TIMP-1 antisense gene transfection attenuates the invasive potential of pancreatic cancer cells in vitro and inhibits tumor growth in vivo

BACKGROUND: TIMP-1 overexpression decreases the invasive potential of pancreatic cancer cells. By tissue inhibitors of metalloproteinase (TIMP)-1 antisense gene transfection, we expected to produce aggressive pancreatic cancer cells with increased in vitro and in vivo invasive potential. METHODS: PANC-1 cells were transfected with either TIMP-1 gene (CD-1), antisense TIMP-1 gene (AS-3), or empty vector (MB-3). The in vitro cell growth kinetics and invasive potential of each cell line were compared. Total and active matrix metalloproteinase (MMP)-2 levels were determined. Each cell line was then implanted in athymic mice and the resultant tumors were compared for size, weight, MMP activity, and TIMP-1 expression. RESULTS: TIMP-1 modulation did not affect cell proliferation in vitro, but its underexpression and, to a lesser extent, overexpression resulted in attenuated tumor growth in vivo. AS-3 cells showed marked decreases in cell invasion and MMP-2 activity in vitro and in vivo. CONCLUSION: TIMP-1 manipulation, particularly underexpression, greatly reduces the invasive potential of pancreatic cancer by limiting MMP-2 activity without affecting in vitro cell growth. TIMP-1 is a reasonable molecular target in pancreatic cancer therapy.     

白细胞介素12基因转染抑制人胰腺癌细胞血管生成及肿瘤的生长

目的　探讨白细胞介素 (IL) 12基因转染人胰腺癌细胞对肿瘤的抑制作用及机制。方法　用逆转录病毒MFG作为载体 ,通过逆转录PCR和磷酸钙沉淀法将鼠IL 12基因转染导入人胰腺癌细胞PK 1,得到PK1/IL 12细胞。单纯用MFG感染PK1所得细胞命名为PK1/MFG。用ELISA方法检测IL 12产生量 ,用NK细胞抗体阻断NK细胞作用 ,比较活体及离体条件下两种细胞的增长曲线。活体显微镜下观察肿瘤细胞的血管生成及IL 12的抑制效应。结果　PK1/IL 12细胞株的IL 12产生量为 1 12 μg·ml-1·48h-1。离体条件下两种细胞的生长曲线相似 ,但在活体条件下PK1/IL 12细胞的生长被显著抑制 (P <0 0 1) ,其血管生成被完全阻断 ,正常毛细血管亦被破坏。结论　IL 12基因转染可以抑制人胰腺癌细胞的生长。在免疫缺陷条件下 ,IL 12的抗血管生成作用足以抑制肿瘤细胞的血管生成及肿瘤的生长。IL 12的血管生成抑制效应缺乏特异性。     

FTY720 inhibits tumor growth and angiogenesis

De novo malignancies and recurrence of tumors are some of the biggest threats to allograft recipients subjected to chronic immunosuppression. FTY720, a synthetic myriocin analogue, is an immunosuppressant that induces apoptosis of activated lymphocytes and prevents infiltration of lymphocytes into allografts, thereby prolonging allograft survival in a dose-dependent manner. Additionally, FTY720 was shown to prevent tumor growth and metastasis. Therefore, we examined the effect of FTY720 on angiogenesis in a HUVEC spheroid model. To substantiate our in vitro findings the effect of FTY720 was also tested in C57/B16 mice subcutaneously injected with Lewis Lung Carcinoma (LLC1) cells. After establishment of a palpable tumor the animals were treated daily with either saline or 1, 5, or 10 mg/kg FTY720. Subsequently, the tumor size was measured, periodically. In our experiments FTY720 showed a strong antiangiogenic effect, overcoming the stimulating effect of VEGF (20 ng/mL) even at subnanomolar concentrations. In vivo, FTY720 showed a dose-dependent inhibition of subcutaneous tumors, and the tumor size of animals treated with 10 mg/kg FTY720 was less than half of the size of tumors in control animals. In conclusion, FTY-720 demonstrated a strong antiangiogenic effect in vitro and a substantial antitumor effect in vivo. Presumably, the stabilizing effect of surrounding pericytes limits the effect of FTY720 in our mouse model. Therefore, a combination of FTY720 with an mTOR inhibitor might be the most favorable immunosuppressive drug combination for allograft recipients at risk for tumor development.     

