低强度聚焦超声结合原卟啉IX诱导肿瘤细胞不同死亡模式及其机制研究
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摘要
研究背景和目的：
恶性肿瘤是危害人类健康的主要疾病之一,近几年其发病率还在呈不断上升之势,而目前应用于癌症临床治疗的手术、放疗、化疗等方法难以从根本上防治肿瘤。因此,探索肿瘤治疗新方法和技术是当前生命科学和医学的研究热点和亟待解决的重大问题。1989,日本学者Umemura S等人首次提出一种利用超声结合声敏剂联合抗肿瘤的新方法,并称之为声动力学疗法(Sonodynamic Therapy, SDT)。SDT是指对肿瘤患者静脉注射一定剂量的声敏剂,如血卟啉及其衍生物等,然后用一定频率和强度的超声照射肿瘤部位,使聚集在肿瘤部位的声敏剂产生抗肿瘤因子杀伤肿瘤细胞和抑制肿瘤生长,达到治疗肿瘤的目的。由于声敏剂的无毒或低毒性及其在肿瘤组织的聚集性,加之超声的可聚焦性、穿透性和照射部位的选择性,与单纯超声相比,SDT在降低了单纯超声致细胞死亡的强度阈值的同时,特异性地杀伤肿瘤细胞而避免对周围正常组织的损害。尤其对一些难于手术、深部组织肿瘤治疗或需静脉化疗的患者,SDT具有较强的靶向性、安全性和微创性,因此SDT抗肿瘤研究具有重要的理论意义和潜在的临床应用价值。
SDT作为抗癌研究另辟蹊径的新探索,涉及生物、医学、物理和化学诸多领域,在肿瘤治疗方面有多学科交叉的优势,但也增加了研究的难度和复杂性。尽管国内外相关学者从不同学科角度开展了许多基础研究,但由于SDT抗癌系统的多因素和复杂性,目前关于SDT抗肿瘤的作用机制仍无定论。本研究是在前期实验中发现SDT可诱导肿瘤细胞凋亡和细胞自噬现象的工作基础上,追踪近年凸显的细胞自噬研究热点及其在SDT抗肿瘤研究尚无报道的动态,利用对正常细胞安全的低强度聚焦超声和对细胞低毒、抑癌效果明显且有特殊亚细胞定位特性的原卟啉Ⅸ(PpⅨ)为声敏剂,选用在体、离体培养的小鼠腹水型肿瘤细胞S180, H-22, EAC和白血病细胞L1210为细胞模型,通过多种现代细胞分子生物学研究手段和技术,从显微结构、超微结构和分子水平不同层次上,研究了声动力学疗法诱导肿瘤细胞的不同死亡模式及差异,重点研究细胞自噬在SDT抗肿瘤中的作用,初步阐明了细胞自噬与细胞凋亡、细胞存活的关系,并探讨了在SDT抗肿瘤研究中如何利用自噬的有利因素增强SDT抗癌疗效,为声动力诱导肿瘤细胞死亡模式及细胞分子生物学机制研究和临床应用提供有价值的理论依据和科学设想。
研究方法和结果：
1.声敏剂理化属性分析利用紫外和荧光分光光度法对PpⅨ的光谱特性,声致荧光光谱测定,超声空化影响因素等进行研究。结果显示：①不同溶剂中的PpⅨ吸收谱存在差异：PBS缓冲液和1640培养液中不同浓度的PpⅨ吸收谱型基本相似,有5个明显的吸收峰,PpⅨ最大吸收位于36511nm左右；完全培养基和甲醇萃取液中PpⅨ吸收谱相似,当PpⅨ浓度≤20μg/ml时,有一强吸收带出现在402nm处,当PpⅨ浓度升高到40μg/ml时,其强吸收带左移并且呈不规则谱型。②不同溶剂中超声处理对PpⅨ吸收谱的影响不同：在PBS溶液中,超声处理降低了PpⅨ的最大吸收峰值,但对其它二级波峰影响不大；在1640溶液中,超声处理显著降低了PpⅨ最大吸收值,且在542nm处其峰值升高,可能是超声处理PpⅨ产生次级产物在该波长处有较强吸收所致；在完全培养基中超声处理对PpⅨ吸收谱基本没有影响,可能与完全培养基中PpⅨ与血清有较强结合能力,溶液粘滞度高,降低超声空化作用相关。③在PBS和1640溶液中,超声处理后PpⅨ最大荧光发射强度均有所降低,其中在1640溶液中表现更为明显。④采用TA法检测声空化产生羟自由基,研究发现超声空化依赖于超声强度,而不同浓度的PpⅨ对超声产生自由基的影响是先增加后降低,1μg/ml PpⅨ促使超声产生自由基含量增到最大,并且超声空化产生的羟自由基可以被叠氮钠、组氨酸、甘露醇、EDTA和过氧化氢酶不同程度地抑制,而不被SOD所抑制。
2.不同腹水型肿瘤细胞对超声敏感性分析以小鼠腹水型肿瘤细胞S180,H-22和EAC为实验模型,比较了不同细胞对低强度聚焦超声应答效应的不同,并从细胞类型、细胞膜特性差异等方面具体分析其可能的影响因素。结果显示：①随处理强度的增加,超声导致不同肿瘤细胞存活率下降的趋势基本一致,尤其S180和H-22细胞存活率的下降趋势非常明显,在辐照强度为3W/cm2存在明显的强度阈值。②超声诱导细胞存活率的降低受到细胞密度效应的影响,高密度细胞表现出一定的‘保护’效应。③三种肿瘤细胞对超声的敏感性依次为S180>H-22>EAC,细胞密度为1×106cells/ml可能是超声辐照离体细胞悬液的一个临界阈值,辐照强度3W/cm2为超声作用S180和H-22肿瘤细胞的强度阈值。不同细胞对超声的敏感性差异可能与肿瘤细胞的组织来源、恶性程度及细胞本身的结构特性等相关。④对不同肿瘤细胞在超声照射后细胞结构敏感靶点的研究中,发现线粒体是低强度超声产生抗癌效应的敏感位点,细胞膜损伤亦是超声导致细胞死亡的主要原因,并且不同肿瘤细胞之间存在差异,其中S180细胞表现最为明显。
3.内、外源PpⅨ在肿瘤细胞中的药代动力学变化和定位研究通过荧光分光光度法和激光共聚焦显微镜对内、外源PpⅨ在离体培养S180肿瘤细胞中的代谢和亚细胞定位情况及其动态变化进行研究,利用MTT检测比较在同等实验条件下内、外源性PpⅨ在SDT抗肿瘤中的作用。研究发现：①外源性PpIX在S180肿瘤细胞内的代谢是一个动态变化过程,细胞内PpIX含量在45min达到最高,之后略有下降,60mmin后趋于稳定。②由ALA生成的内源性PpIX (ALA-PpIX)在细胞内的含量随孵育时间延长而逐渐增多,同时释放到胞外的含量也逐渐升高,并且当加入的ALA浓度超过一定阈值时,所生成的内源性PpIX含量不会随ALA浓度升高而增加。③激光共聚焦显微镜观察结果提示外源性PpIX主要分布在细胞膜,内源性PpIX在合成初期主要定位于线粒体,随孵育时间延长,生成的PpIX由线粒体向胞浆逐渐扩散。④选择相同声敏剂浓度(ALA-PpIX和PpIX),用MTT检测比较ALA-SDT和PpIX-SDT对S180肿瘤细胞的损伤作用。结果表明外源性PpIX比内源性PpIX更有利于增强超声的细胞毒作用。
4.SDT抗癌效应与细胞膜、线粒体生物学属性变化的分析在优化实验参数的基础上,研究超声结合PpIX对S180肿瘤细胞膜、线粒体结构与功能的影响。实验结果显示：①超声处理后大分子荧光物质FD500在细胞内含量升高,LDH释放到细胞外液中的含量也升高,提示细胞膜通透性增强,胞膜完整性受到破坏,并且声敏剂PpIX显著加剧了超声的作用。②在对细胞膜蛋白的检测中发现SDT处理后即时取材,Na+-K+-ATPase和Ca2+-ATPase活性明显降低,唾液酸含量急剧下降,表明膜蛋白结构和功能受损。③超微结构观察证实了细胞膜在SDT处理过程中亦有形态学变化。④研究发现SDT处理后即时细胞内钙离子浓度升高,2h后趋于稳定；质膜电位立即大幅度降低,然后于1h内迅速恢复稳态。其原因是SDT处理一方面破坏了S180肿瘤细胞膜结构的完整性并改变了膜对离子选择性通透的能力,使胞内钙离子浓度升高；另一方面破坏了细胞膜上的糖蛋白结构主要是唾液酸(细胞膜负电荷的主要来源)含量下降,Na+-K+-ATPaes和Ca2+-ATPase舌性降低,促使质膜去极化。⑤细胞内活性氧含量在SDT处理后0.5h显著升高,1h达到最高,然后逐渐下降到正常水平,而细胞内还原性GSH水平在处理后也明显下降(p<0.01),提示声空化产生活性氧自由基在S180细胞损伤中起重要作用。⑥SDT处理后8h, SDT组细胞线粒体膜电位水平极显著降低(p<0.01)；细胞色素C氧化酶活性也明显下降(p<0.01); SDT处理后12h,细胞相对存活率达到最低,提示线粒体结构与功能变化也可能是导致细胞存活能力降低的主要原因之一。
5.SDT处理对DNA损伤与细胞周期的影响与对照相比,在单纯PpIX、单纯超声、超声结合PpIX作用于S180细胞的研究中发现：①DNA损伤是细胞受损的早期事件,处理后1h,三个实验组都可诱发S180细胞DNA严重受损,其中超声联合PpIX组表现最为显著。②超声处理后20h, PpIX组和超声结合PpIX组细胞均出现了S期阻滞,40h后阻滞现象基本被解除。推测周期阻滞的原因主要由胞内PpIX引起,超声增强了PpIX在细胞内的富集,所以表现出更明显的S期阻滞,但经过一个细胞周期,胞内PpIX经过代谢使周期阻滞得以解除。
6.SDT诱导S180细胞凋亡与细胞自噬研究选择固定SDT参数(1.1MHz,3W/cm2,1min,1μg/ml PpIX),对PpIX-SDT诱导S180细胞死亡过程中是否存在自噬现象以及自噬在细胞存活中的作用进行了研究。结果表明：①通过透射电镜观察、免疫印迹和免疫荧光技术证明了SDT可诱导S180细胞自噬发生,并且自噬发生在SDT作用后的早期阶段,随孵育时间延长逐渐减弱。②细胞凋亡的典型特征如Bax转定位、细胞色素C释放、Caspase-3活化、染色质凝集等呈时间依赖性变化,大约需要4-8h。③利用细胞自噬和凋亡的抑制剂研究自噬在SDT诱导细胞死亡中的作用,结果提示自噬体(AVOs)的形成是细胞凋亡的上游事件,自噬抑制剂3-MA和BaAl增强了SDT对线粒体膜电位的破坏,SDT联合自噬抑制剂特别是BaA1显著增强了SDT诱导Caspase-3活化水平; Caspase抑制剂z-VAD降低了SDT诱导Caspase-3活性的升高,但不能抑制线粒体膜电位变化水平,表明线粒体膜电位下降发生在Caspase上游或者不依赖于Caspase的活化；DAPI染色结果进一步证实了自噬抑制剂有利于促进SDT诱导的S180细胞凋亡,而凋亡抑制剂z-VAD减弱了SDT诱导的细胞凋亡。
