摘要
在发展中国家,宫颈癌(Cervical Cancer)是女性癌症导致的死亡中最常见的恶性肿瘤,全世界范围内大约每年有500,000新发病例,其中一半以上的新发病例及死亡病例是发生在亚洲-太平洋地区,而在发达国家仅仅占到15%左右。我国是宫颈癌的高发地区,其发病率位居世界第二位。目前宫颈癌的治疗方式主要有手术治疗、放射治疗以及化学治疗。随着宫颈癌筛查项目的广泛开展以及职业女性生育年龄的推迟,有生育要求的年轻浸润性宫颈癌患者的比例逐年升高,因此保留宫颈癌患者生育功能的治疗变得越来越重要。而对于那些合并多种内科疾病、不能耐受放化疗及手术的老年宫颈癌患者,也迫切的需要一种创伤小、效果好的治疗方式。
光动力疗法(photodynamic therapy, PDT)是近年来备受瞩目的一种新兴的治疗技术,它交叉融合了临床医学、光学及光电子学等学科发展起来。其作用原理是基于光敏剂(photosensitizer, PS)能优先积聚在增长旺盛的细胞和组织中,通过相应波长的激光照射,潴留在靶组织中的基态光敏剂吸收光子能量能够从基态跃迁到激发单线态,然后通过体系间窜跃跃迁到激发三线态,处在激发三线态的光敏剂能够激发两类互相竞争的光氧化反应,Type Ⅰ是光子直接从光敏剂转移至细胞内的细胞器底物,产生自由基,这些自由基能够与分子氧反应产生多种活性氧物质(radical oxygen species, ROS),如过氧化氢,超氧化物和羟自由基。Type Ⅱ是三线态光敏剂直接与氧分子反应产生另一些ROS,单线态氧(1O2)。这两种类型的光氧化反应能够导致细胞内多种分子的氧化反应,触发细胞的凋亡、坏死或者自噬等多种途径导致细胞死亡。其中,单线态氧是光动力作用诱导肿瘤的主要损伤形式。与常规的手术、放化疗等疗法相比,光动力疗法具有创伤小,毒性小、能够重复使用,可保护器官的完整性、保留妇女的生育功能等优点。目前已开始应用于多种良恶性肿瘤的治疗。
四甲基吡啶卟啉(5,10,15,20-tetra-(N-methyl-4-pyridyl) porphyrin,TMPyP4)是一种水溶性光敏剂,为四价阳离子的卟啉类衍生物。TMPyP4是一种高效的光敏剂,能选择性的进入到癌细胞的细胞核中,而对正常的上皮细胞几乎没有毒性作用,在PDT过程中并能产生大量的1O2对癌细胞的DNA产生切割作用而杀伤肿瘤细胞。PDT的光损伤效果有赖于光敏剂的种类以及相应波长的激光的穿透能力。TMPyP4的最长激发波长在420nm左右,因吸收系数小常导致光动力效应深度不能满足较大肿瘤的治疗需要,影响了光动力治疗的疗效。适配体(aptamer)是一种简单的、人工合成的、小分子的DNA或RNA寡聚核苷酸。它作为一种非免疫源性的抗体替代物,可以折叠成三维空间结构,高亲合性、高特异性地与靶蛋白结合。AS1411适配体(AS1411aptamer)是基于26个碱基的富含鸟嘌呤的寡聚核苷酸。它可以形成稳定的G-四联体二聚物,高亲和力、高特异性地与细胞表面的核仁素结合,通过核仁素的穿梭作用进入细胞核内干扰DNA的正常复制和抑制细胞增殖。AS1411已经证实几乎在所有的肿瘤细胞中均能发挥明显增殖抑制的作用,但是在相同的浓度时对正常的上皮细胞作用微弱。而且与其他的化疗药物不同的是,AS1411的作用缓慢,当加入到细胞中的时候,并不是立即发挥细胞毒作用,而是先引起细胞分裂的停滞及细胞周期的阻滞,而明显的细胞凋亡要在给药后7天左右才能出现。目前,AS1411作为一种新型的抗癌药物已经在路易斯维尔大学的James Graham Brown癌症研究中心完成了一期临床试验,2007年Antisoma公司宣布开始AS1411用于治疗急性骨髓细胞白血病和肾细胞癌治疗的二期临床试验。试验结果显示AS1411的安全性及对各种实体癌特别是肾细胞癌具有很好的抗癌效果,而后期的临床试验也显示：AS1411对肿瘤细胞的抑制作用有时间尺度效应(单一剂量的AS1411给予后数月才能出现肿瘤体积的缩小)和非常罕见的临床获益持续时间(许多患者能够获得长期的病情稳定期)。