肿瘤血管生成与大肠癌肝转移

目的分析大肠癌肿瘤血管生成与转移、预后的关系。方法用免疫组化法检测６１例大肠癌手术标本肿瘤组织微血管密度（ＭＶＤ）、血管内皮生长因子（ＶＥＧＦ）的表达。结果ＭＶＤ计数为（２７０±８４）、ＶＥＧＦ表达为（５９７５±１２３６）％,二者与肝、淋巴结转移明显相关（Ｐ＜００１、Ｐ＜００５）。ＭＶＤ计数与术后生存率明显相关（Ｐ＜００１）。结论ＭＶＤ计数与大肠癌肝转移、生存率明显相关,可作为一个新的具有参考意义的预后判断指标。     

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.     

血管生成抑制剂SU5416对大鼠胰腺癌生长和转移的抑制作用

目的研究血管生成抑制剂SU5 4 16对大鼠胰腺癌生长和转移的抑制作用。方法采用二甲基苯丙葱直接置入大鼠胰腺 ,建立临床类似胰腺癌的大鼠模型。将 6 0只胰腺癌SD大鼠随机分为 4组 ,分别自腹腔注射生理盐水 (对照组 )、5氟脲嘧啶 (5 Fu组 )、SU5 4 16制剂 (SU5 4 16组 )和5 Fu SU5 4 16联合用药 (联合组 ) ,隔日 1次。连续 13周后处死大鼠 ,剖腹测量原位肿瘤瘤重、抑瘤率、肿瘤微血管密度、胰腺癌细胞凋亡指数 ,同时观察腹膜、肝脏及毗邻脏器转移和腹水情况。结果对照组、5 Fu组、SU5 4 16组和 5 Fu SU5 4 16联合组诱发肿瘤瘤重分别为 (1 15± 0 2 1)g、(0 6 8±0 4 2 ) g、(0 31± 0 11)g、(0 19± 0 0 6 ) g ;抑瘤率分别为 0、4 8%、80 %、85 % ;肿瘤微血管密度分别为12 3± 3 2、11 4± 3 8、2 1± 1 5、1 8± 1 1;凋亡指数分别为 (2 6± 1 9) %、(5 7± 3 1) %、(13 2±4 3) %、(2 1 1± 7 2 ) % ;腹膜转移率分别为 83%、4 6 %、2 5 %、0 ;肝转移率分别为 6 7%、2 7%、17%、0。实验组与对照组比较差异具有显著意义 (P <0 0 5 )。结论SU5 4 16因能抑制依赖于血管表皮生长因子刺激的血管表皮细胞增殖而抑制胰腺癌的生长和转移 ,化疗药物联合应用具有协同作用。     

MMP-2 and TIMP-1 are derived from, not in response to, pancreatic cancer.

INTRODUCTION: Genetic therapy aimed at disturbing the balance between matrix metalloproteinases (MMP) and their natural tissue inhibitors (TIMP) in treatment of pancreatic cancer requires an understanding of whether MMP and TIMP are tumor- or host-derived. This study was undertaken to determine whether production of MMP-2 and TIMP-1 is by, or in response to, pancreatic cancer. METHODS: PANC-1 (poorly differentiated human pancreatic cancer) or CD-1 (PANC cells transfected to overproduce TIMP-1) cells were implanted into the pancreata of 20 nude mice. After sacrifice, tumors and peritumoral stroma underwent immunohistochemical staining for human and murine MMP-2 and TIMP-1. Normal murine pancreas served as control. All stains were reviewed in a "blinded" manner by a pathologist and graded relative to normal control pancreata. RESULTS: Control pancreata displayed faint murine MMP-2 and TIMP-1 staining and no human MMP-2 or TIMP-1. MMP-2 was most prominent in peritumoral stroma, while TIMP-1 was most prominent in tumors. CD-1 tumors contained very high levels of TIMP-1 compared to PANC-1 tumors and control pancreata. Tumoral and peritumoral MMP-2 were overwhelmingly human. As well, tumoral TIMP-1 was predominantly human. CONCLUSIONS: In a murine model for human pancreatic cancer, nearly all TIMP-1 and MMP-2 expression is tumor-derived (i.e., human). Pharmacologic and gene therapy aimed at disturbing the MMP/TIMP balance in pancreatic cancer should be targeted toward tumor-specific mechanisms and warrants continued investigation.     

