肺癌低灌注与乏氧的检测、干预及机制探讨
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摘要
第一部分肺癌低灌注与乏氧的检测与干预
肿瘤血管生成(angiogenesis)是指新生血管在肿瘤现有血管基础上形成的过程。研究证实,如果没有新生血管的支持,实体组织中的肿瘤生长直径不会超过2mm。Folkman J.等在转基因动物肿瘤模型的实验中发现,在原位癌向浸润癌发展的过程中,往往伴随着新生血管生成的开始。随后大量的临床研究也证实,血管生成与肿瘤的生长、恶变、转移,以及肿瘤病人的预后有着密切的关系。在过去的几年里,肿瘤的抗血管生成治疗引起了人们极大的研究兴趣,各种抗血管生成药物现已进入各期临床试验阶段。检测肿瘤内部血管生成可通过CT或MRI灌注显像或者SPECT等,肝脏、脑肿瘤的研究较多,而肺部肿瘤灌注的研究很少,目前的研究注意力集中在肿瘤的高灌注,针对肿瘤低灌注特别是肺部肿瘤低灌注的研究尚未见报道。
肿瘤低灌注与高灌注同样有着重要意义,微观方面,①肿瘤内低灌注限制了化疗药物的运送,导致药物在瘤体内不均匀分布达不到有效的最大治疗水平的血药浓度,特别对血管内半衰期短的药物更明显;②低灌注直接导致乏氧,糖酵解增加细胞内变酸,对一些化疗药物不敏感,乏氧促使细胞在基因和蛋白质水平发生改变使低灌注区对多种化疗药物耐药;③乏氧的瘤细胞也易于转移,近期的研究表明急性乏氧更易增加转移机会,新生血管结构缺陷,通透性增加以促进了肿瘤的转移。从宏观角度考虑,肿瘤的发生、生长、转移是肿瘤与宿主相互选择与适应的结果,低灌注与乏氧是肿瘤原发部位不能满足其生长需要,使得选择耐受乏氧的瘤细胞,并使瘤细胞脱落转移至其他部位寻找适宜生存的空间。单独抑制血管生成,我们很难阻断促血管生成的所有途径,而且这可能加速上述的选择,使得部分瘤细胞耐药、抵抗放疗,易于转移。
乏氧,作为低灌注的一个主要后果,分为急性和慢性乏氧,通过乏氧诱导因子(HIF)促进血管生成因子等基因的表达,促进血管生成,抑制抑癌基因如野生型P53表达,激活癌基因表达,肿瘤的恶性程度更高,耐受放化疗和化疗。乏氧的检测主要有PET和MRI两种方法,前者采用硝基咪唑类示踪剂,因其毒性较大,空间分辨率低(仅4-5mm),费用昂贵,应用前景不如MRI。最早用于脑功能研究的血氧水平依赖成像(BOLD)技术相对成熟安全,是较好的选择。针对乏氧的治疗措施包括适形放疗,乏氧细胞毒药物,乏氧细胞增敏剂,卡波金吸入等,这些方法多是针对改善肿瘤对放疗耐受,而改善乏氧区对化疗耐受的研究鲜见报道。
七叶一枝花,为百合科植物华重楼(Paris polyphylla Smith var.chinensisHara)的干燥根茎,含甾体皂苷称为蚤休苷及蚤体土宁苷,后者水解后生成薯蓣苷。有用于抗肿瘤治疗的记载。其水提物及正丁醇提取物有体外抗肿瘤作用。我们的预试验显示七叶一枝花提取物可能对乏氧造成的肿瘤进展有特殊效果。
综上所述,肺癌低灌注乏氧区对于其生长、恶变、转移的重要意义,我们提出血管生成及氧供调节治疗(而不是单纯的抗血管治疗)的假设,通过兔肺移植瘤模型,改良验证检测低灌注和乏氧的方法,从而使预测转移等预后提供帮助;探索七叶一枝花调节肿瘤内部血流灌注,平衡氧供的可行性,以提高肺癌的治疗效果。
目的
1.建立兔肺移植瘤模型,观察乏氧和灌注对预后的影响。
2.观察乏氧低灌注区域内瘤细胞生物学特性。
3.初步探讨七叶一枝花的抗肿瘤作用特别是对低灌注及乏氧的影响。
方法
1.所有实验选用新西兰大白兔。采用瘤组织或细胞悬液直接注入肺及后下肢肌肉内建立移植瘤模型。
2.验证MRI灌注显像及BOLD氧水平显像的准确性。3只荷瘤兔行MRI灌注及BOLD检测后与OxyLite氧电极结果相比较。并比较灌注显像与BOLD氧水平显像的一致性。
3.MRI BOLD及灌注显象与肿瘤预后相关性检测。1)选取10只雄性新西兰大白兔全身麻醉后仰卧位固定,将0.1ml瘤组织悬液经右前胸壁穿刺注入。2) 10天后行胸部MRI检查,观察成瘤情况。3) 20天后行MRI灌注扫描及BOLD显像。4) 30天时复查常规MRI测量肿瘤大小。5) 45天复查MRI测量肿瘤大小,观察转移情况。继续喂养带瘤兔至死亡。
4.观察低灌注区和乏氧区瘤组织相对非低灌注、乏氧区生长和转移的差异。取3周左右下肢带瘤兔一只,行MRI灌注、BOLD及CT扫描,选取低灌注/乏氧区(A区)及非低灌注/乏氧区(B区),将MR与CT图像融合后,在CT引导下穿刺分别制成细胞悬液A、B备用。
4.1将上述细胞悬液A、B在低氧(1%O_2,5%CO_2)及正常氧分压下培养48h。四氮唑盐(MTT)检测细胞增值及ELISA组蛋白-DNA片断检测调亡,实时定量RT-PCR检测VEGF、HIF表达。
4.2取20只健康兔,按体重性别配比分为A、B两组,将AB悬液分别接种于右上肺内,每10天扫描观察其生长速度及转移情况,一月取活检标本通过realtime RT-PCR检测VEGF、HIF表达。
5.观察七叶一枝花低灌注区及乏氧区的影响
5.1以方法4中的描述制备乏氧区细胞悬液,加入七叶一枝花水提液后在低氧及正常氧分压下培养48h,检测瘤细胞增殖及凋亡的变化。同时行实时定量RT-PCR检测VEGF、HIF表达的改变。
5.2取50只健康兔,随机分为5组(D.高剂量七叶一枝花灌胃、E.高剂量七叶一枝花灌胃加5-FU化疗,F.5-FU化疗,G.低剂量七叶一枝花灌胃,H.对照)按照前述方法观察七叶一枝花对肿瘤生长、转移及乏氧灌注的影响。
结果
1.瘤组织悬液穿刺注入法肺癌模型建立成功率92%。有6%早期出现胸水或胸壁结节,予以剔出。
2.BOLD结果与OxyLite氧电极结果一致性较好。
3.a MRI灌注与BOLD有良好相关性,二者分布区域一致。b高灌注区体积比例与肿瘤大小,生长速度呈正相关,c低灌注区体积及程变与转移高度正相关,与生存期成负相关。灌注值离均差大小与转移率呈正相关,与生存期呈负相关。BOLD高信号区比例与肿瘤转移、生存期明显相关。
4.1)来源于乏氧区的瘤组织(A组)与源于非乏氧区的瘤组织(B组)在低氧条件下培养生长活性显著高于非乏氧区对照细胞。RT-PCR结果显示A组HIFI显著高于B组,VEGF亦高于B组,但不如HIFI升高显著。MTT法细胞增殖速度A组高于B组(P＜0.05)调亡A组低于B组(P＜0.01)。
2) A组移植瘤生长速度快于B组A组荷瘤兔生存期短于B组(P＜0.01)。A组转移率高于B组,平均转移时间亦早。一月时活检标本mRNA水平显示A组HIF-1,VEGF均高于B组(P＜0.05)。
5.1)七叶一枝花提取液可显著降低正常氧分压下瘤细胞增值率并增加瘤细胞凋亡,这种作用在低氧条件下更为明显。RT-PCR结果显示七叶一枝花提取液可降低低氧条件下瘤细胞VEGF、HIF表达的水平。
2)单纯七叶一枝花治疗组肿瘤大小与对照组相比明显减小,但不如5-FU组明显,但前者转移率低于5FU治疗(F)组。七叶一枝花治疗组(D、E、G组)乏氧低灌注区面积比例较其他两组显著降低。前者灌注值离均差亦有明显降低。E组治疗效果最佳。
结论
1.VX_2瘤组织悬液穿刺法建立兔肺移植瘤方法稳定可靠。
2.MRI检测乏氧及低灌注面积比例及离均差大小是预测肿瘤预后的满意指标。
3.乏氧低灌注区的肿瘤细胞有独特生物学特性,是治疗和预防复发的重点。
4.七叶一枝花是一种有希望的干预肿瘤低灌注和乏氧的中药。
Part ONE Detection and interference of low perfusion and hypoxia in lung cancer.
