VEGF反义核酸抑制人胰腺癌细胞的增殖

目的：探讨VEGF121反义核酸对体外培养胰腺癌细胞增殖的影响,以求证明VEGF反义核酸具有抑制肿瘤生长的作用。方法：将稳定转染有VEGF反义核酸pcDNA3-ASVEGF121的胰腺癌细胞系BxPC-3在体外条件下培养,采用ELISA法检测其培养上清液中的VEGF表达水平。检测肿瘤细胞生长曲线,通过克隆形成试验检测肿瘤细胞的体外增值能力。采用Annexin Ⅴ-FITC和PI双标记法以流式细胞仪检测肿瘤细胞凋亡情况。结果：VEGF反义核酸转染BxPC-3细胞后,肿瘤细胞培养液上清VEGF蛋白水平受到明显抑制。生长曲线显示肿瘤细胞生长明显受到抑制,并且肿瘤细胞克隆形成率降低。流式细胞仪检测显示VEGF反义核酸能促进细胞凋亡。结论：人反义VEGF121能显著抑制胰腺癌细胞的VEGF表达,并能抑制肿瘤细胞的生长,促进凋亡。     

血管生长抑素基因抑制胰腺癌裸鼠移植瘤血管生成的实验研究

目的:探讨人血管生长抑素基因对胰腺癌裸鼠移植瘤生长及血管生成的影响。方法:建立人胰腺癌细胞株BXPC-3裸鼠移植瘤模型,应用人血管生长抑素基因质粒转染胰腺癌裸鼠移植瘤,观察移植瘤生长情况。采用免疫组织化学技术检测肿瘤血管内皮生长因子(VEGF)表达情况和微血管密度(MVD)差异,透射电子显微镜观察肿瘤细胞情况。结果:治疗组肿瘤体积明显小于空白对照组和空质粒组(P<0.01),VEGF主要表达在肿瘤细胞的胞质内,治疗组、空白对照组和空质粒组平均灰度分别为179.57±5.22、150.87±3.44和163.40±3.54;阳性单位分别为14.94±2.26、31.26±2.72和29.21±2.95;MVD分别为10.60±74.56、19.33±2.52和18.67±5.29,治疗组与空白对照组和空质粒组比较,差异有统计学意义(P均<0.05)。电子显微镜下治疗组可见大量肿瘤细胞坏死。结论:人血管生长抑素基因能明显抑制胰腺癌裸鼠移植瘤血管生成,促进肿瘤细胞坏死,进而抑制肿瘤生长。     

人反义VEGF121真核表达质粒的构建及其对胰腺癌细胞VEGF表达的影响

血管内皮生长因子(VEGF)能特异性地刺激血管内皮细胞增殖，在肿瘤的血管生成中发挥着关键作用。胰腺癌作为实体瘤其发生、发展与肿瘤血管生成有着密切的联系。本研究采用分子生物学方法构建人反义VEGF121真核表达质粒，并转染人胰腺癌细胞系BxPC-3，为通过抑制VEGF引起的血管生成来抑制胰腺癌的增殖提供了实验基础。     

COX-2特异性抑制剂NS-398对胰腺癌生长及肿瘤血管 生成影响

目的:探讨COX-2特异性抑制剂NS-398对胰腺癌生长的影响及其机制。方法:分别用q RT-PCR与Western blot检测不同人胰腺癌细胞株(Bx PC-3、SWl990、Capan-2、Aspc-1、PANC-1)中COX-2及VEGF表达,并用MTT法检测NS-398在体外对人胰腺癌细胞增殖抑制作用;用体外实验最敏感细胞株建立裸鼠胰腺癌原位移植瘤模型,并随机将荷瘤鼠分为实验组和对照组,分别用NS-398与生理盐水处理,比较两组移植瘤的生长情况,并检测肿瘤组织中COX-2、VEGF蛋白表达及肿瘤微血管密度(MVD)。结果:各胰腺癌细胞中均有COX-2及VEGF表达,NS-398呈时间与浓度依赖性抑制各胰腺癌细胞的体外增殖,其中Bxpc-3细胞COX-2与VEGF表达量最高,且对NS-398最敏感。用Bxpc-3细胞建立原位移植瘤的实验组与对照组裸鼠比较,平均肿瘤体积明显减小(20.215 2 mm~3 vs.204.444 4 mm~3),瘤组织中COX-2与VEGF表达及MVD均明显降低(均P0.05)。结论:NS-398对胰腺癌的生长有抑制作用,其机制可能是通过COX-2途径降低VEGF基因表达从而抑制肿瘤血管生成有关。     