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。     

埃罗替尼和塞来昔布抑制胆管癌生长和血管生成

目的:观察表皮生长因子受体(EGFR)酪氨酸激酶抑制剂埃罗替尼与环氧合酶(COX-2)抑制剂塞来昔布对胆管癌荷瘤裸鼠的肿瘤生长协同抑制作用。方法:联合EGFR酪氨酸激酶抑制剂(EGFR-selective tyrosine kinaseinhibitor,EGFR TKI)埃罗替尼和COX-2抑制剂塞来昔布作用于胆管癌细胞株QBC939荷瘤裸鼠,评价药物体内作用效果。结果:埃罗替尼、塞来昔布联合用药显著抑制肿瘤生长,与对照组、埃罗替尼、塞来昔布单药组相比,均有显著性差异。抑制肿瘤生长的作用伴随EGFR下游活性蛋白p-MAPK的下调和VEGF、Ki-67表达的降低。肿瘤组织微血管密度降低。结论:埃罗替尼、塞来昔布抑制胆管癌荷瘤生长,抑制肿瘤细胞增殖,同时抑制肿瘤新生血管。两者具协同作用。靶向抑制EGFR和COX-2通路可以作为胆管癌的潜在治疗方法。     

Hypoxia and angiogenesis in pancreatic cancer

BACKGROUND: Pancreatic cancer remains one of the most lethal of all solid tumours of the gastrointestinal tract. It is characterized by late diagnosis, aggressive local invasion, early metastasis and resistance to chemoradiotherapy. Increasing knowledge regarding the molecular events behind the growth and invasion of pancreatic cancer may lead to new targets for intervention. METHODS: A search of Pubmed and Medline databases was undertaken using the keywords pancreatic cancer, gastrointestinal cancer, hypoxia, angiogenesis and anti-angiogenesis therapy. RESULTS: Hypoxia is the driving force behind angiogenesis in pancreatic cancers. Research into angiogenesis has shown many different sites that can be targeted by agents such as tyrosine kinase inhibitors. CONCLUSION: Anti-angiogenic therapy could be an important adjunct to conventional chemotherapy treatment of gastrointestinal neoplasia.     

alphastatin抑制胃癌细胞裸鼠移植瘤血管生成的实验研究

目的探讨alphastatin对裸鼠体内新生血管形成和人胃癌细胞裸鼠移植瘤血管生成的抑制作用及机制。方法裸鼠皮下注射基质胶溶液形成基质胶体。测定alphastatin对基质胶体内新生血管的抑制作用。人胃癌BGC823细胞接种于裸鼠皮下形成移植瘤。分别于裸鼠腹腔注射磷酸盐缓冲液（PBS）和不同剂量的alphastatin（100nmol／L,0．25mg·kg^-1·d^-1;1000nmol／L,2．5mg·kg^-1·d^-1）,测定各组瘤体大小、体质量并进行病理学分析,测定微血管密度（MVD）计数。分离瘤体内血管内皮细胞．经alphastatin处理后,提取细胞内蛋白,进行细胞鞘氨醇激酶（SPK）活性测定。结果裸鼠移植瘤实验中．与PBS对照组相比,两个不同剂量alphastatin实验组裸鼠移植瘤的体积和体质量均得到了不同程度的抑制[体积：（1145．96±29．89）μm^3、（612．65±23．45）μm^3比（1771±31．05）μm^3,P〈0．05;瘤体质量：（0．31±0．03）g、（0．12±0．02）g比（0．67±0．02）g,P〈0．05]。体外实验病理学证实．alphastatin减少了瘤体内MVD的数目．降低了移植瘤体内血管内皮细胞SPK活性．上述作用均呈现一定的量-效关系。结论alphastatin具有明显抑制人胃癌细胞裸鼠移植瘤血管生成的作用．这种效应与降低血管内皮细胞SPK活性、减少1-磷酸鞘氨醇（SIP）的生成有密切关系。     