本研究旨在探讨TMPyP4光动力治疗(TMPyP4/PDT)联合适配体AS1411对宫颈癌Caski细胞增殖抑制作用,并进一步探讨TMPyP4/PDT联合AS1411协同促进宫颈癌Caski细胞凋亡过程中细胞内与凋亡相关的蛋白表达的变化,阐明TMPyP4/PDT联合AS1411促进宫颈癌细胞凋亡的分子机制。以期两者的联合治疗在临床宫颈癌的光动力治疗中能够延长宫颈癌的光动力治疗时效、减少光敏剂的用量。本课题进行了两部分的研究,第一部分：TMPyP4/PDT及AS1411对宫颈癌Caski细胞株增殖抑制作用。第二部分：TMPyP4光动力治疗联合AS1411协同促进宫颈癌Caski细胞凋亡及其分子机制研究。
第一部分TMPyP4/PDT及AS1411对宫颈癌细胞株Caski的增殖抑制作用
研究目的
研究TMPyP4/PDT及AS1411对宫颈癌Caski细胞的增殖抑制作用。
研究方法
1.免疫荧光染色及流式细胞仪检测宫颈癌Caski细胞的核仁素的表达。
2.应用荧光显微镜检测(?)TMPyP4进入到宫颈癌Caski细胞中的荧光定位实验。
3.MTT法检钡(?)TMPyP4/PDT、AS1411及AS1411+TMPyP4/PDT对宫颈癌Caski细胞的增殖抑制作用。
结果：
1.宫颈癌Caski细胞膜表面、细胞浆及细胞核内均有核仁素的表达。
2.TMPyP4能够进入到宫颈癌Caski细胞的细胞质及细胞核中。
3.在3J/cm2激光能量密度的照射下,TMPyP4/PDT对宫颈癌Caski细胞的增殖抑制率随着TMPyP4浓度的增加而显著增高。
4.MTT结果显示TMPyP4/PDT (TMPyP4浓度为9μM,激光能量密度为3J/cm2)与5μM AS1411分别及联合作用对宫颈癌Caski细胞均有增殖抑制作用。单纯TMPyP4/PDT组能明显抑制宫颈癌Caski细胞的增生,其增殖抑制作用在PDT后24h最高,随着PDT后时间的延长,其对宫颈癌Caski细胞的增殖抑制率有所下降。单纯AS1411治疗组对宫颈癌Caski细胞在治疗后24h无明显增殖抑制作用,而在36h及48h显示对宫颈癌Caski细胞逐渐增强的增殖抑制作用。AS1411+TMPyP4/PDT联合治疗组对宫颈癌Caski细胞的增殖抑制作用具有时间依赖性,随着作用时间的延长,联合组对细胞生长的抑制作用越明显,联合治疗后48h达到高峰。
结论
1.TMPyP4/PDT中,TMPyP4能进入到宫颈癌Caski细胞的细胞浆及细胞核中,并呈剂量依赖的方式抑制宫颈癌细胞的增殖。
2.宫颈癌Caski细胞膜表面有核仁素的表达,AS1411通过细胞膜表面的核仁素抑制宫颈癌细胞的增殖,但是其抑制作用要在用药后36-48h方能起效。
3. TMPyP4/PDT中,随PDT后时间的延长,部分细胞活性恢复,细胞的增殖抑制率降低,TMPyP4/PDT联合AS1411治疗中,AS1411作用后24h就有明显增强的抑制细胞增殖的作用,且能够持续至用药后48h。
第二部分TMPy/PDT联合AS1411协同促进宫颈癌Caski细胞凋亡及分子机制的研究
研究目的
探讨TMPyP4/PDT联合AS1411协同促进宫颈癌Caski细胞凋亡及其分子机制。
研究方法
1.应用膜联蛋白V-硫氰酸荧光素(AnnexinV-FITC)/碘化丙啶(PI)双染色结合流式细胞术检测单纯AS1411组、单纯TMPyP4/PDT组、TMPyP4/PDT+AS1411组后24h、36h及48h细胞的凋亡率。
2.应用荧光显微镜观察AO/EB染色不同处理后的宫颈癌Caski细胞的形态学变化。
3.应用Westen-blot检测单纯AS1411组、单纯TMPyP4/PDT组、AS1411+TMPyP4/PDT组后24h、36h及48h细胞内Nf-kb、C23、Bax和Bcl-2蛋白的表达的变化。
结果：
