反义缺氧诱导因子-1α治疗胰腺癌及其作用机制的实验研究
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摘要
胰腺癌是人体最常见的恶性肿瘤之一，其患病率在世界范围呈上升趋势。胰腺癌由于恶性程度高、手术切除率低、早期诊断困难，治疗的效果不尽人意。近年来各种手术方式的改进以及化疗、放疗方案等综合措施的引进，手术并发症和死亡率有所降低，但5年生存率仍提高不多。随着免疫和分子生物学在肿瘤的发生、发展和转移机制研究的不断深入，为胰腺癌的治疗提供了新的希望。
近年来发现，缺氧是影响化疗效果而且干扰了肿瘤对放疗的反应的一个重要因素。胰腺癌血供很差，在严重缺氧时还能继续增殖的原因可能在于持续缺氧的状态诱导了胰腺癌中缺氧诱导因子(HIF)-1α的表达。研究表明，血管内皮生长因子(VEGF)和糖酵解过程中多种关键酶基因都是受HIF-1α调控的，HIF-1α通过促进肿瘤组织的血管生成和能量代谢加速肿瘤组织的生长，同时也促进了肿瘤的侵袭和转移。HIF-1α在胰腺癌中广泛表达，对胰腺癌的血管生成、发生发展起到了很大的作用。而已有实验表明，人类凋亡抑制蛋白(human inhibitor of apoptosis protein，hIAP)家族成员之一survivin在胰腺癌中高表达，且与化疗敏感性有关；整合素家族中β_1-integrin在胰腺癌侵袭、转移过程中也起着重要的作用。
本研究旨在观察反义HIF-1α对胰腺癌生长、转移、化疗敏感性的影响及其与survivin、β_1-integrin的关系。本实验共分为以下四部分。
第一部分缺氧诱导因子-1α在胰腺癌中的表达和意义
目的
本研究旨在检测胰腺癌中缺氧诱导因子-1α(HIF-1α)的表达，探讨HIF-1α的表达与临床特征的关系，从而深入探讨其在胰腺癌发生发展中的作用。
材料和方法
运用免疫组织化学技术(streptavidin-peroxidase，SP)检测研究48例胰腺癌及癌旁组织中HIF-1α的表达。
结果
1．48例胰腺癌标本中27例表达HIF-1α蛋白，而在癌旁组织未检测到HIF-1α的表达(P＜0.01)。
2．HIF-1α蛋白的表达与胰腺癌患者的性别、年龄和肿瘤大小无关，与胰腺癌的分化程度及是否转移有关(P＜0.05)。
结论
1．HIF-1α在胰腺癌中高度表达，在癌旁胰腺组织未见表达，可能与胰腺癌的发生发展或与治疗效果有关，但其具体作用及作用机制均有待进一步深入的研究。
2．HIF-1α与胰腺癌患者预后的密切相关。
第二部分重组表达质粒pcDNA3.1(+)／反义HIF-1α的构建及其在人胰腺癌细胞株BxPc-3中的稳定表达
目的
第一部分显示，胰腺癌高表达HIF-1α蛋白，本部分拟构建HIF-1α基因的反义表达核苷酸，经脂质体转染人胰腺癌细胞株BxPc-3，建立稳定表达反义HIF-1α基因的细胞模型，为反义封闭治疗奠定基础。
方法
1．采用PCR方法扩增pCR3.1-FLAG-HIF-1α质粒中反义HIF-1α基因功能片段。
2．定向克隆至真核表达载体pcDNA3.1(+)上。
3．转染至BxPc-3细胞内，经G418筛选，建立稳定表达HIF-1α反义基因的细胞株。
4．RT-PCR和Western blot分析HIF-1α的表达，建立稳定表达HIF-1α反义基因的细胞株。
结果
1．酶切鉴定，凝胶电泳分析，两条带大小分别为721bp和5.4kb，与预期结果符合。
2．脂质体包裹转染BxPc-3后，RT-PCR和Western blot分析证实实验组HIF-1αmRNA表达为0.2367±0.0094，蛋白表达为0.1798±0.0063，与对照组相比明显降低(P＜0.05)。
结论
1．本研究成功构建了HIF-1α的反义核苷酸，与预期结果符合。
2．经脂质体途径转染BxPc-3细胞，该反义核苷酸能够抑制HIF-1α的表达。
3．该反义核苷酸的成功构建为下一步实验研究奠定了基础。
第三部分反义HIF-1α增加化疗药物敏感性的实验研究
目的
本研究拟利用构建的反义HIF-1α，转染BxPc-3细胞后，观察BxPc-3细胞对化疗药物敏感性的变化及其与survivin的关系。
方法
1．将HIF-1α反义核苷酸转染至BxPc-3细胞，经G418筛选后，建立稳定表达反义HIF-1α的BxPc-3细胞株，加入不同浓度的氟尿嘧啶，阿霉素，吉西他宾。
2．MTT检测各组的增殖活性。
3．运用流式细胞仪检测各组细胞凋亡指数。
4．RT-PCR和Western blot分析各组survivin的表达。
结果
1．与对照组相比，实验组的生长抑制率、凋亡率与剂量成正比，高剂量引起高抑制(P＜0.05)。
2．实验组survivin的mRNA表达为0.4601±0.0122，蛋白表达为0.3659±0.0117，与对照组相比明显降低(P＜0.05)。
结论
转染反义HIF-1α基因可明显增强氟尿嘧啶，阿霉素，吉西他宾对胰腺癌细胞的杀伤力，并伴有survivin表达量的明显下调，这表明反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性，因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。
第四部分反义HIF-1α对胰腺癌生长、转移影响的实验研究
目的
本研究拟利用构建的反义HIF-1α，转染BxPc-3细胞后，观察其对胰腺癌生长、转移的影响及其与β_1-integrin的关系。
方法
1．将HIF-1α反义核苷酸转染至BxPc-3细胞，经G418筛选后，建立稳定表达反义HIF-1α的BxPc-3细胞株，Transwell侵袭室方法检测BxPc-3细胞侵袭能力。
2．RT-PCR和Western blot分析各组β_1-integrin的表达。
3．裸鼠皮下接种各组处理细胞8周后，观察其肿瘤生长情况。
结果
1．实验组迁移的细胞数为110.64±16.4，远少于对照组(P＜0.05)。
2．实验组β_1-integrin的mRNA表达为0.2568±0.0115，蛋白表达为0.2962±0.0167，与对照组相比明显降低(P＜0.05)。
3．实验组肿瘤的体积、重量、生长速度远低于对照组(P＜0.05)。
结论
转染反义HIF-1α基因可明显抑制胰腺癌细胞的侵袭力，并伴有β_1-integrin表达量的明显下调且抑制了皮下移植瘤的生长，这表明反义HIF-1α可能通过阻断β_1-integrin的表达而抑制胰腺癌的生长和转移。因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。
本课题提出以下新见解：
1．HIF-1α在胰腺癌中高度表达，在癌旁胰腺组织未见表达，提示HIF-1α在胰腺癌的发生发展或或治疗效果中发生重要的作用。
2．本文探讨了HIF-1α的表达与胰腺癌患者临床特征的关系，结果显示HIF-1α蛋白的表达与胰腺癌患者的性别、年龄和肿瘤大小无关，而与胰腺癌的分化程度及是否转移有关，提示HIF-1α与胰腺癌患者预后的密切相关。
3．本研究成功构建了HIF-1α的反义核苷酸，与预期结果符合。经脂质体途径转染BxPc-3细胞，可在BxPc-3中稳定表达，该反义核苷酸能够抑制HIF-1α的mRNA和蛋白的表达。
4．缺氧状态下，反义HIF-1α基因可明显增强氟尿嘧啶，阿霉素，吉西他宾对胰腺癌细胞的杀伤力，并伴有survivin表达量的明显下调，这表明反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性。
5．缺氧状态下，反义HIF-1α基因可明显抑制胰腺癌细胞的侵袭力，并伴有β_1-integrin表达量的明显下调且抑制了皮下移植瘤的生长，这表明反义HIF-1α可能通过阻断β_1-integrin的表达而抑制胰腺癌的生长和转移。因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric complex, composed of an α and a β subunit, both belonging to the basic helix-loop-helix (bHLH)/Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. The bHLH domain mediates dimerization and DNA binding in many transcription factors. PAS is an additional dimerization motif. Whereas HIF-1β is a common subunit of multiple bHLH proteins, HIF-1α is the unique, O_2-regulated subunit that determines HIF-1 activity. Although HIF-1β is quite stable in normoxic conditions, HIF-1α is extremely unstable and quickly degraded by the ubiquitin-proteasome system. HIF-1α plays an important role in solid tumor formation in vivo by promoting angiogenesis and anaerobic metabolism. Several studies have demonstrated that HIF-1α can be detected in most types of human tumors examined, including bladder, breast, colon, glial, hepatocellular, ovarian, pancreatic, prostate, renal and oesophageal tumors. The expression of HIF-1α is inducible by oxygen concentrations below 6%, and the activation of hypoxia-responsive elements by HIF-1 has been reported to correlate with decreasing oxygen concentrations in vitro.
Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is characterized by a unique structure that discriminates it from other members of the IAP family. It contains only a single BIR repeat and lacks a carboxy terminal RING finger domain. Survivin is expressed in the G_2/M phase of cell cycle in a cycle-regulated manner. It directly binds to and inhibits both Caspase-3 and Caspase-7 activity, leading to arrest of apoptosis. Survivin expression is not detectable in differentiated normal adult cells of any organ, but it is abundantly expressed in embryonic tissues and in a wide range of cancer tissues including neuroblastoma, cholangiocarcinoma, colorectal, stomach, breast and uterine cervical carcinomas. It has been demonstrated recently that survivin is also frequently expressed in malignant pancreatic ductal tumors and pancreatic adenocarcinoma. Furthermore, the prognostic value of survivin expression has been reported in several human cancers. Studies demonstrating resistance of survivin-transfected cells to anticancer drug-induced apoptosis and sensitization to chemotherapy by survivin antisense treatment have shown that survivin is implicated in sensitization to chemotherapy.
The integrin family of cell-surface heterodimeric glycoproteins composed of a and β subunits function primarily as receptors for extracellular matrix ligands, which regulate many aspects of cell physiology, including morphology, adhesion, migration, proliferation, and differentiation. Integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes. Many cancers show abnormalities of integrin function as a result of transformation by oncogenes. More importantly, the growth of several tumors depends on β_1-integrin function. Previous studies have demonstrated that β_1-integrin is a poor prognostic factor in patients with cancers. Evidences show an essential role of β_1-integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.
On the other hand, the prognosis for patients with pancreatic cancer remains poor, prompting the search for new treatment strategies. Although treatments such as pre-operative chemotherapy and chemoradiation therapy are currently used for patients with advanced pancreatic cancer, their results are not satisfactory. Even in early-stage disease, we have experienced many patients who developed local recurrence of tumor or distant metastasis within a short period after operation.
In pancreatic cancer, the relationship of HIF-1α, survivin and β_1-integrin is not very clear at present. This study aimed to observe the effect of antisense HIF-1α on progression, metastasis and chemosensitivity of pancreatic cancer. Part 1 Expression and significance of HIF-1α in pancreatic cancer Objective
To investigate the expression of HIF-1α, the relationship with clinical features, and determine the role in the development of pancreatic cancer.
Methods
The expression of HIF-1α in tumor tissues and non-cancerous adjacent pancreas tissues was detected by SP immunohistochemical technique.
