摘要
乏氧是多数实质性肿瘤微环境的基本特征之一。肿瘤细胞的快速增殖导致了乏氧微环境的建立,因此,乏氧环境信号成为影响肿瘤生物学行为的重要因素。乏氧环境下肿瘤血管形成增强、乏氧肿瘤细胞对放化疗的抵抗性升高,其恶性表型呈现显著增强。近年来,乏氧对肿瘤生物学特性影响及其调控机制的研究已成为肿瘤研究的热点领域,基于肿瘤乏氧特性的肿瘤治疗新策略已显示出了良好的前景。在肿瘤对乏氧微环境的适应过程中,诸多的肿瘤或宿主源性的因子参与其特性改变的调节,其中乏氧诱导因子-1α(hypoxia-inducible factor-1 alpha,HIF-1α)可能是肿瘤乏氧适应的关键调节因子。大量的研究已证实HIF-1α信号通路在许多肿瘤恶性表型的调节中起关键作用,但在口腔癌及舌鳞癌细胞系Tca8113细胞中的表达以及其对乏氧适应调控的机制报道甚少。
大量的研究发现许多人类实体肿瘤中存在HIF-1的过表达,并被证实是调控肿瘤乏氧适应的关键核心转录调节因子。HIF-1基因是由HIF-1α和HIF-1β各两个亚单位组成的杂二聚体蛋白,其中α亚单位是唯一的O_2调节亚单位。HIF-1α的稳定性、亚细胞定位及其促转录功能受细胞内氧浓度的调节。乏氧环境下,肿瘤细胞可通过上调HIF-1α的表达或减少蛋白的降解,导致其蛋白水平的升高及稳定性的增强,随后HIF-1α进入细胞核内与β亚单位聚合成异源二聚体,此二聚体特异性地与靶基因的乏氧反应元件(hypoxia-responsive elements,HRE)结合,发挥转录因子的作用。目前的研究发现HIF-1α直接调控的下游与乏氧适应反应有关的靶基因约100余个,HIF-1α通过不同的信号通路及机制调控肿瘤细胞的多种生物学行为,如细胞糖酵解、促血管生成及细胞的增殖、凋亡及侵袭等过程。新近的研究发现由于不同肿瘤的高度异质性,HIF-1α信号通路对肿瘤生长和演进的影响及调控机制可能存在较大的区别。
舌癌是口腔癌中发病率最高的恶性肿瘤。在过去的三十年中,我国口腔癌的发病率呈逐年上升的趋势。舌癌的侵袭、转移及对放化疗的抵抗是导致治疗失败的重要因素,而乏氧微环境是关键影响因素之一。已证明人口腔癌组织中存在乏氧微环境,并发现HIF-1α过表达与口腔癌放化疗抵抗性密切相关,但HIF-1α对舌鳞癌细胞生物学行为(增殖、凋亡及侵袭)的影响及调控机制尚不清楚。新近的研究发现,肾上腺髓质素(Adrenomedullin,ADM)参与肿瘤生长及凋亡等生物特性调控,且其可能也是一个重要的乏氧诱导基因,参与肿瘤细胞乏氧适应的调控。有研究发现乏氧条件下HIF-1α和ADM表达均有上调,我们的前期研究也证实乏氧刺激可致舌鳞癌细胞系Tca8113细胞HIF-1α及ADM mRNA含量均显著上调,然而二者之间的关联,以及HIF-1α是否通过调节ADM的表达而致肿瘤细胞的恶性表型增强目前尚无报道。本研究拟以舌鳞癌细胞系Tca8113细胞为研究对象,采用siRNA干扰技术沉默HIF-1α基因,探讨其对Tca8113细胞增殖、凋亡及侵袭特性的影响,并探讨HIF-1α对ADM表达的调节作用。揭示HIF-1α在舌鳞癌细胞系Tca8113细胞的表达特点,及其调控新机制将对制订更为合理的基于乏氧特征的肿瘤治疗策略提供新的思路与有效靶点。
第一部分乏氧对舌鳞癌细胞系Tca8113生物学行为及HIF-1α/ADM表达的影响
目的:
观察乏氧对舌鳞癌细胞系Tca8113细胞增殖、凋亡及侵袭性的影响,以及对HIF-1α及ADM基因的表达的影响。
方法
1.细胞培养:口腔鳞状细胞癌细胞系Tca8113在37℃,5%CO_2饱和湿度条件下,培养于含10%胎牛血清,100 IU/ml青霉素、100μg/ml链霉素的RPMI1640培养液中。乏氧培养时,将80%融合的Tca8113细胞移至乏氧培养箱(1%O_2、5%CO_2、94%N_2混合气体)继续培养4h,24h或48h。
2.real -time PCR方法检测Tca8113细胞中HIF-1αmRNA的表达情况:收集经常氧或乏氧培养4h及24h的Tca8113细胞,Trizol方法提取细胞总RNA,在一定的反应体系中逆转录成cDNA,real -time PCR方法检测常氧及乏氧培养条件下HIF-1α在mRNA水平的表达情况。
3.Western-blot检测HIF-1α蛋白表达情况:收集经常氧或乏氧培养4h及24h的Tca8113细胞,提取细胞总蛋白,Western-blot检测常氧及乏氧培养条件下HIF-1α蛋白表达情况。
4.real-time PCR方法检测乏氧对ADM表达的影响:收集经常氧或乏氧培养2h、4h、8h、24h的Tca8113细胞,Trizol方法提取细胞总RNA,在一定的反应体系中逆转录成cDNA,real -time PCR方法检测常氧及乏氧培养条件下ADM在mRNA水平的表达情况。
5.MTT方法检测乏氧对Tca8113细胞增殖的影响:取指数生长期细胞消化计数后按6×10~3/孔接种于96孔板中常氧培养过夜,然后乏氧培养6h、12h、24h、48h或72h,以常氧培养为对照,MTT方法检测细胞增殖情况。
6.流式细胞仪检测乏氧对Tca8113细胞周期和细胞凋亡率的影响:取指数生长期细胞消化计数后按2×10~5/孔接种于6孔板中。待细胞融合至约80%,置入乏氧培养箱培养4h,24h或48h后收集细胞,PI染色后流式细胞仪检测细胞周期变化。乏氧培养4h,24h或48h后收集细胞,Annexin V-PI染色后流式细胞仪检测细胞凋亡率变化。
7.CytoMatrix~(TM)细胞黏附试剂盒检测乏氧对Tca8113细胞黏附能力的影响:收集乏氧培养4h或24h的Tca8113细胞,取细胞悬液100μl(1×10~5个细胞)加入CytoMatrix~(TM)细胞黏附试剂盒中各孔,以常氧培养为对照,用490nm波长测定每孔的光吸收值(A)。
8.细胞侵袭分析试剂盒检测乏氧对Tca8113细胞侵袭能力的影响:指数生长期细胞消化计数后,取细胞悬液300μl(3×10~5个细胞)加入细胞侵袭分析试剂盒的Boyden小室的上室,在下室加入500μl含10%胎牛血清的RPMI1640培养液,常氧或乏氧培养4h或24h。光镜下计数侵袭到滤膜背面的穿膜细胞数,评价细胞的侵袭能力。
结果
1.乏氧抑制Tca8113细胞的增殖:乏氧培养48h或72h后,Tca8113细胞增殖显著抑制(0.391±0.051 vs 0.537±0.046,P＜0.01;0.255±0.031 vs 0.852±0.078,P＜0.001)。