Background
Angiogenesis is defined as the process of developing new blood vessels and important to tumor formation and progress.Exiting research shows that the solid tumor cannot growth bigger than 2 mm in diameter without any support of vascular. Folkman J.et al found in transgenic animal model that there are angiogenesis accompanied with the progress from in situ to infiltrating stage.More and more clinical researches confirmed the relationship between tumor growth,metastasis, prognosis and angiogenesis.Recent years,anti-angiogenesis therapies attract more attention than ever.Many kinds of anti-angiogenesis substances are in their way. There are several ways to assay angiogenesis in tumor such as CT/MRI perfusion, SPECT.Most of them related to tumor of brain and liver while less of them for lung cancer.Current researches mainly focused on high perfusion area in tumor.Few of them focused on low perfusion.
Low perfusion in lung cancer is of same importance as high perfusion.In microscopic facet,A.Low perfusion decrease the transportation of chemotherapy drug to solid tumor,which leads to uneven distribution of drug and low blood drug level in tumor.B.Low perfusion result in hypoxia,which leads to many changes in gene and protein levels that make tumor cell resistance to chemotherapy and radiotherapy.C.Tumor cells in hypoxia area are more liable to metastasis.In macroscopic facet,the growth and metastasis of tumor is the result of selection and adaption between the tumor and host.The tumor cells in low perfusion and hypoxia area can not get enough nutrition would necessarily select hypoxia resistance cells too survive and make part of them go far away to find new survival space.If we just use anti-angiogenesis drug,it is hard to block all the factors modulate angiogenesis.This may even accelerate the selection of more powerful tumor cell.You kill most tumor cells but got a few super-power cells for relapse in future.
Hypoxic areas arise as a result of an imbalance between the supply and consumption of oxygen.Hypoxia-induced proteome and/or genome changes may promote tumor progression via mechanisms enabling cells to overcome nutritive deprivation,to escape from the hostile environment and to favor unrestricted growth. Sustained hypoxia may also lead to cellular changes resulting in a more clinically aggressive phenotype.The tissue hypoxia assay methods include positron emission tomography(PET),magnetic resonance imaging(19F MRI and BOLD MRI).PET is too expensive and need toxic probe,which limit its application.BOLD MRI is designed for brain function imageing.It is safe and convenient.The current anti-hypoxia therapy methods are mainly designed for radiotherapy.Few article report new way to improve chemotherapy resistance in hypoxia.
Rhizomes of Paris polyphylla Smith vat.chinensis Hara has been used to treat cancer in China for many years.Its extractions such as polyphyllin D have been proved to have anti-tumor effect in vitro.
To sum up,low perfusion and hypoxia in lung cancer are important to tumor growth,progressing and metastasis.Our hypothesis is angiogenesis and oxygen modulation other than simple anti-angiogenesis will work better to cure cancer.Paris polyphylla may have such effect.MRI perfusion and BOLD will be used as good way for lung cancer prognosis.
Objective
1.To establish a rabbit xenograft lung cancer model,assess effect of low perfusion and hypoxia on tumor prognosis.
2.To observe the characteristic of tumor cell in low perfusion and hypoxia area.
3.To observe the anti-tumor effect of Paris polyphylla,especially its effect on perfusion and hypoxia.
Methods
1.New Zealand rabbit were used in all experiments.VX2 lung cancer model was established in rabbits by tissue clot suspension injection.
2.To verify the accuracy of MRI perfusion and BOLD result,3 tumor-bearing rabbits were used.OxyLite probes were inserted into 4 spots for every tumor guided by MRI image to measure the oxygen partial pressure at same time.
3.To test the relation between MRI perfusion/BOLD result and prognosis.10 lung cancer rabbit models were established as described.MRI scan was carried out on 10.20.30 and 45 days after injection.MRI perfusion/BOLD were done on 20th day.
4.To observe the characteristic difference between tumor cells from low perfusion/hypoxia area and non-hypoxia area.MRI perfusion.BOLD and CT were carried out on a hind limb tumor bearing rabbit.Samples from low perfusion/hypoxia area and non-hypoxia area were taken by CT guided biopsy and made to A and B tumor cell suspension.
4.1 The cell suspension A and B were subcultured in hypoxic and non-hypoxic condition for 48 hours.Real time RT-PCR,MTT and ELISA for apoptosis were carried out.
4.2 20 rabbits were separate to two group(A and B) and given suspension A and B injection accordingly.MRI was carried out every ten days.CT guided biopsy were used to take sample for real time RT-PCR.
5.To observe the effects of Paris polyphylla on VX2 tumor growth,especially on hypoxia and perfusion,hypoxic cell suspension was prepared as step4.
A.the hypoxic cells were subcultured for 48h in both hypoxic and non-hypoxic condition with and without Paris polyphylla extraction added.Cell proliferation,apoptosis and gene expression were assayed as described above.
B.50 rabbits were separated to five groups(group D:High dosage of Paris polyphylla.E:High dosage of Paris polyphylla plus 5-FU.F:5-FU.G:Low dosage of Paris polyphylla.H.control).The therapy effects were evaluated as described.
Result:
1.90%of rabbits undergone the suspension injection established lung cancer as desired.
2.There is good correlation between results of MRI BOLD and OxyLite probe.
3.High perfusion ratio is in positive correlation with tumor size and growth rate. Low perfusion ratio is in positive correlation with tumor metastasis and in negative correlation with life span.The area of high BOLD signal correlates to tumor metastasis and life span.
4.Under hypoxic condition,tumor cells from hypoxic area(group A) has higher proliferation rate than that from non-hypoxic area(group B).Real time RT-PCR shows that HIF1and VEGF expression in Group A were higher than group B.
5.1) Paris polyphylla extraction has decreased the cell proliferation rate in normal oxygen level.This effect is more obvious in hypoxic condition.HIF1 and VEGF expression levels were down regulated in hypoxic condition by Paris polyphylla extraction.
2) Paris polyphylla treated tumor bearing rabbit(group D) got small tumor size compared to control(group G),but not as much as in 5-FU treated rabbit (group F).Hypoxic area by MRI BOLD is less in Paris polyphylla treated groups(group D,E,G) compared with group F and H.Group E has the best outcome.
Conclusion
1.Tissue clot suspension injection is a reliable method to establish VX2 rabbit lung cancer model.
2.Low perfusion and hypoxia area by MRI is useful for tumor prognosis.
3.Tumor cells in Low perfusion and hypoxia area have special characteristics and should be key point for relapse prevention.
4.Paris polyphylla is a hopeful Chinese herb to modulate perfusion and hypoxia.
PART TWO Periostin and dentin matrix protein 1(DMP1) are candidate genes for hypoxia and perfusion regulation.