1.TMPyP4/PDT能够诱导宫颈癌Caski细胞凋亡,其凋亡率在PDT作用后24h、36h及48h均显著高于正常对照组(p<0.05)。AS1411作用后24h、36h对宫颈癌Caski细胞凋亡没有明显影响(p>0.05)；48h单纯AS1411可以轻度诱导宫颈癌Caski细胞凋亡(p<0.05)。 AS1411+TMPyP4/PDT联合治疗能明显诱导宫颈癌Caski细胞凋亡,随着作用时间的延长24h、36h、48h,均能显示出增强的诱导细胞凋亡的能力,与正常对照组相比,具有统计学差异p<0.05),宫颈癌Caski细胞凋亡在联合作用24h时,其凋亡率与单纯TMPyP4/PDT之间无显著性差异,随着联合作用时间延长至36h、48h,联合治疗组所致的细胞凋亡明显高于单纯TMPyP4/PDT,两组间凋亡率比较有显著性差异p<0.05),联合治疗组48h细胞凋亡率显著高于单纯TMPyP4/PDT24h的细胞凋亡率(p<0.05)。
2. AO/EB染色结果显示,宫颈癌Caski细胞TMPyP4/PDT治疗后24h、36h、48h均有显著的晚期凋亡细胞出现。AS1411作用后48h有少量凋亡细胞的出现,表现为早期凋亡。联合治疗组24h、36h、48h均有显著的晚期凋亡细胞的出现。
3.单纯AS1411在24h对宫颈癌Caski细胞内促凋亡蛋白Bax、抗凋亡蛋白Bcl-2及细胞内NF-κB和C23蛋白影响不大,随着作用时间的延长,其明显下调C23蛋白、NF-κB蛋白及抗凋亡蛋白Bcl-2的表达,在48h达到高峰。TMPyP4/PDT在24h可明显下调宫颈癌Caski细胞内抗凋亡蛋白Bcl-2、NF-κB及C23蛋白表达,但是随着作用时间的延长至48h,Bcl-2蛋白的表达逐渐恢复。其对促凋亡蛋白Bax的表达影响不大。AS1411+TMPyP4/PDT联合治疗组中,联合治疗明显下调宫颈癌Caski细胞内抗凋亡蛋白Bcl-2、NF-κB及C23蛋白表达,其下调作用可延长至治疗48h,并显示出轻度增强促凋亡蛋白Bax的表达的作用。
结论
TMPyP4/PDT联合AS1411有协同促进宫颈癌Caski细胞凋亡的作用,能够增强光动力治疗效果,减少光敏剂的用量,缩短AS1411发挥抗肿瘤作用的时间。其作用机制可能与调节宫颈癌Caski细胞内抗凋亡蛋白Bcl-2、NF-κB、C23蛋白及Bax蛋白的表达有关。
Cervical cancer causes the most cancer deaths among women in developing countries. Five hundred thousand cases of cervical cancer are diagnosed yearly worldwide. Half of these cases occur in Asia-Oceania, and only15%occur in developed countries. The incidence of cervical cancer in our country ranks second in the world. For cervical cancer, the conventional cancer treatments includes chemotherapy, radiation therapy and surgical intervention, which are effective but substantially invasive for patients. With the Papanicolaou smearing test and the recent trend toward delaying childbearing for career women, fertility-sparing options have become more important for the management of this younger invasive cervical cancer patients with fertility requirement. In addition, an effective and minimally-invasive therapeutic modality is also required for those patients who are so elderly that they cannot undergo conventional cancer treatments.