2.乏氧可致Tca8113细胞周期阻滞于G_1期:乏氧培养24或48h后,与常氧组比较,其G_0/G_1期细胞分别为((73.37±4.01)%vs(66.59±5.25)%,P＜0.05;(87.62±6.54)%vs(61.88±4.38)%,P＜0.01),有显著性差异。
3.乏氧促进Tca8113细胞的凋亡率:乏氧培养4h或24h后,细胞凋亡率与常氧组比较,无显著性差异;乏氧培养48h后,Tca8113细胞凋亡率明显增加,与常氧组比较有显著性差异(11.56±1.82)%vs(4.37±0.59)%,P＜0.01)。
4.乏氧增强Tca8113细胞的黏附和侵袭能力:乏氧培养4h或24h后,Tca8113细胞黏附能力增强,与常氧对照组相比有显著性差异((126.0±9.3)%,(128.0±10.8)%vs(100.0±8.7)%,P＜0.05)。乏氧培养24h后,Tca8113细胞侵袭能力增强,与常氧对照组相比有显著性差异((133.0±11.6)%vs(100.0±9.2)%,P＜0.05)。
5.乏氧不影响Tca8113细胞HIF-1α的转录但其蛋白含量增加:常氧培养条件下,Tca8113细胞中有HIF-1αmRNA和蛋白的基础表达。乏氧培养4h或24h,Tca8113细胞中HIF-1αmRNA表达没有明显增加(P＞0.05)。但乏氧培养4h后HIF-1α蛋白表达量增加,乏氧培养24h后更为显著。
6.乏氧上调Tca8113细胞ADM mRNA的表达:乏氧培养2h后,ADM mRNA的表达开始增加;乏氧培养24h后ADM mRNA的表达较常氧对照组增加17.5倍(P＜0.01)。
结论
1.研究阐述了乏氧微环境对Tca8113细胞生物学行为的影响,证实慢性乏氧(48h)可抑制Tca8113细胞的增殖,促进其凋亡,并能导致Tca8113细胞黏附和侵袭能力增加。乏氧还可导致Tca8113细胞周期阻滞在G_1期。
2.研究发现乏氧不影响Tca8113细胞HIF-1α的转录水平活性但其蛋白含量显著增多,证实乏氧对Tca8113细胞细胞生物学行为的影响是在转录后水平。
3.本研究首次发现乏氧刺激上调Tca8113细胞ADM mRNA的转录活性,推测其上调可能与HIF-1α蛋白含量的增加有关。
第二部分沉默HIF-1α对Tca8113细胞ADM表达及生物学行为的影响
目的
利用siRNA技术特异性阻断HIF-1α基因表达,研究HIF-1α对Tca8113细胞生物学行为的影响,以及对下游调控基因ADM表达的影响。探讨HIF-1α/ADM通路对Tca8113细胞增殖及侵袭性的影响及HIF-1α/ADM作为舌癌生物和基因治疗的潜在分子靶点的可能。
方法
1.针对HIF-1α的siRNA片段(siRNA_(HIF-1α))的转染:取指数生长期细胞消化计数后按2×10~5个/孔接种于6孔细胞培养板中,细胞融合至40-50%时,应用Lipofectamine~(TM) 2000将50nM siRNA_(HIF-1α)转染入Tca8113细胞。
2.干扰效率的检测:转染24h后Tca8113细胞常氧或乏氧培养24h,real-timePCR及Western blot方法检测常氧或乏氧培养条件下siRNA_(HIF-1α)对HIF-1αmRNA和蛋白表达的阻断效率。
3.免疫荧光检测siRNA_(HIF-1α)对HIF-1α蛋白表达的影响:取指数生长期细胞消化计数,按8×10~4个/孔接种于放置一张6×6mm盖玻片的12孔细胞培养板中,40%融合后将50nM siRNA_(HIF-1α)转染入Tca8113细胞,24h后置入乏氧培养箱培养24h。免疫荧光检测siRNA_(HIF-1α)转染后乏氧培养条件下HIF-1α蛋白表达的情况。
4.MTT方法检测siRN_(HIF-1α)对Tca8113细胞增殖的影响:取指数生长期细胞消化计数后按6×10~3个/孔接种于96孔细胞培养板中,约40%融合后转染siRNA_(HIF-1α)。常氧或乏氧培养24、48、72h后,MTT方法检测siRNA_(HIF-1α)对细胞增殖的影响。
5.流式细胞仪检测siRNA_(HIF-1α)对细胞凋亡率的影响:取指数生长期细胞消化计数后按2×10~5个/孔接种于6孔细胞培养板中,约40%融合后转染50nMsiRNA_(HIF-1α),乏氧培养24h后收集细胞,Annexin V/PI双染后利用流式细胞仪检测细胞凋亡率的变化。
6.CytoMatrix~(TM)细胞黏附试剂盒检测siRNA_(HIF-1α)对Tca8113细胞黏附能力的影响:经转染处理的Tca8113细胞常氧或乏氧培养24h,收集细胞,取细胞悬液100μl(1×10~5个细胞)加入CytoMatrix~(TM)细胞黏附试剂盒中各孔,用490nm波长测定每孔的光吸收值(A)。
7.细胞侵袭分析试剂盒检测siRNA_(HIF-1α)对Tca8113细胞侵袭能力的影响:取转染后的Tca8113细胞悬液300μl(3×10~5个细胞)加入细胞侵袭分析试剂盒的Boyden小室的上室,在下室加入500μl含10%胎牛血清的RPMI1640培养液,常氧或乏氧培养24h。光镜下计数侵袭到滤膜背面的穿膜细胞数,评价细胞的侵袭能力。
8.real-time PCR方法检测siRNA_(HIF-1α)转染对ADM表达的影响:转染后的Tca8113细胞常氧或乏氧培养24h,real-time PCR方法检测ADM在mRNA水平的表达情况。
结果
1.siRNA_(HIF-1α)可以有效沉默Tca8113细胞HIF-1α基因:siRNA转染后常氧或乏氧培养24h,干扰组Tca8113细胞HIF-1αm RNA的表达均较对照组显著下调,蛋白含量亦显著降低。
2.沉默HIF-1α抑制Tca8113细胞的增殖:转染siRNA_(HIF-1α)的Tca8113细胞乏氧培养24h后,增殖较对照组明显抑制(0.252±0.029 vs 0.322±0.031,P＜0.05),而常氧培养条件下增殖无明显抑制。转染48h或72h后无论在常氧或乏氧条件下Tca8113细胞增殖均显著抑制(P＜0.01)。
3.沉默HIF-1α诱导Tca8113细胞的凋亡:siRNA_(HIF-1α)转染的Tca8113细胞常氧或乏氧培养24h,凋亡细胞与对照组相比显著增加((12.15±1.02)%vs(4.68±0.56)%,(17.77±1.22)%vs(6.43±0.55)%,P＜0.01)。
4.沉默HIF-1α抑制Tca8113细胞的黏附和侵袭能力:Tca8113细胞转染siRNA_(HIF-1α)后常氧或乏氧培养24h,细胞黏附能力下降至对照组的74%和63.6%,细胞侵袭能力下降至对照组的65%和55.8%。