血管内皮生长因子反义寡核苷酸抑制胆囊癌血管生成的研究

目的探讨Oligofectamine介导的血管内皮生长因子(vascular endothelial cell growthfactor,VEGF)反义寡核苷酸(antisense oligodeoxynucleotides,ASODN)转染对人胆囊癌细胞GBC-SD裸鼠移植瘤的VEGF表达和血管形成的影响。方法裸鼠接种GBC-SD细胞建立移植瘤模型,随机分为A、B、C三组,各组裸鼠在接种1周后,分别给于200μl磷酸缓冲液(PBS)、VEGFSODN100μg Oligofectamine0.5μl及VEGF ASODN100μg Oligofectamine0.5μl,每3天1次,连续治疗5周,观察各组裸鼠的成瘤性、体积及瘤重的变化,各组移植瘤中VEGF表达及微血管密度(MVD)变化。结果三组裸鼠3周时的成瘤率,C组显著低于A、B两组(P<0.05),6周时各组的成瘤率无差异。各组移植瘤6周时的体积和瘤重相比C组显著低于A、B两组(P<0.05),C组的抑瘤率为(50.79±9.19)%。A、B两组移植瘤VEGF表达及MVD均无显著性差异(P>0.05),而C组移植瘤VEGF的表达较A、B两组明显减弱(P<0.05),MVD明显小于A、B两组(P<0.05)。结论VEGF ASODN在体内能显著抑制人胆囊癌裸鼠移植瘤的增殖,降低其在裸鼠体内的成瘤性,抑制VEGF在蛋白水平的表达,减少裸鼠移植瘤中血管的形成,降低微血管密度。     

p14ARF基因对胰腺癌血管生成的影响

目的探讨外源性p14ARF基因对胰腺癌PC-3细胞血管生成的影响。方法将转染了外源性p14ARF基因的人胰腺癌PC-3细胞株接种裸鼠形成实体瘤，用RT-PCR和Westernblot法观察VEGFmRNA及VEGF蛋白的表达，并对实体肿瘤的微血管密度进行检测。结果转染p14ARF基因后的PC-3细胞实体瘤中VEGFmRNA及VEGF蛋白的表达下调，血管密度降低。结论p14ARF基因能下调内源性VEGF的表达，有着抗血管生成的治疗意义。     

β1整合素反义寡核苷酸对人胰腺癌裸鼠皮下移植瘤的治疗作用

目的探讨β1整合素反义寡核苷酸（ASODN）对裸鼠人胰腺癌移植瘤生长和基因表达的影响.方法建立裸鼠人胰腺癌皮下移植瘤模型,随机分为3组：对照组（皮下注射脂质体和转染液）、非特异序列（RODN）组（皮下注射RODN、脂质体和转染液）和ASODN组（皮下注射ASODN、脂质体和转染液）.治疗后计算抑瘤率和肿瘤缩小率,检测肿瘤中β1整合素mRNA和蛋白表达.结果RODN组、ASODN组抑瘤率分别为4.75%和72.70%,ASODN组肿瘤缩小10.91%.与其他两组比较,ASODN组裸鼠肿瘤中β1整合素mRNA和蛋白表达水平明显降低.结论靶向β1整合素的反义寡核苷酸对人胰腺癌裸鼠皮下移植瘤的生长具有一定的抑制作用,可能为胰腺癌治疗提供新的方法.     

Survivin反义核酸治疗人大肠癌裸鼠皮下种植瘤的实验研究

目的探讨Survivin反义寡核苷酸对裸鼠人大肠癌皮下种植瘤体生长的抑制作用及其机制。方法接种人大肠癌细胞制作裸鼠皮下大肠癌种植瘤模型,分别用Survivin反义寡核苷酸、正义核苷酸和生理盐水对皮下瘤体内直接进行注射治疗,检测肿瘤的生长状况、瘤体体积及瘤重变化,计算抑制率。病理切片免疫组化检测瘤体E-CD表达。结果经Survivin硫代反义寡核苷酸治疗组裸鼠皮下移植瘤生长受到抑制,肿瘤重量明显小于对照组,肿瘤体积明显小于对照组和正义组(P<0.05),裸鼠皮下移植瘤E-CD表达增强。结论Survivin反义核酸可以抑制大肠癌裸鼠移植瘤的生长,Survivin反义核酸抑制大肠癌裸鼠移植瘤生长的机理可能与E-CD基因表达变化有关。     