Photodynamic therapy (PDT) is a promising and effective approach which has gradually gained much attention. The developing of PDT combines cross clinical medicine, optics and photonics science and other disciplines. PDT involves the administration of a given photosensitizer (PS), its selective accumulation in active proliferation tissue, and when irradiated by light of appropriate wavelength, the PS absorbs a photon and first transforms from its ground singlet state to an excited singlet state, and then undergoes intersystem crossing to a long-lived triplet state. The triplet state PS stimulates two competing photochemical pathways, generally called type I and type II reactions. The type I reaction involves electron/proton transfer directly from the PS to cellular organic substrates, yielding free radicals or radical ions. These radicals react with molecular oxygen to produce reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion and hydroxyl radical. The type II reaction involves direct energy transfer from the triplet state PS to molecular oxygen, forming another ROS, singlet oxygen. Both types of reactions cause oxidation of various cellular molecules and can induce cell death via multiple pathways of apoptosis, necrosis and/or autophagy. In particular, singlet oxygen is thought to be the primary photochemical product and the pivotal mediator of the cell death induced by PDT. PDT is minimally-invasive and could reserve complete organic structure and preserves a women's fertility function for the cervical cancer patients, and could be repeated multiple times due to low risk of side-effects compared with the conventional cancer treatments. On account of the growing interest in PDT, PDT has been introduced to the therapy for many benign and malignant diseases and has been shown to be useful in further medical applications.
5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) is a water-soluble, tetra-cationic porphyrin derivative that has been studied as a dye for use in PDT. It has been revealed as a potent photosensitizer which could selectively toxic for carcinoma cells relative to normal epithelial cells, enter into the nucleus of carcinoma cells with a quantum yield of1O2formation and provoke DNA photodamage in vitro and in vivo. However, the efficiency of PDT depends upon the PS and the limited penetration of the laser. The maximum excitation wavelength of TMPyP4is435nm, and has a poor penetration of the tissue.That can not satisfy the need for the treatment of the large cancer tissue and affect the efficacy of photodynamic therapy, and further study of improving the anticancer efficacy of TMPyP4/PDT is desirable. Aptamers are small, simple, and synthetic DNA or RNA oligonucleotides that can fold into a three-dimensional structure as a nonimmunogenic alternative to bind to their target proteins with high affinity and specificity.The AS1411aptamer is based on a26-base guanine-rich oligonucleotide which forms a dimeric G-quadruplex structure to target nucleolin receptors on the cell membrane surface with high affinity and specificity which were normally seen over expressed on the tumor cells. Upon binding nucleolin, AS-1411is capable of gaining entry into the cells and induces the inhibition of cell proliferation when the nucleolin receptors are shuttled from the plasma membrane into the cytoplasm and nucleus. AS1411was found to display antiproliferative activity in almost every cancer cell that was tested, and appears to have broad therapeutic potential. But it had very little effect on normal cells at these concentrations.Unlike most chemotherapy agents,AS1411does not cause rapid cytotoxicity when added to cells.Instead, cytostasis occurs as a result of cell division being inhibited and cell cycle being arrested, and induction of cell death occurs only after prolonged exposure to AS1411(about7days). At present, AS1411as a novel anti-cancer drug has completed Phase I clinical trials at the University of Louisville's James Graham Brown Cancer Center, and at2007Antisom company has initiated two phase II clinical trials, one in acute myeloid leukemia and one in renal cell carcinoma. The results show the safty and anti-cancer therapeutic potentional of AS1411, and the following clinical trials also show several feature of the cilinal effects of AS1411, such as the timing of responses(tumor shrinkage over a period of several months after a single dose of AS1411) and the duration of clinical benefit (several patients with long term disease stabilization or responses) are unusual.