Results
HIF-1α was expressed in 27 of 48 cases (56.3%). In contrast, no expression of HIF-1α in adjacent histologically noncancerous pancreas was detectable (P<0.01) . Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level (P<0.05).
Conclusion
High expression of HIF-1α in pancreatic cancer suggests it may play an important role in the development of pancreatic cancer. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients. Part 2 Reconstruction of eukaryotic gene expression plasmid pcDNA3.1(+) /antisense HIF-1α and its stable expression in human pancreatic cancer cell line BxPc-3
Objective
To further explore the role of HIF-1α, antisense plasmid of HIF-1α was constructed, and transfected into human pancreatic cancer cell line BxPc-3.
Methods
Antisense HIF-1α DNA structure was amplified by PCR and inserted into eukaryotic plasmid pcDNA3.1(+). The expression plasmid pcDNA3.1(+)/antisense HIF-1α was transfected into human pancreatic cancer cell line BxPc-3 through Dosper liposomal transfection reagent. Cells stably expressing antisense HIF-1α were screened with G418. BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O_2 for 4 hours. The expression of HIF-1α was detected by RT-PCR and Western blot.
Results
The constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two fragments at 721bp and 5.4kb, were respectively found, which was consistent with what expected. Eukaryotic plasmid was transfected into BxPc-3 cells through Dosper liposomal transfection reagent. HIF-1α mRNA (0.2367±0.0094) and protein (0.1798±0.0063) expression in the experimental group were significantly depressed by this plasmid, compared with the control (P<0.05).
Conclusion
BxPc-3 with stable antisense HIF-1α expression was successfully constructed, which could be applied to explore further. Part 3 An antisense plasmid targeting HIF-1α expression sensitizes pancreatic cancer cells to chemotherapy
Objective
The apoptosis induced by chemotherapeutic agent was inhibited through HIF-1α expression. Antisense plasmid of HIF-1α was constructed and transfected into BxPc-3. The change of sensitizion to chemotherapy and the relationship with survivin was detected.
Methods
BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O_2 for 4 hours. Different dosages of chemotherapy agents (5-fluorouracil, doxorubicin, and gemcitabine) were added into BxPc-3 cells after antisense HIF-1α transfected. The growth inhibition rates were measured by MTT, apoptosis rates were measured by flow cytometry and the expression of survivin was detected by RT-PCR and Western blot.
Results
Higher dosages (100, 200, and 400 mg/L of 5-fluorouracil; 0.05, 0.075, and 0.1 mg/L of doxorubicin; and 10~(-9), 10~(-8), and 10~(-7) mol/L of gemcitabine) caused a greater increase of inhibition in the experimental group than in control (P<0.05). survivin mRNA (0.4601±0.0122) and protein (0.3659±0.0117) expression in the experimental group were markly down-regulated compared with the control (P<0.05).
Conclusion
Our data demonstrate that antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. Part 4 An antisense plasmid targeting HIF-1α expression restrains the progression and metastasis of pancreatic cancer
Objective
To observe the effect of antisense HIF-1α on the progression and metastasis of pancreatic cancer and the relationship with β_1 integrin.
Methods
BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O_2 for 4 hours. The expression of β_1 integrin was detected by RT-PCR and Western blot. The migration of BxPc-3 cells was assayed using transwell cell culture chambers. Subcutaneous transplantation of BxPc-3 cells in nude mice for 8 weeks was to assess progression and metastasis of pancreatic cancer.
Results
β_1 integrin mRNA (0.2568±0.0115) and protein (0.2962±0.0167) expression in the experimental group were markly down-regulated compared with the control (P<0.05).The number of migrated BxPc-3 cells in the experimental group was 110.6±16.4, far less than in control (P<0.05). In vivo, the tumor size and weight in the experimental group were significantly lower than those in control (P<0.05).
Conclusion
Our data demonstrate that antisense HIF-1α inhibits expressions of β_1 integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. In summary, the following points of view outcome:
1. High expression of HIF-1α in pancreatic cancer, but not in non-cancerous adjacent pancreas tissues, suggests it may play an important role in the development of pancreatic cancer.
2. The relationship between HIF-1α expression and the clinical features of pancreatic cancer was discussed. Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients.
3. BxPc-3 with stable antisense HIF-1α expression was successfully constructed. The mRNA and protein expression of HIF-1α was successfully inhibited by the antisense HIF-1α plasmid under hypoxia.
4. Under hypoxia, antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Therefore, HIF-1α may play a very important role in chemosensitivity of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.
5. Under hypoxia, antisense HIF-1α inhibits expressions of β_1 integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.
近年来发现，缺氧是影响化疗效果而且干扰了肿瘤对放疗的反应的一个重要因素。胰腺癌血供很差，在严重缺氧时还能继续增殖的原因可能在于持续缺氧的状态诱导了胰腺癌中缺氧诱导因子(HIF)-1α的表达。研究表明，血管内皮生长因子(VEGF)和糖酵解过程中多种关键酶基因都是受HIF-1α调控的，HIF-1α通过促进肿瘤组织的血管生成和能量代谢加速肿瘤组织的生长，同时也促进了肿瘤的侵袭和转移。HIF-1α在胰腺癌中广泛表达，对胰腺癌的血管生成、发生发展起到了很大的作用。而已有实验表明，人类凋亡抑制蛋白(human inhibitor of apoptosis protein，hIAP)家族成员之一survivin在胰腺癌中高表达，且与化疗敏感性有关；整合素家族中β_1-integrin在胰腺癌侵袭、转移过程中也起着重要的作用。
本研究旨在观察反义HIF-1α对胰腺癌生长、转移、化疗敏感性的影响及其与survivin、β_1-integrin的关系。本实验共分为以下四部分。
第一部分缺氧诱导因子-1α在胰腺癌中的表达和意义
目的
本研究旨在检测胰腺癌中缺氧诱导因子-1α(HIF-1α)的表达，探讨HIF-1α的表达与临床特征的关系，从而深入探讨其在胰腺癌发生发展中的作用。
材料和方法
运用免疫组织化学技术(streptavidin-peroxidase，SP)检测研究48例胰腺癌及癌旁组织中HIF-1α的表达。
结果
1．48例胰腺癌标本中27例表达HIF-1α蛋白，而在癌旁组织未检测到HIF-1α的表达(P＜0.01)。
2．HIF-1α蛋白的表达与胰腺癌患者的性别、年龄和肿瘤大小无关，与胰腺癌的分化程度及是否转移有关(P＜0.05)。
结论
1．HIF-1α在胰腺癌中高度表达，在癌旁胰腺组织未见表达，可能与胰腺癌的发生发展或与治疗效果有关，但其具体作用及作用机制均有待进一步深入的研究。
2．HIF-1α与胰腺癌患者预后的密切相关。
第二部分重组表达质粒pcDNA3.1(+)／反义HIF-1α的构建及其在人胰腺癌细胞株BxPc-3中的稳定表达
目的
第一部分显示，胰腺癌高表达HIF-1α蛋白，本部分拟构建HIF-1α基因的反义表达核苷酸，经脂质体转染人胰腺癌细胞株BxPc-3，建立稳定表达反义HIF-1α基因的细胞模型，为反义封闭治疗奠定基础。
方法
1．采用PCR方法扩增pCR3.1-FLAG-HIF-1α质粒中反义HIF-1α基因功能片段。
2．定向克隆至真核表达载体pcDNA3.1(+)上。
3．转染至BxPc-3细胞内，经G418筛选，建立稳定表达HIF-1α反义基因的细胞株。
4．RT-PCR和Western blot分析HIF-1α的表达，建立稳定表达HIF-1α反义基因的细胞株。
结果
1．酶切鉴定，凝胶电泳分析，两条带大小分别为721bp和5.4kb，与预期结果符合。
2．脂质体包裹转染BxPc-3后，RT-PCR和Western blot分析证实实验组HIF-1αmRNA表达为0.2367±0.0094，蛋白表达为0.1798±0.0063，与对照组相比明显降低(P＜0.05)。
结论
1．本研究成功构建了HIF-1α的反义核苷酸，与预期结果符合。
2．经脂质体途径转染BxPc-3细胞，该反义核苷酸能够抑制HIF-1α的表达。
3．该反义核苷酸的成功构建为下一步实验研究奠定了基础。
第三部分反义HIF-1α增加化疗药物敏感性的实验研究
目的
本研究拟利用构建的反义HIF-1α，转染BxPc-3细胞后，观察BxPc-3细胞对化疗药物敏感性的变化及其与survivin的关系。
方法
1．将HIF-1α反义核苷酸转染至BxPc-3细胞，经G418筛选后，建立稳定表达反义HIF-1α的BxPc-3细胞株，加入不同浓度的氟尿嘧啶，阿霉素，吉西他宾。
2．MTT检测各组的增殖活性。
3．运用流式细胞仪检测各组细胞凋亡指数。
4．RT-PCR和Western blot分析各组survivin的表达。
结果
1．与对照组相比，实验组的生长抑制率、凋亡率与剂量成正比，高剂量引起高抑制(P＜0.05)。
2．实验组survivin的mRNA表达为0.4601±0.0122，蛋白表达为0.3659±0.0117，与对照组相比明显降低(P＜0.05)。
结论
转染反义HIF-1α基因可明显增强氟尿嘧啶，阿霉素，吉西他宾对胰腺癌细胞的杀伤力，并伴有survivin表达量的明显下调，这表明反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性，因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。
第四部分反义HIF-1α对胰腺癌生长、转移影响的实验研究
目的
本研究拟利用构建的反义HIF-1α，转染BxPc-3细胞后，观察其对胰腺癌生长、转移的影响及其与β_1-integrin的关系。
方法
1．将HIF-1α反义核苷酸转染至BxPc-3细胞，经G418筛选后，建立稳定表达反义HIF-1α的BxPc-3细胞株，Transwell侵袭室方法检测BxPc-3细胞侵袭能力。
2．RT-PCR和Western blot分析各组β_1-integrin的表达。
3．裸鼠皮下接种各组处理细胞8周后，观察其肿瘤生长情况。
结果
1．实验组迁移的细胞数为110.64±16.4，远少于对照组(P＜0.05)。
2．实验组β_1-integrin的mRNA表达为0.2568±0.0115，蛋白表达为0.2962±0.0167，与对照组相比明显降低(P＜0.05)。
3．实验组肿瘤的体积、重量、生长速度远低于对照组(P＜0.05)。
结论
转染反义HIF-1α基因可明显抑制胰腺癌细胞的侵袭力，并伴有β_1-integrin表达量的明显下调且抑制了皮下移植瘤的生长，这表明反义HIF-1α可能通过阻断β_1-integrin的表达而抑制胰腺癌的生长和转移。因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。
本课题提出以下新见解：
1．HIF-1α在胰腺癌中高度表达，在癌旁胰腺组织未见表达，提示HIF-1α在胰腺癌的发生发展或或治疗效果中发生重要的作用。
2．本文探讨了HIF-1α的表达与胰腺癌患者临床特征的关系，结果显示HIF-1α蛋白的表达与胰腺癌患者的性别、年龄和肿瘤大小无关，而与胰腺癌的分化程度及是否转移有关，提示HIF-1α与胰腺癌患者预后的密切相关。
3．本研究成功构建了HIF-1α的反义核苷酸，与预期结果符合。经脂质体途径转染BxPc-3细胞，可在BxPc-3中稳定表达，该反义核苷酸能够抑制HIF-1α的mRNA和蛋白的表达。
4．缺氧状态下，反义HIF-1α基因可明显增强氟尿嘧啶，阿霉素，吉西他宾对胰腺癌细胞的杀伤力，并伴有survivin表达量的明显下调，这表明反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性。
5．缺氧状态下，反义HIF-1α基因可明显抑制胰腺癌细胞的侵袭力，并伴有β_1-integrin表达量的明显下调且抑制了皮下移植瘤的生长，这表明反义HIF-1α可能通过阻断β_1-integrin的表达而抑制胰腺癌的生长和转移。因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric complex, composed of an α and a β subunit, both belonging to the basic helix-loop-helix (bHLH)/Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. The bHLH domain mediates dimerization and DNA binding in many transcription factors. PAS is an additional dimerization motif. Whereas HIF-1β is a common subunit of multiple bHLH proteins, HIF-1α is the unique, O_2-regulated subunit that determines HIF-1 activity. Although HIF-1β is quite stable in normoxic conditions, HIF-1α is extremely unstable and quickly degraded by the ubiquitin-proteasome system. HIF-1α plays an important role in solid tumor formation in vivo by promoting angiogenesis and anaerobic metabolism. Several studies have demonstrated that HIF-1α can be detected in most types of human tumors examined, including bladder, breast, colon, glial, hepatocellular, ovarian, pancreatic, prostate, renal and oesophageal tumors. The expression of HIF-1α is inducible by oxygen concentrations below 6%, and the activation of hypoxia-responsive elements by HIF-1 has been reported to correlate with decreasing oxygen concentrations in vitro.
Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is characterized by a unique structure that discriminates it from other members of the IAP family. It contains only a single BIR repeat and lacks a carboxy terminal RING finger domain. Survivin is expressed in the G_2/M phase of cell cycle in a cycle-regulated manner. It directly binds to and inhibits both Caspase-3 and Caspase-7 activity, leading to arrest of apoptosis. Survivin expression is not detectable in differentiated normal adult cells of any organ, but it is abundantly expressed in embryonic tissues and in a wide range of cancer tissues including neuroblastoma, cholangiocarcinoma, colorectal, stomach, breast and uterine cervical carcinomas. It has been demonstrated recently that survivin is also frequently expressed in malignant pancreatic ductal tumors and pancreatic adenocarcinoma. Furthermore, the prognostic value of survivin expression has been reported in several human cancers. Studies demonstrating resistance of survivin-transfected cells to anticancer drug-induced apoptosis and sensitization to chemotherapy by survivin antisense treatment have shown that survivin is implicated in sensitization to chemotherapy.
The integrin family of cell-surface heterodimeric glycoproteins composed of a and β subunits function primarily as receptors for extracellular matrix ligands, which regulate many aspects of cell physiology, including morphology, adhesion, migration, proliferation, and differentiation. Integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes. Many cancers show abnormalities of integrin function as a result of transformation by oncogenes. More importantly, the growth of several tumors depends on β_1-integrin function. Previous studies have demonstrated that β_1-integrin is a poor prognostic factor in patients with cancers. Evidences show an essential role of β_1-integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.
On the other hand, the prognosis for patients with pancreatic cancer remains poor, prompting the search for new treatment strategies. Although treatments such as pre-operative chemotherapy and chemoradiation therapy are currently used for patients with advanced pancreatic cancer, their results are not satisfactory. Even in early-stage disease, we have experienced many patients who developed local recurrence of tumor or distant metastasis within a short period after operation.
In pancreatic cancer, the relationship of HIF-1α, survivin and β_1-integrin is not very clear at present. This study aimed to observe the effect of antisense HIF-1α on progression, metastasis and chemosensitivity of pancreatic cancer. Part 1 Expression and significance of HIF-1α in pancreatic cancer Objective
To investigate the expression of HIF-1α, the relationship with clinical features, and determine the role in the development of pancreatic cancer.
Methods
The expression of HIF-1α in tumor tissues and non-cancerous adjacent pancreas tissues was detected by SP immunohistochemical technique.
Results
HIF-1α was expressed in 27 of 48 cases (56.3%). In contrast, no expression of HIF-1α in adjacent histologically noncancerous pancreas was detectable (P<0.01) . Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level (P<0.05).
Conclusion
High expression of HIF-1α in pancreatic cancer suggests it may play an important role in the development of pancreatic cancer. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients. Part 2 Reconstruction of eukaryotic gene expression plasmid pcDNA3.1(+) /antisense HIF-1α and its stable expression in human pancreatic cancer cell line BxPc-3
Objective
To further explore the role of HIF-1α, antisense plasmid of HIF-1α was constructed, and transfected into human pancreatic cancer cell line BxPc-3.
Methods
Antisense HIF-1α DNA structure was amplified by PCR and inserted into eukaryotic plasmid pcDNA3.1(+). The expression plasmid pcDNA3.1(+)/antisense HIF-1α was transfected into human pancreatic cancer cell line BxPc-3 through Dosper liposomal transfection reagent. Cells stably expressing antisense HIF-1α were screened with G418. BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O_2 for 4 hours. The expression of HIF-1α was detected by RT-PCR and Western blot.
Results
The constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two fragments at 721bp and 5.4kb, were respectively found, which was consistent with what expected. Eukaryotic plasmid was transfected into BxPc-3 cells through Dosper liposomal transfection reagent. HIF-1α mRNA (0.2367±0.0094) and protein (0.1798±0.0063) expression in the experimental group were significantly depressed by this plasmid, compared with the control (P<0.05).
Conclusion
BxPc-3 with stable antisense HIF-1α expression was successfully constructed, which could be applied to explore further. Part 3 An antisense plasmid targeting HIF-1α expression sensitizes pancreatic cancer cells to chemotherapy
Objective
The apoptosis induced by chemotherapeutic agent was inhibited through HIF-1α expression. Antisense plasmid of HIF-1α was constructed and transfected into BxPc-3. The change of sensitizion to chemotherapy and the relationship with survivin was detected.
Methods
BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O_2 for 4 hours. Different dosages of chemotherapy agents (5-fluorouracil, doxorubicin, and gemcitabine) were added into BxPc-3 cells after antisense HIF-1α transfected. The growth inhibition rates were measured by MTT, apoptosis rates were measured by flow cytometry and the expression of survivin was detected by RT-PCR and Western blot.
Results
Higher dosages (100, 200, and 400 mg/L of 5-fluorouracil; 0.05, 0.075, and 0.1 mg/L of doxorubicin; and 10~(-9), 10~(-8), and 10~(-7) mol/L of gemcitabine) caused a greater increase of inhibition in the experimental group than in control (P<0.05). survivin mRNA (0.4601±0.0122) and protein (0.3659±0.0117) expression in the experimental group were markly down-regulated compared with the control (P<0.05).
Conclusion
Our data demonstrate that antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. Part 4 An antisense plasmid targeting HIF-1α expression restrains the progression and metastasis of pancreatic cancer
Objective
To observe the effect of antisense HIF-1α on the progression and metastasis of pancreatic cancer and the relationship with β_1 integrin.
Methods
BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O_2 for 4 hours. The expression of β_1 integrin was detected by RT-PCR and Western blot. The migration of BxPc-3 cells was assayed using transwell cell culture chambers. Subcutaneous transplantation of BxPc-3 cells in nude mice for 8 weeks was to assess progression and metastasis of pancreatic cancer.
Results
β_1 integrin mRNA (0.2568±0.0115) and protein (0.2962±0.0167) expression in the experimental group were markly down-regulated compared with the control (P<0.05).The number of migrated BxPc-3 cells in the experimental group was 110.6±16.4, far less than in control (P<0.05). In vivo, the tumor size and weight in the experimental group were significantly lower than those in control (P<0.05).
Conclusion
Our data demonstrate that antisense HIF-1α inhibits expressions of β_1 integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. In summary, the following points of view outcome:
1. High expression of HIF-1α in pancreatic cancer, but not in non-cancerous adjacent pancreas tissues, suggests it may play an important role in the development of pancreatic cancer.
2. The relationship between HIF-1α expression and the clinical features of pancreatic cancer was discussed. Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients.
3. BxPc-3 with stable antisense HIF-1α expression was successfully constructed. The mRNA and protein expression of HIF-1α was successfully inhibited by the antisense HIF-1α plasmid under hypoxia.
4. Under hypoxia, antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Therefore, HIF-1α may play a very important role in chemosensitivity of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.
5. Under hypoxia, antisense HIF-1α inhibits expressions of β_1 integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.
引文
1. Patrick HM, Christopher WP, Peter JR. Activation of the HIF pathway in cancer. Current Opin Gen Dev. 2001; 11: 293-299.
2. Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O_2 tension. Am J Physiol. 1996; 271: C1172-C1180.
3. Kallio PJ, Wilson WJ, O'Brien S, et al. Regulation of the hypoxia-inducible transcription factor l alpha by the ubiquitin-proteasome pathway. J Biol Chem. 1999; 274: 6519-6525.
4. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 1998; 17: 3005-3015.
5. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-lalpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Path. 2000; 157: 411-421.
6. Kurokawa T, Miyamoto M, Kato K, et al. Overexpression of hypoxia-inducible-factor 1 alpha (HIF-lalpha) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer. 2003; 89: 1042-1047.
7. Andrew LK, Stream W, Jeffery M, et al. Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nature Med. 2000; 12: 1335-1340.
8. Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci USA. 1997; 94: 8104-8109.
9. Boring C, Squires T, Tong T, et al. Cancer Statistics. Cancer J Clin. 1993; 43: 7-26.
10. Flanders TY, Foulkes WD. Pancreatic adenocarcinoma: epidemiology and genetics. J Med Genet. 1996; 33: 889-898.
11. Sener SF, Fremgen A, Menck HR, et al. Pancreatic cancer: a report of treatment and survival trends for 100,313 patients diagnosed from 1985-1995, using the National Cancer Database. J Am Coll Surg. 1999; 189: 1-7.
1. Kopper L, Kovalszky I. Antisense tumor therapy (a dream under construction). In Vivo. 1994; 8: 781-786.
2. Zhang WW, Roth JA. Anti-oncogene and tumor suppressor gene therapy--examples from a lung cancer animal model. In Vivo. 1994; 8: 755-769.
3. Carter G, Lemoine NR. Antisense technology for cancer therapy: does it make sense? Br J Cancer. 1993; 67: 869-876.
4. Weiss B, Davidkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci. 1999; 55: 334-358.
5. Peng KW, Russell SJ. Viral vector targeting. Curr Opin Biotechnol. 1999; 10: 454-457.
6. Kost TA. Expression vectors and delivery systems tools for determining gene function and gene therapy. Curr Opin Biotechnol. 1999; 10: 409-410.
7. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 1999; 10: 440-447.
8. Bueler H. Adeno-associated viral vectors for gene transfer and gene therapy. Biol Chem. 1999; 380: 613-622.
9. Rolling F, Samulski RJ. AAV as a viral vector for human gene therapy. Generation of recombinant virus. Mol Biotechnol. 1995; 3: 9-15.
10. Pitard B, Aguerre O, Airiau M, et al. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer. Proc Natl Acad Sci USA. 1997; 94: 14412-14417.
11. Miwa T, Emi N, Nonami T, et al. Gene transfer with cationic lipid into human hepatocellular carcinoma in nude mice. Hepatogastroenterology. 1999; 46: 825-829.
12. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 1998; 17: 3005-3015.
13. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Path. 2000; 157.: 411-421.
14. Kurokawa T, Miyamoto M, Kato K, et al. Overexpression of hypoxia-inducible-factor 1 alpha (HIF-lalpha) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer. 2003; 89: 1042-1047.
1. Shibaji T, Nagao M, Ikeda N, et al. Prognostic significance of HIF-1 alpha overexpression in human pancreatic cancer. Anticancer Res. 2003, 23: 4721-4727.
2. Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer. 2002, 86: 886-892.
3. Teicher BA. Hypoxia and drug resistance. Cancer Metast Rev. 1994; 13: 139-168.
4. Moulder JE, Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metast Rev. 1987; 5: 313-341.
5. Nunez G, Benedict MA, Hu Y, et al. Caspases: the proteases of the apoptotic pathway. Oncogene. 1998; 17: 3237-3245.
6. Thomberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281: 1312-1316.
7. Micoud F, Mandrand B, Malcus-Vocanson C. Comparison of several techniques for the detection of apoptotic astrocytes in vitro. Cell Prolif. 2001; 34: 99-113.
8. Raber MN, Barlogie B, Latreille J, et al. Ploidy, proliferative activity and estrogen receptor content in human breast cancer. Cytometry. 1982; 3: 36-41.
9. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992; 119: 493-501.
10. Silverstein MJ, Handel N, Gamagami P, et al. Breast cancer diagnosis and prognosis in women following augmentation with silicone gel-filled prostheses. Eur JCancer. 1992; 28: 635-640.
11. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980; 284: 555-556.
12. Dimmeler S, Haendeler J, Nehls M, et al. Suppression of apoptosis by nitric oxide via inhibition of interleukin-lbeta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-1ike proteases. J Exp Med. 1997; 185: 601-607.
13. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65: 55-63.
14. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986; 89: 271-277.
15. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998; 396: 580-583.
16. Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 1998; 58: 5315-5320.
17. Suzuki A, Ito T, Kawano H, et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with CDK4 to resist Fas-mediated cell death. Oncogene. 2000; 19: 1346-1353.
18. Ambrosini G, Adida C, Altieri DC. A novel antiapoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997; 3: 917-921.
19. Yamamoto T, Tanigawa N. The role of survivin as a new target of diagnosis and treatment in human cancer. Med Electron Microsc. 2001; 34: 207-212.
20. Adida C, Berrebi D, Peuchmaur M, et al. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet. 1998, 351: 882-883.
21. Kawasaki H, Altieri DC, Lu CD, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998; 58: 5071-5074.
22. Lu CD, Altieri DC, Tanigawa N. Expression of novel anti-apoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res. 1998; 58:1808-1812.
23. Tanaka K, Iwamoto S, Gon G, et al. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000; 6: 127-134.
24. Satoh K, Kaneko K, Hirota M, et al. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer. 2001; 92: 271-278.
25. Sarela AI , Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J cancer. 2002; 86: 886-892.
26. Altieri DC, Marchisio C. Survivin Apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest. 1999; 79: 1327-1333.
27. Cohen C, Lohmann CM, Cotsonis G, et al. Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis. Mod Pathol. 2003; 16: 574-583.
28. Ikeguchi M, Ueda T, Sakatani T, et al. Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma. Diagn Mol Pathol. 2002; 11: 33-40.
29. Ikehara M, Oshita F, Kameda Y, et al. Expression of survivin correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep. 2002; 9: 835-838.
30. Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer. 2002; 87: 883-887.
31. Takai N, Miyazaki T, Nishida M, et al. Survivin expression correlates with clinical stage, histological grade, invasive behavior and survival rate in endometrial carcinoma. Cancer Lett. 2002; 184: 105-116.
32. Kappler M, Kotzsch M, Bartel F, et al. Elevated expression level of survivin protein in soft-tissue sarcomas is a strong independent predictor of survival. Clin Cancer Res. 2003; 9: 1098-1104.
33. Kennedy SM, O'Driscoll L, Purcell R, et al. Prognostic importance of survivin in breast cancer. BrJ Cancer. 2003; 88: 1077-1083.
34. Olie RA, Simoes-Wust AP, Baumann B, et al. A novel antisense nucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res. 2000; 60: 2805-2809.
35. Ikeguchi M, Nakamura S, Kaibara N. Quantitative analysis of expression levels of bax, bcl-2, and survivin in cancer cells during cisplatin treatment. Oncol Rep. 2002; 9:1121-1126.
36. Zaffaroni N, Pennati M, Colella G, et al. Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer. Cell Mol Life Sci. 2002; 59: 1406-1412.
37. Zaffaroni N, Daidone MG Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat. 2002; 5: 65-72.
38. Xiang R, Mizutani N, Luo Y, et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res. 2005; 65: 553-561.
39. Altieri D. Blocking survivin to kill cancer cells. Methods Mol Biol. 2003; 223: 533-542.
40. Yamamoto T, Manome Y, Nakamura M, et al. Downregulation of survivin expression by induction of the effector cell protease receptor-1 reduces tumor growth potential and results in an increased sensitivity to anticancer agents in human colon cancer. Eur J Cancer. 2002; 38: 2316-2324.
41. Chen T, Tian FZ, Cai ZH, et al. The signal transduction pathway related to hepatocellular carcinoma apoptosis induced by survivin antisense oligonucleotide. Chin JMed. 2003; 83: 425-429.
42. Asanuma K, Moriai R, Yajima T, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res. 2000; 91: 1204-1209.
43. Asanuma K, Kobayashi D, Furuya D, et al. A role for survivin in radioresistance of pancreatic cancer cells. JpnJ Cancer Res. 2002; 93: 1057-1062.
44. Rodel C, Haas J, Groth A, Grabenbauer GG, Sauer R, Rodel F. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys. 2003; 55: 1341-1347.
45. Pennati M, Binda M, Colella G, Folini M, Citti L, Villa R, Daidone MG, Zaffaroni N. Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression. J Invest Dermatol. 2003; 120: 648-654.
46. Wei HY, Chen LB, Wang CY. Correlation of mRNA expression of hypoxia inducible factor-1 alpha to biological features of pancreatic cancer. A i Zheng. 2005; 24: 184-188.
1. Shibaji T, Nagao M, Ikeda N, et al. Prognostic significance of HIF-1 alpha overexpression in human pancreatic cancer. Anticancer Res. 2003; 23: 4721-4727.
2. Arao S, Masumoto A, Otsuki M. Betal integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells. Pancreas. 2000; 20: 129-137.
3. Buchholz M, Biebl A, Neebetae A, et al. SERPINE2 (protease nexin Ⅰ) promotes extracellular matrix production and local invasion of pancreatic tumors in vivo. Cancer Res. 2003; 63: 4945-4951.
4. Bourguignon LY, Zhu H, Shao L, et al. CD44 interaction with tiaml promotes Racl signaling and hyaluronic acid-mediated breast tumor cell migration, J Biol Chem. 2000; 275: 1829-1838.
5. Luparello C, Sirchia R, Pupello D. PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression. J Cell Sci. 2003; 116: 2421-2430.
6. Li DQ, Wang ZB, Bai J, et al. Effects of mifepristone on invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo. World J Gastroenterol. 2004; 10: 1726-1729.
7. Hou M, Tan L, Wang X, et al. Antisense Tiaml down-regulates the invasiveness of 95D cells in vitro. Acta Biochim Biophys Sin. 2004; 36: 537-540.
8. Teicher BA. Hypoxia and drug resistance. Cancer Metast Rev. 1994; 13: 139-168.
9. Moulder JE, Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metast Rev. 1987; 5: 313-341.
10. Evan G, Littlewood T. A matter of life and cell death. Science. 1998; 281: 1317-1322.
11. Liotta LA. Tumor invasion and metastases: role of the basement membrane. Am J Pathol. 1986; 117: 339-348.
12. Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987; 47: 3239-3245.
13. Kleinman HK, McGarvey ML, Hassell JR, et al. Basement membrane complexes with biological activity. Biochem. 1986; 25: 312-318.
14. Vukicevic S, Kleinman HK, Luyten FP, et al. Identification of multip le active growth factors in basement membrane matrigel suggests caution in interp retation of cellular activity related to extracelmlarmatrix components. Exp Cell Res. 1992; 202: 1-8.
15. Mackay AR. Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase type IV collagenase in preparations of laminin and Matrigel. Biotechniques. 1993; 15: 1048-1051.
16. McGuire PG, Seeds NW. The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells. J Cell Biochem. 1989; 40: 215-227.
17. Kleinman HK, McGarvey ML, Liotta LA, et al. Isolation and characterization of type IV p rocollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982; 21: 6188-6193.
18. Bloomfield KL, Baldwin BL, Harkin DG, et al. Modification of the Boyden chamber to improve uniformity of cell invasion of matrigel-coated membranes. Biotechniques. 2001; 31: 1242-1246.
19. Vipra MR, Chiplonkar JM. Vital stain cell invasion in modified Boyden chamber assay. Biotechniques. 2002; 33: 1200-1202.
20. Giancotti FG, Ruoslahti E. Intgrin signaling. Science. 1999; 285: 1028-1033.
21. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer. 2002; 2: 91-100.
22. Green LJ, Mould AP, Humphries MJ. The integrin beta subunit. Int J Biochem Cell Biol. 1998; 30: 179-184.
23. Koukoulis GK, Patriarca C, Gould VE. Adhesion molecules and tumor metastasis. Hum Pathol. 1998; 29: 889-892.
24. Adachi M, Taki T, Higashiyama M, et al. Significance of integrin alpha5 gene expression as a prognostic factor in node-negative non-small cell lung cancer. Clin Cancer Res. 2000; 6: 96-101.
25. Blaschke F, Stawowy P, Goetze S, et al. Hypoxia activates β_1-integdn via ERK 1/2 and p38 MAP kinase in human vascular smooth musele cells. Biochem Biophys Res Commun. 2002; 296: 890-896.
1. Xiong HQ. Molecular targeting therapy for pancreatic cancer. Cancer Chemother Pharmacol. 2004; 54: S69-77.
2. Lebedeva IV, Sarkar D, Su ZZ, et al. Molecular target-based therapy of pancreatic cancer. Cancer Res. 2006; 66: 2403-2413.
3. Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nat Rev Cancer. 2002; 2: 897-909.
4. Jaffee EM, Hruban RH, Canto M, Kern SE. Focus on pancreas cancer. Cancer Cell. 2002; 2: 25-28.
5. Rosenberg L. Pancreatic cancer: a review of emerging therapies. Drugs. 2000; 59: 1071-1089.
6. Mulvihill S, Warren R, Venook A, et al. Safety and feasibility of injection with an E1B-55 kDa gene deleted, replication selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase Ⅰ trial. Gene Ther. 2001; 8: 308-315.
7. Tango Y, Fujiwara T, Itoshima T, et al. Adenovirus-mediated p14ARF gene transfer cooperates with Ad5CMV-p53 to induce apoptosis in human cancer cells. Hum Gene Ther. 2002; 13: 1373-1382.
8. Hecht JR, Bedford R, Abbruzzese JL, et al. A Phase IPII Trial of Intraturnoral Endoscopic Ultrasound Injection of ONYX-015 with Intravenous Gemcitabine in Unresectable Pancreatic Carcinoma. Clin Cancer Res. 2003; 9: 555-561.