血管内皮生长因子反义核糖核酸对人肺癌的抗血管生成作用

目的探讨血管内皮生长因子(VEGF)反义RNA封闭内源性VEGF表达对人肺癌裸鼠皮下移植瘤血管生成的影响。方法采用30只裸鼠建立人肺癌皮下移植瘤模型,通过原位分子杂交法测定VEGFmRNA、免疫组织化学法测定VEGF表达和微血管密度计数,比较治疗组和空载组、对照组间的差异,研究以脂质体介导VEGF反义cDNA经局部多点注射封闭内源性VEGF表达的抗血管生成作用。结果治疗组肿瘤组织的VEGFmRNA阳性细胞数为(21.83±13.47)个/0.05mm2、VEGF蛋白阳性细胞数(20.76±15.69)个/0.05mm2、微血管密度计数(MVD)为(2.30±1.95)条/0.20mm2,与对照组、空载组的差异均有统计学意义(P<0.01),VEGF反义RNA治疗能够有效的封闭内源性VEGF的生成、使肿瘤组织微血管生成减少,实验结束时瘤体大小与对照组和空载组的差异均有统计学意义(P<0.001)。结论VEGF反义RNA治疗是一种有效的肿瘤抗血管生成基因治疗方法。     

反义HIF-1α基因治疗人肝癌裸鼠移植瘤的实验研究

摘要：目的:探讨反义HIF-1α基因对人肝癌裸鼠皮下移植瘤的影响及其相关机制。方法:使用人原发性肝癌细胞株SMMC-7721建立人肝癌裸鼠皮下移植瘤动物模型。待肿瘤生长至直径约0.4cm时，将荷瘤裸鼠随机分为3组，分别注射生理盐水、质粒PcDNA3和HIF-1α/PcDNA3B，质粒用脂质体DOTAP介导转染细胞。观察各组动物的肿瘤生长曲线；取肿瘤标本作免疫组织化学检查（SABC法）及蛋白质印迹检查，检测各组肿瘤的VEGF和HIF-1α表达及微血管密度（MVD）和细胞凋亡。结果:HIF-1α/PcDNA3B治疗组各时点的肿瘤体积，以及肿瘤组织中HIF-1α蛋白、MVD，VEGF表达均低于对照组，而细胞凋亡指数高于对照组，差异均有显著性（P＜0.05）。结论:通过阻断癌细胞的缺氧适应途径，反义HIF-1α基因治疗肝癌有抑制肿瘤生长、抑制肿瘤血管生成及诱导细胞凋亡的作用。     

VEGF antisense therapy inhibits tumor growth and improves survival in experimental pancreatic cancer

BACKGROUND: Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, is overexpressed in pancreatic cancer. This study evaluated VEGF production in pancreatic cancer cells and the effect of VEGF antisense on growth and angiogenesis of human pancreatic cancer in a nude mouse model. METHODS: In vitro: VEGF in cell culture supernatant of pancreatic cancer cells (AsPC-1, poorly differentiated; HPAF-2, moderately differentiated) was assessed by enzyme-linked immunosorbent assay. In vivo: A VEGF antisense oligonucleotide (AS-3) was synthesized. One-mm(3) fragments of subcutaneous pancreatic cancer donor tumors were implanted into the pancreas of nude mice also receiving AS-3 (10 mg/kg/day) or vehicle intraperitoneally for 14 weeks. Primary tumor volume, metastasis, and VEGF in plasma and ascites were determined at autopsy. Microvessel density was analyzed in CD31-stained tumors. RESULTS: In vitro: Both pancreatic cancer cell lines secreted VEGF protein (AsPC-1, 4200 +/- 40 pg/10(6) cells; HPAF-2, 8120 +/- 60 pg/10(6) cells). In vivo: AS-3 reduced tumor volume in the HPAF-2 group (860 +/- 140 vs 3830 +/- 590 mm(3)) and metastatic spread in both groups (AsPC-1, 6.5 +/- 0.8 vs 16.7 +/- 0.9 points; HPAF-2, 2.5 +/- 0.2 vs 8.3 +/- 1.5 points). Tumor volume was not different in the AsPC-1 group (1050 +/- 80 vs 1400 +/- 150 mm(3)). Survival was increased in the AsPC-1 group. Plasma levels of VEGF and microvessel density in tumors were significantly reduced in treated animals. Only control animals (50%) developed ascites with high VEGF concentrations. CONCLUSIONS: Human pancreatic cancer cells secrete VEGF at biologically relevant high levels. AS-3 therapy normalizes plasma VEGF and decreases neoangiogenesis, thereby reducing tumor growth and metastasis and improving survival. AS-3-treated animals developed no ascites, suggesting decreased vascular permeability by reducing VEGF expression in pancreatic cancer cells.     