5.沉默HIF-1α抑制乏氧诱导的ADM mRNA的表达:Tca8113细胞转染siRNA_(HIF-1α)后乏氧培养24h,ADM mRNA的表达较对照组无明显增加((0.04±0008)vs(0.37±0.04).P＞0.05)。
结论
1.siRNA_(HIF-1α)介导的HIF-1α沉默能够引起Tca8113细胞凋亡率增加,从而抑制细胞的增殖,并能抑制Tca8113细胞的黏附和侵袭能力,提示HIF-1α在Tca8113细胞生物学行为的调节中可能起关键作用。
2.本研究首次证实沉默Tca8113细胞HIF-1α能抑制下游ADM基因的表达,提示HIF-1α可能通过调控ADM基因的表达参与其生物学行为及乏氧适应的调节。
3.HIF-1α/ADM通路对Tca8113细胞乏氧适应调节机制的揭示,对制订基于乏氧设计策略的舌癌生物治疗疗法提供新的思路和新靶点。
Background:
The oxygen supply to cells and tissues is pivotal in maintaining their function and integrity.Rapid growth of tumors often results in the development of a hypoxic microenvironment.Therefore,signal of hypoxic microenvironment has become an important factor that affects the biological characteristics of solid tumor.Under hypoxic microenvironment,the tumors present more progressive phenotype in abnormal neovascularization,the invasion,metastasis and resistance to chemo- and radiotherapies.In recent years,many researches focus on the effect of hypoxia on the biological characteristics of tumor,and the possible mechanisms.New strategy of tumor treatment based on hypoxia has shown a good future.During adaptation of tumor to the hypoxic microenvironment,a wide variety of tumor- and host-derived factors are regulated.Among those factors,hypoxia-inducible factor-1 alpha(HIF-1α) might be a central transcriptional regulator of tumor microenvironment.A lot of studies have proved that these signal pathways such as HIF-1αpathway are playing an important role in the regulation of tumor's malignant phenotype.However,very few reports are regarding the expression of HIF-1αin oral carcinoma and tongue squamous cell carcinoma cells line Tca8113,and their mechanisms by which to adapt the hypoxic microenvironment.
Over-expression of HIF-1αwidely presents in the solid tumor and it has been proved as a key transcription factor to adapt to the hypoxic microenvironment.HIF-1 is a heterodimeric transcription factor composed of a strictly regulatedαsubunit and a constitutiveβsubunit(HIF-1β/ARNT).The stability and function as a transcription fcctor of HIF-1α,is regulated by level of O_2.Under hypoxic condition,protein degradation of HIF-1αis reduced and it is allowed to translocate into the nucleus where it dimerizes with ARNT.HIF-1 binds to hypoxia-responsive elements(HRE) located in the promoter and enhancer regions of hypoxia-regulated genes,causing their transactivation.According to the studies up to today,HIF-1αcan regulate more than 100 genes,which are involved in adaptive responses to hypoxia,to regulate many biological behaviors of cell,such as the process of glycolytic enzymes,growth factors and vasoactive peptides,angiogenesis factors,and genes involved in apoptosis, invasion and proliferation.Since different kind of tumor has high heterogeneity,the effects of HIF-1αand its possible mechanisms may be quite different.