9. Wesseling JG, Yamamoto M, Adachi Y, et al. Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic carcinoma. Cancer Gene Ther. 2001; 8: 990-996.
10. Ohashi M, Kanai F, Tanaka T, et al. In vivo adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing pancreatic cancer. Jpn J Cancer Res. 1998; 89: 457-462.
11. Carrio M, Romagosa A, Mercade E, et al. Enhanced pancreatic tumor regression by a combination of adenovirus and retrovirus-mediated delivery of the herpes simplex virus thymicline kinase gene. Gen Ther. 1999; 6: 547-553.
12. McNeish IA, Green NK, Gilligan MG, et al. Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954. Gene Ther. 1998; 5: 1061-1069.
13. Miki K, Xu M, Gupta A, et al . Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate. Cancer Res. 2001; 61: 6805-6810.
14. Calbo J, Marotta M, Cascallo M, et al. Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer. Cancer Gene Ther. 2001; 8: 740-750.
15. Hwang RF,Gordon EM,Anderson WF, et al . Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene. Surgery. 1998; 124: 143-150.
16. Muscarella P, Knobloch TJ, Ulrich AB, et al. Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16( INK-a) and p15 ( INK-b) cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells. Gene. 2001; 278: 235-243.
17. Adjei AA, Dy GK, Erlichman C, et al. A phase I trial of ISIS 2503, an antisense inhibitor of H-ras ,in combination with gemcitabine in patients with advanced cancer. Clin Cancer Res. 2003; 9: 115-123.
18. Sato T, Yamauchi N, Sasaki H, et al . An apoptosis-inducing gene therapy for pancreatic cancer with a combination of 55-KDA tumor necrosis factor (TNF) receptor gene transfection and mutein TNF administration. Cancer Res. 1998; 58: 1677-1683.
19. Detjen KM, Farwig K, Welzel M, et al. Interferon gamma inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis. Gut. 2001; 49: 251-262.
20. Putzer BM, Rodicker F, Hitt MM, et al. Improved treatment of pancreatic cancer by IL-12 and B7. 1 costimulation: antitumor efficacy and immunoregulation in a nonimmunogenic tumor model. Mol Ther. 2002; 5: 405-412.
21. Zalatnai A, Szegedi Z, Bocsi J. Flow cytometric evidence of apoptosis in human pancreatic cancer xenografts treated with sandosta (octreotide). Anticancer Res. 2000; 20:1663-1666.
22. Hofsli E. The somatostatin receptor family-2a window against new diagnosis and therapy of cancer. Tidsskr Nor Laegeforen. 2002; 122: 487-491.
23. Fisher WE, Wu Y, Amaya F, et al. Somatostatin receptor subtype 2 gene therapy inhibits pancreatic cancer in vitro. J Surg Res. 2002; 105:58-64.
24. Kasuya H, Nishiyama Y, Nomoto S. Itraperitoneal delivery of hrR3 and ganciclover prolongs smwival in mice with disseminated pancreatic cancer. J Surg Oncol. 1999; 72: 136-141.
25. Yew PR, Berk AJ. Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature. 1992; 357: 82-85.
26. Goodrum FD, Ornelles DA. p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lyric infection. J Virol. 1998; 72: 9479-9490.
27. Matthew H, Daniel E, Shinako BS, et al. Gene therapy of pancreatic cancer with green fluorescent protein and tumor necrosis factor-related apoptosis-inducing ligand fusion gene expression driven by a human telomerase reverse transcriptase promoter. Ann Surg Oncol. 2003; 10: 762-772.
28. Baker AH,Georege SJ, Zattsman AB, et al. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP3. Br J Cancer. 1999; 79: 1347-1355.
29. Rooney PH, Murray GI, Stevenson DA, et al. Comparative genomic hybridization and chromosomal instability in solid tumors. Br J Cancer. 1999; 80: 862-873.
30. Gilliam AD, Watson SA. Emerging biological therapies for pancreatic carcinoma. Eur J Surg Oncol. 2002; 28: 370-378.
1. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992; 12: 5447-5454.
2. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor-1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular 02 tension. Proe Natl Acad Sci USA. 1995; 92: 5510-5514.
3. Semenza GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochern Pharmacol. 2000; 59: 47-53.
4. Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J. 2002; 16: 1151-1162.
5. Semenza GL. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol Med. 2001; 7: 345-350.
6. Jewell UR, Kvietikova I, Scheid A, et al. Induction of HIF-1α in response to hypoxia is instantaneous. FASEBJ. 2001; 15: 1312-1314.
7. Salceda S, Caro J. Hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem. 1997; 272: 22642-22647.
8. Huang LE, Gu J, Schau M, et al. Regulation of hypoxia-inducible factor-Is is mediated by an oxygen-dependent domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci. 1998; 95: 7987-7992.
9. Maxwell PH, Wiesener MS, Chang GW, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999; 399: 271-275.
10. Ivan M, Kaelin WG Jr. The yon Hippel-Lindau tumor suppressor protein. Curr Opin Genet Dev. 2001; 11: 27-34.
11. Ivan M, Kondo K, Yang H, et al. HIF-1α targeted for VHL-mediated destruction by proline hydroxylation: implications for 02 sensing. Science. 2001; 292: 464-468.
12. Norman M, Carsten W, Patrick H, et al. Independent function of two destruction domains in hypoxia-inducible factor-1α chains activated by prolyl hydroxylation. EMBO J. 2001; 20: 5197-5206.
13. Ravi R, Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor lalpha. Genes Dev. 2000; 14: 34-44.
14. Hammond EM, Denko NC, Dorie MJ, et al. Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol. 2002; 22: 1834-1843.
15. Lars OH, Assaf F, Stefan F, et al. DNA-binding site of p53. Proc Natl Acad Sci. 2002; 99: 10305-10311.
16. Chen D, Li M, Luo J, et al. Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem. 2003; 278: 13595-13598.
17. Alvarez-Tejado M, Alfranca A, Aragones J, et al. Lack of evidence for the involvement of the phosphoinositide 3-kinase/ Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J Biol Chem. 2002; 277: 13508-13517.
18. Zhong H, Agani F, Baccala AA, et al. Increased expression of hypoxia inducible factor-alpha in rat and human prostate cancer. Cancer Res. 1998; 58: 5280-5284.
19. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem. 2002; 277: 38205-38211.
20. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev. 2000; 14: 391-396.
21. Laughner E, Taghavi P, Semenza GL, et al. HER2(neu) signaling increases the rate of hypoxia-inducible factor-lalpha (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell biol. 2001; 21: 3995-4004.
22. Gradin K, Takasaki C, Fujii-Kuriyama Y, et al. The transcriptional activation function of the HIF-like factor requires phosphorylation at a conserved threonine. JBiol Chem. 2002; 277: 23508-23514.
23. Gu J, Milligan J, Huang LE. Molecular mechanism of hypoxia-inducible factor 1 alpha -p300 interaction. A leucinerich interface regulated by a single cysteine. J Biol Chem. 2001; 276: 3550-3554.
24. Lee JW, Lee YC, Na SY, et al. Transcriptional coregulators of the nuclear receptor superfamily: coactivators and corepressors. Cell Mol Life Sci. 2001; 58: 289-297.
25. Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-lalpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 2001; 15: 2675-2686.
26. Lando D, Peer D J, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science. 2002; 295: 858-861.
27. Freedman SJ, Sun ZY, Poy F, et al. Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1 alpha. Proc Natl Acad Sci USA. 2002; 99: 5367-5372.
28. Jorge L, Ruas H, Lorenz P, et al. Functional analysis of hypoxia-inducible factor-1-mediated transactivation. J Biol Chem. 2002; 277: 38723-38729.
29. Maxwell PH, Pugh CW, Ratcliffe PJ. The pVHL-HIF-1 system. A key mediator of oxygen homeostasis. Adv Exp Med Biol. 2001; 502: 365-376.
30. Powis G, Kirkpatrick L. Hypoxia inducible factor-lalpha as a cancer drug target. Mol Cancer Ther. 2004; 3: 647-654.
31. Heather ER, Michelle P, Wayne MN. Hypoxia-inducible factor-laipha is a positive factor in solid tumor growth. Cancer Res. 2000; 60: 4010-4016.
32. Vaupel P. The role of hypoxia-induced factors in tumor progression. Oncologist. 2004; 9: 10-17.
33. Palayoor ST, Tofilon PJ, Coleman CN. Ibuprofen-mediated reduction of hypoxia-inducible factors HIF-lalpha and HIF-2alpha in prostate cancer cells. Clin Cancer Res. 2003; 9: 3150-3157.
34. Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell. 2003; 3: 363-375.
35. Greijer AE, van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol. 2004; 57: 1009-1014.
36. Fan LF, Diao LM, Chen DJ, et al. Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer. Ai Zheng. 2002; 21: 254-258.
37. Piret JP, Mottet D, Raes M, et al. COC12, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2. Ann N Y Acad Sci. 2002; 973: 443-447.
38. Ghafar MA, Anastasiadis AG, Chen MW, et al. Acute hypoxia increases the aggressive characteristics and survival properties of prostate cancer cells. Prostate. 2003; 54: 58-67.
39. Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res. 2002; 62: 3387-3394.
40. Talks KL, Turley H, Gatter KC. The expression and distribution of the hypoxia-inducible Factors HIF-1α and HIF-2α in normal human tissues, cancer, and tumor-associated macrophages. Am J Pathol. 2000; 157: 411-421.
41. Wartenberg M, Ling FC, Muschen M, et al. Regulation of the multidrug resistance MDR transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species. FASEB J. 2003; 17: 503-505.
42. Ruan H, Wang J, Hu L, et al. Killing of brain tumor cells by hypoxia-responsive element mediated expression of BAX. Neoplasia. 1999; 1: 431-437.
43. Ruan H, Su H, Hu L, et al. A hypoxia-regulated adeno-associated virus vector for cancer-specific gene therapy. Neoplasia. 2001; 3: 255-263.
44. Brown JM. Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies. Mol Med Today. 2000; 6: 157-162.
45. Kruger EA, Blagosklonny MV, Dixon SC, et al. UCN-01, a protein kinase C inhibitor, inhibits endothelial cell proliferation and angiogenic hypoxic response. Invasion Metastasis. 1998; 18: 209-218.
46. Mabjeesh NJ, Post DE, Willard MT, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1 alpha protein via the proteosome pathway in prostate cancer cells. Cancer Res. 2002; 62: 2478-2482.
47. Sodhi A, Montaner S, Patel V, et al. The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor lalpha. Cancer Res. 2000; 60: 4873-4880.
48. Kung AL, Wang S, Klco JM, et al. Livingston DM Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nat Med. 2000; 6: 1335-1340.
1. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell. 1997; 88: 347-354.
2. Worf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem. 1999; 274: 20049-20052.
3. Takahashi R, Deveraux Q, Tamm I, et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem. 1998; 273: 7787-7790.
4. Ambrosini G, Adida C, Sirugo G, et al. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem. 1998; 273: 11177-11182.
5. Nunez G, Benedict MA, Hu Y, et al. Caspases: the proteases of the apoptotic pathway. Oncogene. 1998; 17: 3237-3245.
6. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281: 1312-1316.
7. Ambrosini G, Adida C, Altieri DC. A novel antiapoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997; 3: 917-921.
8. Adida C, Crotty PL, McGrath J, et al. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol. 1998; 152: 43-49.
9. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998; 396: 580-583.