Background:
Hypoxia is very important for tumor growth and metastasis.Periostin is a new molecule found increased in many cancers.Periostin is a secreted cell adhesion protein of relatively unknown function that has homology with the insect growth cone guidance protein fasciclin-Ⅰ.Periostin is thought to function as a homophilic adhesion molecule during bone formation and can support osteoblastic cell line attachment and spreading.Purified recombinant periostin has been shown to be a ligand forα_vβ_3 andα_vβ_5 integrins and proved to promoteα_vβ_3 andα_vβintegrin-dependent cell adhesion and enhance cell motility.Multiple reports have also demonstrated elevated periostin levels in neuroblastoma,epithelial ovarian cancer and in non-small cell lung carcinoma that had undergone epithelial-mesenchymal transformation(EMT) and metastasized.Moreover,it has been shown that periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Yet, periosin's roles in hypoxia are largely unclear.
Dentin matrix protein 1(DMP1) is an acidic phosphoglycoprotein and member of the integrin-binding SIBLING protein family.All of the SIBLINGs have been shown over the years to be up-regulated in many different primary tumors.DMP1, historically thought to be expressed in only bones and teeth,has been shown to be expressed in some normal ductal epithelial tissues recently.Several studies have shown that expression of DMP1 increased in a number of cancerous tissues,including lung,breast,uterus et al.It was reported that there is a coordinate increase in MMP-9 and DMP1 expression in lung and kidney cancer.There is one study shows that DMP1 may accelerate cancer metastasis by bridging MMP-9.
Real time RT-PCR super-array was carried out on Lewis cell cultured under hypoxic condition shows that both periostin and DMP1 expression level increased significantly.We hypothesized periostin and DMP1 play an important role in the process of tumor adaptation to hypoxia and low perfusion.
PARTⅡA If periostin and DMP1 is involved in hypoxic effect on tumor growth?
Objectives:
1.To address hypoxia's effects on the expression of periostin and DMP1 in rabbit VX2 lung cancer model.
2.To identify periosin's effects on tumor growth.
Methods:
1.The VX2 carcinoma was maintained through serial transplantation into the hind limb muscle of the New Zealand white rabbit.VX2 lung cancer model was established in rabbits by tissue clot suspension injection under CT guidance.
2.CT/MRI perfusion scans and blood oxygenation level-dependent(BOLD) MRI were used to assay the oxygen level in tumor.CT guided biopsy were used to get specimen in both hypoxic and non hypoxic area according to the BOLD/perfusion image.
3.Total RNA was extracted using Trizle.Real-time RT-PCR was performed for periostin,DMP1,HIF-1αand VEGF.
4.HIF-1 expression vectors were transfected to Lewis cells cultured in non-hypoxic conditions.Periostin expression level was assessed by real time RT PCR and ELISA.
5.Lewis cells were cultured in hypoxic(1%O_2,5%CO_2) and(21%O_2,5%CO_2) conditions.Periostin expression vector were transected to both group.
Methylthiazolete-trazolium(MTT) assay were carried out.
Results:
1.High BOLD signal,which means low oxygen level,mainly distribute in central area in vivo.mRNA levels of periostin DMP1 in hypoxia area were 4.5 and 3.9 fold in comparison to non hypoxia area.HIF-1 VEGF mRNA levels were also increased in hypoxia area.
2.When the VX2 cell from hypoxic area of rabbit tumor cultured in vitro,periostin mRNA level was 6 times higher than that from normoxic area,the periostin protein level higher too.In hypoxic compared to normal condition.DMP1 level was higher than cells from normoxic area.
3.Overexpression of HIF-1 in non-hypoxic condition can up-regulate periostin level by about 6 folds compared to pcDNA3 control.DMP1 mRNA level was increased by about 3 folds.Overexpression of HIF-1 increased the relative growth rate of Lewis lung cancer cells,this effect can be blocked partially by periostin neutralize antibody(Figure 4 B,P＜0.05).
4.Overexpression of periostin inceased cell relative growth rate in both hypoxic and normoxic condition in Levis cells,but the effect was more significant under hypoxic condition.
Conclusion:
1.Periostin is responsive to hypoxia and low perfusion.
2.Periostin is likely a potent positive regulator of tumor growth in response to hypoxia and possibly a downstream factor of HIF-1.
3.DMP1 is another possible molecular in hypoxia regulation process.
PARTⅡB The possible molecular mechanism in periostin's effect on tumor.
Objectives:
1.To address how periostin be regulated under hypoxic condition.
2.To explore the signal pathway through which periostin exert its effects on tumor growth.
Methods:
1.Construction of 6.3kb periostin promoter for Dual-Luciferase(?) reporter assay.
A.Producing PCR Product from periostin Bac clone using AccuPrime~(TM) Taq DNA Polymerase.
B.Cloning this 6.3kb promoter into pCR(?)2.1 using TA Cloning(?) Kit.Then the plasmid were amplified and collected for digestion. C.Cloning the 6.3kb amplified from pCR(?)2.1into the promoterless pGL3-Basic vector using TA Cloning(?) Kit.pGL3-periostin vector was amplified and purified for later use.
2.To observe the effect of TGF-β1on periostin by Dual-Luciferase(?) reporter assay kit.
A.pGL3-periostin and pRL-TK plasmid were co-transfect in Levis and VX_2 cells,pGL3-basic and pRL-TK plasmid were co-transfect as negative control.
B.TGF-β1protein in serial concentration(2.5,5,10,20ng/ml) was added to the medium 12h after the transfection.
C.Promoter activity was assayed by luminometry followed the instruction.
3.To observe the effect of TGF-β1 on periostin,recombinant TGF-β1 was added to the culture medium of Lewis cells and real time RT-PCR was carried out.
4.To observe the recombinant periostin protein's effect on AKT/PKB signal pathway.
A.Preparation of periostin protein.Baculovirus Expression System was used to produce recombinant periostin protein.SF9 insect cell was used in this system.
B.The periostin protein in conditioned medium was purified by Heparin Sepharose CL-6B column.The diluted conditioned medium was applied to a Heparin Sepharose column equilibrated with 10 mM phosphate buffer containing 0.15 M NaCl.After the column was washed with the same buffer,proteins were eluted with a step-wise gradient from 0-1500 mM NaCl in 6M urea/Tris-hcl.Western Blot was carried out to confirm the product.
C.Levis cell was cultured in 21%O_2 condition with 100 ng/ml of periostin or BSA added.Cell lysates were analyzed by Western blot with the anti-Akt1 /PKB and anti-pS473-Akt1/PKB.
D.Periostin expression vector were transected to Lewis cells.Cell lysates were analyzed by Western blot as step C.
Results:
1.6.3kb promoter was amplified successfully by PCR.We got 3 clones of bacteria containing 6.3kb periostin-pCR2.1 vector one of them was selected for next step and made into stock.6.3kb periostin-pGL3 vector was built up and confirmed by two different group of restriction endonuclease.
2.Periostin promoter relative activity was increased in all the 4 concentrations. There is a dose-effect relation.The periostin mRNA expression levels were increased by 3,5,9 and 11 times respectively by 2.5,5.0,10 and 20ng/ml of TGF-β1.
3.Periostin protein was purified from conditioned medium successfully.Western blot shows periostin recombinant protein can increase the phosphorylation of Akt1/PKB on Ser473 significantly.Over expression of periostin has similar effect.
Conclusion:
1.TGF-β1 can up-regulate periostin expression in vitro and is a possible molecular modulating periostin expression under hypoxic condition.It is a bridge molecular between HIF-1 and periostin
2.Periostin can activate Akt1/PKB signal pathway to exert its effect on tumor growth.