Vitamin E succinate decreases lung cancer tumor growth in mice

Background.In vitro studies have shown that Vitamin E Succinate (VES) arrests lung cancer cellular proliferation; however, in vivo studies have not been performed. This study examined in vivo effects of VES on lung cancer. Methods. An in vitro dose-response curve of human A549 lung cancer tumors to VES was established. A549 tumors were established in the right submammary fat pads of athymic nude mice (C57/BL/6J-Hfh11nu). Seven days postinjection, mice were separated into VES and control groups. VES mice (n = 12) underwent daily intraperitoneal (IP) injection of 0.1 Ml VES in polyethylene glycol and dimethysulfoxide (7% DMSO, 93% PEG); control mice (n = 11) were injected with vehicle only. At 27 days, harvested tumors were measured and weighed. Lungs were stained for metastases using hematoxylin-eosin. Tumor volume and weights were compared using a two-sample t test. Tumor growth curves were compared using a mixed model analysis of variance. Results.In vitro studies demonstrated dose-dependent inhibition of A549 cell proliferation by VES (IC₅₀ 18 μg/ml). Final tumor volumes and weights differed significantly between VES and control mice with volumes of 292.9 ± 31.4 mm³ vs.192.6 ± 20.4 mm³ (P = 0.01) and weights of 255.7 ± 37.0 mg versus 168.6 ± 20.0 mg (P = 0.05), respectively. Tumor growth curves differed significantly (P < 0.001). Both groups of mice showed pulmonary metastases. Conclusions. Intraperitoneal VES was associated with decreased A549 tumor volume, weight, and growth; however, despite reduced tumor growth, pulmonary metastases were seen in VES-treated mice. Nonetheless, these results suggest that lung cancer patients may benefit from inclusion in eventual clinical studies using VES.     

Vitamin E succinate decreases lung cancer tumor growth in mice

BACKGROUND: In vitro studies have shown that Vitamin E succinate (VES) arrests lung cancer proliferation; however, in vivo studies have not been performed. This study examined in vivo effects of VES on lung cancer. METHODS: An in vitro dose-response curve of human A549 lung cancer tumors to VES was established. A549 tumors were established in the right submammary fat pads of athymic nude mice (C57/BL/6J-Hfh11nu). Seven days after injection, mice were separated into VES and control groups. VES mice (n = 12) underwent daily intraperitoneal (IP) injection of VES (150 mg/kg in 7% dimethyl sulfoxide, 93% polyethylene glycol); control mice (n = 11) were injected with vehicle only. At 27 days, harvested tumors were measured and weighed. Lungs were stained for metastases using hematoxylin-eosin. Tumor volume and weights were compared using a two-sample t test. Tumor growth curves were compared using a mixed model analysis of variance. RESULTS: In vitro studies demonstrated dose-dependent manner inhibition of A549 cell proliferation by VES (IC(50) 18 mug/mL). Tumor volumes and weights differed significantly between VES and control mice with volumes of 192.6 +/- 20.4 mm(3)versus 292.9 +/- 31.4 mm(3) (P = 0.01) and weights of 168.6 +/- 20.0 mg versus 255.7 +/- 37.0 mg, respectively (P = 0.05). Tumor growth differed significantly (P < 0.001). Both groups of mice showed pulmonary metastases. CONCLUSIONS: Lung cancer cells appear to respond to VES, albeit incompletely. Because tumor cell response is seen, lung cancer patients may derive some benefit from VES and should be considered in eventual clinical studies using this vitamin E derivative.     