7.SDT诱导小鼠白血病细胞L1210的不同死亡模式及机制研究实验以小鼠白血病细胞L1210为研究模型,筛选出PpIX-SDT诱导肿瘤细胞发生自噬和凋亡的参数阈值,通过形态学观察、生化分析和分子检测等鉴定细胞自噬和凋亡的典型特征,然后通过抑制剂实验检测细胞自噬与细胞凋亡、细胞存活之间的关系,最后对SDT作用的可能机制进行探讨。其主要研究方法和结果如下：
①SDT诱导细胞自噬活性的提高依赖很大范围内PpIX剂量和超声强度,结合细胞存活能力检测结果,选择最佳SDT参数为PpIX浓度1μg/ml,超声强度为1W/cm2。单纯PpIX和单纯超声产生很轻微的细胞毒性作用,而二者的协同作用产生明显抗肿瘤效应,使细胞存活率下降了将近40%。
②透射电镜观察证实了自噬体产生；Western blot检测自噬体标志蛋白LC3-Ⅱ提示细胞自噬活性依赖于SDT处理后的孵育时间,最短检测到自噬发生时间是超声处理后0.5h,且随孵育时间延迟自噬现象减弱；其它自噬相关蛋白Beclin1, UVRAG, VPS34和Lamp2等不同程度的表达增强分析均可证实自噬的发生。
③激光共聚焦显微镜观察到的LC3和溶酶体标志蛋白Lamp2、Cathepsin B共定位,进一步证实了在SDT处理后自噬溶酶体的形成。
④SDT处理后6h,扫描电镜观察到细胞体积缩小和膜表面起泡现象；DAPI荧光染色显示染色质凝集；与对照组相比,SDT处理组细胞内Caspase-3活性明显增强,且可以被Caspase光谱型抑制剂z-VAD所抑制；PARP作为Capase-3经典底物,其剪切片段(89kDa)也间接证明了Caspase-3活化。
⑤免疫荧光和流式细胞仪分析显示,SDT处理后,细胞凋亡的其它特征如线粒体膜电位丧失、Bax/Bak转定位、Bcl-2蛋白表达受损、细胞色素C释放等非常明显并且呈时间依赖性,提示线粒体依赖性凋亡通路在SDT抑制L1210白血病细胞增殖方面起重要作用。
⑥抑制剂实验表明SDT诱导细胞内AVOs的形成发生在凋亡的早期；自噬抑制剂3-MA或BaAl增强了SDT诱导的细胞凋亡和细胞存活率的下降；Caspase抑制剂z-VAD抑制了细胞凋亡但并不能最终抑制SDT引发的细胞死亡,提示SDT诱导细胞死亡存在多种模式和途径。
⑦SDT处理后受损的线粒体与自噬体标记蛋白LC3和Atg5明显共定位,且可以被自噬抑制剂BaAl所抑制,提示线粒体损伤是细胞自噬启动的诱因之一。而自噬抑制剂BaA1存在时,细胞内Bax的转定位和线粒体膜电位的下降程度均增强,则表明抑制自噬而伴随的细胞凋亡增强与线粒体损伤密切相关。
⑧SDT处理后细胞内ROS显著升高,处理后0.5h,加入ROS清除剂NAC在降低ROS水平的同时,发现LC3-Ⅱ表达水平明显降低并且几乎完全抑制了线粒体和Atg5的共定位,从而表明细胞内ROS水平与细胞自噬活性变化有关。
⑨DNA损伤是L1210细胞应答SDT作用的早期事件,并且在SDT处理后4h内快速被修复。SDT引发的DNA损伤可以被NAC和线粒体MPTP孔抑制剂CsA所抑制,被自噬抑制剂BaAl增强,由此推测细胞自噬在保护DNA损伤方面有一定作用,可能与自噬清除氧化受损的线粒体维持细胞内代谢平衡相关。
⑩ROS清除剂NAC可以部分抑制Caspase-3的活化和PARP的剪切,MTT检测显示NAC保护了SDT诱导的细胞存活能力的下降。
结论：
1.超声激活PpIX的作用机制与超声空化相关。超声空化可引起PpIX分子结构的变化,可使PpIX的最大吸收强度和最大荧光发射强度降低,其程度与超声作用介质、超声强度以及PpIX浓度有关,合适的PpIX浓度可以增强超声的空化作用,并且超声空化产生高温高压裂解水分子过程中涉及Fenton反应,有H2O2、OH·、O21等分子参与。
2.不同细胞由于其结构和功能等方面的差异对超声辐照的敏感性存在差异,研究比较三种小鼠腹水瘤细胞对低强度超声应答效应的敏感性顺序依次为S180>H-22>EAC,这可能与肿瘤细胞的恶性程度以及细胞膜特性差异等因素相关。
3.离体培养的S180肿瘤细胞对PpIX有一定的选择性吸收和滞留作用,加药后45min为超声激活PpIX杀伤S180肿瘤细胞的最佳声照处理时间点。外源性PpIX主要定位与细胞膜,内源性PpIX则定位于线粒体,外源性PpIX比内源性PpIX更有利于增强超声对S180的细胞毒效应。
4. PpⅨ-SDT诱导细胞死亡的靶位点主要是细胞膜和线粒体,通过超声空化直接产生机械剪切力或间接产生活性氧自由基引发一系列生物学效应,作用于细胞膜的功能蛋白、膜酶和胞内重要细胞器如线粒体等最终导致细胞死亡。
5. PpⅨ-SDT导致DNA损伤和细胞周期阻滞,从而在一定程度上抑制肿瘤细胞快速增殖,反映了SDT对肿瘤细胞的多效性。
6.在一定实验条件下,PpⅨ-SDT诱导S180细胞凋亡和自噬的发生呈时间依赖性。自噬发生在凋亡之前,细胞自噬本身不足以引发细胞死亡,它在SDT效应中起保护作用,可能与其清除受损线粒体阻止细胞凋亡应答相关。3-MA将细胞自噬阻止在早期阶段,可以通过细胞凋亡和坏死增强SDT诱导的细胞死亡,并且坏死占主要地位。Ba A1将细胞自噬阻止在晚期阶段,也可以增强SDT诱导的细胞死亡,但效果弱于3-MA。因此,无论将细胞自噬阻止在任何阶段,都可以促进SDT诱导的细胞死亡。研究结果为SDT诱导的细胞死亡模式提供新的思路,提示在本研究系统中,SDT联合细胞自噬抑制剂可以增强SDT抗肿瘤疗效。
7.由于不同肿瘤细胞对SDT敏感性的差异,研究首先筛选出诱导L1210细胞存活能力降低并且产生自噬现象的最优SDT作用参数组合,自噬发生过程中的关键作用蛋白在SDT处理后呈时间依赖性变化。
8.SDT对快速增殖的小鼠白血病细胞L1210有显著的增殖抑制作用,其机制与诱导细胞的线粒体凋亡途径有关。
9.SDT作用于离体培养的L1210细胞后,自噬潮出现细胞凋亡的早期,用自噬特异性药物抑制剂可以抑制自噬体的形成,增强SDT诱导的细胞凋亡。
10.细胞内活性氧在自噬和凋亡启动过程中起重要作用,并且线粒体损伤和DNA损伤与自噬发生相关。
Background and Objective:
Malignant tumor is one of the most deadly diseases for human beings, and its incidence rate is still rising in recent years. While, at present, the usually used methods for cancer therapy including radiotherapy, chemotherapy, surgical therapy, etc., can not prevent and cure cancer effectively. Therefore, it needs to explore new approaches for cancer therapy, and which should be the hotspots in the fields of life sciences and medical research. In1989, Sonodynamic therapy (SDT) was firstly proposed by Umemura and colleagues to describe the synergistic effects of ultrasound and sono-sensitizers on tumor treatment. SDT is a relatively new approach for cancer treatment, which involves the administration of a sono-sensitizer, such as hematoporphyrin and its derivatives, then followed by local activation by ultrasound exposure to induce tissue or cell destruction and produce significant anti-tumor effects. A series of in vivo and in vitro experiments have demonstrated that the porphyrins alone had no or very low cytotoxicity and ultrasound, especially focused ultrasound, can be precisely focused on the target volume, which made it possible to effectively activate the cytotoxicity of sonosensitizers that preferentially accumulate in tumor sites while with minimal damage to peripheral healthy tissues, this indicates that SDT has potential value for cancer therapy. In particular, for some difficult surgery, deep tissue tumors, or some patients required intravenous chemotherapy, SDT has strong targeting and security. So, SDT has important theoretical significance and clinical application.