10. Grossman D, Kim PJ, Blanc-Brude OP, et al. Transgenic expression of survivin in keratinocytes counteracts UVB induced apoptosis and cooperates with loss of p53. J Clin Invest. 2001; 108: 991.
11. Shin S, Sung BJ, Cho YS, et al. An anti-apoptotic protein human survivin is a direct inhibitor of Caspase-3 and -7. Biochemistry. 2001; 40: 1117-1123.
12. Suzuki A, Ito T, Kawano H, et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with CDK4 to resist Fas-mediated cell death. Oncogene. 2000; 19: 1346-1353.
13. Jiang X, Wilford C, Duensing S, et al. Participation of surviving mitotic and apoptotic activities. J Cell Biochem. 2001; 83: 1.
14. Zhao J, Tenev T, Martins LM, et al. The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J Cell Sci. 2000; 113: 4363-4371.
15. Giodini A, Kallio MJ, Wall NR, et al. Regulation of microtubule stability and mitotic progression by survivin. Cancer Res. 2002; 62: 2462-2467.
16. Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer. 2002; 87: 883-887.
17. Kato J, Kuwabara Y, Mitani M, et al. Expression of survivin in esophageal cancer: correlation with the prognosis and response to chemotherapy. Int J Cancer. 2001; 95: 92-95.
18. Yu T, Leung WK, Ebert MP, et al. Increased expression of suvivin in gastric cancer patients and in first degree relatives. Br J Cancer. 2002; 87: 91.
19. Wakana Y, Kasuya K, Katayanagi S, et al. Effect of survivin on cell proliferation and apoptosis in gastric cancer. Oncol Rep. 2002; 9: 1213-1218.
20. Okada E, Murai Y, Matsui K, et al. Survivin expression in tumor cell nuclei is predictive of a favorable prognosis in gastric cancer patients. Cancer Letts. 2001; 163: 109-116.
21. Ikeguchi M, Liu J, Kaibara N. Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment. Apoptosis. 2002; 7: 23-29.
22. Ikeguchi M, Ueta T, Yamane Y, et al. Inducible nitric oxide synthase and survivin messenger RNA expression in hepatocellular carcinoma. Clin Cancer Res. 2002; 8: 3131-3136.
23. Ito T, Shiraki K, Sugimoto K, et al. Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology. 2000; 31: 1080-1085.
24. Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J cancer. 2002; 86: 886-892.
25. Asanuma K, Moriai R, Yajima T, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res. 2000; 91: 1204-1209.
26. Agui T, McConkey DJ, Tanigawa N. Comparative study of various biological parameters, including expression of survivin, between primary and metastatic human colonic adenocarcinomas. Anticancer Res. 2002; 22: 1769-1776.
27. Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998; 58: 5071-5074.
28. Zaffaroni N, Daidone MG. Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat. 2002; 5: 65-72.
29. Altieri D. Blocking survivin to kill cancer cells. Methods Mol Biol. 2003; 223: 533-542.
30. Olie RA, Simoes-Wust AP, Baumann B, et al. A novel antisense nucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res. 2000; 60: 2805-2809.
31. Yamamoto T, Manome Y, Nakamura M, et al. Downregulation of survivin expression by induction of the effector cell protease receptor-1 reduces tumor growth potential and results in an increased sensitivity to anticancer agents in human colon cancer. Eur J Cancer. 2002; 38: 2316-2324.
32. Chen T, Tian FZ, Cai ZH, et al. The signal transduction pathway related to hepatocellular carcinoma apoptosis induced by survivin antisense oligonucleotide. Chin J Med. 2003; 83: 425-429.
33. Asanuma K, Kobayashi D, Furuya D, et al. A role for survivin in radioresistance of pancreatic cancer cells. Jpn J Cancer Res. 2002; 93: 1057-1062.
34. Rodel C, Haas J, Groth A, et al. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys. 2003; 55: 1341-1347.
35. Pennati M, Binda M, Colella G, et al. Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression. J Invest Dermatol. 2003; 120: 648-654.
36. Mesri M, Wall NR, Li J, et al. Cancer gene therapy using a survivin mutant adenovirus. J Clin Invest. 2001; 108: 981-990.
2. Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O_2 tension. Am J Physiol. 1996; 271: C1172-C1180.
3. Kallio PJ, Wilson WJ, O'Brien S, et al. Regulation of the hypoxia-inducible transcription factor l alpha by the ubiquitin-proteasome pathway. J Biol Chem. 1999; 274: 6519-6525.
4. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 1998; 17: 3005-3015.
5. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-lalpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Path. 2000; 157: 411-421.
6. Kurokawa T, Miyamoto M, Kato K, et al. Overexpression of hypoxia-inducible-factor 1 alpha (HIF-lalpha) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer. 2003; 89: 1042-1047.
7. Andrew LK, Stream W, Jeffery M, et al. Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nature Med. 2000; 12: 1335-1340.
8. Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci USA. 1997; 94: 8104-8109.
9. Boring C, Squires T, Tong T, et al. Cancer Statistics. Cancer J Clin. 1993; 43: 7-26.
10. Flanders TY, Foulkes WD. Pancreatic adenocarcinoma: epidemiology and genetics. J Med Genet. 1996; 33: 889-898.
11. Sener SF, Fremgen A, Menck HR, et al. Pancreatic cancer: a report of treatment and survival trends for 100,313 patients diagnosed from 1985-1995, using the National Cancer Database. J Am Coll Surg. 1999; 189: 1-7.
1. Kopper L, Kovalszky I. Antisense tumor therapy (a dream under construction). In Vivo. 1994; 8: 781-786.
2. Zhang WW, Roth JA. Anti-oncogene and tumor suppressor gene therapy--examples from a lung cancer animal model. In Vivo. 1994; 8: 755-769.
3. Carter G, Lemoine NR. Antisense technology for cancer therapy: does it make sense? Br J Cancer. 1993; 67: 869-876.
4. Weiss B, Davidkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci. 1999; 55: 334-358.
5. Peng KW, Russell SJ. Viral vector targeting. Curr Opin Biotechnol. 1999; 10: 454-457.
6. Kost TA. Expression vectors and delivery systems tools for determining gene function and gene therapy. Curr Opin Biotechnol. 1999; 10: 409-410.
7. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 1999; 10: 440-447.
8. Bueler H. Adeno-associated viral vectors for gene transfer and gene therapy. Biol Chem. 1999; 380: 613-622.
9. Rolling F, Samulski RJ. AAV as a viral vector for human gene therapy. Generation of recombinant virus. Mol Biotechnol. 1995; 3: 9-15.
10. Pitard B, Aguerre O, Airiau M, et al. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer. Proc Natl Acad Sci USA. 1997; 94: 14412-14417.
11. Miwa T, Emi N, Nonami T, et al. Gene transfer with cationic lipid into human hepatocellular carcinoma in nude mice. Hepatogastroenterology. 1999; 46: 825-829.
12. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 1998; 17: 3005-3015.
13. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Path. 2000; 157.: 411-421.
14. Kurokawa T, Miyamoto M, Kato K, et al. Overexpression of hypoxia-inducible-factor 1 alpha (HIF-lalpha) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer. 2003; 89: 1042-1047.
1. Shibaji T, Nagao M, Ikeda N, et al. Prognostic significance of HIF-1 alpha overexpression in human pancreatic cancer. Anticancer Res. 2003, 23: 4721-4727.
2. Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer. 2002, 86: 886-892.
3. Teicher BA. Hypoxia and drug resistance. Cancer Metast Rev. 1994; 13: 139-168.
4. Moulder JE, Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metast Rev. 1987; 5: 313-341.
5. Nunez G, Benedict MA, Hu Y, et al. Caspases: the proteases of the apoptotic pathway. Oncogene. 1998; 17: 3237-3245.
6. Thomberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281: 1312-1316.
7. Micoud F, Mandrand B, Malcus-Vocanson C. Comparison of several techniques for the detection of apoptotic astrocytes in vitro. Cell Prolif. 2001; 34: 99-113.
8. Raber MN, Barlogie B, Latreille J, et al. Ploidy, proliferative activity and estrogen receptor content in human breast cancer. Cytometry. 1982; 3: 36-41.
9. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992; 119: 493-501.
10. Silverstein MJ, Handel N, Gamagami P, et al. Breast cancer diagnosis and prognosis in women following augmentation with silicone gel-filled prostheses. Eur JCancer. 1992; 28: 635-640.
11. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980; 284: 555-556.
12. Dimmeler S, Haendeler J, Nehls M, et al. Suppression of apoptosis by nitric oxide via inhibition of interleukin-lbeta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-1ike proteases. J Exp Med. 1997; 185: 601-607.
13. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65: 55-63.
14. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986; 89: 271-277.
15. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998; 396: 580-583.
16. Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 1998; 58: 5315-5320.
17. Suzuki A, Ito T, Kawano H, et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with CDK4 to resist Fas-mediated cell death. Oncogene. 2000; 19: 1346-1353.
18. Ambrosini G, Adida C, Altieri DC. A novel antiapoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997; 3: 917-921.
19. Yamamoto T, Tanigawa N. The role of survivin as a new target of diagnosis and treatment in human cancer. Med Electron Microsc. 2001; 34: 207-212.
20. Adida C, Berrebi D, Peuchmaur M, et al. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet. 1998, 351: 882-883.
21. Kawasaki H, Altieri DC, Lu CD, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998; 58: 5071-5074.
22. Lu CD, Altieri DC, Tanigawa N. Expression of novel anti-apoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res. 1998; 58:1808-1812.
23. Tanaka K, Iwamoto S, Gon G, et al. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000; 6: 127-134.
24. Satoh K, Kaneko K, Hirota M, et al. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer. 2001; 92: 271-278.
25. Sarela AI , Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J cancer. 2002; 86: 886-892.
26. Altieri DC, Marchisio C. Survivin Apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest. 1999; 79: 1327-1333.
27. Cohen C, Lohmann CM, Cotsonis G, et al. Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis. Mod Pathol. 2003; 16: 574-583.
28. Ikeguchi M, Ueda T, Sakatani T, et al. Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma. Diagn Mol Pathol. 2002; 11: 33-40.
29. Ikehara M, Oshita F, Kameda Y, et al. Expression of survivin correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep. 2002; 9: 835-838.
30. Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer. 2002; 87: 883-887.
31. Takai N, Miyazaki T, Nishida M, et al. Survivin expression correlates with clinical stage, histological grade, invasive behavior and survival rate in endometrial carcinoma. Cancer Lett. 2002; 184: 105-116.
32. Kappler M, Kotzsch M, Bartel F, et al. Elevated expression level of survivin protein in soft-tissue sarcomas is a strong independent predictor of survival. Clin Cancer Res. 2003; 9: 1098-1104.
33. Kennedy SM, O'Driscoll L, Purcell R, et al. Prognostic importance of survivin in breast cancer. BrJ Cancer. 2003; 88: 1077-1083.
34. Olie RA, Simoes-Wust AP, Baumann B, et al. A novel antisense nucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res. 2000; 60: 2805-2809.
35. Ikeguchi M, Nakamura S, Kaibara N. Quantitative analysis of expression levels of bax, bcl-2, and survivin in cancer cells during cisplatin treatment. Oncol Rep. 2002; 9:1121-1126.
36. Zaffaroni N, Pennati M, Colella G, et al. Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer. Cell Mol Life Sci. 2002; 59: 1406-1412.