Carcinoma of the oral tongue is the most common malignancy of the oral cavity. Over the past three decades,the incidence of all oral cancers has increased in china. The most important reasons that caused failure in treatment to carcinoma of the oral tongue are invasion,metastasis and resistance to chemo- and radiotherapies.And hypoxic microenvironment is one of the key affective factors.It was proved that the hypoxic microenvironment was present in human oral tumor,and high levels of HIF-1αin human squamous cell carcinomas seemed correlated with tumor resistance to radio- and chemotherapy.Nonetheless,its effects on the phenotype of human tongue squamous cell carcinoma cells under hypoxic condition have not yet been sufficiently studied.Recent studies have discovered that Adrenomedullin(ADM) involves in regulating of biological characteristics of tumor,such as proliferation and apoptosis.Also it may be an important hypoxic regulated gene involved in hypoxic adaption of tumor cells.It was reported that under hypoxic conditions,the expression of HIF-1αand ADM are both up-regulated.Our study also proved that hypoxia will obviously increase the content of HIF-1αprotein and ADM mRNA in the tongue squamous cell carcinoma cells line Tca8113.However,there is no report regarding the relationship of them,and whether HIF-1αwill cause more progressive phenotype in tumor cells mediated by regulating the expression of ADM.In the present study,we are using tongue squamous cell carcinoma cells line Tca8113,and using siRNA_(HIF-1α) to study the effects of HIF-1αto proliferation,apoptosis and invasion,as well as study the expression of ADM regulated by HIF-1α.We would like to discover the expression of HIF-1αin the tongue squamous cell carcinoma cells line Tca8113,and the new regulative pathway of HIF-1α,with a view to find an efficient target to work out more reasonable treatment strategy,target to hypoxic characteristic.
SectionⅠ: Influence of hypoxia on the biological characteristics of tongue squamous cell carcinoma cells line Tca8113 and the expression of HIF-1α/ADM
Objective
To observe the hypoxia- induced changes of biological characteristics in the tongue squamous cell carcinoma cells line Tca8113 and to investigate the expression of HIF-1αand ADM in Tca8113 cells.
Meterial and Methods
1.Cell culture:Tongue squamous cell carcinoma cells line(Tca8113 cells) were routinely cultured in RPMI1640 containing 10%foetal bovine serum(FBS),penicillin (100 units/ml),and streptomycin(100μg/ml) at 37℃in a humidified air atmosphere containing 5%carbon dioxide.For hypoxic culture,Tca8113 cells were cultured at 37℃in 94%N_2,5%CO_2 and 1%O_2 for 4h,24h or 48h when growing to 80% confluence.
2.Real-time RT-PCR analysis for HIF-1α:The HIF-1αmRNA expression was assayed quantitatively by real-time reverse transcriptionpolymerase chain reaction (real-time RT-PCR).Tca8113 cells were incubated under normoxic or hypoxic condition for 4h or 24h and harvested,total RNA was extracted by TRIZOL according to the manufacturer's instructions.Synthesis of first-strand cDNA was carried out with Revert Aid~(TM) First Strand cDNA Synthesis Kit.The relative amount of PCR product was calculated as threshold cycle(CT value) of the sample divided by that of humanβ-actin.
3.Western blot analysis:Tca8113 cells were incubated under normoxic or hypoxic condition and cellular protein extracts were prepared.HIF-1αexpression induced by hypoxia was measured on protein level by Western blot.
4.Real-time RT-PCR analysis for ADM mRNA:Tca8113 cells(2×10~5) were incubated under hypoxic condition for 2h,4h,8h,or 24h and harvested.Total RNA was extracted by TRIZOL.The expression of ADM mRNA was assayed quantitatively by real-time PCR.
5.Cell proliferation assay:The Tca8113 cells were incubated overnight at a density of 6,000 cells/well in 96-well plates.Then Tca8113 cells were incubated under hypoxic condition for 6h,12h,24h,48h,or 72h respectively.20μl of MTT was added each well and color development was measured on a microplate reader at 570 nm.
6.Cell cycle and apoptosis analysis:Tca8113 cells were plated at a concentration of 2×10~5 cells per well in six-well plates.Growing to 80%confluence,the cells were incubated under hypoxic condition for 4h,24h or 48h and stained with PI.DNA content was detected on a FACS Calibur.After incubated under hypoxic condition for 4h,24h or 48h,Tca8113 cells were collected and labeled with Annexin V-biotin followed by PI.Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.
7.Adhesion assay by CytoMatrix~(TM) cell adhesion kit:Tca8113 cells were collected after incubated under hypoxic condition for 4h or 24h.1×10~5 cells were added each well of CytoMatrix~(TM) cell adhesion kit.Absorbance was measured at A490 nm.
8.Invasion assay by cell invasion assay kit:The Tca8113 cells(3×10~5 cells) were added into the transwell inserts and were incubated for 24 hours under hypoxic condition.After incubation,the invaded cells were counted in five fields for each filter under a light microscope at 20×magnification.
Results
1.Proliferation of Tca8113 cells were inhibited by hypoxia.The proliferation of Tca8113 cells was inhibited significantly after the cells were cultured under hypoxic condition for 48h or 72h compared with the normoxic control(0.391±0.051 vs 0.537±0.046,P<0.01;0.255±0.031 vs 0.852±0.078,P<0.001).
2.Hypoxia induces G_1 phase cell cycle arrest in Tca8113 cells.The cells in G_0/G_1 phase were increased significantly under hypoxic condition for 24h or 48h(73.37±4.01 vs 66.59±5.25,P<0.05;87.62±6.54 vs 61.88±4.38,P<0.01).
3.Apoptosis can be induced by hypoxia.No significant increase in the percentage of apoptotic cells was found after the Tca8113 cells were cultured under hypoxic condition for 4h or 24h when compared with normoxic incubated cells.