SDT as a new cancer therapy, has the advantage of a number of interdisciplinary, involving biology, medicine, physics and chemistry fields, but this also increases the difficulty and complexity of the study. Recently, SDT has been widely investigated, mainly focusing on the mechanisms of killing effects by using different ultrasound parameters and different sonosensitizers. But until now, the exact mechanism about SDT is still unclear because of its multiple factors. Our previous study has found that SDT can induce cell apoptosis and necrosis. However, as the development of life sciences and the knowledge of cell death modes, the cytotoxicity of SDT protocols, cannot be totally explained by the induction of apoptosis or necrosis. Autophagy is a relatively newly described cellular response to various cancer therapies. Now, literature search indicates that no prior information on the potential role of autophagy in the efficacy of SDT. Therefore, in the current study, it is very interesting for us to evaluate whether autophagy occur following SDT at the experimental conditions, and to determine the function of SDT induced autophagy in the fate of tumor cells.
We chose focus ultrasound at a relatively safe intensity with minimal damage to normal cells, and protoporphyrin IX (PpIX) known to have low cytotoxicity with special sub-cellular localizations, to study SDT induced cell death in tumor cell lines like S180, H-22, EAC and L1210. Following PpIX-SDT, hallmarks of apoptosis and autophagy were detected by morphological observation, biochemical analysis and molecular measurements. The relationship between autophagy and apoptosis was further obtained by applying pharmacological inhibition studies. The potential role of autophagy in the SDT induced cell death was also evaluated. The available findings shed new insights into SDT induced cell death, and further propose some ideas about how to use the favorable factor of autophagy to enhance the anti-tumor effect of SDT, which provide useful information for SDT mediated cancer therapy from the clinical views.
Methods and Results:
1. Physical and chemical properties of sono-sensitizer. The ultraviolet-visible absorbance spectra and fluorescence emission spectra of PpIX before and after ultrasound exposure were recorded on a spectrophotometer and a spectrofluorimeter, respectively. The acoustic cavitation measurement was also studied.①The results showed that PpIX has different spectra pattern in different solvents. In PBS and1640medium, PpIX has five distinct absorption peaks, the maximum (max) peak was about at365nm; in complete cultured medium and methanol-water (v/v,9:1), the max peak was nearly at402nm when PpIX concentration was≤20μg/ml, while the absorbance pattern became irregular when PpIX concentration was up to40μg/ml.②Ultrasound treatment had different effects on PpIX absorption pattern in different solvents.In PBS buffer, ultrasound treatment decreased the max absorption peak of PpIX, but had no influence on other peaks. In1640, ultrasound treatment not only significantly reduced the max absorption value of PpIX, but also increased the peak at542nm, which may be due to some secondary products have strong absorption in this wavelength after sonication. In complete culture medium, ultrasound treatment did not produce any effect on PpIX, this may be due to PpIX has great affinity with serum and the higher viscosity in the solution reduced ultrasonic cavitation.③In PBS and1640, ultrasound treatment decreased the maximum fluorescence emission intensity, and which was more obvious in1640 medium.④Using TA dosimetry method, it was possible to evaluate the efficiency of irradiation parameters on the cavitation activity in ultrasound fields by monitoring hydroxyl radicals. The findings suggest cavitation dependents on the ultrasound intensity, and PpIX at different concentrations can cause different results on OH-radical, the max content of OH· was obtained when PpⅨ was at1μg/ml PpIX. The ultrasound exposure produced OH·radicals could be inhibited by NaN3, histidine, mannitol, EDTA and catalase, but not by SOD.
2. Different sensitivities of ascites tumor cells to ultrasound exposure. A direct comparison among the different types of tumor cells (S180, H-22and EAC) was made, and the effects of ultrasound on cellular responses were evaluated, and the potential mechanism underlying different senstivities was also investigated.①The results showed that there were similar trends for three cell types exposed in vitro to potentially sonolytic ultrasound. The relative cell survival decreased as ultrasound intensity increased, which was very obvious in S180cells and H-22cells, and the sonication threshold was approximately at3W/cm2.②There also appeared to be a common dependency of lysis on density among different cell types, at higher cell concentrations with no obvious cell death, whereas at lower densities, most of cells were damaged. The density threshold seemed to be around1×106cells/ml.③The relative cell lysis was in an order of S180>H-22>EAC. There are several possible explanations for this apparent discrepancy in relation to the results:cell types are different, raising the potential structural membrane dissimilarities and hence, different sonolytic potentials.④Different cellular responses to a given ultrasound exposure were measured. The data implied that mitochondria may act as sensitive indicators for cell injury after irradiation, and the plasma membrane can be the critical target for ultrasound induced cell death. S180exhibited the most sensitive response to ultrasound induced cell damage.
3. The pharmacokinetics and localization patterns of endo-or exo-generous PpIX in S180cells. The5-aminolaevulinic acid (ALA)-derived endogenous PpIX and exogenous PpIX pharmacokinetic profiles were determined by the fluorescence intensity of cell extractions with a fluorescence spectrophotometer based on the standard curve. The changes of their sub-cellular localization patterns with prolonged incubation time were evaluated by laser scanning confocal microscope. The cytotoxic effects of5-ALA mediated sonodynamic therapy (ALA-SDT) and exogenous PpIX mediated sonodynamic therapy (PpIX-SDT) were also evaluated by MTT assay.①Results showed that for exogenous PpIX, the pharmacokinetic was in a dose dependent manner and a plateau was found in intra-and extracellular content after45min of administration, followed by slightly decreasing, and saturated after60min.②The amount of ALA-derived endogenous intracellular PpIX showed a linear accumulation with incubation time, which was independent of ALA concentration, so did the extracellular PpIX level.③Fluorescent images revealed that the exogenous PpIX was mainly accumulated in plasma membrane, whereas the ALA-derived PpIX was initially localized in the mitochondria and released from mitochondria to cytosol at later time points.④In order to compare the sonodynamic cytotoxicity induced by PpIX and ALA, the similar amount of endogenous PpIX induced by ALA and exogenous PpIX was applied to cells under the same incubation conditions, then exposed to ultrasound treatment. Cell survival was evaluated upon irradiation by the MTT assay as described. The result showed exogenous PpIX has more potential to enhance the ultrasound induced cytotoxicity than ALA derived endogenous PpIX.
4. SDT anti-tumor effect and the biological changes of cell membrane and mitochondrial. We performed some initial experiments to evaluate the ultrasound activation requirements of PpIX. After PpIX-SDT, several potential sensitive targets such as cell membrane and mitochondria were studied from the biological views.①After sonication, FD500fluorescent molecules in the cell and LDH released into the extracellular medium were increased, indicating the cell membrane integrity was damaged and PpIX efficiently mediated the ultrasound induced cytotoxicity.②Immediately after SDT, the activities of Na+-K+-ATPase and Ca2+-ATPase and the sialic acid content were obviously decreased, thus indicating the membrane proteins were seriously injured.③Ultra-structural observations confirmed the membrane had morphological changes after SDT treatment.④The results displayed intracellular Ca2+concentration instantly increased after exposure and resumed to normal level at2h post-treatment; the plasma membrane potential significantly decreased immediately after sonication, then quickly come to a nearly control value within1h. We speculate that on the one hand, SDT enhanced the cell membrane permeability and thus damaged its selective capacity to transport ions, causing intracellular Ca2+increased; on the other hand, SDT damaged the membrane proteins such as decreasing SA content and Na+-K+-ATPase and Ca2+-ATPase activities, promoting the plasma membrane depolarization.⑤Intracellular ROS generation increased quickly immediately after treatment and reached maximum at1h post-irradiation, then decreased gradually to normal level. And, cellular glutathione level also decreased remarkably, implying free radicals produced by acoustic cavitation play important role in S180cell damaging process.⑥The mitochondrial membrane potential and cytochrome c oxidase activity were significantly declined at8h post SDT treatment, and the cell viability decreased to its lowest level at12h post SDT treatment, this indicated that the mitochondria structural and functional damage may play important role in SDT induced cell death.
5. SDT treatment on DNA damage and cell cycle arrest.①DNA damage is an early event in cell damage. At1h after treatment, compared with control, PpIX alone, ultrasound alone and SDT can cause significant DNA damage in S180cells, and the combined group showed more seriously DNA damage than any alone treatment group.②There were obvious S phase arrest in both PpIX group and SDT group cells at20h after treatment, and which was relieved after a cell cycle (at40h after treatment), this may be due to the reduced intracellular PpIX content after longer incubation time. Compared with PpIX group, more remarkable S phase arrest in SDT group, this might be caused by more PpIX accumulation in S180cells after ultrasound exposure.