肿瘤血管生成(angiogenesis)是指新生血管在肿瘤现有血管基础上形成的过程。研究证实,如果没有新生血管的支持,实体组织中的肿瘤生长直径不会超过2mm。Folkman J.等在转基因动物肿瘤模型的实验中发现,在原位癌向浸润癌发展的过程中,往往伴随着新生血管生成的开始。随后大量的临床研究也证实,血管生成与肿瘤的生长、恶变、转移,以及肿瘤病人的预后有着密切的关系。在过去的几年里,肿瘤的抗血管生成治疗引起了人们极大的研究兴趣,各种抗血管生成药物现已进入各期临床试验阶段。检测肿瘤内部血管生成可通过CT或MRI灌注显像或者SPECT等,肝脏、脑肿瘤的研究较多,而肺部肿瘤灌注的研究很少,目前的研究注意力集中在肿瘤的高灌注,针对肿瘤低灌注特别是肺部肿瘤低灌注的研究尚未见报道。
肿瘤低灌注与高灌注同样有着重要意义,微观方面,①肿瘤内低灌注限制了化疗药物的运送,导致药物在瘤体内不均匀分布达不到有效的最大治疗水平的血药浓度,特别对血管内半衰期短的药物更明显;②低灌注直接导致乏氧,糖酵解增加细胞内变酸,对一些化疗药物不敏感,乏氧促使细胞在基因和蛋白质水平发生改变使低灌注区对多种化疗药物耐药;③乏氧的瘤细胞也易于转移,近期的研究表明急性乏氧更易增加转移机会,新生血管结构缺陷,通透性增加以促进了肿瘤的转移。从宏观角度考虑,肿瘤的发生、生长、转移是肿瘤与宿主相互选择与适应的结果,低灌注与乏氧是肿瘤原发部位不能满足其生长需要,使得选择耐受乏氧的瘤细胞,并使瘤细胞脱落转移至其他部位寻找适宜生存的空间。单独抑制血管生成,我们很难阻断促血管生成的所有途径,而且这可能加速上述的选择,使得部分瘤细胞耐药、抵抗放疗,易于转移。
乏氧,作为低灌注的一个主要后果,分为急性和慢性乏氧,通过乏氧诱导因子(HIF)促进血管生成因子等基因的表达,促进血管生成,抑制抑癌基因如野生型P53表达,激活癌基因表达,肿瘤的恶性程度更高,耐受放化疗和化疗。乏氧的检测主要有PET和MRI两种方法,前者采用硝基咪唑类示踪剂,因其毒性较大,空间分辨率低(仅4-5mm),费用昂贵,应用前景不如MRI。最早用于脑功能研究的血氧水平依赖成像(BOLD)技术相对成熟安全,是较好的选择。针对乏氧的治疗措施包括适形放疗,乏氧细胞毒药物,乏氧细胞增敏剂,卡波金吸入等,这些方法多是针对改善肿瘤对放疗耐受,而改善乏氧区对化疗耐受的研究鲜见报道。
七叶一枝花,为百合科植物华重楼(Paris polyphylla Smith var.chinensisHara)的干燥根茎,含甾体皂苷称为蚤休苷及蚤体土宁苷,后者水解后生成薯蓣苷。有用于抗肿瘤治疗的记载。其水提物及正丁醇提取物有体外抗肿瘤作用。我们的预试验显示七叶一枝花提取物可能对乏氧造成的肿瘤进展有特殊效果。
综上所述,肺癌低灌注乏氧区对于其生长、恶变、转移的重要意义,我们提出血管生成及氧供调节治疗(而不是单纯的抗血管治疗)的假设,通过兔肺移植瘤模型,改良验证检测低灌注和乏氧的方法,从而使预测转移等预后提供帮助;探索七叶一枝花调节肿瘤内部血流灌注,平衡氧供的可行性,以提高肺癌的治疗效果。
目的
1.建立兔肺移植瘤模型,观察乏氧和灌注对预后的影响。
2.观察乏氧低灌注区域内瘤细胞生物学特性。
3.初步探讨七叶一枝花的抗肿瘤作用特别是对低灌注及乏氧的影响。
方法
1.所有实验选用新西兰大白兔。采用瘤组织或细胞悬液直接注入肺及后下肢肌肉内建立移植瘤模型。
2.验证MRI灌注显像及BOLD氧水平显像的准确性。3只荷瘤兔行MRI灌注及BOLD检测后与OxyLite氧电极结果相比较。并比较灌注显像与BOLD氧水平显像的一致性。
3.MRI BOLD及灌注显象与肿瘤预后相关性检测。1)选取10只雄性新西兰大白兔全身麻醉后仰卧位固定,将0.1ml瘤组织悬液经右前胸壁穿刺注入。2) 10天后行胸部MRI检查,观察成瘤情况。3) 20天后行MRI灌注扫描及BOLD显像。4) 30天时复查常规MRI测量肿瘤大小。5) 45天复查MRI测量肿瘤大小,观察转移情况。继续喂养带瘤兔至死亡。
4.观察低灌注区和乏氧区瘤组织相对非低灌注、乏氧区生长和转移的差异。取3周左右下肢带瘤兔一只,行MRI灌注、BOLD及CT扫描,选取低灌注/乏氧区(A区)及非低灌注/乏氧区(B区),将MR与CT图像融合后,在CT引导下穿刺分别制成细胞悬液A、B备用。
4.1将上述细胞悬液A、B在低氧(1%O_2,5%CO_2)及正常氧分压下培养48h。四氮唑盐(MTT)检测细胞增值及ELISA组蛋白-DNA片断检测调亡,实时定量RT-PCR检测VEGF、HIF表达。
4.2取20只健康兔,按体重性别配比分为A、B两组,将AB悬液分别接种于右上肺内,每10天扫描观察其生长速度及转移情况,一月取活检标本通过realtime RT-PCR检测VEGF、HIF表达。
5.观察七叶一枝花低灌注区及乏氧区的影响
5.1以方法4中的描述制备乏氧区细胞悬液,加入七叶一枝花水提液后在低氧及正常氧分压下培养48h,检测瘤细胞增殖及凋亡的变化。同时行实时定量RT-PCR检测VEGF、HIF表达的改变。
5.2取50只健康兔,随机分为5组(D.高剂量七叶一枝花灌胃、E.高剂量七叶一枝花灌胃加5-FU化疗,F.5-FU化疗,G.低剂量七叶一枝花灌胃,H.对照)按照前述方法观察七叶一枝花对肿瘤生长、转移及乏氧灌注的影响。
结果
1.瘤组织悬液穿刺注入法肺癌模型建立成功率92%。有6%早期出现胸水或胸壁结节,予以剔出。
2.BOLD结果与OxyLite氧电极结果一致性较好。
3.a MRI灌注与BOLD有良好相关性,二者分布区域一致。b高灌注区体积比例与肿瘤大小,生长速度呈正相关,c低灌注区体积及程变与转移高度正相关,与生存期成负相关。灌注值离均差大小与转移率呈正相关,与生存期呈负相关。BOLD高信号区比例与肿瘤转移、生存期明显相关。
4.1)来源于乏氧区的瘤组织(A组)与源于非乏氧区的瘤组织(B组)在低氧条件下培养生长活性显著高于非乏氧区对照细胞。RT-PCR结果显示A组HIFI显著高于B组,VEGF亦高于B组,但不如HIFI升高显著。MTT法细胞增殖速度A组高于B组(P＜0.05)调亡A组低于B组(P＜0.01)。
2) A组移植瘤生长速度快于B组A组荷瘤兔生存期短于B组(P＜0.01)。A组转移率高于B组,平均转移时间亦早。一月时活检标本mRNA水平显示A组HIF-1,VEGF均高于B组(P＜0.05)。
5.1)七叶一枝花提取液可显著降低正常氧分压下瘤细胞增值率并增加瘤细胞凋亡,这种作用在低氧条件下更为明显。RT-PCR结果显示七叶一枝花提取液可降低低氧条件下瘤细胞VEGF、HIF表达的水平。
2)单纯七叶一枝花治疗组肿瘤大小与对照组相比明显减小,但不如5-FU组明显,但前者转移率低于5FU治疗(F)组。七叶一枝花治疗组(D、E、G组)乏氧低灌注区面积比例较其他两组显著降低。前者灌注值离均差亦有明显降低。E组治疗效果最佳。
结论
1.VX_2瘤组织悬液穿刺法建立兔肺移植瘤方法稳定可靠。
2.MRI检测乏氧及低灌注面积比例及离均差大小是预测肿瘤预后的满意指标。
3.乏氧低灌注区的肿瘤细胞有独特生物学特性,是治疗和预防复发的重点。
4.七叶一枝花是一种有希望的干预肿瘤低灌注和乏氧的中药。
Part ONE Detection and interference of low perfusion and hypoxia in lung cancer.