VEGF-RII influences the prognosis of pancreatic cancer

OBJECTIVE: To evaluate whether the vascular endothelial growth factor (VEGF) pathway can be used as a target for effective treatment of pancreatic cancer. SUMMARY BACKGROUND DATA: VEGF and its receptors (VEGF-RI and -RII) are the predominant regulators of tumor neoangiogenesis, a key element for tumor growth and progression. However, VEGF receptor expression has been thought to be limited to endothelial cells, limiting the possibility of targeting it for therapy of pancreatic cancer. METHODS: Protein localization and mRNA were studied in pancreatic cancer specimens, normal pancreas, human pancreatic cancer cell lines, and an endothelial cell line. Cell proliferation was determined by [ H] thymidine uptake. Both VEGF receptors were genetically eliminated by antisense technology. The same approach was used in a murine model of pancreatic cancer in a therapeutic approach. RESULTS: VEGF-RI mRNA and VEGF-RII mRNA were expressed in 17 and 15 of 24 pancreatic cancer samples, respectively. VEGF receptors were found not only in blood vessels but also in pancreatic cancer cells. VEGF-RII expression correlated with poor tumor differentiation and was associated with poorer survival, while VEGF-RI expression did not correlate. VEGF treatment led to extensive growth stimulation in six of seven pancreatic cancer cell lines, which was completely inhibited by antisense treatment against VEGF-RII. Liposome-mediated gene transfer in nude mice with pancreatic tumors markedly reduced local tumor growth and decreased metastatic tumor spread. CONCLUSIONS: The VEGF/VEGF-RII pathway regulates angiogenesis and local tumor growth and spread in pancreatic cancer. Genetic targeting of VEGF-RII blocks local growth and metastatic spread of pancreatic cancer cells in vivo and therefore offers a potential new therapeutic option for patients with this disease.     

Pancreatic cancer growth is inhibited by blockade of VEGF-RII

BACKGROUND: Angiogenesis is important in the development and progression of pancreatic cancer. Therefore antiangiogenic therapy targeting endothelial cells may represent a promising therapeutic option. The aim of the study was to evaluate antiangiogenic therapy as a potential therapeutic option in pancreatic cancer. METHODS: Replication-deficient retroviruses encoding truncated VEGF-RII were used to block vascular endothelial growth factor (VEGF) signaling. Tumor growth of 3 pancreatic cancer cell lines was assayed in a nude mouse model in which each pancreatic cancer cell line was subcutaneously inoculated together with retrovirus-producing cells. Expression of VEGF was assayed by RT-PCR and by enzyme-linked immunosorbent assay. Oxygen tension in tumors was determined polarographically. RESULTS: All 3 pancreatic cancer cell lines expressed VEGF mRNA, with the highest VEGF secretion seen in MIA PaCa-2 cells. In vivo therapeutic intervention through dominant negative inhibition of VEGF-RII significantly reduced the growth rate of subcutaneous tumors and inhibited tumor neoangiogenesis. Tumor oxygenation, however, was not altered in xenograft tumors treated with dominant negative retroviruses. CONCLUSION: The ligand/receptor system consisting of VEGF and VEGF-RII seems to be of biologic significance in the pathogenesis of pancreatic cancer growth. Therefore therapeutic intervention in this angiogenic system by a retroviral-based gene transfer technology represents a rational and feasible new technique to inhibit tumor growth.     

Pim-3 promotes the growth of human pancreatic cancer in the orthotopic nude mouse model through vascular endothelium growth factor

Background

As one of the most lethal cancers, pancreatic cancer presents poor prognosis with an overall 5-y survival of less than 5%. We previously reported that Pim-3, a member of the proto-oncogene Pim family that encodes serine/threonine kinases, is aberrantly expressed in human pancreatic cancer lesions. In the current study, we investigated the role of Pim-3 in promoting tumor growth and angiogenesis in an orthotopic nude mouse model of human pancreatic cancer.

Methods

We constructed retroviral vectors for human Pim-3 and a kinase-dead mutant of human Pim-3 (K69M); the retroviral supernatants generated from these vectors were then used to infect the human pancreatic cancer cell line MiaPaCa-2 to establish stable cell lines. We assessed cell proliferation using CCK-8, tumor growth, and angiogenesis in vivo in an orthotopic mouse model of pancreatic cancer. While tumor size was measured using magnetic resonance imaging, the tumor tissues were excised for protein extraction and histological analysis to detect vascular endothelium growth factor (VEGF) expression and vessel density.