6. SDT induced cell apoptosis and autophagy in S180cells. Based on previous study, this study was to determine whether autophagy may exist after PpIX-SDT in in vitro S180cells and to investigate its relationship with apoptosis.①Under the optimal SDT conditions, autophagy was indentified by transmission electron microscopy, immunoblot and immunofluorescence observations. Autophagy flux occurred in the early step of cell damage following SDT, and gradually decreased with the incubation time.②The apoptotic features such as Bax redistribution, cytochrome c release, caspase-3activation and chromatin condensation were prominent and time dependent, which required4-8hours.③The relationship between autophagy and apoptosis was studied by applying pharmacological inhibition of autophagy or apoptosis. Data showed the autophagy inhibitors either3-methyladenine (3-MA) or Bafilomycin Al (Ba Al) led to increased dissipation of mitochondria potential. SDT treatment combined with autophagy inhibitor especially Ba Al, significantly enhanced Caspase-3activity and the ultimate cell death. Whereas the pan-caspase inhibitor, z-VAD-fmk partially prevented SDT induced cytotoxicity and Caspase-3activation, but did not obviously improve the mitochondria depolarization, suggesting that the MMP loss was likely to occur upstream and independently from caspases. DAPI staining further confirmed that autophagy inhibitors enhanced SDT induced cell apoptosis; while, the pan-caspase inhibitor z-VAD-fmk weakened SDT induced cell apoptosis.
7. Different cell death modes and its potential mechanisms in murine leukemia L1210cells following SDT. In the study, it is very interesting for us to evaluate the autophagic and apoptotic responses to PpIX-SDT in murine leukemia L1210cells. Following SDT, hallmarks of apoptosis and autophagy were detected by morphological observation, biochemical analysis and molecular measurements. The relationship between autophagy and apoptosis was further obtained by applying pharmacological inhibition studies. The potential mechanisms of SDT induced cellular responses were also evaluated. The main research methods and results are as follows:
①Results show that SDT induced autophagy was a general phenomenon at a wide range of PpIX concentrations and ultrasound intensities. However, taken the viability assay together, we chose the optimal SDT does in which PpIX (1μg/ml) alone and ultrasound alone (1W/cm2) caused slight cytotoxicity, while the synergistic effect of them can produce significant anti-tumor effect (cell viability declined about40%).
②Under the given exposure conditions, hallmarks of autophagy were confirmed. Autophagosome formation was examined by TEM observation and LC3-Ⅱ generation, furthermore, the extent of induction of autophagy was time dependent and occurred as early as0.5h post SDT. Additional markers of autophagy associated proteins Beclin1, UVRAG and Lamp2also showed enhanced expression levels following SDT.
③Confocal microscopy also revealed co-localizations of LC3(an AVOs marker) with the lysosomal marker proteins Lamp2and Cathepsin B, supporting the formation of autolysosomes.
④After6h of incubation, SEM observation provided the classical cellular shrinking and membrane belebbing following SDT. DAPI staining demonstrated the condensed chromatin by fluorescence observation. SDT could induce significant caspase-3activation compared with control, and was ensured by decreased level with the pan caspase inhibitor z-VAD-fink. Consistent with the findings, the cleavage assay of PARP, a classical caspase-3substrate, confirmed similar changes of89kDa PARP fragments in cells.
⑤As the immunofluorescence and FACS analysis revealed, after SDT, apoptotic features such as dissipation of mitochondria potential, Bax/Bak redistribution, sonodamage of Bcl-2and Cyto c release were prominent and time dependent, which suggested mitochondria dependent apoptosis pathway was involved.
⑥The inhibitor studies suggested that AVOs are formed upstream and independently of the caspase dependent death mechanism; the autophagy inhibitors either3-methyladenine (3-MA) or bafilomycin Al (Ba Al) enhanced the anti-tumor effect of SDT through induction of apoptosis and necrosis, while the pan-caspase inhibitor z-VAD-fmk decreased cell apoptosis but did not protect SDT induced cell death. The findings implied multiple cell death modes occurred following SDT.
⑦Results demonstrated mitochondria damage was an early event following SDT, and the damaged mitochondria co-localized rapidly with autophagosome markers LC3and Atg5, which were inhibited by Ba Al, suggesting mitochondria damage might play a role in initiation of autophagy. In addition, pretreatment with Ba Al clearly enhanced SDT induced Bax redistribution onto mitochondria, indicating the increased cell apoptosis by inhibiting autophagy might be related with the more seriously damaged mitochondria.
⑧The current study demonstrated obvious ROS formation immediately after treatment, and the presence of ROS scavenger NAC (N-acetylcysteine) significantly decreased ROS generation. NAC also visibly reduced the LC3-Ⅱ levels and almost completely inhibited the co-localization of mitochondria and Atg5at0.5h post exposure, thus preventing the damaged mitochondria being enclosed by AVOs.
⑨The comet assay showed intracellular DNA damage occurred early and repaired quickly within4hours following SDT, and the induced DNA damage could be mostly suppressed by cyclosporine A (an inhibitor for mitochondria permeability transition pore) and NAC. Otherwise, the autophagy inhibitor Ba Al slightly enhanced SDT induced DNA damage. Therefore, we speculate that autophagy may play a role in preventing DNA damage, presumably through its cellular housekeeping role in removing sources of oxidative stress such as defective mitochondria.
⑩Blockage of ROS production partially protected SDT induced Caspase-3activation and PARP cleavage. The ultimate role of ROS in SDT induced cell death as determined by MTT assay, showed NAC slightly protected SDT induced loss of cell viability.
Conclusion:
1. The mechanism of ultrasound activating PpIX was related to acoustic cavitation. Cavitation destroyed PpIX molecules, and reduced its maximum absorption and fluorescence emission intensity, which was dependent on ultrasonic medium, ultrasound intensity and PpIX does. PpIX concentration has great relationship with OH-production, at lower dose, it potentiated OH·radicals, while at higher does, it can inhibite OH-radicals. Cavitation can produce high-temperature and high-pressure to split water molecules, and Fenten reaction, H2O2, OH·, O21were involved during the process.
2. The results showed that cellular responses of different cells were distinct, of interest to note, the aggressive S180cells were much more sensitive than others, whereas EAC cells were relatively more resistant to ultrasound irradiation. The direct comparisons among different types of cells indicate that the sono-sensitization seems to depend on the physiological and chemical properties of tumor cells. Perhaps sections of cell membrane became destabilized following the initial radical attack and LPO reaction, which caused S180cells more susceptible to mechanical stresses during sonolysis.
3. PpIX has great preferential accumulation in in vitro cultured S180cells. Sono-sensitization with PpIX involved a45min drug-loading incubation at37℃, allowing sufficient time for cell uptake of the sensitizer to reach a maximum level, then, cells were exposed to ultrasound. Exogenous PpⅨ was mainly accumulated in plasma membrane, whereas endogenous PpⅨ was initially localized in the mitochondria. Exogenous PpIX has more potential than ALA derived endogenous PpIX in SDT induced cytotoxicity in S180cells.
4. PpIX mediated sonodynamic therapy can trigger a series of bio-effects by both direct mechanical stress and indirect chemical actions. The main damaging targets are the cell membrane proteins, membrane enzymes and cellular other organelles such as mitochondria, then eventually lead to cell death.
5. PpIX-SDT led to DNA damage and cell cycle arrest, which to some extent, inhibited the rapid proliferation of tumor cells, reflecting the pleiotropic effect of SDT on tumor cells
6. The experiments confirmed that, at optimal SDT dose, both apoptosis and autophagy occurred in a time-dependent manner. Autophagy occurred earlier than apoptosis. Autophagy by itself was not sufficient to induce cell death, which played a protective role, perhaps by promoting the rapid remove of sono-damaged mitochondria, and thereby preventing some apoptotic response following SDT. And, inhibition of the early stage of autophagy, using3-MA, sensitized cells to SDT-induced cell death through apoptosis and necrosis, and the latter was more than the former. Similarly, inhibition of the later stage of autophagy, using Ba Al, also enhanced SDT induced cell death, but was less efficient than3-MA, which mainly promoted cell apoptosis. Therefore, irrespective of the stage at which autophagy was inhibited, disabled autophagy accelerated SDT induced cell death. Therefore, the findings can be incorporated into a more general hypothesis suggesting the efficiency of killing cancer cells by sonodynamic therapy may be enhanced by the simultaneous treatment with autophagy inhibitors.
7. Because different tumor cell lines have different sensitivities to ultrasound irradiation, this study firstly selected the optimal SDT parameters in which SDT treatment can reduce the cell viability of L1210cells and simultaneously induce autophagy occurrence. Some key proteins in the process of autophagy showed time dependent changes after SDT treatment.
8. SDT can significantly inhibit the rapid proliferation of murine leukemia L1210cells, and the corresponding mechanism may be associated with the induced mitochondrial--dependent apoptosis pathway.
9. Following SDT, autophagy flux well before cell apoptosis. The relative percentages of cells undergoing apoptosis and autophagy following SDT could be experimentally manipulated. Pre-incubation with autophagy inhibitors prior to SDT promoted the appearance of apoptosis and suppressed AVOs formation.
10. ROS play important role in initiating cell autophagic and apoptotic responses. The mitochondria damage and DNA damage had great relationship with the occurrence of autophagy.