Background
Angiogenesis is defined as the process of developing new blood vessels and important to tumor formation and progress.Exiting research shows that the solid tumor cannot growth bigger than 2 mm in diameter without any support of vascular. Folkman J.et al found in transgenic animal model that there are angiogenesis accompanied with the progress from in situ to infiltrating stage.More and more clinical researches confirmed the relationship between tumor growth,metastasis, prognosis and angiogenesis.Recent years,anti-angiogenesis therapies attract more attention than ever.Many kinds of anti-angiogenesis substances are in their way. There are several ways to assay angiogenesis in tumor such as CT/MRI perfusion, SPECT.Most of them related to tumor of brain and liver while less of them for lung cancer.Current researches mainly focused on high perfusion area in tumor.Few of them focused on low perfusion.
Low perfusion in lung cancer is of same importance as high perfusion.In microscopic facet,A.Low perfusion decrease the transportation of chemotherapy drug to solid tumor,which leads to uneven distribution of drug and low blood drug level in tumor.B.Low perfusion result in hypoxia,which leads to many changes in gene and protein levels that make tumor cell resistance to chemotherapy and radiotherapy.C.Tumor cells in hypoxia area are more liable to metastasis.In macroscopic facet,the growth and metastasis of tumor is the result of selection and adaption between the tumor and host.The tumor cells in low perfusion and hypoxia area can not get enough nutrition would necessarily select hypoxia resistance cells too survive and make part of them go far away to find new survival space.If we just use anti-angiogenesis drug,it is hard to block all the factors modulate angiogenesis.This may even accelerate the selection of more powerful tumor cell.You kill most tumor cells but got a few super-power cells for relapse in future.
Hypoxic areas arise as a result of an imbalance between the supply and consumption of oxygen.Hypoxia-induced proteome and/or genome changes may promote tumor progression via mechanisms enabling cells to overcome nutritive deprivation,to escape from the hostile environment and to favor unrestricted growth. Sustained hypoxia may also lead to cellular changes resulting in a more clinically aggressive phenotype.The tissue hypoxia assay methods include positron emission tomography(PET),magnetic resonance imaging(19F MRI and BOLD MRI).PET is too expensive and need toxic probe,which limit its application.BOLD MRI is designed for brain function imageing.It is safe and convenient.The current anti-hypoxia therapy methods are mainly designed for radiotherapy.Few article report new way to improve chemotherapy resistance in hypoxia.
Rhizomes of Paris polyphylla Smith vat.chinensis Hara has been used to treat cancer in China for many years.Its extractions such as polyphyllin D have been proved to have anti-tumor effect in vitro.
To sum up,low perfusion and hypoxia in lung cancer are important to tumor growth,progressing and metastasis.Our hypothesis is angiogenesis and oxygen modulation other than simple anti-angiogenesis will work better to cure cancer.Paris polyphylla may have such effect.MRI perfusion and BOLD will be used as good way for lung cancer prognosis.
Objective
1.To establish a rabbit xenograft lung cancer model,assess effect of low perfusion and hypoxia on tumor prognosis.
2.To observe the characteristic of tumor cell in low perfusion and hypoxia area.
3.To observe the anti-tumor effect of Paris polyphylla,especially its effect on perfusion and hypoxia.
Methods
1.New Zealand rabbit were used in all experiments.VX2 lung cancer model was established in rabbits by tissue clot suspension injection.
2.To verify the accuracy of MRI perfusion and BOLD result,3 tumor-bearing rabbits were used.OxyLite probes were inserted into 4 spots for every tumor guided by MRI image to measure the oxygen partial pressure at same time.
3.To test the relation between MRI perfusion/BOLD result and prognosis.10 lung cancer rabbit models were established as described.MRI scan was carried out on 10.20.30 and 45 days after injection.MRI perfusion/BOLD were done on 20th day.
4.To observe the characteristic difference between tumor cells from low perfusion/hypoxia area and non-hypoxia area.MRI perfusion.BOLD and CT were carried out on a hind limb tumor bearing rabbit.Samples from low perfusion/hypoxia area and non-hypoxia area were taken by CT guided biopsy and made to A and B tumor cell suspension.
4.1 The cell suspension A and B were subcultured in hypoxic and non-hypoxic condition for 48 hours.Real time RT-PCR,MTT and ELISA for apoptosis were carried out.
4.2 20 rabbits were separate to two group(A and B) and given suspension A and B injection accordingly.MRI was carried out every ten days.CT guided biopsy were used to take sample for real time RT-PCR.
5.To observe the effects of Paris polyphylla on VX2 tumor growth,especially on hypoxia and perfusion,hypoxic cell suspension was prepared as step4.
A.the hypoxic cells were subcultured for 48h in both hypoxic and non-hypoxic condition with and without Paris polyphylla extraction added.Cell proliferation,apoptosis and gene expression were assayed as described above.
B.50 rabbits were separated to five groups(group D:High dosage of Paris polyphylla.E:High dosage of Paris polyphylla plus 5-FU.F:5-FU.G:Low dosage of Paris polyphylla.H.control).The therapy effects were evaluated as described.
Result:
1.90%of rabbits undergone the suspension injection established lung cancer as desired.
2.There is good correlation between results of MRI BOLD and OxyLite probe.
3.High perfusion ratio is in positive correlation with tumor size and growth rate. Low perfusion ratio is in positive correlation with tumor metastasis and in negative correlation with life span.The area of high BOLD signal correlates to tumor metastasis and life span.
4.Under hypoxic condition,tumor cells from hypoxic area(group A) has higher proliferation rate than that from non-hypoxic area(group B).Real time RT-PCR shows that HIF1and VEGF expression in Group A were higher than group B.
5.1) Paris polyphylla extraction has decreased the cell proliferation rate in normal oxygen level.This effect is more obvious in hypoxic condition.HIF1 and VEGF expression levels were down regulated in hypoxic condition by Paris polyphylla extraction.
2) Paris polyphylla treated tumor bearing rabbit(group D) got small tumor size compared to control(group G),but not as much as in 5-FU treated rabbit (group F).Hypoxic area by MRI BOLD is less in Paris polyphylla treated groups(group D,E,G) compared with group F and H.Group E has the best outcome.
Conclusion
1.Tissue clot suspension injection is a reliable method to establish VX2 rabbit lung cancer model.
2.Low perfusion and hypoxia area by MRI is useful for tumor prognosis.
3.Tumor cells in Low perfusion and hypoxia area have special characteristics and should be key point for relapse prevention.
4.Paris polyphylla is a hopeful Chinese herb to modulate perfusion and hypoxia.
PART TWO Periostin and dentin matrix protein 1(DMP1) are candidate genes for hypoxia and perfusion regulation.
Background:
Hypoxia is very important for tumor growth and metastasis.Periostin is a new molecule found increased in many cancers.Periostin is a secreted cell adhesion protein of relatively unknown function that has homology with the insect growth cone guidance protein fasciclin-Ⅰ.Periostin is thought to function as a homophilic adhesion molecule during bone formation and can support osteoblastic cell line attachment and spreading.Purified recombinant periostin has been shown to be a ligand forα_vβ_3 andα_vβ_5 integrins and proved to promoteα_vβ_3 andα_vβintegrin-dependent cell adhesion and enhance cell motility.Multiple reports have also demonstrated elevated periostin levels in neuroblastoma,epithelial ovarian cancer and in non-small cell lung carcinoma that had undergone epithelial-mesenchymal transformation(EMT) and metastasized.Moreover,it has been shown that periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Yet, periosin's roles in hypoxia are largely unclear.
Dentin matrix protein 1(DMP1) is an acidic phosphoglycoprotein and member of the integrin-binding SIBLING protein family.All of the SIBLINGs have been shown over the years to be up-regulated in many different primary tumors.DMP1, historically thought to be expressed in only bones and teeth,has been shown to be expressed in some normal ductal epithelial tissues recently.Several studies have shown that expression of DMP1 increased in a number of cancerous tissues,including lung,breast,uterus et al.It was reported that there is a coordinate increase in MMP-9 and DMP1 expression in lung and kidney cancer.There is one study shows that DMP1 may accelerate cancer metastasis by bridging MMP-9.