Our study aimed to investigate the effect of TMPyP4photodynamic therapy (TMPyP4/PDT) in combination with AS1411on proliferation inhibition and apoptosis of cervical cancer Caski cells, and to establish theory foundation for studying the mechanism of the improved TMPyP4/PDT efficacy of cervical cancer Caski cells in vitro. We expected the combination treatment of TMPyP4/PDT+AS1411could prolong the duration of photodynamic therapy, reduce the amount of photosensitizer while shorten AS1411exert anti-tumor effects of time in clinical cervical cancer therapy, and provided a new idea for the treatment of cervical cancer. Our study was consisted of two parts. The first part:TMPyP4/PDT and AS1411had inhibitions of cell proliferation on cervical cancer Caski cells. The second part:In order to provide new strategies that could improve TMPyP4/PDT on cervical cancer Caski cells,we studied the synergistic anticancer mechanism of TMPyP4/PDT combined with AS1411on apoptosis of cervical cancer Caski cells.
Part1The antiproliferative effects of TMPyP4/PDT and AS1411on cervical cancer Caski cells
Objective:
To investigate the antiproliferative effects of TMPyP4/PDT and AS141alone or incombination on cervical cancer Caski cells
Methods:
1.Expression of nucleolin on the surface of cervical cancer Caski cells was detected by immunofluorescence and flow cytometry.
2. The intracellular localization of TMPyP4in cercival cancer Caski cells was assessed using fluorescence microscopy.
3. Antiproliferative effects of TMPyP4/PDT and AS1411, respectively and in combination, on growth of cervical cancer Caski cells were assessed using MTT assay.
Results
1. The expressions of nucleolin were detected on the cell membrane, cytoplasm and nucleus.
2.Fluorescence microscopy showed that TMPyP4could localize in the cytoplasm and nuclei of cervical cancer Caski cells.
3. The proliferation inhibition rate of TMPyP4/PDT increased in Caski cells in a dose-dependent manner following exposure to3J/cm2laser irradiation energy density.
4.MTT showed that TMPyP4/PDT (the concentration of TMPyP4is9μM and the laser irradiation energy density is3J/cm2) and AS1411(the concentration of AS1411is5μM) respectively, and in combination all could induce antiproliferative effects on Caski cells. Simple TMPyP4/PDT can inhibit cancer cell proliferation, and the antiproliferative effects was most obvious after PDT24h. With the extension of time after PDT, partial cells restore their avtivity and the cell proliferation inhibition rate slightly declined. No significant antiproliferative effects was found in Caski cells after simple AS1411treatment24h. AS1411showed slightly and gradually increased antiproliferative effects after simple AS1411treatment36h and48h.In combination treatment group, the antiproliferative effects were found in a time-dependent manner.The most obvious antiproliferative effects were found after combination treatment48h.
Conclusions
1. During TMPyP4/PDT, TMPyP4can enter into the cytoplasm and nucleus of Caski cells, and inhibit the proliferation of Caksi cells in a dose-dependent manner.
2.The expression of nucleolin was found on the membrane surface of cervical cancer Caski cells, and AS1411could inhibit the proliferation of Caski cells by the shuttle of nucleolin on the membrane surface.But its antiproliferative effects only were showed after exposure36h-48h.
3.During TMPyP4/PDT, with the extension of time after PDT, partial cells restore their avtivity and the cell proliferation inhibition rate slightly declined.But in TMPyP4/PDT combined with AS1411treatment group, the significantly enhanced antiproliferative effects were found after AS1411treatment24h,and the inhibition could can be sustained after AS1411treatment48h.
Part2The Synergistic Mechanism of TMPyP4/PDT Combined with AS1411on Apoptosis of Cervical Cancer Caski Cells
Objective:
To explore the synergistic molecular mechanism of TMPyP4/PDT combined with AS1411on apoptosis of cervical cancer Caski cells.
Methods:
1. The cell apoptosis rate of simple AS1411group, simple TMPyP4/PDT group, TMPyP4/PDT+AS1411group after treatment24h,36h and48h was observed by flow cytometry with annexin V-FITC (AnnexinV-FITC)/propidium iodide (PI) double staining.
2. The AO/EB staining morphological changes of cervical cancer Caski cells after different treatments were detected by fluorescence microscope.
3. The effect of simple AS1411, simple TMPyP4/PDT, TMPyP4/PDT+AS1411on expression of proteins related with apoptosis in cervical cancer Caski cells was analyzed by Western blot.