恶性肿瘤是危害人类健康的主要疾病之一,近几年其发病率还在呈不断上升之势,而目前应用于癌症临床治疗的手术、放疗、化疗等方法难以从根本上防治肿瘤。因此,探索肿瘤治疗新方法和技术是当前生命科学和医学的研究热点和亟待解决的重大问题。1989,日本学者Umemura S等人首次提出一种利用超声结合声敏剂联合抗肿瘤的新方法,并称之为声动力学疗法(Sonodynamic Therapy, SDT)。SDT是指对肿瘤患者静脉注射一定剂量的声敏剂,如血卟啉及其衍生物等,然后用一定频率和强度的超声照射肿瘤部位,使聚集在肿瘤部位的声敏剂产生抗肿瘤因子杀伤肿瘤细胞和抑制肿瘤生长,达到治疗肿瘤的目的。由于声敏剂的无毒或低毒性及其在肿瘤组织的聚集性,加之超声的可聚焦性、穿透性和照射部位的选择性,与单纯超声相比,SDT在降低了单纯超声致细胞死亡的强度阈值的同时,特异性地杀伤肿瘤细胞而避免对周围正常组织的损害。尤其对一些难于手术、深部组织肿瘤治疗或需静脉化疗的患者,SDT具有较强的靶向性、安全性和微创性,因此SDT抗肿瘤研究具有重要的理论意义和潜在的临床应用价值。
SDT作为抗癌研究另辟蹊径的新探索,涉及生物、医学、物理和化学诸多领域,在肿瘤治疗方面有多学科交叉的优势,但也增加了研究的难度和复杂性。尽管国内外相关学者从不同学科角度开展了许多基础研究,但由于SDT抗癌系统的多因素和复杂性,目前关于SDT抗肿瘤的作用机制仍无定论。本研究是在前期实验中发现SDT可诱导肿瘤细胞凋亡和细胞自噬现象的工作基础上,追踪近年凸显的细胞自噬研究热点及其在SDT抗肿瘤研究尚无报道的动态,利用对正常细胞安全的低强度聚焦超声和对细胞低毒、抑癌效果明显且有特殊亚细胞定位特性的原卟啉Ⅸ(PpⅨ)为声敏剂,选用在体、离体培养的小鼠腹水型肿瘤细胞S180, H-22, EAC和白血病细胞L1210为细胞模型,通过多种现代细胞分子生物学研究手段和技术,从显微结构、超微结构和分子水平不同层次上,研究了声动力学疗法诱导肿瘤细胞的不同死亡模式及差异,重点研究细胞自噬在SDT抗肿瘤中的作用,初步阐明了细胞自噬与细胞凋亡、细胞存活的关系,并探讨了在SDT抗肿瘤研究中如何利用自噬的有利因素增强SDT抗癌疗效,为声动力诱导肿瘤细胞死亡模式及细胞分子生物学机制研究和临床应用提供有价值的理论依据和科学设想。
研究方法和结果：
1.声敏剂理化属性分析利用紫外和荧光分光光度法对PpⅨ的光谱特性,声致荧光光谱测定,超声空化影响因素等进行研究。结果显示：①不同溶剂中的PpⅨ吸收谱存在差异：PBS缓冲液和1640培养液中不同浓度的PpⅨ吸收谱型基本相似,有5个明显的吸收峰,PpⅨ最大吸收位于36511nm左右；完全培养基和甲醇萃取液中PpⅨ吸收谱相似,当PpⅨ浓度≤20μg/ml时,有一强吸收带出现在402nm处,当PpⅨ浓度升高到40μg/ml时,其强吸收带左移并且呈不规则谱型。②不同溶剂中超声处理对PpⅨ吸收谱的影响不同：在PBS溶液中,超声处理降低了PpⅨ的最大吸收峰值,但对其它二级波峰影响不大；在1640溶液中,超声处理显著降低了PpⅨ最大吸收值,且在542nm处其峰值升高,可能是超声处理PpⅨ产生次级产物在该波长处有较强吸收所致；在完全培养基中超声处理对PpⅨ吸收谱基本没有影响,可能与完全培养基中PpⅨ与血清有较强结合能力,溶液粘滞度高,降低超声空化作用相关。③在PBS和1640溶液中,超声处理后PpⅨ最大荧光发射强度均有所降低,其中在1640溶液中表现更为明显。④采用TA法检测声空化产生羟自由基,研究发现超声空化依赖于超声强度,而不同浓度的PpⅨ对超声产生自由基的影响是先增加后降低,1μg/ml PpⅨ促使超声产生自由基含量增到最大,并且超声空化产生的羟自由基可以被叠氮钠、组氨酸、甘露醇、EDTA和过氧化氢酶不同程度地抑制,而不被SOD所抑制。
2.不同腹水型肿瘤细胞对超声敏感性分析以小鼠腹水型肿瘤细胞S180,H-22和EAC为实验模型,比较了不同细胞对低强度聚焦超声应答效应的不同,并从细胞类型、细胞膜特性差异等方面具体分析其可能的影响因素。结果显示：①随处理强度的增加,超声导致不同肿瘤细胞存活率下降的趋势基本一致,尤其S180和H-22细胞存活率的下降趋势非常明显,在辐照强度为3W/cm2存在明显的强度阈值。②超声诱导细胞存活率的降低受到细胞密度效应的影响,高密度细胞表现出一定的‘保护’效应。③三种肿瘤细胞对超声的敏感性依次为S180>H-22>EAC,细胞密度为1×106cells/ml可能是超声辐照离体细胞悬液的一个临界阈值,辐照强度3W/cm2为超声作用S180和H-22肿瘤细胞的强度阈值。不同细胞对超声的敏感性差异可能与肿瘤细胞的组织来源、恶性程度及细胞本身的结构特性等相关。④对不同肿瘤细胞在超声照射后细胞结构敏感靶点的研究中,发现线粒体是低强度超声产生抗癌效应的敏感位点,细胞膜损伤亦是超声导致细胞死亡的主要原因,并且不同肿瘤细胞之间存在差异,其中S180细胞表现最为明显。
3.内、外源PpⅨ在肿瘤细胞中的药代动力学变化和定位研究通过荧光分光光度法和激光共聚焦显微镜对内、外源PpⅨ在离体培养S180肿瘤细胞中的代谢和亚细胞定位情况及其动态变化进行研究,利用MTT检测比较在同等实验条件下内、外源性PpⅨ在SDT抗肿瘤中的作用。研究发现：①外源性PpIX在S180肿瘤细胞内的代谢是一个动态变化过程,细胞内PpIX含量在45min达到最高,之后略有下降,60mmin后趋于稳定。②由ALA生成的内源性PpIX (ALA-PpIX)在细胞内的含量随孵育时间延长而逐渐增多,同时释放到胞外的含量也逐渐升高,并且当加入的ALA浓度超过一定阈值时,所生成的内源性PpIX含量不会随ALA浓度升高而增加。③激光共聚焦显微镜观察结果提示外源性PpIX主要分布在细胞膜,内源性PpIX在合成初期主要定位于线粒体,随孵育时间延长,生成的PpIX由线粒体向胞浆逐渐扩散。④选择相同声敏剂浓度(ALA-PpIX和PpIX),用MTT检测比较ALA-SDT和PpIX-SDT对S180肿瘤细胞的损伤作用。结果表明外源性PpIX比内源性PpIX更有利于增强超声的细胞毒作用。
4.SDT抗癌效应与细胞膜、线粒体生物学属性变化的分析在优化实验参数的基础上,研究超声结合PpIX对S180肿瘤细胞膜、线粒体结构与功能的影响。实验结果显示：①超声处理后大分子荧光物质FD500在细胞内含量升高,LDH释放到细胞外液中的含量也升高,提示细胞膜通透性增强,胞膜完整性受到破坏,并且声敏剂PpIX显著加剧了超声的作用。②在对细胞膜蛋白的检测中发现SDT处理后即时取材,Na+-K+-ATPase和Ca2+-ATPase活性明显降低,唾液酸含量急剧下降,表明膜蛋白结构和功能受损。③超微结构观察证实了细胞膜在SDT处理过程中亦有形态学变化。④研究发现SDT处理后即时细胞内钙离子浓度升高,2h后趋于稳定；质膜电位立即大幅度降低,然后于1h内迅速恢复稳态。其原因是SDT处理一方面破坏了S180肿瘤细胞膜结构的完整性并改变了膜对离子选择性通透的能力,使胞内钙离子浓度升高；另一方面破坏了细胞膜上的糖蛋白结构主要是唾液酸(细胞膜负电荷的主要来源)含量下降,Na+-K+-ATPaes和Ca2+-ATPase舌性降低,促使质膜去极化。⑤细胞内活性氧含量在SDT处理后0.5h显著升高,1h达到最高,然后逐渐下降到正常水平,而细胞内还原性GSH水平在处理后也明显下降(p<0.01),提示声空化产生活性氧自由基在S180细胞损伤中起重要作用。⑥SDT处理后8h, SDT组细胞线粒体膜电位水平极显著降低(p<0.01)；细胞色素C氧化酶活性也明显下降(p<0.01); SDT处理后12h,细胞相对存活率达到最低,提示线粒体结构与功能变化也可能是导致细胞存活能力降低的主要原因之一。
5.SDT处理对DNA损伤与细胞周期的影响与对照相比,在单纯PpIX、单纯超声、超声结合PpIX作用于S180细胞的研究中发现：①DNA损伤是细胞受损的早期事件,处理后1h,三个实验组都可诱发S180细胞DNA严重受损,其中超声联合PpIX组表现最为显著。②超声处理后20h, PpIX组和超声结合PpIX组细胞均出现了S期阻滞,40h后阻滞现象基本被解除。推测周期阻滞的原因主要由胞内PpIX引起,超声增强了PpIX在细胞内的富集,所以表现出更明显的S期阻滞,但经过一个细胞周期,胞内PpIX经过代谢使周期阻滞得以解除。
6.SDT诱导S180细胞凋亡与细胞自噬研究选择固定SDT参数(1.1MHz,3W/cm2,1min,1μg/ml PpIX),对PpIX-SDT诱导S180细胞死亡过程中是否存在自噬现象以及自噬在细胞存活中的作用进行了研究。结果表明：①通过透射电镜观察、免疫印迹和免疫荧光技术证明了SDT可诱导S180细胞自噬发生,并且自噬发生在SDT作用后的早期阶段,随孵育时间延长逐渐减弱。②细胞凋亡的典型特征如Bax转定位、细胞色素C释放、Caspase-3活化、染色质凝集等呈时间依赖性变化,大约需要4-8h。③利用细胞自噬和凋亡的抑制剂研究自噬在SDT诱导细胞死亡中的作用,结果提示自噬体(AVOs)的形成是细胞凋亡的上游事件,自噬抑制剂3-MA和BaAl增强了SDT对线粒体膜电位的破坏,SDT联合自噬抑制剂特别是BaA1显著增强了SDT诱导Caspase-3活化水平; Caspase抑制剂z-VAD降低了SDT诱导Caspase-3活性的升高,但不能抑制线粒体膜电位变化水平,表明线粒体膜电位下降发生在Caspase上游或者不依赖于Caspase的活化；DAPI染色结果进一步证实了自噬抑制剂有利于促进SDT诱导的S180细胞凋亡,而凋亡抑制剂z-VAD减弱了SDT诱导的细胞凋亡。