Real time RT-PCR super-array was carried out on Lewis cell cultured under hypoxic condition shows that both periostin and DMP1 expression level increased significantly.We hypothesized periostin and DMP1 play an important role in the process of tumor adaptation to hypoxia and low perfusion.
PARTⅡA If periostin and DMP1 is involved in hypoxic effect on tumor growth?
Objectives:
1.To address hypoxia's effects on the expression of periostin and DMP1 in rabbit VX2 lung cancer model.
2.To identify periosin's effects on tumor growth.
Methods:
1.The VX2 carcinoma was maintained through serial transplantation into the hind limb muscle of the New Zealand white rabbit.VX2 lung cancer model was established in rabbits by tissue clot suspension injection under CT guidance.
2.CT/MRI perfusion scans and blood oxygenation level-dependent(BOLD) MRI were used to assay the oxygen level in tumor.CT guided biopsy were used to get specimen in both hypoxic and non hypoxic area according to the BOLD/perfusion image.
3.Total RNA was extracted using Trizle.Real-time RT-PCR was performed for periostin,DMP1,HIF-1αand VEGF.
4.HIF-1 expression vectors were transfected to Lewis cells cultured in non-hypoxic conditions.Periostin expression level was assessed by real time RT PCR and ELISA.
5.Lewis cells were cultured in hypoxic(1%O_2,5%CO_2) and(21%O_2,5%CO_2) conditions.Periostin expression vector were transected to both group.
Methylthiazolete-trazolium(MTT) assay were carried out.
Results:
1.High BOLD signal,which means low oxygen level,mainly distribute in central area in vivo.mRNA levels of periostin DMP1 in hypoxia area were 4.5 and 3.9 fold in comparison to non hypoxia area.HIF-1 VEGF mRNA levels were also increased in hypoxia area.
2.When the VX2 cell from hypoxic area of rabbit tumor cultured in vitro,periostin mRNA level was 6 times higher than that from normoxic area,the periostin protein level higher too.In hypoxic compared to normal condition.DMP1 level was higher than cells from normoxic area.
3.Overexpression of HIF-1 in non-hypoxic condition can up-regulate periostin level by about 6 folds compared to pcDNA3 control.DMP1 mRNA level was increased by about 3 folds.Overexpression of HIF-1 increased the relative growth rate of Lewis lung cancer cells,this effect can be blocked partially by periostin neutralize antibody(Figure 4 B,P＜0.05).
4.Overexpression of periostin inceased cell relative growth rate in both hypoxic and normoxic condition in Levis cells,but the effect was more significant under hypoxic condition.
Conclusion:
1.Periostin is responsive to hypoxia and low perfusion.
2.Periostin is likely a potent positive regulator of tumor growth in response to hypoxia and possibly a downstream factor of HIF-1.
3.DMP1 is another possible molecular in hypoxia regulation process.
PARTⅡB The possible molecular mechanism in periostin's effect on tumor.
Objectives:
1.To address how periostin be regulated under hypoxic condition.
2.To explore the signal pathway through which periostin exert its effects on tumor growth.
Methods:
1.Construction of 6.3kb periostin promoter for Dual-Luciferase(?) reporter assay.
A.Producing PCR Product from periostin Bac clone using AccuPrime~(TM) Taq DNA Polymerase.
B.Cloning this 6.3kb promoter into pCR(?)2.1 using TA Cloning(?) Kit.Then the plasmid were amplified and collected for digestion. C.Cloning the 6.3kb amplified from pCR(?)2.1into the promoterless pGL3-Basic vector using TA Cloning(?) Kit.pGL3-periostin vector was amplified and purified for later use.
2.To observe the effect of TGF-β1on periostin by Dual-Luciferase(?) reporter assay kit.
A.pGL3-periostin and pRL-TK plasmid were co-transfect in Levis and VX_2 cells,pGL3-basic and pRL-TK plasmid were co-transfect as negative control.
B.TGF-β1protein in serial concentration(2.5,5,10,20ng/ml) was added to the medium 12h after the transfection.
C.Promoter activity was assayed by luminometry followed the instruction.
3.To observe the effect of TGF-β1 on periostin,recombinant TGF-β1 was added to the culture medium of Lewis cells and real time RT-PCR was carried out.
4.To observe the recombinant periostin protein's effect on AKT/PKB signal pathway.
A.Preparation of periostin protein.Baculovirus Expression System was used to produce recombinant periostin protein.SF9 insect cell was used in this system.
B.The periostin protein in conditioned medium was purified by Heparin Sepharose CL-6B column.The diluted conditioned medium was applied to a Heparin Sepharose column equilibrated with 10 mM phosphate buffer containing 0.15 M NaCl.After the column was washed with the same buffer,proteins were eluted with a step-wise gradient from 0-1500 mM NaCl in 6M urea/Tris-hcl.Western Blot was carried out to confirm the product.
C.Levis cell was cultured in 21%O_2 condition with 100 ng/ml of periostin or BSA added.Cell lysates were analyzed by Western blot with the anti-Akt1 /PKB and anti-pS473-Akt1/PKB.
D.Periostin expression vector were transected to Lewis cells.Cell lysates were analyzed by Western blot as step C.
Results:
1.6.3kb promoter was amplified successfully by PCR.We got 3 clones of bacteria containing 6.3kb periostin-pCR2.1 vector one of them was selected for next step and made into stock.6.3kb periostin-pGL3 vector was built up and confirmed by two different group of restriction endonuclease.
2.Periostin promoter relative activity was increased in all the 4 concentrations. There is a dose-effect relation.The periostin mRNA expression levels were increased by 3,5,9 and 11 times respectively by 2.5,5.0,10 and 20ng/ml of TGF-β1.
3.Periostin protein was purified from conditioned medium successfully.Western blot shows periostin recombinant protein can increase the phosphorylation of Akt1/PKB on Ser473 significantly.Over expression of periostin has similar effect.
Conclusion:
1.TGF-β1 can up-regulate periostin expression in vitro and is a possible molecular modulating periostin expression under hypoxic condition.It is a bridge molecular between HIF-1 and periostin
2.Periostin can activate Akt1/PKB signal pathway to exert its effect on tumor growth.
引文
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7.Powers, W.E. and L.J. Tolmach, A multicomponent x-ray survival curve for mouse lymphosarcoma cells irradiated in vivo. Nature, 1963. 197: p. 710-1.
8.Fouassier, L., et al., Hypoxia-induced changes in the expression of rat hepatobiliary transporter genes. Am J Physiol Gastrointest Liver Physiol, 2007. 293(1): p. G25-35.
9.Moulik, S. and A. Chatterjee, Vascular endothelial growth factor (VEGF) & tumour angiogenesis. Indian J Med Res, 2007. 125(6): p. 715-6.
10.Roskoski, R., Jr., Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol, 2007. 62(3): p. 179-213.
11 .Holmquist-Mengelbier, L., et al., Recruitment of HIF-1 alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype. Cancer Cell, 2006.10(5): p. 413-23.
12.Padhani, A.R., et al., Imaging oxygenation of human tumours. Eur Radiol, 2007.17(4): p. 861-72.
13.Vaupel, P. and A. Mayer, Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev, 2007. 26(2): p. 225-39.
14.Yu, M., et al., Oxygen carriers and cancer chemo- and radiotherapy sensitization: bench to bedside and back. Cancer Treat Rev, 2007. 33(8): p. 757-61.
15.Foley, R.N., Erythropoietin: physiology and molecular mechanisms. Heart Fail Rev,2008.
16.Leo, C., L.C. Horn, and M. Hockel, Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer. Int J Gynecol Cancer, 2006. 16(3): p. 1314-20.
17.Vaupel, P., A. Mayer, and M. Hockel, Impact of hemoglobin levels on tumor oxygenation: the higher, the better? Strahlenther Onkol, 2006.182(2): p. 63-71.