4.The adhesion and invasion potency of Tca8113 cells was stimulated by hypoxia. Adhesion of Tca8113 cells to Matrigel-coated cell culture plates in vitro was increased significantly under hypoxic condition for 4h or 24h((126.0±9.3)%,(128.0±10.8)% vs(100.0±8.7)%,P<0.05).When the cells were cultured under hypoxic condition for 24h,the invasion potency of Tca8113 cells was stimulated significantly compared with the normoxic control.((133.0±11.6)%vs(100.0±9.2)%,P<0.05).
5.Hypoxia induced the accumulation of HIF-1αprotein.mRNA and protein was stably expressed in Tca8113 cells under normoxic condition analysed by real-time RT-PCR and Western blot.Exposed to the hypoxic environment for 4h or 24h,no significant increase in HIF-1αmRNA transcript was observed.However,hypoxia induced a rapid and sustained accumulation of HIF-1αprotein in Tca8113 cells up to 24 hours.
6.The expression of ADM mRNA in Tca8113 cells was induced by hypoxia.The expression of ADM mRNA increases when incubated under hypoxic condition up to 24h.
Conclusion
1.The study focused on the changes of biological characteristics of Tca8113 cells under hypoxic condition.It was proved that chronic hypoxia(for 48h) inhibited the proliferation and induced apoptosis of Tca8113 cells.The adhesion and invasion potency were stimulated and G_1 phase cell cycle arrest was induced by hypoxia in Tca8113 cells.
2.Hypoxia had no effect on the transcription of HIF-1αmRNA but caused a rapid and sustained accumulation of HIF-1αprotein in Tca8113 cells.It was suggested that the hypoxia induced post -transcription regulation of HIF-1αexpression.
3.We firstly found that the expression of ADM mRNA was induced by hypoxia in Tca8113 cells.HIF- 1αmight play a role in the up-regulation of ADM.
Section II The effect of HIF-1αgene silence on the expression of ADM and the phenotype of Tca8113 cells
Objective
To investigate the inhibitory effects of RNA interference(RNAi) mediated by siRNA_(HIF-1α) on expression of HIF-1αand ADM and the regulation of HIF-1α/ADM pathway in the proliferation and invasion of the tongue squamous cell carcinoma cells line Tca8113.
Meterial and Methods
1.siRNA transfection:Tca8113 cells were plated at a concentration of 2×10~5 cells per well in six-well plates.Growing to 40-50%confluence,the cells were transfected with the siRNA_(HIF-1α)(50nM) premixed with the Lipofectamine~(TM) 2000.
2.The assay of potency of the siRNA_(HIF-1α) to block HIF-1αexpression:Transfected Tca8113 cells were incubated under normoxic or hypoxic condition for 24h.HIF-1αexpression was measured on mRNA level by real-time PCR and protein level by Western blot.
3.Immunofluorescence assay:The Tca8113 cells(8×10~4 in 1ml culture medium) were plated onto chamber slides and allowed to attach overnight.When growing to 40%confluence,the cells were transfected with 50 nM siRNA_(HIF-1α) for 24h and incubated at 1%oxygen for 24h.The samples were incubated with mouse anti-human HIF-1αmonoclonal antibodies.Expression of HIF-1αwas detected with fluorescence microscopy.
4.Cell proliferation assay:For cell proliferation assay of 50nM siRNA_(HIF-1α) transfected Tca8113 cells,transfected cells were incubated under normoxic or hypoxic condition for 24h,48h,or 72h respectively.Then cell proliferation was analyzed by MTT colorimetric assay.20μl of MTT was added each well and color development was measured on a microplate reader at 570nm.
5.Analysis of cell cycle and apoptosis of transfected Tca8113 cells:For analysing the cell cycle,the transfected Tca8113 cells were incubated under hypoxic condition for 24 of 48h.Propidium iodine was added to the cells for 30 minutes in the dark prior to FACS analysis.Transfected Tca8113 cells were incubated under normoxic or hypoxic condition for 24h.After incubation with Annexin V-FlITC antibody and PI in the dark,the cells were analysed by a flow cytometer.
6.Adhesion assay of siRNA_(HIF-1α) transfected Tca8113 cells:The transfected cells were incubated under hypoxic condition for 24h.1×10~5 cells were added each well of CytoMatrix~(TM) cell adhesion kit.Absorbance was measured at A490 nm.
7.Invasion assay of siRNA_(HIF-1α) transfected Tca8113 cells:The siRNA_(HIF-1α) transfected Tca8113 cells(3×10~5 cells) were added into the transwell inserts and were incubated for 24 hours under normoxic or hypoxic condition.After incubation, the invaded cells were counted in five fields for each filter under a light microscope at 20×magnification.
8.Expression of ADM mRNA in siRNA_(HIF-1α) transfected Tca8113 cells:The transfected Tca8113 cells were incubated under hypoxic condition for 24h.Total RNA was extracted by TRIZOL.The expression of ADM mRNA was assayed quantitatively by real-time PCR.
Results
1.HIF-1αexpression was suppressed by siRNA_(HIF-1α):Reduction of HIF-1αmRNA expression by approximately 90%was found in siRNA_(HIF-1α) treated cells,compared to siRNA_(Irr) treated cells or mock transfection control in both normoxia and hypoxia (P<0.01).Likewise,the results of western blot and immunofluorescence showed that hypoxia-induced HIF-1αprotein was significantly suppressed in Tca8113 cells treated with siRNA_(HIF-1α) in comparison to mock transfection controls.
2.Knockdown of HIF-1αinhibits proliferation of Tca8113 cells:In the first 24 hours,the proliferation of Tca8113 cells treated with siRNA_(HIF-1α) was not inhibited significantly compared with the mock transfected control under normoxic condition. However,the proliferation was inhibited significantly under hypoxic condition (0.252±0.029 vs 0.322±0.031,P<0.05).At 48h and 72h,siRNA_(HIF-1α) significantly inhibited the proliferation of Tca8113 cells under hypoxic and normoxic condition compared to the mock transfected control(P<0.01).