7.SDT诱导小鼠白血病细胞L1210的不同死亡模式及机制研究实验以小鼠白血病细胞L1210为研究模型,筛选出PpIX-SDT诱导肿瘤细胞发生自噬和凋亡的参数阈值,通过形态学观察、生化分析和分子检测等鉴定细胞自噬和凋亡的典型特征,然后通过抑制剂实验检测细胞自噬与细胞凋亡、细胞存活之间的关系,最后对SDT作用的可能机制进行探讨。其主要研究方法和结果如下：
①SDT诱导细胞自噬活性的提高依赖很大范围内PpIX剂量和超声强度,结合细胞存活能力检测结果,选择最佳SDT参数为PpIX浓度1μg/ml,超声强度为1W/cm2。单纯PpIX和单纯超声产生很轻微的细胞毒性作用,而二者的协同作用产生明显抗肿瘤效应,使细胞存活率下降了将近40%。
②透射电镜观察证实了自噬体产生；Western blot检测自噬体标志蛋白LC3-Ⅱ提示细胞自噬活性依赖于SDT处理后的孵育时间,最短检测到自噬发生时间是超声处理后0.5h,且随孵育时间延迟自噬现象减弱；其它自噬相关蛋白Beclin1, UVRAG, VPS34和Lamp2等不同程度的表达增强分析均可证实自噬的发生。
③激光共聚焦显微镜观察到的LC3和溶酶体标志蛋白Lamp2、Cathepsin B共定位,进一步证实了在SDT处理后自噬溶酶体的形成。
④SDT处理后6h,扫描电镜观察到细胞体积缩小和膜表面起泡现象；DAPI荧光染色显示染色质凝集；与对照组相比,SDT处理组细胞内Caspase-3活性明显增强,且可以被Caspase光谱型抑制剂z-VAD所抑制；PARP作为Capase-3经典底物,其剪切片段(89kDa)也间接证明了Caspase-3活化。
⑤免疫荧光和流式细胞仪分析显示,SDT处理后,细胞凋亡的其它特征如线粒体膜电位丧失、Bax/Bak转定位、Bcl-2蛋白表达受损、细胞色素C释放等非常明显并且呈时间依赖性,提示线粒体依赖性凋亡通路在SDT抑制L1210白血病细胞增殖方面起重要作用。
⑥抑制剂实验表明SDT诱导细胞内AVOs的形成发生在凋亡的早期；自噬抑制剂3-MA或BaAl增强了SDT诱导的细胞凋亡和细胞存活率的下降；Caspase抑制剂z-VAD抑制了细胞凋亡但并不能最终抑制SDT引发的细胞死亡,提示SDT诱导细胞死亡存在多种模式和途径。
⑦SDT处理后受损的线粒体与自噬体标记蛋白LC3和Atg5明显共定位,且可以被自噬抑制剂BaAl所抑制,提示线粒体损伤是细胞自噬启动的诱因之一。而自噬抑制剂BaA1存在时,细胞内Bax的转定位和线粒体膜电位的下降程度均增强,则表明抑制自噬而伴随的细胞凋亡增强与线粒体损伤密切相关。
⑧SDT处理后细胞内ROS显著升高,处理后0.5h,加入ROS清除剂NAC在降低ROS水平的同时,发现LC3-Ⅱ表达水平明显降低并且几乎完全抑制了线粒体和Atg5的共定位,从而表明细胞内ROS水平与细胞自噬活性变化有关。
⑨DNA损伤是L1210细胞应答SDT作用的早期事件,并且在SDT处理后4h内快速被修复。SDT引发的DNA损伤可以被NAC和线粒体MPTP孔抑制剂CsA所抑制,被自噬抑制剂BaAl增强,由此推测细胞自噬在保护DNA损伤方面有一定作用,可能与自噬清除氧化受损的线粒体维持细胞内代谢平衡相关。
⑩ROS清除剂NAC可以部分抑制Caspase-3的活化和PARP的剪切,MTT检测显示NAC保护了SDT诱导的细胞存活能力的下降。
结论：
1.超声激活PpIX的作用机制与超声空化相关。超声空化可引起PpIX分子结构的变化,可使PpIX的最大吸收强度和最大荧光发射强度降低,其程度与超声作用介质、超声强度以及PpIX浓度有关,合适的PpIX浓度可以增强超声的空化作用,并且超声空化产生高温高压裂解水分子过程中涉及Fenton反应,有H2O2、OH·、O21等分子参与。
2.不同细胞由于其结构和功能等方面的差异对超声辐照的敏感性存在差异,研究比较三种小鼠腹水瘤细胞对低强度超声应答效应的敏感性顺序依次为S180>H-22>EAC,这可能与肿瘤细胞的恶性程度以及细胞膜特性差异等因素相关。
3.离体培养的S180肿瘤细胞对PpIX有一定的选择性吸收和滞留作用,加药后45min为超声激活PpIX杀伤S180肿瘤细胞的最佳声照处理时间点。外源性PpIX主要定位与细胞膜,内源性PpIX则定位于线粒体,外源性PpIX比内源性PpIX更有利于增强超声对S180的细胞毒效应。
4. PpⅨ-SDT诱导细胞死亡的靶位点主要是细胞膜和线粒体,通过超声空化直接产生机械剪切力或间接产生活性氧自由基引发一系列生物学效应,作用于细胞膜的功能蛋白、膜酶和胞内重要细胞器如线粒体等最终导致细胞死亡。
5. PpⅨ-SDT导致DNA损伤和细胞周期阻滞,从而在一定程度上抑制肿瘤细胞快速增殖,反映了SDT对肿瘤细胞的多效性。
6.在一定实验条件下,PpⅨ-SDT诱导S180细胞凋亡和自噬的发生呈时间依赖性。自噬发生在凋亡之前,细胞自噬本身不足以引发细胞死亡,它在SDT效应中起保护作用,可能与其清除受损线粒体阻止细胞凋亡应答相关。3-MA将细胞自噬阻止在早期阶段,可以通过细胞凋亡和坏死增强SDT诱导的细胞死亡,并且坏死占主要地位。Ba A1将细胞自噬阻止在晚期阶段,也可以增强SDT诱导的细胞死亡,但效果弱于3-MA。因此,无论将细胞自噬阻止在任何阶段,都可以促进SDT诱导的细胞死亡。研究结果为SDT诱导的细胞死亡模式提供新的思路,提示在本研究系统中,SDT联合细胞自噬抑制剂可以增强SDT抗肿瘤疗效。
7.由于不同肿瘤细胞对SDT敏感性的差异,研究首先筛选出诱导L1210细胞存活能力降低并且产生自噬现象的最优SDT作用参数组合,自噬发生过程中的关键作用蛋白在SDT处理后呈时间依赖性变化。
8.SDT对快速增殖的小鼠白血病细胞L1210有显著的增殖抑制作用,其机制与诱导细胞的线粒体凋亡途径有关。
9.SDT作用于离体培养的L1210细胞后,自噬潮出现细胞凋亡的早期,用自噬特异性药物抑制剂可以抑制自噬体的形成,增强SDT诱导的细胞凋亡。
10.细胞内活性氧在自噬和凋亡启动过程中起重要作用,并且线粒体损伤和DNA损伤与自噬发生相关。
Background and Objective:
Malignant tumor is one of the most deadly diseases for human beings, and its incidence rate is still rising in recent years. While, at present, the usually used methods for cancer therapy including radiotherapy, chemotherapy, surgical therapy, etc., can not prevent and cure cancer effectively. Therefore, it needs to explore new approaches for cancer therapy, and which should be the hotspots in the fields of life sciences and medical research. In1989, Sonodynamic therapy (SDT) was firstly proposed by Umemura and colleagues to describe the synergistic effects of ultrasound and sono-sensitizers on tumor treatment. SDT is a relatively new approach for cancer treatment, which involves the administration of a sono-sensitizer, such as hematoporphyrin and its derivatives, then followed by local activation by ultrasound exposure to induce tissue or cell destruction and produce significant anti-tumor effects. A series of in vivo and in vitro experiments have demonstrated that the porphyrins alone had no or very low cytotoxicity and ultrasound, especially focused ultrasound, can be precisely focused on the target volume, which made it possible to effectively activate the cytotoxicity of sonosensitizers that preferentially accumulate in tumor sites while with minimal damage to peripheral healthy tissues, this indicates that SDT has potential value for cancer therapy. In particular, for some difficult surgery, deep tissue tumors, or some patients required intravenous chemotherapy, SDT has strong targeting and security. So, SDT has important theoretical significance and clinical application.