18.Auer, F., et al., Technical improvement of pO_2 measurements in breast cancer:investigation of the feasibility in patients and in vitro validation of the method.Strahlenther Onkol, 2007. 183(5): p. 265-70.
19.Khan, N., et al., Repetitive tissue pO2 measurements by electron paramagnetic resonance oximetry: current status and future potential for experimental and clinical studies. Antioxid Redox Signal, 2007. 9(8): p. 1169-82.
20.Galanis, A., et al., Reactive oxygen species and HIF-1 signalling in cancer. Cancer Lett, 2008.
21.Chan, N., et al., Chronic hypoxia decreases synthesis of homologous recombination proteins to offset chemoresistance and radioresistance. Cancer Res,2008. 68(2): p. 605-14.
22.Kirkpatrick, J.P., L.I. Cardenas-Navia, and M.W. Dewhirst, Predicting the effect of temporal variations in PO2 on tumor radiosensitivity. Int J Radiat Oncol Biol Phys,2004. 59(3): p. 822-33.
23.Sprong, D., et al., Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status. Int J Radiat Oncol Biol Phys, 2006. 64(2): p.562-72.
24.Auberger, T., et al., Oxygen tension in transplanted mouse osteosarcomas during fractionated high-LET- and low-LET radiotherapy-predictive aspects for choosing beam quality? Strahlenther Onkol, 1999. 175 Suppl 2: p. 52-6.
25.Dasu, A. and J. Denekamp, Superfractionation as a potential hypoxic cell radiosensitizer: prediction of an optimum dose per fraction.Int J Radiat Oncol Biol Phys, 1999. 43(5): p. 1083-94.
26.Kraut, E.H., et al., Evaluation of topotecan in resistant and relapsing multiple myeloma: a Southwest Oncology Group study. J Clin Oncol, 1998. 16(2): p. 589-92.
27.Wincewicz, A., et al., Cumulative expression of HIF-1-alpha, Bax, Bcl-xL and P53 in human colorectal cancer. Pathology, 2007. 39(3): p. 334-8.
28.Zhang, L., P. Subarsky, and R.P. Hill, Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells. Int J Radiat Biol, 2007. 83(7): p. 443-56.
29.Sansone, P., et al., The p53 codon 72 proline allele is endowed with enhanced cell-death inducing potential in cancer cells exposed to hypoxia. Br J Cancer, 2007.96(8): p. 1302-8.
30.Sprague, L.D., et al., Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells. Oncol Rep, 2007. 17(5): p. 1259-68.
31.Ambalavanan, N., et al., Role of matrix metalloproteinase-2 in newborn mouse lungs under hypoxic conditions. Pediatr Res, 2008. 63(1): p. 26-32.
32.Du, R., et al., HIF1 alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell, 2008.13(3): p. 206-20.
33.Ahn, J.K., et al., Role of hypoxia-inducible factor-1 {alpha} in hypoxia-induced expressions of IL-8, MMP-1 and MMP-3 in rheumatoid fibroblast-like synoviocytes. Rheumatology (Oxford), 2008.
34.Hollborn, M., et al., Positive feedback regulation between MMP-9 and VEGF in human RPE cells. Invest Ophthalmol Vis Sci, 2007. 48(9): p. 4360-7.
35.Textor, B., et al., c-Jun and JunB are essential for hypoglycemia-mediated VEGF induction. Ann N Y Acad Sci, 2006.1091: p. 310-8.
36.Hamilton, K.L., S. Gupta, and A.A. Knowlton, Estrogen and regulation of heat shock protein expression in female cardiomyocytes: cross-talk with NF kappa B signaling. J Mol Cell Cardiol, 2004. 36(4): p. 577-84.
37.Reynolds, E.O., et al., New non-invasive methods for the investigation of cerebral oxidative metabolism and haemodynamics in newborn infants. Ann Med, 1991.23(6): p. 681-6.
38.Shi, Y.H., et al., Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells. Pathology, 2007. 39(4): p. 396-400.
39.Comerford, K.M., et al., Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res, 2002. 62(12): p. 3387-94.
40.Ryu, J.S., et al., Fractionated irradiation leads to restoration of drug sensitivity in MDR cells that correlates with down-regulation of P-gp and DNA-dependent protein kinase activity. Radiat Res, 2004. 162(5): p. 527-35.
41.Sprague, L.D., et al., Effect of reoxygenation on the hypoxia-induced up-regulation of serine protease inhibitor PAI-1 in head and neck cancer cells. Oncology, 2006.71(3-4): p. 282-91.
42.Lu, Q., et al., Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery. Cancer Biol Ther,2007. 7(2).
43.Yao, L.Q., et al., Characteristics and differentiated mechanism of vascular endothelial cells-like derived from epithelial ovarian cancer cells induced by hypoxia. Int J Oncol, 2007. 30(5): p. 1069-75.
44.Beraud, C. and W.C. Greene, Interaction of HTLV-I Tax with the human proteasome: implications for NF-kappa B induction. J Acquir Immune Defic Syndr Hum Retrovirol, 1996. 13 Suppl 1: p. S76-84.
45.Beraud, C., W.J. Henzel, and P.A. Baeuerle, Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-kappaB activation. Proc Natl Acad Sci U S A, 1999. 96(2): p. 429-34.
46.Scholbach, T., et al., New method of dynamic color doppler signal quantification in metastatic lymph nodes compared to direct polarographic measurements of tissue oxygenation. Int J Cancer, 2005. 114(6): p. 957-62.
47.Nordsmark, M., et al., Differential risk assessments from five hypoxia specific assays: The basis for biologically adapted individualized radiotherapy in advanced head and neck cancer patients. Radiother Oncol, 2007. 83(3): p. 389-97.
48.Chen, X., et al., Dynamics of MRI-Guided thermal ablation of VX2 tumor in paraspinal muscle of rabbits. IEEE Trans Biomed Eng, 2008. 55(3): p. 1004-14.
49.Howe, F.A., et al., Issues in flow and oxygenation dependent contrast (FLOOD) imaging of tumours. NMR Biomed, 2001. 14(7-8): p. 497-506.
50.Hsu, Y.Y., et al., Blood oxygenation level-dependent MRI of cerebral gliomas during breath holding. J Magn Reson Imaging, 2004. 19(2): p. 160-7.
51.Landuyt, W., et al., BOLD contrast fMRI of whole rodent tumour during air or carbogen breathing using echo-planar imaging at 1.5 T. Eur Radiol, 2001. 11 (11): p.2332-40.
52.Al-Hallaq, H.A., et al., MRI measurements correctly predict the relative effects of tumor oxygenating agents on hypoxic fraction in rodent BA1112 tumors. Int J Radiat Oncol Biol Phys,2000.47(2):p.481-8.
53.Padhani,A.,PET imaging of tumour hypoxia.Cancer Imaging,2006.6:p.S117-21.
54.Choi,U.H.,et al.,Hypoxia-inducible expression of vascular endothelial growth factor for the treatment of spinal cord injury in a rat model.J Neurosurg Spine,2007.7(1):p.54-60.
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5.Hubbard, W.C., et al., The site of VX2 tumor transplantation affects the
development of hypercalcemia in rabbits. Prostaglandins, 1980. 19(6): p. 881-9. 6.Thomlinson, R.H. and L.H. Gray, The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer, 1955. 9(4): p.539-49.
7.Powers, W.E. and L.J. Tolmach, A multicomponent x-ray survival curve for mouse lymphosarcoma cells irradiated in vivo. Nature, 1963. 197: p. 710-1.
8.Fouassier, L., et al., Hypoxia-induced changes in the expression of rat hepatobiliary transporter genes. Am J Physiol Gastrointest Liver Physiol, 2007. 293(1): p. G25-35.
9.Moulik, S. and A. Chatterjee, Vascular endothelial growth factor (VEGF) & tumour angiogenesis. Indian J Med Res, 2007. 125(6): p. 715-6.
10.Roskoski, R., Jr., Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev Oncol Hematol, 2007. 62(3): p. 179-213.