Results
1. TMPyP4/PDT could induce cervical cancer Caski cell apoptosis, and the percentage of apoptotic cells was significantly higher than the control group after PDT24h,36h and48h (VS control group, p<0.05). It had no significant effects on the percentage of apoptotic cells of Caski cells after AS141124h and36h (V*S control group, p>0.05), and after AS141148h, it displayed a mild effect on induced apoptosis (VS control group,p<0.05). TMPyP4/PDT+AS1411combination therapy can significantly induce apoptosis in Caski cells, and the percentage of apoptotic cells increased with prolonged exposure to AS1411(VS control group,p<0.05). When exposure to AS141lafter48h, the percentage, of apoptotic cells in combined treatment group was significantly higher than simple TMPyP/PDT group (p<0.05). Furthermore the the percentage of apoptotic cells in combined treatment group after AS141148h was significantly higher than that of simple TMPyP/PDT group24h.
2. The results of AO/EB staining showed that Caski cells showed typtical changes of late apoptosis after TMPyP4/PDT24h,36h,48h. Only early apoptotic morphological changes in a small number of Caski cells were observed after AS141148h. During24h-48h of TMPyP4/PDT+AS1411combination treatment, it can be detected typical late apoptotic morphological changes on Caski cells.
3. When exposure to AS1411after24h, it has little effect on the expressions of Bax protein, Bcl-2protein, NF-κB protein and C23protein, but with extention of exposure time, it could down-regulate the expressions of Bcl-2protein, NF-κB protein and C23protein and it was obviously after48h. TMPyP4/PDT could down-regulate the expressions of Bcl-2protein, NF-κB protein and C23protein in cervical Caski cells after24h, and after48h, the expressions of Bcl-2protein gradually restored. TMPyP4/PDT had little effect on the expressions of Bax protein. In combination treatment group it could remarkably down-regulate the expressions of Bcl-2protein, NF-κB protein and C23protein in cervical Caski cells and the down-regulation could prolonged after48h,and could show slightly up-regulating expressions of Bax protein
Conclusions
TMPyP4/PDT+AS1411combination treatments have synergistic inducing apoptosis on Caski cells.Those can enhance the effect of photodynamic therapy, reduce the amount of photosensitizer and shorten AS1411exert anti-tumor effects of time.The mechanism of ynergistic inducing apoptosis might have relation to regulating the expressions of Bcl-2protein, NF-κB protein, C23protein and Bax protein in cervical cancer cells.
引文
1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA:a cancer journal for clinicians.2011;61:69-90
2. Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics,2003. CA Cancer J Clin.2003;53:5-26
3. Gomer CJ, Dougherty TJ. Determination of [3h]-and [14c]hematoporphyrin derivative distribution in malignant and normal tissue. Cancer research. 1979;39:146-151
4. Plaetzer K, Krammer B, Berlanda J, Berr F, Kiesslich T. Photophysics and photochemistry of photodynamic therapy:Fundamental aspects. Lasers Med Sci.2009;24:259-268
5. Cuenca RE, Allison RR, Sibata C, Downie GH. Breast cancer with chest wall progression:Treatment with photodynamic therapy. Ann Surg Oncol. 2004;11:322-327
6. Du KL, Mick R, Busch TM, Zhu TC, Finlay JC, Yu G, Yodh AQ Malkowicz SB, Smith D, Whittington R, Stripp D, Hahn SM. Preliminary results of interstitial motexafin lutetium-mediated pdt for prostate cancer. Lasers Surg Med.2006;38:427-434
7. Moghissi K, Dixon K, Thorpe JA, Stringer M, Oxtoby C. Photodynamic therapy (pdt) in early central lung cancer:A treatment option for patients ineligible for surgical resection. Thorax.2007;62:391-395