Results

We established an orthotopic nude mouse model of human pancreatic cancer. We observed that Pim-3 promoted the proliferation of human pancreatic cancer cells, both in vitro and in vivo. Moreover, Pim-3 is required for vasculogenesis of primary human pancreatic tumors in vivo and promotion of angiogenesis through the induction of VEGF expression.

Conclusions

Pim-3 can promote tumor growth and angiogenesis by stimulating the VEGF pathway.     

Antitumor effect of reduction of 150-kDa oxygen-regulated protein expression on human prostate cancer cells

BACKGROUND: The 150-kDa oxygen-regulated protein ORP150, a new member of the heat shock protein family that functions as a molecular chaperone in the endoplasmic reticulum, was found to increase in infiltrating cancer cells. Since enhancement of ORP150 expression and the presence of vascular endothelial growth factor (VEGF) in human prostate cancer glands were immunohistochemically demonstrated, we examined whether transduced antisense ORP150 cDNA can reduce angiogenicity and tumorigenicity through suppression of VEGF secretion. METHODS: Human prostate cancer specimens were immunohistochemically stained with fluorescein isothiocyanate (FITC) for ORP150 or vascular endothelial growth factor (VEGF). An adenovirus vector (Ad) carrying antisense ORP150 cDNA (AdCA-Antisense ORP150) was constructed and infected to prostate cancer DU145 cells. Expression of ORP150 in the cells was analyzed with western blotting and secretion of VEGF into the supernatant with an enzyme-linked immunoabsorbent assay (ELISA). Angiogenicity was evaluated by chorioallantoic membrane (CAM) assay. A nude mouse xenograft model was used to examine tumorigenicity. RESULTS: Immunohistochemical study proved that the expression of ORP150 and VEGF was enhanced in the cytoplasm of prostate cancer cells. The Ad showed 100% transduction efficiency and minimum cytotoxicity when the cells were infected at a multiplicity of infection (MOI) of 20 for 24 h. Expression of ORP150 was substantially reduced by the antisense treatment. Secretion of VEGF into the culture supernatant was reduced to 30%. Consequently, the CAM assay showed relatively low angiogenicity, while marked suppression of tumor formation was observed in the xenograft model. CONCLUSION: Adenoviral-mediated antisense ORP150 cDNA transfer is well worth considering as an option for prostate cancer gene therapy.     

反义寡核苷酸联合磁性载药微球靶向治疗耐药裸鼠人胰腺癌

目的 观察辐射及多药耐药基因逆转剂反义寡核苷酸( mdr1ASON)联合5-氟尿嘧啶磁性白蛋白微球(5-Fu-MAMS)靶向治疗耐药裸鼠人胰腺癌模型的效果.方法 构建人胰腺癌耐药细胞株(SW1990/Fu),建立裸鼠人胰腺癌耐药模型,选择适当的磁场,施加于肿瘤表面,瘤内注射mdr1ASON及5-Fu-MAMS,观察肿瘤生长,检测其对耐药裸鼠人胰腺癌的治疗效果.结果 在外加磁场的磁导向作用下,5-Fu-MAMS能定位于肿瘤组织,联合mdr1ASON能显著抑制肿瘤生长,肿瘤体积抑制率达到85.00％,肿瘤重量抑制率达到87.74％,与其他组比较差异有统计学意义(P＜0.01).结论　辐射促转染的mdr1ASON联合磁性载药微球对肿瘤细胞具有较好的耐药逆转作用.     

PCNA、VEGF反义寡核苷酸联合治疗裸鼠肝癌的研究

目的 探讨增殖细胞核抗原（PCNA）反义寡核苷酸和血管内皮生长因子（VEGF）反义寡核苷酸单用或联用对裸鼠人肝癌模型肝癌生长的抑制作用。方法 制作裸鼠皮下肝癌模型24只,分PCNA反义核酸治疗组、VEGF反义核酸治疗组、联合治疗组及对照组,接种后24h之内用PCNA反义核酸和（或）VEGF反义核酸皮下注射进行治疗。观察裸鼠瘤体变化及组织形态学改变。结果 PCNA反义核酸治疗组,VEGF反义核酸治疗组和联合治疗组裸鼠肝癌的生长均受到不同程度的抑制,抑瘤率分别为72．8％、44．9％和87．2％。以联合治疗组的疗效最佳。结论 联合应用PCNA和VEGF2种反义核酸治疗肝癌比应用单种反义核酸有更显著的疗效,2种反义核酸具有协同作用。