11 .Holmquist-Mengelbier, L., et al., Recruitment of HIF-1 alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype. Cancer Cell, 2006.10(5): p. 413-23.
12.Padhani, A.R., et al., Imaging oxygenation of human tumours. Eur Radiol, 2007.17(4): p. 861-72.
13.Vaupel, P. and A. Mayer, Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev, 2007. 26(2): p. 225-39.
14.Yu, M., et al., Oxygen carriers and cancer chemo- and radiotherapy sensitization: bench to bedside and back. Cancer Treat Rev, 2007. 33(8): p. 757-61.
15.Foley, R.N., Erythropoietin: physiology and molecular mechanisms. Heart Fail Rev,2008.
16.Leo, C., L.C. Horn, and M. Hockel, Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer. Int J Gynecol Cancer, 2006. 16(3): p. 1314-20.
17.Vaupel, P., A. Mayer, and M. Hockel, Impact of hemoglobin levels on tumor oxygenation: the higher, the better? Strahlenther Onkol, 2006.182(2): p. 63-71.
18.Auer, F., et al., Technical improvement of pO_2 measurements in breast cancer:investigation of the feasibility in patients and in vitro validation of the method.Strahlenther Onkol, 2007. 183(5): p. 265-70.
19.Khan, N., et al., Repetitive tissue pO2 measurements by electron paramagnetic resonance oximetry: current status and future potential for experimental and clinical studies. Antioxid Redox Signal, 2007. 9(8): p. 1169-82.
20.Galanis, A., et al., Reactive oxygen species and HIF-1 signalling in cancer. Cancer Lett, 2008.
21.Chan, N., et al., Chronic hypoxia decreases synthesis of homologous recombination proteins to offset chemoresistance and radioresistance. Cancer Res,2008. 68(2): p. 605-14.
22.Kirkpatrick, J.P., L.I. Cardenas-Navia, and M.W. Dewhirst, Predicting the effect of temporal variations in PO2 on tumor radiosensitivity. Int J Radiat Oncol Biol Phys,2004. 59(3): p. 822-33.
23.Sprong, D., et al., Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status. Int J Radiat Oncol Biol Phys, 2006. 64(2): p.562-72.
24.Auberger, T., et al., Oxygen tension in transplanted mouse osteosarcomas during fractionated high-LET- and low-LET radiotherapy-predictive aspects for choosing beam quality? Strahlenther Onkol, 1999. 175 Suppl 2: p. 52-6.
25.Dasu, A. and J. Denekamp, Superfractionation as a potential hypoxic cell radiosensitizer: prediction of an optimum dose per fraction.Int J Radiat Oncol Biol Phys, 1999. 43(5): p. 1083-94.
26.Kraut, E.H., et al., Evaluation of topotecan in resistant and relapsing multiple myeloma: a Southwest Oncology Group study. J Clin Oncol, 1998. 16(2): p. 589-92.
27.Wincewicz, A., et al., Cumulative expression of HIF-1-alpha, Bax, Bcl-xL and P53 in human colorectal cancer. Pathology, 2007. 39(3): p. 334-8.
28.Zhang, L., P. Subarsky, and R.P. Hill, Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells. Int J Radiat Biol, 2007. 83(7): p. 443-56.
29.Sansone, P., et al., The p53 codon 72 proline allele is endowed with enhanced cell-death inducing potential in cancer cells exposed to hypoxia. Br J Cancer, 2007.96(8): p. 1302-8.
30.Sprague, L.D., et al., Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells. Oncol Rep, 2007. 17(5): p. 1259-68.
31.Ambalavanan, N., et al., Role of matrix metalloproteinase-2 in newborn mouse lungs under hypoxic conditions. Pediatr Res, 2008. 63(1): p. 26-32.
32.Du, R., et al., HIF1 alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell, 2008.13(3): p. 206-20.
33.Ahn, J.K., et al., Role of hypoxia-inducible factor-1 {alpha} in hypoxia-induced expressions of IL-8, MMP-1 and MMP-3 in rheumatoid fibroblast-like synoviocytes. Rheumatology (Oxford), 2008.
34.Hollborn, M., et al., Positive feedback regulation between MMP-9 and VEGF in human RPE cells. Invest Ophthalmol Vis Sci, 2007. 48(9): p. 4360-7.
35.Textor, B., et al., c-Jun and JunB are essential for hypoglycemia-mediated VEGF induction. Ann N Y Acad Sci, 2006.1091: p. 310-8.
36.Hamilton, K.L., S. Gupta, and A.A. Knowlton, Estrogen and regulation of heat shock protein expression in female cardiomyocytes: cross-talk with NF kappa B signaling. J Mol Cell Cardiol, 2004. 36(4): p. 577-84.
37.Reynolds, E.O., et al., New non-invasive methods for the investigation of cerebral oxidative metabolism and haemodynamics in newborn infants. Ann Med, 1991.23(6): p. 681-6.
38.Shi, Y.H., et al., Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells. Pathology, 2007. 39(4): p. 396-400.
39.Comerford, K.M., et al., Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res, 2002. 62(12): p. 3387-94.
40.Ryu, J.S., et al., Fractionated irradiation leads to restoration of drug sensitivity in MDR cells that correlates with down-regulation of P-gp and DNA-dependent protein kinase activity. Radiat Res, 2004. 162(5): p. 527-35.
41.Sprague, L.D., et al., Effect of reoxygenation on the hypoxia-induced up-regulation of serine protease inhibitor PAI-1 in head and neck cancer cells. Oncology, 2006.71(3-4): p. 282-91.
42.Lu, Q., et al., Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery. Cancer Biol Ther,2007. 7(2).
43.Yao, L.Q., et al., Characteristics and differentiated mechanism of vascular endothelial cells-like derived from epithelial ovarian cancer cells induced by hypoxia. Int J Oncol, 2007. 30(5): p. 1069-75.
44.Beraud, C. and W.C. Greene, Interaction of HTLV-I Tax with the human proteasome: implications for NF-kappa B induction. J Acquir Immune Defic Syndr Hum Retrovirol, 1996. 13 Suppl 1: p. S76-84.
45.Beraud, C., W.J. Henzel, and P.A. Baeuerle, Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-kappaB activation. Proc Natl Acad Sci U S A, 1999. 96(2): p. 429-34.
46.Scholbach, T., et al., New method of dynamic color doppler signal quantification in metastatic lymph nodes compared to direct polarographic measurements of tissue oxygenation. Int J Cancer, 2005. 114(6): p. 957-62.
47.Nordsmark, M., et al., Differential risk assessments from five hypoxia specific assays: The basis for biologically adapted individualized radiotherapy in advanced head and neck cancer patients. Radiother Oncol, 2007. 83(3): p. 389-97.
48.Chen, X., et al., Dynamics of MRI-Guided thermal ablation of VX2 tumor in paraspinal muscle of rabbits. IEEE Trans Biomed Eng, 2008. 55(3): p. 1004-14.
49.Howe, F.A., et al., Issues in flow and oxygenation dependent contrast (FLOOD) imaging of tumours. NMR Biomed, 2001. 14(7-8): p. 497-506.
50.Hsu, Y.Y., et al., Blood oxygenation level-dependent MRI of cerebral gliomas during breath holding. J Magn Reson Imaging, 2004. 19(2): p. 160-7.
51.Landuyt, W., et al., BOLD contrast fMRI of whole rodent tumour during air or carbogen breathing using echo-planar imaging at 1.5 T. Eur Radiol, 2001. 11 (11): p.2332-40.
52.Al-Hallaq, H.A., et al., MRI measurements correctly predict the relative effects of tumor oxygenating agents on hypoxic fraction in rodent BA1112 tumors. Int J Radiat Oncol Biol Phys,2000.47(2):p.481-8.
53.Padhani,A.,PET imaging of tumour hypoxia.Cancer Imaging,2006.6:p.S117-21.
54.Choi,U.H.,et al.,Hypoxia-inducible expression of vascular endothelial growth factor for the treatment of spinal cord injury in a rat model.J Neurosurg Spine,2007.7(1):p.54-60.
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