3.Apoptosis is induced by siRNA_(HIF-1α) in Tca8113 cells:The apoptosis of siRNA_(HIF-1α) transfected Tca8113 cells significant increases as compared to mock transfected control when incubated under normoxic or hypoxic condition for 24h ((12.15±1.02)%vs(4.68±0.56)%;(17.77±1.22)%vs(6.43±0.55)%,P<0.01).
4.Treated with siRNA_(HIF-1α) inhibited adhesion and invasion of Tca8113 cells:The adhesion of siRNA_(HIF-1α) treated Tca8113 cells was decreased to 74%and 63.6%of mock transfected control and the invasion of siRNA_(HIF-1α) treated Tca8113 cells was decreased to 65%and 55.8%of mock transfected control when incubated under hypoxic condition for 24h.
5.The expression of ADM mRNA induced by hypoxia was inhibited by siRNA_(HIF-1α):Exposed to the hypoxic environment for 24h,no significant increase in ADM mRNA transcript was observed in siRNA_(HIF-1α) treated Tca8113 cells.
Conclusion
1.Down-regualtion of HIF-1αmediated by siRNA can inhibit the proliferation, induced the apoptosis of Tca8113 cells and inhibited adhesion and invasion of Tca8113 cells.It was suggested that HIF-1αplayed an important role in the regulation of phenotype of Tca8113 cells.
2.We firstly found that the expression of ADM mRNA induced by hypoxia cound be inhibited completely by siRNA_(HIF-1α).It was suggested that HIF-1αregulated the phenotype of Tca8113 cells to adapt the hypoxic condition mediated by regulating the expression of ADM.
3.It is important to discover the mechanism of HIF-1α/ADM pathway to Tca8113 cells on hypoxic adaption for finding the new treatment strategy target hypoxia.
引文
1. Kademani D. Oral cancer. Mayo Clin Proc. 2007; 82(7): 878-887.
2. Majer M, Jensen RL, Shrieve DC, et al. Biochemotherapy of metastatic melanoma in patients with or without recently diagnosed brain metastases. Cancer. 2007 110(6):1329-1337.
3. Dear R, Wilcken N, Shannon J, et al. Beyond chemotherapy-demystifying the new 'targeted' cancer treatments, Aust Fam Physician. 2004; 37(1-2): 45-49
4. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst, 2001; 93(4): 266-276.
5. Thomlinson RH. Tumour anoxia and the response to radiation. Sci Basis Med Annu Rev, 1965:74-90
6. Park SY, Kim YJ, Gao AC, et al. Hypoxia increases and rogen receptor activity in prostate cancer cells. Cancer Res. 2006; 66(10): 5121-5129
7. Postovit LM, Seftor EA, Seftor RE, et al. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res. 2006; 66(16): 7833-7836.
8. Yasuda H. Solid tumor physiology and hypoxia-induced chemo/radio-resistance: novel strategy for cancer therapy: nitric oxide donor as a therapeutic enhancer. Nitric Oxide. 2008 Sep; 19(2): 205-216.
9. H¨ockel M, Schlenger K, Mitze M. Hypoxia and radiation response in human tumors. Semin Radiat Oncol, 1996; 6: 1-8.
10. Carmeliet P, Jain RK. Angiogenesis in cancer and other disease. Nature, 2000,407: 249-257.
11. Gregg L, Semenza. Regulation of mammalian O_2 homeostasis by hypoxia-inducible factor-1. Annu Rev Cell Dev Biol, 1999; 15: 551-578.
12. Brahimi-Horn MC, Pouyssegur J. Harnessing the hypoxia-inducible factor in cancer and ischemic disease. Biochem Pharmacol, 2007; 73: 450-457.
13. 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: 5447-5554.
14. Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O_2 tension. Proc Natl Acad Sci USA, 1995; 92: 5510-5514.
15. Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp Mol Med, 2004; 36 (1): 1212.
16. Semenza G. L., Jiang B-H., Leung S. W., et al. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem, 1996; 271: 32529-32537.
17. 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(15): 8104-8109.
18. Zhong H, De Marzo A. M , Laughner E, et al. Overexpression of hypoxia-inducible factor la in common human cancers and their metastases. Cancer Res, 1999; 59: 5830-5835.
19. Greijer AE, Delis-van Diemen PM, Fijneman RJ, et al. Presence of HIF-1 and related genes in normal mucosa, adenomas and carcinomas of the colorectum. Virchows Arch. 2008; 452(5): 535-544.
20. Gwak GY, Yoon JH, Kim KM. Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression. J Hepatol, 2005 Mar; 42(3): 358-364
21. Kurokawa T, Miyamoto M, Kato K, et al. Overexpression of hypoxia-inducible-factor 1 alpha (HIF-1 alpha) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer, 2003, 89(6): 1042-1047.
22. Carmeliet P, Dor Y, Herbert JM, et al. Role of HIF-1α in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Nature, 1998; 394: 485-490
23. Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Hypoxia-inducible factor (HIF 1A and HIF 2A), angiogenesis, and chemoradiotherapy outcome of squamous cell head-and-neck cancer. Int J Radiat Oncol Biol Phys, 2002, 53(5): 1192-1202.
24. Aebersold DM, Burri P, Beer KT, et al. Expression of hypoxia-inducible factor-1 alpha: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res, 2001; 61: 2911-2916.
25. Mohamed KM, Le A, Duong H, et al. Correlation between VEGF and HIF-1 alpha expression in hunmn oral squanlous cell carcinonla. Exp Mol Pathol, 2004; 76(2): 143-152.
26. Sasabe E, Tatemoto Y, Li D, et al. Mechanism of HIF-1 alpha-dependent suppression of hypoxia induced apoptosis in squamous cell carcinOma cells. Cancer Sci, 2005,96: 394-402.
27. Shemirani B, Crowe DL. Hypoxic induction of HIF-1 alpha and VEGF expression in head and neck squamous cell carcinoma lines is mediated by stress activated protein kinases. Oral Oncol, 2002, 38(3): 251-257.