SDT as a new cancer therapy, has the advantage of a number of interdisciplinary, involving biology, medicine, physics and chemistry fields, but this also increases the difficulty and complexity of the study. Recently, SDT has been widely investigated, mainly focusing on the mechanisms of killing effects by using different ultrasound parameters and different sonosensitizers. But until now, the exact mechanism about SDT is still unclear because of its multiple factors. Our previous study has found that SDT can induce cell apoptosis and necrosis. However, as the development of life sciences and the knowledge of cell death modes, the cytotoxicity of SDT protocols, cannot be totally explained by the induction of apoptosis or necrosis. Autophagy is a relatively newly described cellular response to various cancer therapies. Now, literature search indicates that no prior information on the potential role of autophagy in the efficacy of SDT. Therefore, in the current study, it is very interesting for us to evaluate whether autophagy occur following SDT at the experimental conditions, and to determine the function of SDT induced autophagy in the fate of tumor cells.
We chose focus ultrasound at a relatively safe intensity with minimal damage to normal cells, and protoporphyrin IX (PpIX) known to have low cytotoxicity with special sub-cellular localizations, to study SDT induced cell death in tumor cell lines like S180, H-22, EAC and L1210. Following PpIX-SDT, hallmarks of apoptosis and autophagy were detected by morphological observation, biochemical analysis and molecular measurements. The relationship between autophagy and apoptosis was further obtained by applying pharmacological inhibition studies. The potential role of autophagy in the SDT induced cell death was also evaluated. The available findings shed new insights into SDT induced cell death, and further propose some ideas about how to use the favorable factor of autophagy to enhance the anti-tumor effect of SDT, which provide useful information for SDT mediated cancer therapy from the clinical views.
Methods and Results:
1. Physical and chemical properties of sono-sensitizer. The ultraviolet-visible absorbance spectra and fluorescence emission spectra of PpIX before and after ultrasound exposure were recorded on a spectrophotometer and a spectrofluorimeter, respectively. The acoustic cavitation measurement was also studied.①The results showed that PpIX has different spectra pattern in different solvents. In PBS and1640medium, PpIX has five distinct absorption peaks, the maximum (max) peak was about at365nm; in complete cultured medium and methanol-water (v/v,9:1), the max peak was nearly at402nm when PpIX concentration was≤20μg/ml, while the absorbance pattern became irregular when PpIX concentration was up to40μg/ml.②Ultrasound treatment had different effects on PpIX absorption pattern in different solvents.In PBS buffer, ultrasound treatment decreased the max absorption peak of PpIX, but had no influence on other peaks. In1640, ultrasound treatment not only significantly reduced the max absorption value of PpIX, but also increased the peak at542nm, which may be due to some secondary products have strong absorption in this wavelength after sonication. In complete culture medium, ultrasound treatment did not produce any effect on PpIX, this may be due to PpIX has great affinity with serum and the higher viscosity in the solution reduced ultrasonic cavitation.③In PBS and1640, ultrasound treatment decreased the maximum fluorescence emission intensity, and which was more obvious in1640 medium.④Using TA dosimetry method, it was possible to evaluate the efficiency of irradiation parameters on the cavitation activity in ultrasound fields by monitoring hydroxyl radicals. The findings suggest cavitation dependents on the ultrasound intensity, and PpIX at different concentrations can cause different results on OH-radical, the max content of OH· was obtained when PpⅨ was at1μg/ml PpIX. The ultrasound exposure produced OH·radicals could be inhibited by NaN3, histidine, mannitol, EDTA and catalase, but not by SOD.
2. Different sensitivities of ascites tumor cells to ultrasound exposure. A direct comparison among the different types of tumor cells (S180, H-22and EAC) was made, and the effects of ultrasound on cellular responses were evaluated, and the potential mechanism underlying different senstivities was also investigated.①The results showed that there were similar trends for three cell types exposed in vitro to potentially sonolytic ultrasound. The relative cell survival decreased as ultrasound intensity increased, which was very obvious in S180cells and H-22cells, and the sonication threshold was approximately at3W/cm2.②There also appeared to be a common dependency of lysis on density among different cell types, at higher cell concentrations with no obvious cell death, whereas at lower densities, most of cells were damaged. The density threshold seemed to be around1×106cells/ml.③The relative cell lysis was in an order of S180>H-22>EAC. There are several possible explanations for this apparent discrepancy in relation to the results:cell types are different, raising the potential structural membrane dissimilarities and hence, different sonolytic potentials.④Different cellular responses to a given ultrasound exposure were measured. The data implied that mitochondria may act as sensitive indicators for cell injury after irradiation, and the plasma membrane can be the critical target for ultrasound induced cell death. S180exhibited the most sensitive response to ultrasound induced cell damage.
3. The pharmacokinetics and localization patterns of endo-or exo-generous PpIX in S180cells. The5-aminolaevulinic acid (ALA)-derived endogenous PpIX and exogenous PpIX pharmacokinetic profiles were determined by the fluorescence intensity of cell extractions with a fluorescence spectrophotometer based on the standard curve. The changes of their sub-cellular localization patterns with prolonged incubation time were evaluated by laser scanning confocal microscope. The cytotoxic effects of5-ALA mediated sonodynamic therapy (ALA-SDT) and exogenous PpIX mediated sonodynamic therapy (PpIX-SDT) were also evaluated by MTT assay.①Results showed that for exogenous PpIX, the pharmacokinetic was in a dose dependent manner and a plateau was found in intra-and extracellular content after45min of administration, followed by slightly decreasing, and saturated after60min.②The amount of ALA-derived endogenous intracellular PpIX showed a linear accumulation with incubation time, which was independent of ALA concentration, so did the extracellular PpIX level.③Fluorescent images revealed that the exogenous PpIX was mainly accumulated in plasma membrane, whereas the ALA-derived PpIX was initially localized in the mitochondria and released from mitochondria to cytosol at later time points.④In order to compare the sonodynamic cytotoxicity induced by PpIX and ALA, the similar amount of endogenous PpIX induced by ALA and exogenous PpIX was applied to cells under the same incubation conditions, then exposed to ultrasound treatment. Cell survival was evaluated upon irradiation by the MTT assay as described. The result showed exogenous PpIX has more potential to enhance the ultrasound induced cytotoxicity than ALA derived endogenous PpIX.
4. SDT anti-tumor effect and the biological changes of cell membrane and mitochondrial. We performed some initial experiments to evaluate the ultrasound activation requirements of PpIX. After PpIX-SDT, several potential sensitive targets such as cell membrane and mitochondria were studied from the biological views.①After sonication, FD500fluorescent molecules in the cell and LDH released into the extracellular medium were increased, indicating the cell membrane integrity was damaged and PpIX efficiently mediated the ultrasound induced cytotoxicity.②Immediately after SDT, the activities of Na+-K+-ATPase and Ca2+-ATPase and the sialic acid content were obviously decreased, thus indicating the membrane proteins were seriously injured.③Ultra-structural observations confirmed the membrane had morphological changes after SDT treatment.④The results displayed intracellular Ca2+concentration instantly increased after exposure and resumed to normal level at2h post-treatment; the plasma membrane potential significantly decreased immediately after sonication, then quickly come to a nearly control value within1h. We speculate that on the one hand, SDT enhanced the cell membrane permeability and thus damaged its selective capacity to transport ions, causing intracellular Ca2+increased; on the other hand, SDT damaged the membrane proteins such as decreasing SA content and Na+-K+-ATPase and Ca2+-ATPase activities, promoting the plasma membrane depolarization.⑤Intracellular ROS generation increased quickly immediately after treatment and reached maximum at1h post-irradiation, then decreased gradually to normal level. And, cellular glutathione level also decreased remarkably, implying free radicals produced by acoustic cavitation play important role in S180cell damaging process.⑥The mitochondrial membrane potential and cytochrome c oxidase activity were significantly declined at8h post SDT treatment, and the cell viability decreased to its lowest level at12h post SDT treatment, this indicated that the mitochondria structural and functional damage may play important role in SDT induced cell death.
5. SDT treatment on DNA damage and cell cycle arrest.①DNA damage is an early event in cell damage. At1h after treatment, compared with control, PpIX alone, ultrasound alone and SDT can cause significant DNA damage in S180cells, and the combined group showed more seriously DNA damage than any alone treatment group.②There were obvious S phase arrest in both PpIX group and SDT group cells at20h after treatment, and which was relieved after a cell cycle (at40h after treatment), this may be due to the reduced intracellular PpIX content after longer incubation time. Compared with PpIX group, more remarkable S phase arrest in SDT group, this might be caused by more PpIX accumulation in S180cells after ultrasound exposure.
