CK-7（＋）CA125（＋）Survivin（＋）组（P＜0.01,P＜0.05）.结论 CK-7、CA125、p53、Survivin、MMP-2及TIMP-2等分子指标的表达向无癌方向呈递减趋势,这些肿瘤相关基因表达全部阴性或主要指标CK-7、CA125、Survivin阴性均可作为筛选癌旁残余正常卵巢组织的标准,流式细胞术可作为筛查残留癌灶及隐匿转移的有效方法;人卵巢上皮性肿瘤裸鼠冻融卵巢组织移植安全、可行.     

表没食子儿茶素没食子酸酯诱导人胃癌细胞裸鼠移植瘤凋亡的研究

背景与目的探讨绿茶提取物表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)诱导人胃癌细胞裸鼠移植瘤凋亡及分子机制。方法建立人胃腺癌(SGC7901)细胞裸鼠异种移植瘤模型,用不同剂量的EGCG进行治疗,并设对照,用流式细胞分析术检测肿瘤组织中细胞凋亡情况,免疫组织化学方法检测肿瘤组织的凋亡相关基因Bcl-2、Bax、Caspase-3的表达情况。结果裸鼠异种移植瘤治疗实验结果显示,EGCG对移植瘤生长有明显抑制作用(其中20mg/kg、EGCG抑制率54.64%与对照组比较P<0.05);PI染色FCM分析发现,EGCG20mg/kg能诱导移植瘤细胞凋亡率17.2%与对照组比P<0.05;SP免疫组织化学结果表明EGCG可上调移植瘤细胞中Bax、Caspase-3蛋白的表达,下调Bcl-2蛋白的表达。结论表没食子儿茶素没食子酸酯(EGCG)具有体内诱导人胃腺癌细胞凋亡的作用,其机制可能与Bcl-2/Bax比值降低导致Caspase-3的活化从而启动细胞凋亡有关。     

Radioimmunoimaging of human bladder tumor xenografts in nude mice by using monoclonal antibodies

Monoclonal antibodies HBJ127 and HBJ8, raised against T24 human bladder cancer cells, predominantly react with the cells in proliferating stages and with a portion of epithelial tumor cells, respectively. To investigate the in vivo localization of these monoclonal antibodies, the antibodies were labeled with radioiodine and indium-111 (111In) and injected into nude mice transplanted with human bladder tumors. The BT-11 bladder tumor had the highest concentration of radioiodinated HBJ127 and HBJ8 monoclonal antibodies, with 11.6 and 14.3% of the injected dose per gram and with a tumor-to-blood ratio of 2.6 and 1.6, respectively, at 4 days after the administration. An irrelevant monoclonal antibody did not show any specific accumulation in the BT-11 tumor. The 111In-labeled HBJ127 antibody was also localized in the tumor with a higher tumor-to-blood ratio than the radioiodinated antibody. The xenografted BT-11 tumor was successfully visualized with the radiolabeled HBJ127 and HBJ8 antibodies by scintigraphy. These monoclonal antibodies and the human bladder tumor xenografts may provide a good model for radioimmunoimaging and possibly therapy.     

双调蛋白反义核酸表达对裸鼠乳腺癌血管生成的抑制作用

目的 研究双调蛋白（AR）反义RNA表达对乳腺癌血管生成的抑制作用。方法 乳腺癌NS2T2A1细胞经AR反义cDNA质粒转染后,经筛选获得表达AR反义RNA的AR-AS1及AR-AS3克隆,转染空载体获得NS2T2A1 V对照细胞,接种裸鼠皮下形成肿瘤。研究条件培养液对人微血管内皮细胞（HMEC）增殖的影响,以EL1SA法测定细胞血管内皮生长因子（VEGF）分泌量。以定量RT-PCR分析肿瘤组织的VEGF表达水平。以免疫组化法标记CD31研究肿瘤内血管数量。结果 HMEC在AR-AS1和AR-AS3细胞条件培养液中的增殖比例明显降低。AR-AS1和AR-AS3细胞VEGF分泌量亦降低。AR-AS1和AR-AS3肿瘤的血管数量仅有对照组的50％左右,且VEGF表达显著降低。结论 AR反义RNA表达可有效抑制乳腺癌的血管生成,应作为新治疗靶点进一步研究．