28. Bremian PA, Quintero M, Hunterian Lecture. Regulation of hypoxia-inducible factor 1 (HIF-1) by nitric oxide in oral squamous cell carcinoma. Ann R Coil Surg Engl. 2005; 87: 153-158
29. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res, 1989; 49: 6449-6465.
30. Sutherland RM. Tumor hypoxia and gene expression. Acta oncol, 1998; 37: 567-574
31. Dachs GU, Tozer GM. Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer, 2000; 36: 1649-1660
32. Graeber TG, Osmanian C, Jacks T, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature, 1996; 379(6560): 88-91
33. Gatenby RA, Kessler HB, Rosenblum JS, et al. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int J Radiat Oncol Biol Phys, 1988; 14(5): 831-838.
34. Brizel DM, Sibley GS, Prosnitz LR, et al. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck. Int J Radiat Oncol Biol Phys, 1997; 38(2):285-289.
35.邱蔚六.口腔癌诊治的现状和趋向.中华口腔医学杂志,2002;37(2):81-83.
36.Warburg O.On the origin of cancer cells.Science,1956;124(3215):269-270
37.Amellem O.,Pettersen E.O.Cell inactivation and cell cycle inhibition as induced by extreme hypoxia:the possible role of cell cycle arrest as a protection against hypoxia-induced lethal damage.Cell Prolif,1991;24:127-141.
38.Serrano M.,Hannon G.J.and Beach D.A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4.Nature,1993;366:704-707.
39.Liotta LA,Stetler-Stevenson WG.Tumor invasive and metastases:an imbalance of positive and negative regulation.Cancer Res,1991 Sep 15;51(18 Suppl):5054s-5059s
40.Pugh CW,Ratcliffe PJ.Regulation of angiogenesis by hypoxia:role of the HIF system.Nat Med,2003;9:677-684.
41.Mazure NM,Brahimi-Horn MC,Pouyssegur J.Protein kinases and the hypoxia-inducible factor-1,two switches in angiogenesis.Curr Pharm Des,2003,9:531-541.
42.Forsythe A,Jiang H,Iyer V,et al.Activation of vascular endothelial growth factor gene transcrip tion by hypoxia-inducible factor 1.Mol Cell Biol.1996;16:4604-4613.
43.Manalo D.J,Rowan A,Lavoie T,et al.Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1,Blood,2005;105:659-669.
44.Damert A,Machein M,Breier G,et al.Up-regulation of vascular endothelial growth factor expression in a rat glioma is conferred by two distinct hypoxia-driven mechanisms.Cancer Res,1997;57(17):3860-3864.
45.Semenza GL.Regulation of cancer cell metabolism by hypoxia-inducible factor 1.Semin Cancer Biol.2009;19(1):12-16.
46.Guillemin K,Krasnow MA.The hypoxic response:huffing and HIFing.Cell,1997;89:9-12.
47.Hao Zhu.Oxygen sensing and signaling:impact on the regulation of physiologically important genes.Respir Physiol,1999;115:239-247.
48.Shoshani T,Faerman A,Mett I,et al.Klentificatien of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol Cell Biol, 2002; 22(7): 2283-2293.
49. Jiang B-H., Rue E., Wang G. L., et al. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem. 1996, 271: 17771-17778.
50. Gregg L.S. Hypoxia, clonal selection, and the role of HIF-1 in tumor progression. Crit Rev Biochem Mol Biol. 2000; 35(2):71-103.
51. Rolfs A, Kvietikova I, Gassmann M, et al. Oxygen-regulated transferring expression is mediated by hypoxia-inducible factor-1. J Biol Chem, 1997; 272: 20055-20062.
52. Forsythe A, Jiang H, Iyer V, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996; 16: 4604-4613.
53. Melillo G, Musso T, Sica A, et al. A hypoxia-responsive element mediates a noval pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med, 1995; 182:1683-1693.
54. Lee J, Jiang H, Chin Y, et al. Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J Biol Chem, 1997; 272:5375-5381.
55. Ebert L, Firth D, Ratcliffe J. Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences. J Biol Chem, 1995; 270:29083-29089.
56. Zaman K, Ryu H, Hall D, et al. Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF21/ CREB and increased expression of glycolytic enzymes, p21, and erythropoietin. J Neurosci, 1999; 19: 9821-9830.
57. Scheid A, Wenger H, Schaffer L, et al. Physiologically low oxygen concentrations in fetal skin regulate hypoxia-inducible factor 1 and transforming growth factor-beta 3. FASEB J, 2002; 16: 411-413.
58. FeldserD, Agani F, Iyer V, et al. Reciprocal positive regulation of hypoxia-inducible factor 1 alpha and insulin-like growth factor 2. Cancer Res, 1999; 59:3915-3918.
59. Manalo D.J, Rowan A, Lavoie T, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1, Blood, 2005; 105: 659-669.
60. Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha in normal human tissues, cancers, and tumor- associated macrophages. Am J Pathol, 2000; 157(2): 411-421.
61. Zagzag D, Zhong H, Scaizitti JM, et al. Expression of hypoxia-inducible factor 1 alpha in brain tumors: association with angiogenesis, invasion, and progression. Cancer, 2000; 88: 2606-2618.
62. Turner KJ, Moore JW, Jones A, et al. Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation. Cancer Res, 2002; 62: 2957-2961
63. Akakura N, Kobayashi M, Horiuchi I, et al. Constitutive expression of hypoxia-inducible factor-1 alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient deprivation. Cancer Res, 2001; 61(17): 6548-6554.
64. Kitamura K, Kangawa K, Kawamoto M, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun, 1993; 192: 553-560.
65. Mclatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-likereceptor. Nature, 1998, 393(3): 333.
66. Cormier-Regard S, Nguyen SV, Claycomb WC. Adrenomedullin gene expression is developmentally regulated and induced by hypoxia in rat ventricular cardiac myocytes. J. Biol. Chem, 1998; 273: 17787-17792.
67. Satoh F, Takahashi K, Murakami O, et al. Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. J Clin Endocrinol Metab, 1995; 80: 1750-1752.