Primary tumor dependent inhibition of tumor growth,angiogenesis, and perfusion of secondary breast cancer in bone

The systemic balance of angiogenic and anti‐angiogenic factors has been proposed to play a key‐role in primary tumor growth dependent growth suppression of secondary tumors. Despite the importance of the organ microenvironment to angiogenesis and microcirculation, the influence of a primary tumor on secondary bone tumors has not been investigated so far. Since breast cancer has a high propensity to spread to bone, we used an in vivo xenograft model to determine the impact of growing breast cancer cells (MCF‐7) in the mammary fat pad on the microvascular properties of subsequently inoculated secondary breast cancer tumors in bone. Mice were either treated with a resection of the primary tumor (n = 10) or no surgery (n = 9) and intravital microscopy was performed over 25 days in bone tumors. Tumor growth in bone was temporarily suppressed by the primary tumor on days 10 and 14. While microvascular permeability and vascular diameter decreased in both groups over time, the presence of the primary tumor was accompanied by a decreased tumor perfusion on days 8 and 10 through a reduction in vessels with diameters between 5 and 20 µm. The results imply a potential benefit of a therapeutic regime in which the resection of the primary tumor is combined with an anti‐angiogenic therapy in the perioperative or direct postoperative period. This might result in reduced progression of bone metastasis subsequent to excision of the primary tumor. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1251–1258, 2011     

Specific targeting of tumor vasculature by diphtheria toxin-vascular endothelial growth factor fusion protein reduces angiogenesis and growth of pancreatic cancer

Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), a mediator of neoangiogenesis. The aim of this study was to specifically target and damage the vasculature of pancreatic cancer (PaCa) by fusing VEGF to diphtheria toxin (DT), which inhibits protein synthesis of target cells. DT-VEGF fusion protein was produced in vector pGEX-KG and expressed in E. coli SG12036. Human PaCa cell lines (HPAF-2 and AsPC-1) and human endothelial cells (HUVEC) were exposed to DT-VEGF (10 ng/ml – 10,000 ng/ml). Proliferation was assessed after 3 days. One mm3 fragments of subcutaneous PaCa donor tumors were implanted into the pancreas of nude mice that received either DT-VEGF (200 ώg/kg) every other day) or phosphate-buffered saline intraperitoneally for 14 weeks. Tumor volume, metastatic spread, and animal weight were determined at autopsy. Microvessel density was analyzed in CD31 -stained tumor sections. Proliferation of PaCa cells was inhibited at high concentrations of DT-VEGF (≥1000 ng/ml). DT-VEGF decreased the growth of HUVEC at 10 ng/ml. In vivo, DT-VEGF reduced tumor volume (HPAF-2, 76%; AsPC-1, 53%), microvessel density (HPAF-2, 54%; AsPC-1, 62%), and tumor spread (HPAF-2, 89%; AsPC-1, 50%). Survival was increased (HPAF-2, 7/8 vs. 4/8 animals; AsPC-1, 6/8 vs. 1/8 animals). Weight was not influenced by DT-VEGF. The DT-VEGF effect is due to its toxic action on the tumor vasculature rather than to direct inhibition of PaCa cell growth. DT-VEGF therapy was not associated with systemic side effects. Presented at the Forty-Second Annual Meeting of The Society for Surgery of the Alimentary Tract, Atlanta, Georgia, May 20–23, 2001. Supported by the R.S. Hirshberg Foundation and the Deutsche Forschungsgemeinschaft (grant HO 1843–1).     

重组腺病毒介导TIMP-1基因对肝癌的治疗作用

目的探讨携带人基质金属蛋白酶组织抑制因子(hTIMP)-1的重组腺病毒(Adh- TIMP)-1对人肝癌生长、转移、血管形成以及凋亡的作用。方法构建AdhTIMP-1感染人肝癌细胞并接种于裸鼠皮下观察其成瘤情况;建立荷瘤鼠模型,观察AdhTIMP-1对肿瘤生长的作用;计数荷瘤鼠肺转移结节;CD34免疫组织化学观察肿瘤血管生成;TUNEL法检测肝癌细胞凋亡。结果AdhTIMP-1感染的HepG2细胞成瘤量下降4倍(P<0．01),荷瘤鼠瘤体内注射AdhTIMP-1使肿瘤生长减少45％(P<0．01),组织血管密度减少47％(P<0．05),肺转移结节减少70％(P<0．01),肿瘤组织中细胞凋亡增加3倍(P<0．05)。结论AdhTIMP-1能抑制肝癌的生长、转移及血管形成,诱导肝癌细胞凋亡,可用于肝癌基因治疗的研究。