8. Ackroyd R, Kelty CJ, Brown NJ, Stephenson TJ, Stoddard CJ, Reed MW. Eradication of dysplastic barrett's oesophagus using photodynamic therapy: Long-term follow-up. Endoscopy.2003;35:496-501
9. Villanueva A, Caggiari L, Jori G, Milanesi C. Morphological aspects of an experimental tumour photosensitized with a meso-substituted cationic porphyrin. J Photochem Photobiol B.1994;23:49-56
10. Tada-Oikawa S, Oikawa S, Hirayama J, Hirakawa K, Kawanishi S. DNA damage and apoptosis induced by photosensitization of 5,10,15,20-tetrakis (n-methyl-4-pyridyl)-21h,23h-porphyrin via singlet oxygen generation. Photochem Photobiol.2009;85:1391-1399
11. De Cian A, Lacroix L, Douarre C, Temime-Smaali N, Trentesaux C, Riou JF, Mergny JL. Targeting telomeres and telomerase. Biochimie.2008;90:l 31-155
12. Granotier C, Pennarun G, Riou L, Hoffschir F, Gauthier LR, De Cian A, Gomez D, Mandine E, Riou JF, Mergny JL, Mailliet P, Dutrillaux B, Boussin FD. Preferential binding of a g-quadruplex ligand to human chromosome ends. Nucleic Acids Res.2005;33:4182-4190
13. Zahler AM, Williamson JR, Cech TR, Prescott DM. Inhibition of telomerase by g-quartet DNA structures. Nature.1991;350:718-720
14. Kuimova MK, Yahioglu G, Ogilby PR. Singlet oxygen in a cell:Spatially dependent lifetimes and quenching rate constants. J Am Chem Soc. 2009;131:332-340
15. Storck S, Shukla M, Dimitrov S, Bouvet P. Functions of the histone chaperone nucleolin in diseases. Subcell Biochem.2007;41:125-144
16. Ginisty H, Sicard H, Roger B, Bouvet P. Structure and functions of nucleolin. J Cell Sci.1999;112 (Pt 6):761-772
17. Sinclair JF, O'Brien AD. Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic escherichia coli o157:H7. JBiol Chem.2002;277:2876-2885
18. Teng Y, Girvan AC, Casson LK, Pierce WM, Jr., Qian M, Thomas SD, Bates PJ. As1411 alters the localization of a complex containing protein arginine methyltransferase 5 and nucleolin. Cancer Res.2007;67:10491-10500
19. Soundararajan S, Wang L, Sridharan V, Chen W, Courtenay-Luck N, Jones D, Spicer EK, Fernandes DJ. Plasma membrane nucleolin is a receptor for the anticancer aptamer asl411 in mv4-11 leukemia cells. Mol Pharmacol. 2009;76:984-991
20. Shieh YA, Yang SJ, Wei MF, Shieh MJ. Aptamer-based tumor-targeted drug delivery for photodynamic therapy. ACSNano.2010;4:1433-1442
21. Xu X, Hamhouyia F, Thomas SD, Burke TJ, Girvan AC, McGregor WG, Trent JO, Miller DM, Bates PJ. Inhibition of DNA replication and induction of s phase cell cycle arrest by g-rich oligonucleotides. JBiol Chem. 2001;276:43221-43230
22. Rosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN, Jr., Harzstark AL, Wagle N, Figlin RA, Smith GW, Garraway LA, Choueiri T, Erlandsson F, Laber DA. A phase ii trial of as 1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma. Invest New Drugs. 2014;32:178-187
23. Bates PJ, Laber DA, Miller DM, Thomas SD, Trent JO. Discovery and development of the g-rich oligonucleotide as1411 as a novel treatment for cancer. Exp Mol Pathol.2009;86:151-164
24. Chung CW, Chung KD, Jeong YI, Kang DH.5-aminolevulinic acid-incorporated nanoparticles of methoxy poly(ethylene glycol)-chitosan copolymer for photodynamic therapy. Int JNanomedicine.2013;8:809-819
25. Sato T, Kishi S, Matsumoto H, Mukai R. Comparisons of outcomes with different intervals between adjunctive ranibizumab and photodynamic therapy for polypoidal choroidal vasculopathy. Am J Ophthalmol.2013; 156:95-105 e101
26. Usuda J, Okunaka T, Furukawa K, Tsuchida T, Kuroiwa Y, Ohe Y, Saijo N, Nishio K, Konaka C, Kato H. Increased cytotoxic effects of photodynamic therapy in il-6 gene transfected cells via enhanced apoptosis. Int J Cancer. 2001;93:475-480