37. Zaffaroni N, Daidone MG Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat. 2002; 5: 65-72.
38. Xiang R, Mizutani N, Luo Y, et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res. 2005; 65: 553-561.
39. Altieri D. Blocking survivin to kill cancer cells. Methods Mol Biol. 2003; 223: 533-542.
40. Yamamoto T, Manome Y, Nakamura M, et al. Downregulation of survivin expression by induction of the effector cell protease receptor-1 reduces tumor growth potential and results in an increased sensitivity to anticancer agents in human colon cancer. Eur J Cancer. 2002; 38: 2316-2324.
41. Chen T, Tian FZ, Cai ZH, et al. The signal transduction pathway related to hepatocellular carcinoma apoptosis induced by survivin antisense oligonucleotide. Chin JMed. 2003; 83: 425-429.
42. Asanuma K, Moriai R, Yajima T, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res. 2000; 91: 1204-1209.
43. Asanuma K, Kobayashi D, Furuya D, et al. A role for survivin in radioresistance of pancreatic cancer cells. JpnJ Cancer Res. 2002; 93: 1057-1062.
44. Rodel C, Haas J, Groth A, Grabenbauer GG, Sauer R, Rodel F. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys. 2003; 55: 1341-1347.
45. Pennati M, Binda M, Colella G, Folini M, Citti L, Villa R, Daidone MG, Zaffaroni N. Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression. J Invest Dermatol. 2003; 120: 648-654.
46. Wei HY, Chen LB, Wang CY. Correlation of mRNA expression of hypoxia inducible factor-1 alpha to biological features of pancreatic cancer. A i Zheng. 2005; 24: 184-188.
1. Shibaji T, Nagao M, Ikeda N, et al. Prognostic significance of HIF-1 alpha overexpression in human pancreatic cancer. Anticancer Res. 2003; 23: 4721-4727.
2. Arao S, Masumoto A, Otsuki M. Betal integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells. Pancreas. 2000; 20: 129-137.
3. Buchholz M, Biebl A, Neebetae A, et al. SERPINE2 (protease nexin Ⅰ) promotes extracellular matrix production and local invasion of pancreatic tumors in vivo. Cancer Res. 2003; 63: 4945-4951.
4. Bourguignon LY, Zhu H, Shao L, et al. CD44 interaction with tiaml promotes Racl signaling and hyaluronic acid-mediated breast tumor cell migration, J Biol Chem. 2000; 275: 1829-1838.
5. Luparello C, Sirchia R, Pupello D. PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression. J Cell Sci. 2003; 116: 2421-2430.
6. Li DQ, Wang ZB, Bai J, et al. Effects of mifepristone on invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo. World J Gastroenterol. 2004; 10: 1726-1729.
7. Hou M, Tan L, Wang X, et al. Antisense Tiaml down-regulates the invasiveness of 95D cells in vitro. Acta Biochim Biophys Sin. 2004; 36: 537-540.
8. Teicher BA. Hypoxia and drug resistance. Cancer Metast Rev. 1994; 13: 139-168.
9. Moulder JE, Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metast Rev. 1987; 5: 313-341.
10. Evan G, Littlewood T. A matter of life and cell death. Science. 1998; 281: 1317-1322.
11. Liotta LA. Tumor invasion and metastases: role of the basement membrane. Am J Pathol. 1986; 117: 339-348.
12. Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987; 47: 3239-3245.
13. Kleinman HK, McGarvey ML, Hassell JR, et al. Basement membrane complexes with biological activity. Biochem. 1986; 25: 312-318.
14. Vukicevic S, Kleinman HK, Luyten FP, et al. Identification of multip le active growth factors in basement membrane matrigel suggests caution in interp retation of cellular activity related to extracelmlarmatrix components. Exp Cell Res. 1992; 202: 1-8.
15. Mackay AR. Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase type IV collagenase in preparations of laminin and Matrigel. Biotechniques. 1993; 15: 1048-1051.
16. McGuire PG, Seeds NW. The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells. J Cell Biochem. 1989; 40: 215-227.
17. Kleinman HK, McGarvey ML, Liotta LA, et al. Isolation and characterization of type IV p rocollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982; 21: 6188-6193.
18. Bloomfield KL, Baldwin BL, Harkin DG, et al. Modification of the Boyden chamber to improve uniformity of cell invasion of matrigel-coated membranes. Biotechniques. 2001; 31: 1242-1246.
19. Vipra MR, Chiplonkar JM. Vital stain cell invasion in modified Boyden chamber assay. Biotechniques. 2002; 33: 1200-1202.
20. Giancotti FG, Ruoslahti E. Intgrin signaling. Science. 1999; 285: 1028-1033.
21. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer. 2002; 2: 91-100.
22. Green LJ, Mould AP, Humphries MJ. The integrin beta subunit. Int J Biochem Cell Biol. 1998; 30: 179-184.
23. Koukoulis GK, Patriarca C, Gould VE. Adhesion molecules and tumor metastasis. Hum Pathol. 1998; 29: 889-892.
24. Adachi M, Taki T, Higashiyama M, et al. Significance of integrin alpha5 gene expression as a prognostic factor in node-negative non-small cell lung cancer. Clin Cancer Res. 2000; 6: 96-101.
25. Blaschke F, Stawowy P, Goetze S, et al. Hypoxia activates β_1-integdn via ERK 1/2 and p38 MAP kinase in human vascular smooth musele cells. Biochem Biophys Res Commun. 2002; 296: 890-896.
1. Xiong HQ. Molecular targeting therapy for pancreatic cancer. Cancer Chemother Pharmacol. 2004; 54: S69-77.
2. Lebedeva IV, Sarkar D, Su ZZ, et al. Molecular target-based therapy of pancreatic cancer. Cancer Res. 2006; 66: 2403-2413.
3. Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nat Rev Cancer. 2002; 2: 897-909.
4. Jaffee EM, Hruban RH, Canto M, Kern SE. Focus on pancreas cancer. Cancer Cell. 2002; 2: 25-28.
5. Rosenberg L. Pancreatic cancer: a review of emerging therapies. Drugs. 2000; 59: 1071-1089.
6. Mulvihill S, Warren R, Venook A, et al. Safety and feasibility of injection with an E1B-55 kDa gene deleted, replication selective adenovirus (ONYX-015) into primary carcinomas of the pancreas: a phase Ⅰ trial. Gene Ther. 2001; 8: 308-315.
7. Tango Y, Fujiwara T, Itoshima T, et al. Adenovirus-mediated p14ARF gene transfer cooperates with Ad5CMV-p53 to induce apoptosis in human cancer cells. Hum Gene Ther. 2002; 13: 1373-1382.
8. Hecht JR, Bedford R, Abbruzzese JL, et al. A Phase IPII Trial of Intraturnoral Endoscopic Ultrasound Injection of ONYX-015 with Intravenous Gemcitabine in Unresectable Pancreatic Carcinoma. Clin Cancer Res. 2003; 9: 555-561.
9. Wesseling JG, Yamamoto M, Adachi Y, et al. Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic carcinoma. Cancer Gene Ther. 2001; 8: 990-996.
10. Ohashi M, Kanai F, Tanaka T, et al. In vivo adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing pancreatic cancer. Jpn J Cancer Res. 1998; 89: 457-462.
11. Carrio M, Romagosa A, Mercade E, et al. Enhanced pancreatic tumor regression by a combination of adenovirus and retrovirus-mediated delivery of the herpes simplex virus thymicline kinase gene. Gen Ther. 1999; 6: 547-553.
12. McNeish IA, Green NK, Gilligan MG, et al. Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954. Gene Ther. 1998; 5: 1061-1069.
13. Miki K, Xu M, Gupta A, et al . Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate. Cancer Res. 2001; 61: 6805-6810.
14. Calbo J, Marotta M, Cascallo M, et al. Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer. Cancer Gene Ther. 2001; 8: 740-750.
15. Hwang RF,Gordon EM,Anderson WF, et al . Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene. Surgery. 1998; 124: 143-150.
16. Muscarella P, Knobloch TJ, Ulrich AB, et al. Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16( INK-a) and p15 ( INK-b) cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells. Gene. 2001; 278: 235-243.
17. Adjei AA, Dy GK, Erlichman C, et al. A phase I trial of ISIS 2503, an antisense inhibitor of H-ras ,in combination with gemcitabine in patients with advanced cancer. Clin Cancer Res. 2003; 9: 115-123.
18. Sato T, Yamauchi N, Sasaki H, et al . An apoptosis-inducing gene therapy for pancreatic cancer with a combination of 55-KDA tumor necrosis factor (TNF) receptor gene transfection and mutein TNF administration. Cancer Res. 1998; 58: 1677-1683.
19. Detjen KM, Farwig K, Welzel M, et al. Interferon gamma inhibits growth of human pancreatic carcinoma cells via caspase-1 dependent induction of apoptosis. Gut. 2001; 49: 251-262.
20. Putzer BM, Rodicker F, Hitt MM, et al. Improved treatment of pancreatic cancer by IL-12 and B7. 1 costimulation: antitumor efficacy and immunoregulation in a nonimmunogenic tumor model. Mol Ther. 2002; 5: 405-412.
21. Zalatnai A, Szegedi Z, Bocsi J. Flow cytometric evidence of apoptosis in human pancreatic cancer xenografts treated with sandosta (octreotide). Anticancer Res. 2000; 20:1663-1666.
22. Hofsli E. The somatostatin receptor family-2a window against new diagnosis and therapy of cancer. Tidsskr Nor Laegeforen. 2002; 122: 487-491.
23. Fisher WE, Wu Y, Amaya F, et al. Somatostatin receptor subtype 2 gene therapy inhibits pancreatic cancer in vitro. J Surg Res. 2002; 105:58-64.
24. Kasuya H, Nishiyama Y, Nomoto S. Itraperitoneal delivery of hrR3 and ganciclover prolongs smwival in mice with disseminated pancreatic cancer. J Surg Oncol. 1999; 72: 136-141.
25. Yew PR, Berk AJ. Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein. Nature. 1992; 357: 82-85.
26. Goodrum FD, Ornelles DA. p53 status does not determine outcome of E1B 55-kilodalton mutant adenovirus lyric infection. J Virol. 1998; 72: 9479-9490.
27. Matthew H, Daniel E, Shinako BS, et al. Gene therapy of pancreatic cancer with green fluorescent protein and tumor necrosis factor-related apoptosis-inducing ligand fusion gene expression driven by a human telomerase reverse transcriptase promoter. Ann Surg Oncol. 2003; 10: 762-772.
28. Baker AH,Georege SJ, Zattsman AB, et al. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP3. Br J Cancer. 1999; 79: 1347-1355.
29. Rooney PH, Murray GI, Stevenson DA, et al. Comparative genomic hybridization and chromosomal instability in solid tumors. Br J Cancer. 1999; 80: 862-873.
30. Gilliam AD, Watson SA. Emerging biological therapies for pancreatic carcinoma. Eur J Surg Oncol. 2002; 28: 370-378.
1. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992; 12: 5447-5454.
2. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor-1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular 02 tension. Proe Natl Acad Sci USA. 1995; 92: 5510-5514.
3. Semenza GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochern Pharmacol. 2000; 59: 47-53.
4. Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J. 2002; 16: 1151-1162.