6. SDT induced cell apoptosis and autophagy in S180cells. Based on previous study, this study was to determine whether autophagy may exist after PpIX-SDT in in vitro S180cells and to investigate its relationship with apoptosis.①Under the optimal SDT conditions, autophagy was indentified by transmission electron microscopy, immunoblot and immunofluorescence observations. Autophagy flux occurred in the early step of cell damage following SDT, and gradually decreased with the incubation time.②The apoptotic features such as Bax redistribution, cytochrome c release, caspase-3activation and chromatin condensation were prominent and time dependent, which required4-8hours.③The relationship between autophagy and apoptosis was studied by applying pharmacological inhibition of autophagy or apoptosis. Data showed the autophagy inhibitors either3-methyladenine (3-MA) or Bafilomycin Al (Ba Al) led to increased dissipation of mitochondria potential. SDT treatment combined with autophagy inhibitor especially Ba Al, significantly enhanced Caspase-3activity and the ultimate cell death. Whereas the pan-caspase inhibitor, z-VAD-fmk partially prevented SDT induced cytotoxicity and Caspase-3activation, but did not obviously improve the mitochondria depolarization, suggesting that the MMP loss was likely to occur upstream and independently from caspases. DAPI staining further confirmed that autophagy inhibitors enhanced SDT induced cell apoptosis; while, the pan-caspase inhibitor z-VAD-fmk weakened SDT induced cell apoptosis.
7. Different cell death modes and its potential mechanisms in murine leukemia L1210cells following SDT. In the study, it is very interesting for us to evaluate the autophagic and apoptotic responses to PpIX-SDT in murine leukemia L1210cells. Following SDT, hallmarks of apoptosis and autophagy were detected by morphological observation, biochemical analysis and molecular measurements. The relationship between autophagy and apoptosis was further obtained by applying pharmacological inhibition studies. The potential mechanisms of SDT induced cellular responses were also evaluated. The main research methods and results are as follows:
①Results show that SDT induced autophagy was a general phenomenon at a wide range of PpIX concentrations and ultrasound intensities. However, taken the viability assay together, we chose the optimal SDT does in which PpIX (1μg/ml) alone and ultrasound alone (1W/cm2) caused slight cytotoxicity, while the synergistic effect of them can produce significant anti-tumor effect (cell viability declined about40%).
②Under the given exposure conditions, hallmarks of autophagy were confirmed. Autophagosome formation was examined by TEM observation and LC3-Ⅱ generation, furthermore, the extent of induction of autophagy was time dependent and occurred as early as0.5h post SDT. Additional markers of autophagy associated proteins Beclin1, UVRAG and Lamp2also showed enhanced expression levels following SDT.
③Confocal microscopy also revealed co-localizations of LC3(an AVOs marker) with the lysosomal marker proteins Lamp2and Cathepsin B, supporting the formation of autolysosomes.
④After6h of incubation, SEM observation provided the classical cellular shrinking and membrane belebbing following SDT. DAPI staining demonstrated the condensed chromatin by fluorescence observation. SDT could induce significant caspase-3activation compared with control, and was ensured by decreased level with the pan caspase inhibitor z-VAD-fink. Consistent with the findings, the cleavage assay of PARP, a classical caspase-3substrate, confirmed similar changes of89kDa PARP fragments in cells.
⑤As the immunofluorescence and FACS analysis revealed, after SDT, apoptotic features such as dissipation of mitochondria potential, Bax/Bak redistribution, sonodamage of Bcl-2and Cyto c release were prominent and time dependent, which suggested mitochondria dependent apoptosis pathway was involved.
⑥The inhibitor studies suggested that AVOs are formed upstream and independently of the caspase dependent death mechanism; the autophagy inhibitors either3-methyladenine (3-MA) or bafilomycin Al (Ba Al) enhanced the anti-tumor effect of SDT through induction of apoptosis and necrosis, while the pan-caspase inhibitor z-VAD-fmk decreased cell apoptosis but did not protect SDT induced cell death. The findings implied multiple cell death modes occurred following SDT.
⑦Results demonstrated mitochondria damage was an early event following SDT, and the damaged mitochondria co-localized rapidly with autophagosome markers LC3and Atg5, which were inhibited by Ba Al, suggesting mitochondria damage might play a role in initiation of autophagy. In addition, pretreatment with Ba Al clearly enhanced SDT induced Bax redistribution onto mitochondria, indicating the increased cell apoptosis by inhibiting autophagy might be related with the more seriously damaged mitochondria.
⑧The current study demonstrated obvious ROS formation immediately after treatment, and the presence of ROS scavenger NAC (N-acetylcysteine) significantly decreased ROS generation. NAC also visibly reduced the LC3-Ⅱ levels and almost completely inhibited the co-localization of mitochondria and Atg5at0.5h post exposure, thus preventing the damaged mitochondria being enclosed by AVOs.
⑨The comet assay showed intracellular DNA damage occurred early and repaired quickly within4hours following SDT, and the induced DNA damage could be mostly suppressed by cyclosporine A (an inhibitor for mitochondria permeability transition pore) and NAC. Otherwise, the autophagy inhibitor Ba Al slightly enhanced SDT induced DNA damage. Therefore, we speculate that autophagy may play a role in preventing DNA damage, presumably through its cellular housekeeping role in removing sources of oxidative stress such as defective mitochondria.
⑩Blockage of ROS production partially protected SDT induced Caspase-3activation and PARP cleavage. The ultimate role of ROS in SDT induced cell death as determined by MTT assay, showed NAC slightly protected SDT induced loss of cell viability.
Conclusion:
1. The mechanism of ultrasound activating PpIX was related to acoustic cavitation. Cavitation destroyed PpIX molecules, and reduced its maximum absorption and fluorescence emission intensity, which was dependent on ultrasonic medium, ultrasound intensity and PpIX does. PpIX concentration has great relationship with OH-production, at lower dose, it potentiated OH·radicals, while at higher does, it can inhibite OH-radicals. Cavitation can produce high-temperature and high-pressure to split water molecules, and Fenten reaction, H2O2, OH·, O21were involved during the process.
2. The results showed that cellular responses of different cells were distinct, of interest to note, the aggressive S180cells were much more sensitive than others, whereas EAC cells were relatively more resistant to ultrasound irradiation. The direct comparisons among different types of cells indicate that the sono-sensitization seems to depend on the physiological and chemical properties of tumor cells. Perhaps sections of cell membrane became destabilized following the initial radical attack and LPO reaction, which caused S180cells more susceptible to mechanical stresses during sonolysis.
3. PpIX has great preferential accumulation in in vitro cultured S180cells. Sono-sensitization with PpIX involved a45min drug-loading incubation at37℃, allowing sufficient time for cell uptake of the sensitizer to reach a maximum level, then, cells were exposed to ultrasound. Exogenous PpⅨ was mainly accumulated in plasma membrane, whereas endogenous PpⅨ was initially localized in the mitochondria. Exogenous PpIX has more potential than ALA derived endogenous PpIX in SDT induced cytotoxicity in S180cells.
4. PpIX mediated sonodynamic therapy can trigger a series of bio-effects by both direct mechanical stress and indirect chemical actions. The main damaging targets are the cell membrane proteins, membrane enzymes and cellular other organelles such as mitochondria, then eventually lead to cell death.
5. PpIX-SDT led to DNA damage and cell cycle arrest, which to some extent, inhibited the rapid proliferation of tumor cells, reflecting the pleiotropic effect of SDT on tumor cells
6. The experiments confirmed that, at optimal SDT dose, both apoptosis and autophagy occurred in a time-dependent manner. Autophagy occurred earlier than apoptosis. Autophagy by itself was not sufficient to induce cell death, which played a protective role, perhaps by promoting the rapid remove of sono-damaged mitochondria, and thereby preventing some apoptotic response following SDT. And, inhibition of the early stage of autophagy, using3-MA, sensitized cells to SDT-induced cell death through apoptosis and necrosis, and the latter was more than the former. Similarly, inhibition of the later stage of autophagy, using Ba Al, also enhanced SDT induced cell death, but was less efficient than3-MA, which mainly promoted cell apoptosis. Therefore, irrespective of the stage at which autophagy was inhibited, disabled autophagy accelerated SDT induced cell death. Therefore, the findings can be incorporated into a more general hypothesis suggesting the efficiency of killing cancer cells by sonodynamic therapy may be enhanced by the simultaneous treatment with autophagy inhibitors.
7. Because different tumor cell lines have different sensitivities to ultrasound irradiation, this study firstly selected the optimal SDT parameters in which SDT treatment can reduce the cell viability of L1210cells and simultaneously induce autophagy occurrence. Some key proteins in the process of autophagy showed time dependent changes after SDT treatment.
8. SDT can significantly inhibit the rapid proliferation of murine leukemia L1210cells, and the corresponding mechanism may be associated with the induced mitochondrial--dependent apoptosis pathway.
9. Following SDT, autophagy flux well before cell apoptosis. The relative percentages of cells undergoing apoptosis and autophagy following SDT could be experimentally manipulated. Pre-incubation with autophagy inhibitors prior to SDT promoted the appearance of apoptosis and suppressed AVOs formation.
10. ROS play important role in initiating cell autophagic and apoptotic responses. The mitochondria damage and DNA damage had great relationship with the occurrence of autophagy.
引文
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