68. Martinez A, Miller MJ, Unsworth EJ, et al. Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology, 1995; 136: 4099-4105.
69. Graeber TG, Peterson JF, Tsai M, et al. Hypoxia induces accumulation of p53 protein, but activation of a G1 phase checkpoint by low oxygen conditions is independent of p53 status. Mol Cell Biol, 1994; 14: 6264-6277
70. MoritzW, Meier F, Stroka DM, et al. Apoptosis in hypoxic human pancreatic islets correlates with H IF-1 alpha exp ression. FASEB J, 2002,16 (7): 745-747
71. Kilic M, Kasperczyk H, Fulda S. Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance. Oncogene, 2007,26(14):2027-2038
72. Fujiwara S, Nakagawa K, Harada H,et al. Silencing hypoxia-inducible factor-1 alpha inhibits cell migration and invasion under hypoxic environment in malignant gliomas. Int J Oncol. 2007; 30(4): 793-802
73. Miller MJ, M artinez A, U nswo rth EJ, et al. A drenomedullin expression in human tumor cell lines: It potential role as an autocrine growth factor. Biol Chem, 1996; 271(38): 23345-23351.
74. Albertin G, Carraro G, Petrelli L, et al. Endothelin-1 and adrenomedullin enhance the growth of human adrenocorticalcarcinoma derived SW213 cell line by stimulating proliferation and inhibiting apoptosis. Int J Mol Med, 2005,15(3): 469-474.
75. Ramachandran V, Arumugam T, Hwang RF, et al. Adrenomedullin is expressed in pancreatic cancer and stimulates cell proliferation and invasion in an autocrine manner via the adrenomedullin receptor, ADMR. Cancer Res. 2007, 67(6): 2666-2675.
76. Supriya Kapas, Dean W. Brown, Paula M. Farthing, et al. Adrenomedullin has mitogenic effects on human oral keratinocytes: involvement of cyclic AMP. 1997, 418(3): 287-290.
77. Fujita Y, Mimata H, Nasu N, et al. Involvement of adrenomedullin induced by hypoxia in angiogenesis in human renal cell carcinoma. Int J Urol, 2002; 9(6): 285-295.
78. Keleg S, Kayed H, Jiang X, et al. Adrenomedullin is induced by hypoxia and enhances pancreatic cancer cell invasion. Int J Cancer; 2007; 121(1): 21-32.
79. Oehler MK, Norbury C, Hague S, et al. Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells: a possible promotion mechanism for tumour growth. Oncogene, 2001; 20: 2937-2945.
80. FireA, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditise legans. Nature, 1998; 391(6669): 806-811.
81. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001; 411(6836): 494-498.
82. Couzin J. Breakthrough of the year: Small RNAs make big splash. Science, 2002; 298(5602): 2296-2297.
83. Meister G, Tuschl T. Mechanisms of genes silencing by double-stranded RNA. Nature, 2004; 431(7006): 343-349.
84. Harmon GJ. RNA interference. Nature, 2002; 418 (6894): 244-251.
85. Davenport RJ. Gene silencing: a faster way to shut down genes. Science, 2001; 292 (5521): 1469-1471.
86. Li L, Liu X, Staver M, et al, Evaluating hypoxia-inducible factor-1 alpha as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res, 2005; 65(16): 7249-7258.
87. Yu EZ, Li YY, Liu XH, et al. Antiapoptotic action of hypoxia-inducible factor-1 alpha in human endothelial cells. Lab Invest, 2004; 84(5): 553-561.
88. 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 HepG_2. Ann N Y Acad Sci, 2002; 973: 443-447.
89. KoshijiM, Kageyama Y, Pete EA, et al. HIF-1 alpha induces cell cycle arrest by functionally counteracting Myc. EMBO J, 2004; 23(9): 1949-1956.
90. Liang X, Yang D, Hu J, et al. Hypoxia inducible factor-alpha expression correlates with vascular endothelial growth factor-C expression and lymphangiogenesis/angiogenesis in oral squamous cell carcinoma. Anticancer Res, 2008; 28(3A): 1659-1666
91. Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism. Cancer Res, 1996; 56(23): 5309-5318.
92. Brune B. Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ, 2003; 10 (8): 864-869.
93. Di Iorio R, Marinoni E, Urban G, et al. Fetomaternal adrenomeduUin levels in diabetic pregnancy. Hoim Metab Res, 2001,33: 486-490.
94. Hata K, Takebayashi Y, Akiba S, et al. Expression of the adrenomeduUin gene in epithelialovarian cancer. Mol Hum Reprod, 2000; 6 (10): 867-872.
95. Sakata J, Sh imokubo T, Kitamura K, et al. Mo lecular co lningandbio logical activities of rat adrenomeduUin, a hypo tensive peptide. Biochem Biophys Res Commun, 1993; 195: 921-927.
96. Alfredo Marti'nez, Michele Vos, Liliana Gue'dez, et al. The Effects of AdrenomeduUin Overexpression in Breast Tumor Cells. Journal of the National Cancer Institute, 2002; 94(16): 1226-1237.
97. Ibane Abasolo, Luis M, Montuenga et al. AdrenomeduUin prevents apoptosis in prostate cancer cells. Regulatory Peptides, 2006; 133(1-3): 115-122
98. Li Z., Takeuchi S., Ohara N. Paradoxically abundant expression of Bcl-2 and adrenomedullin in invasive cervical squamous carcinoma. International Journal of Clinical Oncology, 2003; 8(2): 83-89.
99. Hague S, Zhang L, Oehler MK, et al. Expression of the hypoxically regulated angiogenic factor adrenomedullin correlates with Utreine leiomyoma vascular density. Clin Cancer Res, 2000; 6(7): 2808-2814
100. Calvo A, Abasolo I, Jimenez N, et al. AdrenomeduUin and proadrenomedullin N-terminal 20 peptide in the normal prostate and in prostate carcinoma. Microsc Res Tech, 2002; 57: 98-104.