5. Semenza GL. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol Med. 2001; 7: 345-350.
6. Jewell UR, Kvietikova I, Scheid A, et al. Induction of HIF-1α in response to hypoxia is instantaneous. FASEBJ. 2001; 15: 1312-1314.
7. Salceda S, Caro J. Hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem. 1997; 272: 22642-22647.
8. Huang LE, Gu J, Schau M, et al. Regulation of hypoxia-inducible factor-Is is mediated by an oxygen-dependent domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci. 1998; 95: 7987-7992.
9. Maxwell PH, Wiesener MS, Chang GW, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999; 399: 271-275.
10. Ivan M, Kaelin WG Jr. The yon Hippel-Lindau tumor suppressor protein. Curr Opin Genet Dev. 2001; 11: 27-34.
11. Ivan M, Kondo K, Yang H, et al. HIF-1α targeted for VHL-mediated destruction by proline hydroxylation: implications for 02 sensing. Science. 2001; 292: 464-468.
12. Norman M, Carsten W, Patrick H, et al. Independent function of two destruction domains in hypoxia-inducible factor-1α chains activated by prolyl hydroxylation. EMBO J. 2001; 20: 5197-5206.
13. Ravi R, Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor lalpha. Genes Dev. 2000; 14: 34-44.
14. Hammond EM, Denko NC, Dorie MJ, et al. Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol. 2002; 22: 1834-1843.
15. Lars OH, Assaf F, Stefan F, et al. DNA-binding site of p53. Proc Natl Acad Sci. 2002; 99: 10305-10311.
16. Chen D, Li M, Luo J, et al. Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem. 2003; 278: 13595-13598.
17. Alvarez-Tejado M, Alfranca A, Aragones J, et al. Lack of evidence for the involvement of the phosphoinositide 3-kinase/ Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J Biol Chem. 2002; 277: 13508-13517.
18. Zhong H, Agani F, Baccala AA, et al. Increased expression of hypoxia inducible factor-alpha in rat and human prostate cancer. Cancer Res. 1998; 58: 5280-5284.
19. Fukuda R, Hirota K, Fan F, et al. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem. 2002; 277: 38205-38211.
20. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev. 2000; 14: 391-396.
21. Laughner E, Taghavi P, Semenza GL, et al. HER2(neu) signaling increases the rate of hypoxia-inducible factor-lalpha (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell biol. 2001; 21: 3995-4004.
22. Gradin K, Takasaki C, Fujii-Kuriyama Y, et al. The transcriptional activation function of the HIF-like factor requires phosphorylation at a conserved threonine. JBiol Chem. 2002; 277: 23508-23514.
23. Gu J, Milligan J, Huang LE. Molecular mechanism of hypoxia-inducible factor 1 alpha -p300 interaction. A leucinerich interface regulated by a single cysteine. J Biol Chem. 2001; 276: 3550-3554.
24. Lee JW, Lee YC, Na SY, et al. Transcriptional coregulators of the nuclear receptor superfamily: coactivators and corepressors. Cell Mol Life Sci. 2001; 58: 289-297.
25. Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-lalpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 2001; 15: 2675-2686.
26. Lando D, Peer D J, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science. 2002; 295: 858-861.
27. Freedman SJ, Sun ZY, Poy F, et al. Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1 alpha. Proc Natl Acad Sci USA. 2002; 99: 5367-5372.
28. Jorge L, Ruas H, Lorenz P, et al. Functional analysis of hypoxia-inducible factor-1-mediated transactivation. J Biol Chem. 2002; 277: 38723-38729.
29. Maxwell PH, Pugh CW, Ratcliffe PJ. The pVHL-HIF-1 system. A key mediator of oxygen homeostasis. Adv Exp Med Biol. 2001; 502: 365-376.
30. Powis G, Kirkpatrick L. Hypoxia inducible factor-lalpha as a cancer drug target. Mol Cancer Ther. 2004; 3: 647-654.
31. Heather ER, Michelle P, Wayne MN. Hypoxia-inducible factor-laipha is a positive factor in solid tumor growth. Cancer Res. 2000; 60: 4010-4016.
32. Vaupel P. The role of hypoxia-induced factors in tumor progression. Oncologist. 2004; 9: 10-17.
33. Palayoor ST, Tofilon PJ, Coleman CN. Ibuprofen-mediated reduction of hypoxia-inducible factors HIF-lalpha and HIF-2alpha in prostate cancer cells. Clin Cancer Res. 2003; 9: 3150-3157.
34. Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell. 2003; 3: 363-375.
35. Greijer AE, van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol. 2004; 57: 1009-1014.
36. Fan LF, Diao LM, Chen DJ, et al. Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer. Ai Zheng. 2002; 21: 254-258.
37. Piret JP, Mottet D, Raes M, et al. COC12, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2. Ann N Y Acad Sci. 2002; 973: 443-447.
38. Ghafar MA, Anastasiadis AG, Chen MW, et al. Acute hypoxia increases the aggressive characteristics and survival properties of prostate cancer cells. Prostate. 2003; 54: 58-67.
39. Comerford KM, Wallace TJ, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res. 2002; 62: 3387-3394.
40. Talks KL, Turley H, Gatter KC. The expression and distribution of the hypoxia-inducible Factors HIF-1α and HIF-2α in normal human tissues, cancer, and tumor-associated macrophages. Am J Pathol. 2000; 157: 411-421.
41. Wartenberg M, Ling FC, Muschen M, et al. Regulation of the multidrug resistance MDR transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species. FASEB J. 2003; 17: 503-505.
42. Ruan H, Wang J, Hu L, et al. Killing of brain tumor cells by hypoxia-responsive element mediated expression of BAX. Neoplasia. 1999; 1: 431-437.
43. Ruan H, Su H, Hu L, et al. A hypoxia-regulated adeno-associated virus vector for cancer-specific gene therapy. Neoplasia. 2001; 3: 255-263.
44. Brown JM. Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies. Mol Med Today. 2000; 6: 157-162.
45. Kruger EA, Blagosklonny MV, Dixon SC, et al. UCN-01, a protein kinase C inhibitor, inhibits endothelial cell proliferation and angiogenic hypoxic response. Invasion Metastasis. 1998; 18: 209-218.
46. Mabjeesh NJ, Post DE, Willard MT, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1 alpha protein via the proteosome pathway in prostate cancer cells. Cancer Res. 2002; 62: 2478-2482.
47. Sodhi A, Montaner S, Patel V, et al. The Kaposi's sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor lalpha. Cancer Res. 2000; 60: 4873-4880.
48. Kung AL, Wang S, Klco JM, et al. Livingston DM Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nat Med. 2000; 6: 1335-1340.
1. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell. 1997; 88: 347-354.
2. Worf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem. 1999; 274: 20049-20052.
3. Takahashi R, Deveraux Q, Tamm I, et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem. 1998; 273: 7787-7790.
4. Ambrosini G, Adida C, Sirugo G, et al. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem. 1998; 273: 11177-11182.
5. Nunez G, Benedict MA, Hu Y, et al. Caspases: the proteases of the apoptotic pathway. Oncogene. 1998; 17: 3237-3245.
6. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998; 281: 1312-1316.
7. Ambrosini G, Adida C, Altieri DC. A novel antiapoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997; 3: 917-921.
8. Adida C, Crotty PL, McGrath J, et al. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am J Pathol. 1998; 152: 43-49.
9. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998; 396: 580-583.
10. Grossman D, Kim PJ, Blanc-Brude OP, et al. Transgenic expression of survivin in keratinocytes counteracts UVB induced apoptosis and cooperates with loss of p53. J Clin Invest. 2001; 108: 991.
11. Shin S, Sung BJ, Cho YS, et al. An anti-apoptotic protein human survivin is a direct inhibitor of Caspase-3 and -7. Biochemistry. 2001; 40: 1117-1123.
12. Suzuki A, Ito T, Kawano H, et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with CDK4 to resist Fas-mediated cell death. Oncogene. 2000; 19: 1346-1353.
13. Jiang X, Wilford C, Duensing S, et al. Participation of surviving mitotic and apoptotic activities. J Cell Biochem. 2001; 83: 1.
14. Zhao J, Tenev T, Martins LM, et al. The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J Cell Sci. 2000; 113: 4363-4371.
15. Giodini A, Kallio MJ, Wall NR, et al. Regulation of microtubule stability and mitotic progression by survivin. Cancer Res. 2002; 62: 2462-2467.
16. Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer. 2002; 87: 883-887.
17. Kato J, Kuwabara Y, Mitani M, et al. Expression of survivin in esophageal cancer: correlation with the prognosis and response to chemotherapy. Int J Cancer. 2001; 95: 92-95.
18. Yu T, Leung WK, Ebert MP, et al. Increased expression of suvivin in gastric cancer patients and in first degree relatives. Br J Cancer. 2002; 87: 91.
19. Wakana Y, Kasuya K, Katayanagi S, et al. Effect of survivin on cell proliferation and apoptosis in gastric cancer. Oncol Rep. 2002; 9: 1213-1218.
20. Okada E, Murai Y, Matsui K, et al. Survivin expression in tumor cell nuclei is predictive of a favorable prognosis in gastric cancer patients. Cancer Letts. 2001; 163: 109-116.
21. Ikeguchi M, Liu J, Kaibara N. Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment. Apoptosis. 2002; 7: 23-29.
22. Ikeguchi M, Ueta T, Yamane Y, et al. Inducible nitric oxide synthase and survivin messenger RNA expression in hepatocellular carcinoma. Clin Cancer Res. 2002; 8: 3131-3136.
23. Ito T, Shiraki K, Sugimoto K, et al. Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology. 2000; 31: 1080-1085.
24. Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J cancer. 2002; 86: 886-892.
25. Asanuma K, Moriai R, Yajima T, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res. 2000; 91: 1204-1209.
26. Agui T, McConkey DJ, Tanigawa N. Comparative study of various biological parameters, including expression of survivin, between primary and metastatic human colonic adenocarcinomas. Anticancer Res. 2002; 22: 1769-1776.
27. Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998; 58: 5071-5074.
28. Zaffaroni N, Daidone MG. Survivin expression and resistance to anticancer treatments: perspectives for new therapeutic interventions. Drug Resist Updat. 2002; 5: 65-72.
29. Altieri D. Blocking survivin to kill cancer cells. Methods Mol Biol. 2003; 223: 533-542.
30. Olie RA, Simoes-Wust AP, Baumann B, et al. A novel antisense nucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res. 2000; 60: 2805-2809.
31. Yamamoto T, Manome Y, Nakamura M, et al. Downregulation of survivin expression by induction of the effector cell protease receptor-1 reduces tumor growth potential and results in an increased sensitivity to anticancer agents in human colon cancer. Eur J Cancer. 2002; 38: 2316-2324.
32. Chen T, Tian FZ, Cai ZH, et al. The signal transduction pathway related to hepatocellular carcinoma apoptosis induced by survivin antisense oligonucleotide. Chin J Med. 2003; 83: 425-429.
33. Asanuma K, Kobayashi D, Furuya D, et al. A role for survivin in radioresistance of pancreatic cancer cells. Jpn J Cancer Res. 2002; 93: 1057-1062.
34. Rodel C, Haas J, Groth A, et al. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys. 2003; 55: 1341-1347.
35. Pennati M, Binda M, Colella G, et al. Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression. J Invest Dermatol. 2003; 120: 648-654.
36. Mesri M, Wall NR, Li J, et al. Cancer gene therapy using a survivin mutant adenovirus. J Clin Invest. 2001; 108: 981-990.