27. Wu L, Yang L, Huang J, Zhang L, Weng X, Zhang X, Shen C, Zhou X, Zheng C. Cationic ester porphyrins cause high levels of phototoxicity in tumor cells and induction of apoptosis in hela cells. Chem Biodivers.2009;6:1066-1076
28. Hovanessian AG, Puvion-Dutilleul F, Nisole S, Svab J, Perret E, Deng JS, Krust B. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Exp Cell Res.2000;261:312-328
29. Mongelard F, Bouvet P. As-1411, a guanosine-rich oligonucleotide aptamer targeting nucleolin for the potential treatment of cancer, including acute myeloid leukemia. Curr Opin Mol Ther.2010; 12:107-114
30. Ruck A, Kollner T, Dietrich A, Strauss W, Schneckenburger H. Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers. J Photochem Photobiol B. 1992;12:403-412
31. Mettath S, Munson BR, Pandey RK. DNA interaction and photocleavage properties of porphyrins containing cationic substituents at the peripheral position. Bioconjug Chem.1999;10:94-102
32. Armitage B. Photocleavage of nucleic acids. Chem Rev.1998;98:1171-1200
33. Gorman AA, Rodgers MA. Current perspectives of singlet oxygen detection in biological environments. J Photochem Photobiol B.1992;14:159-176
34. Peng Q, Moan J, Nesland JM. Correlation of subcellular and intratumoral photosensitizer localization with ultrastructural features after photodynamic therapy. Ultrastruct Pathol.1996;20:109-129
35. Girvan AC, Teng Y, Casson LK, Thomas SD, Juliger S, Ball MW, Klein JB, Pierce WM, Jr., Barve SS, Bates PJ. Agro100 inhibits activation of nuclear factor-kappab (nf-kappab) by forming a complex with nf-kappab essential modulator (nemo) and nucleolin. Mol Cancer Ther.2006;5:1790-1799
36. Agarwal ML, Larkin HE, Zaidi SI, Mukhtar H, Oleinick NL. Phospholipase activation triggers apoptosis in photosensitized mouse lymphoma cells. Cancer Res.1993;53:5897-5902
37. Gomer CJ, Ferrario A, Hayashi N, Rucker N, Szirth BC, Murphree AL. Molecular, cellular, and tissue responses following photodynamic therapy. Lasers Surg Med.1988;8:450-463
38. Oleinick NL, Evans HH. The photobiology of photodynamic therapy:Cellular targets and mechanisms. Radial Res.1998;150:S146-156
39. Agarwal ML, Clay ME, Harvey EJ, Evans HH, Antunez AR, Oleinick NL. Photodynamic therapy induces rapid cell death by apoptosis in 15178y mouse lymphoma cells. Cancer Res.1991;51:5993-5996
40. Colombo LL, Vanzulli SI, Villanueva A, Canete M, Juarranz A, Stockert JC. Long-term regression of the murine mammary adenocarcinoma, Im3, by repeated photodynamic treatments using meso-tetra (4-n-methylpyridinium) porphine. Int J Oncol.2005;27:1053-1059
41. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer.1994;73:2013-2026
42. Lindsay J, Esposti MD, Gilmore AP. Bcl-2 proteins and mitochondria--specificity in membrane targeting for death. Biochim Biophys Acta. 2011；1813:532-539
43. Almeida RD, Manadas BJ, Carvalho AP, Duarte CB. Intracellular signaling mechanisms in photodynamic therapy. Biochim Biophys Acta. 2004; 1704:59-86
44. Kito S, Morimoto Y, Tanaka T, Haneji T, Ohba T. Cleavage of nucleolin and agnor proteins during apoptosis induced by anticancer drugs in human salivary gland cells. J Oral Pathol Med.2005;34:478-485
45. Otake Y, Sengupta TK, Bandyopadhyay S, Spicer EK, Fernandes DJ. Retinoid-induced apoptosis in hl-60 cells is associated with nucleolin down-regulation and destabilization of bcl-2 mrna. Mol Pharmacol. 2005;67:319-326
46. Soundararajan S, Chen W, Spicer EK, Courtenay-Luck N, Fernandes DJ. The nucleolin targeting aptamer as1411 destabilizes bcl-2 messenger rna in human breast cancer cells. Cancer Res.2008;68:2358-2365
47. Di Segni A, Farin K, Pinkas-Kramarski R. Identification of nucleolin as new erbb receptors-interacting protein. PLoS ONE.2008;3:e2310